Monoclonal Antibodies as Pharmacokinetic Antagonists for the Treatment of (+)-Methamphetamine Addiction

Developing specific medications to treat (+)-methamphetamine (METH) addiction is a difficult challenge because METH has multiple sites of action that are intertwined with normal neurological function. As a result, no small molecule medication for the treatment of METH addiction has made it through the FDA clinical trials process. With the invention of a new generation of protein-based therapies, it is now possible to consider treating drug addiction by an entirely different approach. This new approach is based on the discovery of very high affinity anti-METH monoclonal antibodies (mAbs), which are non-addictive and antagonize METH effects from the blood stream without entering the brain. Due to a very long biological half-life, anti-METH mAbs would only need to be administered once every 2-4 weeks, aiding in patient compliance. As a relapse prevention medication, anti-METH mAbs could reduce or prevent the rewarding effects of a relapse to METH use and thereby improve a patient's probability of remaining in therapy and recovering from their addiction. In this review, we discuss the discovery process of anti-METH mAbs, with a focus on the preclinical development leading to high affinity anti-METH mAb antagonists

The Development and Characterization of an scFv-Fc Fusion-Based Gene Therapy to Reduce the Psychostimulant Effects of Methamphetamine Abuse

Methamphetamine (METH) continues to be among the most addictive and abused drugs in the United States. Unfortunately, there are currently no Food and Drug Administration-approved pharmacological treatments for METH-use disorder. We have previously explored the use of adeno-associated viral (AAV)-mediated gene transfer of an anti-METH monoclonal antibody. Here, we advance our approach by generating a novel anti-METH single-chain variable fragment (scFv)-Fc fusion construct (termed 7F9-Fc) packaged into AAV serotype 8 vector (called AAV-scFv-Fc) and tested in vivo and ex vivo. A range of doses [1 × 10 , 1 × 10 , and 1 × 10 vector copies (vcs)/mouse] were administered to mice, eliciting a dose-dependent expression of 7F9-Fc in serum with peak circulating concentrations of 48, 1785, and 3831 µg/ml, respectively. Expressed 7F9-Fc exhibited high-affinity METH binding, IC = 17 nM. Between days 21 and 35 after vector administration, at both 1 × 10 vc/mouse and 1 × 10 vc/mouse doses, the AAV-7F9-Fc gene therapy significantly decreased the potency of METH in locomotor assays. On day 116 post-AAV administration, mice expressing 7F9-Fc sequestered over 2.5 times more METH in the serum than vehicle-treated mice, and METH concentrations in the brain were reduced by 1.2 times the value for vehicle mice. These data suggest that an AAV-delivered anti-METH Fc fusion antibody could be used to persistently reduce concentrations of METH in the central nervous system. SIGNIFICANCE STATEMENT: In this manuscript, we describe the testing of a novel antimethamphetamine (METH) single-chain variable fragment-Fc fusion protein delivered in mice using gene therapy. The results suggest that the gene therapy delivery system can lead to the production of significant antibody concentrations that mitigate METH's psychostimulant effects in mice over an extended time period

The development and characterization of an scFv-Fc fusion based gene therapy to reduce the psychostimulant effects of methamphetamine abuse

Abstract Methamphetamine (METH) continues to be amongst the most addictive and abused drugs in the US. Unfortunately, there are currently no FDA approved pharmacological treatments for METH substance abuse disorder. As an alternative approach, we have previously explored the use of Adeno-associated viral (AAV) mediated gene transfer of an anti-METH monoclonal antibody. Here, we advance our approach by generating a novel anti-METH scFv-Fc fusion construct (7F9-Fc), packaged into AAV serotype 8 vector (called AAV-scFv-Fc), and tested in vivo and ex vivo. A range of doses (1 × 10 10 . 1 × 10 11 , and 1 × 10 12 vector copies(vc)/mouse) were administered to mice, which exhibited a dose-dependent expression of 7F9-Fc in serum with peak circulating concentrations of 48, 1785, and 3,831 μg/ml. The dose of 1 × 10 12 vc/mouse was further tested in METH locomotor and biodistribution studies to determine the efficacy of the antibody protection. Expressed 7F9-Fc exhibited high affinity binding, 17 nM, to METH. Between days 21 and 35 after vector administration, the 7F9-Fc gene therapy significantly reduced the effects of METH in locomotor assays following administration of moderate and high doses of subcutaneous METH, 3.1 and 9.4 mg/kg respectively. On day 116 post-AAV administration, mice expressing 7F9-Fc sequestered over 2.5 times more METH into the serum than vehicle mice, and METH concentrations in the brain were reduced by 1.2 times compared to vehicle mice. Taken together, these data suggest that a AAV-delivered anti-METH Fc fusion antibody could be a design for persistently reducing concentrations of METH in the CNS. Significance Statement In this manuscript, we describe the use of a novel anti-METH scFv-Fc fusion protein delivered in mice using gene therapy. The results suggest that the gene therapy delivery system can lead to the production of enough antibody to mitigate METH’s psychostimulant effects in mice over an extended time period

Development and testing of AAV-delivered single-chain variable fragments for the treatment of methamphetamine abuse

Methamphetamine (METH) substance abuse disorders have major impact on society, yet no medications have proven successful at preventing METH relapse or cravings. Anti-METH monoclonal antibodies can reduce METH brain concentrations; however, this therapy has limitations, including the need for repeated dosing throughout the course of addiction recovery. An adeno-associated viral (AAV)-delivered DNA sequence for a single-chain variable fragment could offer long-term, continuous expression of anti-METH antibody fragments. For these studies, we injected mice via tail vein with 1 x 10(12) vector genomes of two AAV8 scFv constructs and measured long-term expression of the antibody fragments. Mice expressed each scFv for at least 212 days, achieving micromolar scFv concentrations in serum. In separate experiments 21 days and 50 days after injecting mice with AAV-scFvs mice were challenged with METH in vivo. The circulating scFvs were capable of decreasing brain METH concentrations by up to 60% and sequestering METH in serum for 2 to 3 hrs. These results suggest that AAV-delivered scFv could be a promising therapy to treat methamphetamine abuse

