Introduction: An active metabolite of buprenorphine (BUP), called norbuprenorphine (NorBUP), is implicated in neonatal opioid withdrawal syndrome when BUP is taken during pregnancy. Therefore, reducing or eliminating metabolism of BUP to NorBUP is a novel strategy that will likely lower total fetal exposure to opioids and thus improve offspring outcomes. Precision deuteration alters pharmacokinetics of drugs without altering pharmacodynamics. Here, we report the synthesis and testing of deuterated buprenorphine (BUP-D2). Methods: We determined opioid receptor affinities of BUP-D2 relative to BUP with radioligand competition receptor binding assays, and the potency and efficacy of BUP-D2 relative to BUP to activate G-proteins via opioid receptors with [35S]GTPγS binding assays in homogenates containing the human mu, delta, or kappa opioid receptors. The antinociceptive effects of BUP-D2 and BUP were compared using the warm-water tail withdrawal assay in rats. Blood concentration versus time profiles of BUP, BUP-D2, and NorBUP were measured in rats following intravenous BUP-D2 or BUP injection. Results: The synthesis provided a 48% yield and the product was ≥99% deuterated. Like BUP, BUP-D2 had sub-nanomolar affinity for opioid receptors. BUP-D2 also activated opioid receptors and induced antinociception with equal potency and efficacy as BUP. The maximum concentration and the area under the curve of NorBUP in the blood of rats that received BUP-D2 were over 19- and 10-fold lower, respectively, than in rats that received BUP. Discussion: These results indicate that BUP-D2 retains key pharmacodynamic properties of BUP and resists metabolism to NorBUP and therefore holds promise as an alternative to BUP.

In the United States, at least 1.6 million people use methamphetamine (METH) annually, with over half of these meeting criteria for a substance use disorder. Extended METH use causes a state of chronic neural inflammation, which can lead to lasting detrimental effects if left untreated. Due to the cognitive, behavioral, and financial toll of METH use disorders, further investigation into the mechanism behind the persistence of these effects is warranted. Our lab conducted a pilot behavioral and proteomics study using a rodent model of intravenous (i.v.) METH self-administration to simulate daily METH use. We aimed to identify key protein expression changes in specific brain regions affected by METH-induced inflammation as potential future therapeutic targets. We hypothesized that in a model emulating contingent, chronic METH usage and subsequent long-term washout, we would observe regional and molecular proteomic changes indicating the persistence of METH-induced neural inflammation. Male Sprague-Dawley rats (N = 8) were trained to acquire a METH dose of 0.1 mg/kg via lever-press, and after acquisition criteria were met, underwent dose-substitution testing with METH (0.001-0.32 mg/kg/inf) in daily, 2-hour sessions. This was followed by progressive ratio testing in which rats responded for saline or METH (0.010-0.1 mg/kg/inf) in 12-hour sessions (all rats had 3-4 months access to METH). After a 72-hour washout period post-METH exposure, a subset of the brains (n = 3) were sectioned into hippocampus, striatum, frontal cortex, and cerebellum regions for non-targeted proteomics analysis. Identification of a total of 6,892 proteins was performed using a MaxQuant (Max Planck Institute) database search against Rattus norvegicus (May 2020), and these data were examined via Qiagen's Ingenuity Pathway Analysis tool. Our results displayed upregulation of acute phase response signaling across the four brain regions studied, indicating a METH-induced inflammatory state had been achieved and maintained despite a 72-hour period free of METH self-administration. Four key pro-inflammatory upstream regulators were identified to be upregulated - OSM, inosine, JUN, and IL1B. Inosine is an anti-inflammatory agent. OSM, JUN, and IL1B are activated by the Toll-like Receptor 4 (TLR4) and downstream Peroxisome Proliferator-Activated Receptor-Gamma (PPAR-γ) pathways, each of which have been previously implicated to modulate inflammatory signals in other substances of abuse such as ethanol, cocaine, and heroin. METH has been previously noted to induce microglial activation via a stabilizing interaction with TLR4, though mitigating METH-induced neuroinflammation via modulating regulators downstream of this interaction remains an underdeveloped area of study. Our preliminary findings suggest that TLR4 and PPAR-γ may play a role as important regulators of METH-induced neural inflammatory changes at least 72-hours post-exposure, and are potential targets for METH use disorder therapies.

