Methamphetamine (METH), one of the top drug problems in the United States, has a large number of biological targets, with the most critical targets located in the brain. The range of neurological effects METH can elicit is wide, and the majority of these effects are either rewarding or toxic. Despite this fact, there are no pharmacological treatments for METH abuse that are approved by the FDA. Therefore, we generated an anti-METH single chain antibody fragment (scFv7F9Cys) as a pharmacological treatment option for METH abuse. However, based on its size, scFv7F9Cys will likely have a short half-life in vivo, which would limit the potential use of scFv7F9Cys to the treatment of METH overdose. Thus, the aim of this dissertation was to create a longer lasting form of scFv7F9Cys through conjugation to poly(ethylene) glycol (PEG) (scFv7F9Cys-PEG), to evaluate the change in half-life using pharmacokinetic analysis, and to determine whether the extension in half-life will provide better protection against METH-induced neurological changes.
The goal of the first major aim was to test the in vitro effects of conjugating scFv7F9Cys to three different PEG’s, linear 5 kDa and 20 kDa PEGs, and a branched 40 kDa PEG. ScFv7F9Cys-PEG5K was conjugated but then unable to be purified, and thus was eliminated from future studies. The 40 kDa PEG was conjugated and purified; however, scFv7F9Cys-PEG40K displayed a loss of METH binding activity in vitro. Therefore, it was eliminated from pharmacokinetic analysis. The 20 kDa conjugate was purified and bound to METH with no change in affinity compared to scFv7F9Cys in vitro, suggesting PEGylation did not alter METH binding affinity. Therefore, the evaluation of scFv7F9Cys-PEG20K was extended to in vivo pharmacokinetic studies.
Pharmacokinetic parameters of scFv7F9Cys and scFv7F9Cys-PEG20K were then tested in an in vivo rat model for the second major aim. Rats were administered METH (3.2 mg/kg/day, s.c.) and treated with either scFv7F9Cys, scFv7F9Cys-PEG20K, or saline (30 mg/kg, i.v.) containing a radiolabeled tracer. Blood samples were taken at multiple time points after therapeutic protein administration and analyzed for therapeutic protein concentrations (scintillation counting) and METH concentrations (LC-MS/MS). Our results indicate PEGylation increased the serum half-life of scFv7F9Cys by 27-fold. Furthermore, PEGylation significantly extended scFv7F9Cys’s ability to alter METH disposition back into the serum from a 2 hour increase in METH serum concentrations to a 48 hour increase.
The effects of these pharmacokinetic antagonists on METH neurotoxicity markers have not been tested. Therefore, the purpose of the third major aim was to determine the effects of chronic administration of METH with scFv7F9Cys or scFv7F9Cys-PEG20K on three markers of the dopaminergic system: dopamine transporter (DAT), tyrosine hydroxylase (TH), and VMAT2; and three markers in the glutamatergic system: NMDA receptor (NR2A), AMPA receptor (GluR2), and PSD-95. Brain samples were harvested 144 hours post-pump implantation (METH administration), flash frozen, and pulverized into a fine powder. The pulverized brain tissue was then processed for qRT-PCR and Western blot analysis for all six markers. qRT-PCR results showed a significant reduction in TH, NR2A, GluR2, and PSD-95 mRNA expression in the scFv7F9Cys and scFv7F9Cys-PEG20K treatment groups. However, Western blotting revealed that only TH protein levels were significantly reduced in the scFv7F9Cys-PEG20K group. These unexpected findings will be the basis for further study of the relationship of anti-METH scFvs and neuronal markers of toxicity.
|Advisor:||Peterson, Eric C.|
|Commitee:||Gottschall, Paul E., Owens, S. Michael, Rusch, Nancy J., Waterhouse, Barry D.|
|School:||University of Arkansas for Medical Sciences|
|School Location:||United States -- Arkansas|
|Source:||DAI-B 76/09(E), Dissertation Abstracts International|
|Keywords:||Meth, Methamphetamine, scFv7F9Cys|
Copyright in each Dissertation and Thesis is retained by the author. All Rights Reserved
The supplemental file or files you are about to download were provided to ProQuest by the author as part of a
dissertation or thesis. The supplemental files are provided "AS IS" without warranty. ProQuest is not responsible for the
content, format or impact on the supplemental file(s) on our system. in some cases, the file type may be unknown or
may be a .exe file. We recommend caution as you open such files.
Copyright of the original materials contained in the supplemental file is retained by the author and your access to the
supplemental files is subject to the ProQuest Terms and Conditions of use.
Depending on the size of the file(s) you are downloading, the system may take some time to download them. Please be