Simple Radiometric Method for Accurately Quantitating Epitope Densities of Hapten–Protein Conjugates with Sulfhydryl Linkages

Control of small molecule hapten epitope densities on antigenic carrier proteins is essential for development and testing of optimal conditions for vaccines. Yet, accurate determination of epitope density can be extremely difficult to accomplish, especially with the use of small haptens, large molecular weight carrier proteins, and limited amounts of protein. Here we report a simple radiometric method that uses ¹⁴C-labeled cystine to measure hapten epitope densities during sulfhydryl conjugation of haptens to maleimide activated carrier proteins. The method was developed using a (+)-methamphetamine (METH)-like hapten with a sulfhydryl terminus, and two prototype maleimide activated carrier proteins, bovine serum albumin (BSA) and immunocyanin monomers of keyhole limpet hemocyanin. The method was validated by immunochemical analysis of the hapten–BSA conjugates, and least-squares linear regression analysis of epitope density values determined by the new radiometric method versus values determined by matrix-assisted laser desorption/ionization mass spectrometry. Results showed that radiometric epitope density values correlated extremely well with the mass spectrometrically derived values (<i>r</i>² = 0.98, <i>y</i> = 0.98<i>x</i> + 0.91). This convenient and simple method could be useful during several stages of vaccine development including the optimization and monitoring of conditions for hapten–protein conjugations, and choosing the most effective epitope densities for conjugate vaccines

The pharmacokinetics of methamphetamine self-administration in male and female rats

BACKGROUND: Because methamphetamine (METH) pharmacokinetics after single iv doses show significant differences between male and female rats, we hypothesized that pharmacokinetic differences in METH disposition could be a contributing factor to the patterns of METH self-administration behaviors in rats. METHODS: For the studies, we used a passive (non-contingent) METH dosing schedule consisting of 27 METH iv bolus injections (0.048 mg/kg) over 2 hrs derived from a previous active (contingent) METH self-administration behavioral study in male rats. After METH dosing of male and female Sprague-Dawley rats (n=5/group), METH and amphetamine serum concentrations were determined by LC-MS/MS. Pharmacokinetic analysis, including predictive mathematical simulations of the data, was then conducted. RESULTS: Male and female rats achieved relatively stable METH serum concentrations within 20 min, which remained constant from 20–120 min. While not statistically different, METH clearance and volume of distribution values for females were 25% and 33% lower (respectively) than males. Linear regression analysis of predicted METH concentrations from pharmacokinetic simulations versus observed concentrations showed a substantially better correlation with male data than female data (r(2) = 0.71 vs. 0.56; slope = 0.95 vs. 0.45, respectively). At 120 min, the time of predicted peak METH serum concentrations, female values were 42% higher than expected, while male values were within 3%. CONCLUSIONS: Unlike METH male pharmacokinetic data, the female data was less predictable during multiple METH administrations and produced overall higher than expected METH concentrations. These findings demonstrate that METH pharmacokinetics could contribute to differences in METH self-administration behaviors in rats

Combining Active Immunization with Monoclonal Antibody Therapy To Facilitate Early Initiation of a Long-Acting Anti-Methamphetamine Antibody Response

We hypothesized that an anti-METH mAb could be used in combination with a METH-conjugate vaccine (MCV) to safely improve the overall quality and magnitude of the anti-METH immune response. The benefits would include immediate onset of action (from the mAb), timely increases in the immune responses (from the combined therapy) and duration of antibody response that could last for months (from the MCV). A novel METH-like hapten (METH-SSOO9) was synthesized and then conjugated to immunocyanin monomers of keyhole limpet hemocyanin (IC_KLH) to create the MCV IC_KLH-SOO9. The vaccine, in combination with previously discovered anti-METH mAb7F9, was then tested in rats for safety and potential efficacy. The combination antibody therapy allowed safe achievement of an early high anti-METH antibody response, which persisted throughout the study. Indeed, even after 4 months the METH vaccine antibodies still had the capacity to significantly reduce METH brain concentrations resulting from a 0.56 mg/kg METH dose

Development and testing of AAV-delivered single-chain variable fragments for the treatment of methamphetamine abuse

<div><p>Methamphetamine (METH) substance abuse disorders have major impact on society, yet no medications have proven successful at preventing METH relapse or cravings. Anti-METH monoclonal antibodies can reduce METH brain concentrations; however, this therapy has limitations, including the need for repeated dosing throughout the course of addiction recovery. An adeno-associated viral (AAV)-delivered DNA sequence for a single-chain variable fragment could offer long-term, continuous expression of anti-METH antibody fragments. For these studies, we injected mice via tail vein with 1 x 10¹² vector genomes of two AAV8 scFv constructs and measured long-term expression of the antibody fragments. Mice expressed each scFv for at least 212 days, achieving micromolar scFv concentrations in serum. In separate experiments 21 days and 50 days after injecting mice with AAV-scFvs mice were challenged with METH <i>in vivo</i>. The circulating scFvs were capable of decreasing brain METH concentrations by up to 60% and sequestering METH in serum for 2 to 3 hrs. These results suggest that AAV-delivered scFv could be a promising therapy to treat methamphetamine abuse.</p></div