Bleomycin, a chemotherapy agent that inhibits synthesis of DNA, has been increasingly utilized in sclerotherapy for patients with vascular malformations. A serious long-term risk of intravenous bleomycin is dose-dependent interstitial pneumonitis. Little is known about absorption and circulating levels of bleomycin when used in sclerotherapy for patients with vascular malformations. This is an Institutional Review Board (IRB)-approved prospective study on patients receiving bleomycin sclerotherapy in the management of vascular malformations. Depending on the type of vascular malformation, bleomycin was administered either in the lumen or interstitial space of the involved lesion. A bleomycin assay measured serum bleomycin plasma concentrations versus time at seven intervals following treatment. Pharmacokinetic parameters were obtained for each participant and included peak plasma concentration (C-max), time to reach peak plasma concentration (T-max), volume of distribution (V-d), elimination half-life (t(1/2)), the volume of plasma cleared of the drug per unit time (CL), and total systemic exposure area under the curve (AUC). Fifteen patients were enrolled (5: lymphatic, 4: venous, 6: arteriovenous malformations). Bleomycin was administered interstitially (IS) in 11 patients and intraluminal (IL) in four; median age of 13 years (range: 2-67). Pharmacokinetic analysis revealed terminal elimination half-life (t(1/2 lambda z)) of 88.51 (+/- 23.09) and 111.61 (+/- 37.75) minutes for the IS and IL groups, respectively. V-d was 4.86 L (+/- 6.74) and 1.55 L (+/- 0.54) for the IS and IL groups, respectively. AUC was 53.9 (+/- 23.45) and 129.17 (+/- 93.57) mg min/L for the IS and IL groups, respectively. There were no statistically significant differences in t(1/2 lambda z), V-d, or AUC parameters between groups. Bleomycin is absorbed systemically when used as a sclerosant for vascular malformations when injected either IS or IL.

Current guidelines recommend restricting acetaminophen (APAP) use in patients with cirrhosis, but evidence to support that recommendation is lacking. Prior studies focused on pharmacokinetics (PK) of APAP in cirrhosis but did not rigorously examine clinical outcomes, sensitive biomarkers of liver damage, or serum APAP‐protein adducts, which are a specific marker of toxic bioactivation. Hence, the goal of this pilot study was to test the effects of regularly scheduled APAP dosing in a well‐defined compensated cirrhosis group compared to control subjects without cirrhosis, using the abovementioned outcomes. After a 2‐week washout, 12 subjects with and 12 subjects without cirrhosis received 650 mg APAP twice per day (1.3 g/day) for 4 days, followed by 650 mg on the morning of day 5. Patients were assessed in‐person at study initiation (day 1) and on days 3 and 5. APAP‐protein adducts and both conventional (alanine aminotransferase) and sensitive (glutamate dehydrogenase [GLDH], full‐length keratin 18 [K18], and total high‐mobility group box 1 protein) biomarkers of liver injury were measured in serum on the mornings of days 1, 3, and 5, with detailed PK analysis of APAP, metabolites, and APAP‐protein adducts throughout day 5. No subject experienced adverse clinical outcomes. GLDH and K18 were significantly different at baseline but did not change in either group during APAP administration. In contrast, clearance of APAP‐protein adducts was dramatically delayed in the cirrhosis group. Minor differences for other APAP metabolites were also detected. Conclusion: Short‐term administration of low‐dose APAP (650 mg twice per day, <1 week) is likely safe in patients with compensated cirrhosis. These data provide a foundation for future studies to test higher doses, longer treatment, and subjects who are decompensated, especially in light of the remarkably delayed adduct clearance in subjects with cirrhosis.

3,4‐Methylenedioxymethamphetamine (MDMA) is a popular recreational psychostimulant and hallucinogen typically used in “rave” settings. Despite the “positive” feelings sometimes associated with its use, there are toxicities that accompany its use. In the periphery, MDMA causes immunosuppression that can make users more susceptible to infection. Conversely, in the CNS MDMA initiates a proinflammatory response that is linked to glial activation and the subsequent overproduction of cytokines, chemokines, and even cellular adhesion molecules. A treatment that could potentially mitigate these effects would be beneficial.In our laboratory, we are developing an anti‐MDMA/methamphetamine (METH) dual‐acting antibody gene therapy as a treatment for substance use disorders (SUD). Utilizing the adeno‐associated virus AAV8 as our gene therapy vector, we packaged the single chain‐Fc fusion fragment 7F9‐Fc to create AAV‐7F9‐Fc. AAV‐7F9‐Fc expresses anti‐MDMA/METH antibodies systemically that work as pharmacokinetic antagonists to slow and possibly reduce entry of drug into the brain and other organs. AAV‐7F9‐Fc produces effective long‐term serum concentrations of antibodies in mice (>7 months from a single dose) and has been shown to alter METH‐induced locomotor effects in mice. Because of the chemical backbone similarity between METH and MDMA, we hypothesized that AAV‐7F9‐Fc would also be able to bind to MDMA in serum and reduce the immunomodulatory effects induced by MDMA. If proven effective, this treatment could provide some protection against the toxic effects of METH and MDMA during recovery.We hypothesized that an AAV delivered antibody fusion fragment (AAV‐7F9‐Fc) will provide extended protection from MDMA‐induced neuroinflammation and systemic immune suppression. To begin to test this hypothesis, we are evaluating an acute mouse model of MDMA use. Drug administration followed a binge dosing regimen of an ip injection every 2 hr for 4 consecutive doses. After the MDMA dosing regimen, we measured serum cytokine levels by multiplex analysis, and CNS proinflammatory cytokine and glial cell activation markers by qRT‐PCR. BALB/c mice were divided into nine groups (n=5/group); 1) Saline + Saline, 2) Saline + MDMA (10 mg/kg 2hr × 4), 3) Saline + MDMA (3 mg/kg 2hr × 4), 4) Saline + MDMA (1 mg/kg 2hr × 4) 5) LPS, 6) AAV‐7F9‐Fc + Saline, 7) AAV‐7F9‐Fc + MDMA (10 mg/kg 2hr × 4), 8) Empty Vector + saline & 9) Empty Vector + MDMA (10 mg/kg 2hr × 4). Our results indicate significant decreases (p< 0.05) in the serum concentration of proinflammatory cytokine IL‐1β & IFN‐γ and increases in the anti‐inflammatory cytokine IL‐10 (p< 0.05) in response to the 10 mg/kg MDMA dose. In the striatum, 10 mg/kg MDMA elicited a significant increase in expression of IL‐1β and astrocyte activation marker GFAP. AAV‐7F9‐Fc treatment mitigated the increase of IL‐10 in the serum and IL‐1β in the striatum. These results indicate that in addition to its previously reported protection against METH, AAV‐7F9‐Fc can mitigate an immunomodulatory cascade in the periphery and CNS resulting from MDMA exposure.Support or Funding InformationThis research was funded by NIH/NIDA:R01‐DA036600 & R01 DA036600‐03S1, NIH/NIGMS:T32‐GM106999, and R25GM083297.

Disrupting transmission of Borrelia burgdorferi sensu lato complex (B. burgdorferi) from infected ticks to humans is one strategy to prevent the significant morbidity from Lyme disease. We have previously shown that an anti-OspA human mAb, 2217, prevents transmission of B. burgdorferi from infected ticks in animal models. Maintenance of a protective plasma concentration of a human mAb for tick season presents a significant challenge for a preexposure prophylaxis strategy. Here, we describe the optimization of mAb 2217 by amino acid substitutions (2217LS: M428L and N434S) in the Fc domain. The LS mutation led to a 2-fold increase in half-life in cynomolgus monkeys. In a rhesus macaque model, 2217LS protected animals from tick transmission of spirochetes at a dose of 3 mg/kg. Crystallographic analysis of Fab in complex with OspA revealed that 2217 bound an epitope that was highly conserved among the B. burgdorferi, B. garinii, and B. afzelii species. Unlike most vaccines that may require boosters to achieve protection, our work supports the development of 2217LS as an effective preexposure prophylaxis in Lyme-endemic regions, with a single dose at the beginning of tick season offering immediate protection that remains for the duration of exposure risk.

Background Histone post-translational modifications (PTMs) play an important role in our system by regulating the structure of chromatin and therefore contribute to the regulation of gene and protein expression. Irregularities in histone PTMs can lead to a variety of different diseases including various forms of cancer. Histone modifications are analyzed using high resolution mass spectrometry, which generate large amounts of data that requires sophisticated bioinformatics tools for analysis and visualization. PTMViz is designed for downstream differential abundance analysis and visualization of both protein and/or histone modifications. Results PTMViz provides users with data tables and visualization plots of significantly differentiated proteins and histone PTMs between two sample groups. All the data is packaged into interactive data tables and graphs using the Shiny platform to help the user explore the results in a fast and efficient manner to assess if changes in the system are due to protein abundance changes or epigenetic changes. In the example data provided, we identified several proteins differentially regulated in the dopaminergic pathway between mice treated with methamphetamine compared to a saline control. We also identified histone post-translational modifications including histone H3K9me, H3K27me3, H4K16ac, and that were regulated due to drug exposure. Conclusions Histone modifications play an integral role in the regulation of gene expression. PTMViz provides an interactive platform for analyzing proteins and histone post-translational modifications from mass spectrometry data in order to quickly identify differentially expressed proteins and PTMs.

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 × 1010, 1 × 1011, and 1 × 1012 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, IC50 = 17 nM. Between days 21 and 35 after vector administration, at both 1 × 1011 vc/mouse and 1 × 1012 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 technological advances in mass spectrometry allow us to collect more comprehensive data with higher quality and increasing speed. With the rapidly increasing amount of data generated, the need for streamlining analyses becomes more apparent. Proteomics data is known to be often affected by systemic bias from unknown sources, and failing to adequately normalize the data can lead to erroneous conclusions. To allow researchers to easily evaluate and compare different normalization methods via a user-friendly interface, we have developed "proteiNorm". The current implementation of proteiNorm accommodates preliminary filters on peptide and sample levels followed by an evaluation of several popular normalization methods and visualization of the missing value. The user then selects an adequate normalization method and one of the several imputation methods used for the subsequent comparison of different differential expression methods and estimation of statistical power. The application of proteiNorm and interpretation of its results are demonstrated on two tandem mass tag multiplex (TMT6plex and TMT10plex) and one label-free spike-in mass spectrometry example data set. The three data sets reveal how the normalization methods perform differently on different experimental designs and the need for evaluation of normalization methods for each mass spectrometry experiment. With proteiNorm, we provide a user-friendly tool to identify an adequate normalization method and to select an appropriate method for differential expression analysis.

Methamphetamine (METH) is a potent psychostimulant, with no FDA‐approved pharmacological treatment options. METH can adversely affect brain homeostasis by initiating dysfunction in the Blood Brain Barrier (BBB) and hyperthermia. The BBB is a semipermeable barrier composed of endothelial cells and pericytes along blood capillaries. These cells are held together by tight junction (TJ) proteins (e.g., claudin‐5, occludin, etc) which limit paracellular transport and help regulate body temperature. METH can decrease the expression of these TJ proteins and induce hyperthermia leading to BBB dysfunction. Thus, a novel therapy that could mitigate these effects is needed.A novel treatment under evaluation in our laboratory is an anti‐METH single chain variable antibody‐fusion antibody (7F9‐Fc) packaged into adeno‐associated virus (AAV8) particles. AAV‐7F9‐Fc works as a pharmacokinetic antagonist to slow and reduce entry of METH into the brain. AAV‐7F9‐Fc has achieved long‐term serum concentrations in mice (>7 months from a single dose) and has been shown to alter locomotor effects of METH in mice. This treatment could have clinical potential to attenuate the effects of METH during drug use and aid addiction recovery.We hypothesized that an AAV delivered anti‐METH antibody fragment (AAV‐7F9‐Fc) will mitigate METH‐induced hyperthermia and BBB dysfunction. To begin to test this hypothesis, we recorded baseline temperatures of mice 1 h before administration of either saline or METH (10 mg/kg). Male BALB/c mice were randomized into one of five groups (n=5/group); 1) Saline + Saline, 2) Saline + METH, 3) AAV‐7F9‐Fc + Saline, 4) AAV‐7F9‐Fc + METH, 5) AAV‐empty vector + saline. After administration of either Saline or METH, rectal temperatures were probed for 1 h, and brains were collected at the 3 h time point. Brains were dissected on dry ice into key dopaminergic regions such as the Frontal Cortex and Hippocampus. After dissection, proteins were extracted via RIPA buffer and analyzed for their expression by Western Blot. In Saline+METH treated mice, a 40% decrease in expression of Claudin‐5 and a 45% decrease in expression of Occludin (p < 0.05) were observed in the Frontal Cortex. These results were mitigated in AAV‐7F9‐Fc + METH mice in the Frontal Cortex and Hippocampus. These results indicate two important findings in an acute setting. This indicates that AAV‐7F9‐Fc has the potential to mitigate in an acute administration of METH, 1). the ability of METH to induce Hyperthermia, and 2). cause a reduction of critical tight junction protein expression in the CNS.Support or Funding InformationThis research was funded by NIH/NIDA:R01‐DA036600 & R01 DA036600‐03S1, NIH/NIGMS:T32‐GM106999, and R25GM083297.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

alpha-PVP (alpha-pyrrolidinovalerophenone) and MDPV (3,4-methylenedioxypyrovalerone) are potent abused stimulants that are members of the synthetic cathinone class of drugs. Although these drugs are taken with recreational intent, high doses can lead to unintended adverse effects including agitation, cardiovascular effects, sympathomimetic syndromes, hallucinations, and psychoses. One possible treatment is the use of a vaccine to block or attenuate adverse medical effects. These studies report the preparation of a vaccine that generates high affinity antibodies specific for both drugs and the pharmacological testing of this vaccine in male rats. Alkylation of a hydroxy-alpha-PVP analog with an appropriate thiol-bearing linker afforded the hapten. When hapten-conjugated carrier protein was mixed with adjuvant, the resulting vaccine stimulated production of antibodies in male Sprague Dawley rats that were found to significantly reduce alpha-PVP- and MDPV-induced hyperlocomotion as well as to significantly reduce the concentrations of MDPV drugs in critical organs. The novel vaccine produced high affinity antibodies against MDPV, (R)-MDPV, (S)-MDPV, and alpha-PVP. Cross-reactivity testing against nine structurally similar cathinones showed very limited binding, and no binding to off-target endogenous and exogenous compounds. Antibodies generated by this bi-specific vaccine also significantly shortened the duration of locomotor activity induced by both drugs up to a dose of 5.6 mg/kg in male rats. (C) 2019 Elsevier Ltd. All rights reserved.

Methamphetamine (METH) is a highly abused, addictive drug that can adversely affect the peripheral immune response, causing immune dysregulation in the user. This can lead to an overproduction of cytokines, chemokines, and cellular adhesion molecules causing potential physiologic damage. Thus novel, sustained treatments that focus on mitigating these effects should be evaluated.Our laboratory is combining an anti‐METH single chain variable antibody‐fusion fragment (7F9‐Fc) with adeno‐associated virus (AAV) particles as a potential gene therapy. AAV‐7F9‐Fc works as a pharmacokinetic antagonist to slow and reduce entry of METH into the brain and can achieve lasting serum concentrations in mice (>7 months) from a single treatment. Thus, this therapy has the clinical potential to attenuate the effects of periodic METH relapse during rehabilitation and possibly decrease associated physiological complications such as inflammation.We hypothesized that an AAV‐delivered anti‐METH antibody fragment (AAV‐7F9‐Fc) will provide protection from METH‐induced inflammation. To test this hypothesis, we established a mouse model of METH‐induced early withdrawal inflammation in our laboratory. Male BALB/c mice were divided into six groups (n=5/group); 1) Saline + Saline, 2) Saline + METH, 3) LPS, 4) AAV‐7F9‐Fc + Saline, 5) AAV‐7F9‐Fc + METH, 6) AAV‐empty vector + saline. METH administration followed a dosing regimen of 1 mg/kg i.p. for seven consecutive days with sacrifice after 72 hrs. We performed a MSD Proinflammatory Panel multiplex analysis to measure important serum cytokine and chemokine levels (e.g., IL‐10, IL‐6, IL‐1®). In brain tissues, we measured markers of glial cell activation, glial fibrillary acidic protein (GFAP), ionized calcium binding adaptor molecule (Iba1) and proinflammatory cytokines via qPCR.Our results indicate that this dosing regimen lead to a significant (p < 0.05) increase in proinflammatory mediators (e.g., IL‐1® and IL‐6) in the striatum of Saline + METH mice, but not AAV‐7F9‐Fc + METH mice. In the hippocampus and frontal cortex, the Saline + METH mice showed a significant increase in the mRNA transcripts of IL‐6 & IL‐1® vs those mice treated with AAV‐7F9‐Fc + METH. Noticeably, GFAP was also increased in all observed brain regions of Saline + METH vs those treated with AAV‐7F9‐Fc + METH. Our results also indicate that this dosing regimen lead to a significant increase in serum proinflammatory cytokines (e.g., IL‐1® and IL‐6) vs the AAV‐7F9‐Fc + METH mice. Taken together, these data indicate that a chronic, low dose METH regimen can induce proinflammatory cytokine elevations in serum and brain, increase activation of glial cell markers in brain tissue, and that AAV‐7F9‐Fc treatment has the potential to mitigate some of these proinflammatory effects.Support or Funding InformationFunded by NIH R01‐DA036600, R01 DA036600‐03S1, T32‐GM106999, and R25GM083297This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

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.

There currently are no FDA‐approved pharmacological treatments to aid in treatment of methamphetamine (METH) substance use disorders. To address this, our laboratory has combined short chain variable fragments (scFvs) derived from antibodies with high binding affinity to METH with adeno‐associated viruses (AAV) to develop a long‐lasting potential therapy. These first generation gene therapies showed expression of the anti‐METH scFvs for at least 212 days but achieved an average serum concentration of less than 70.0 μg/ml in mice. We hypothesized that incorporating a constant region (Fc) derived from an IgG antibody would extend serum half‐life and increase circulating concentrations of the anti‐METH antibody fragment, thus providing greater levels of protection against METH in mice. To test this hypothesis, we cloned an anti‐METH scFv‐Fc encoding DNA sequence into custom expression plasmids and packaged into AAV serotype 8 viral vectors to make AAV‐7F9‐Fc. To test this new gene therapy, BALB/C mice were administered 1×1012 vector copies per mouse of either the AAV‐7F9‐Fc or empty AAV8 vector. Mice were allowed to reach peak AAV‐7F9‐Fc expression (~28 days, as determined by ELISA) before testing in locomotor studies. Locomotor activity was used to measure protection from the stimulant effects of METH. Briefly, after a 30‐minute baseline activity was recorded, mice were administered saline, 0.56, 1.0, 3.0, or 5.56 mg/kg METH sc, and locomotor activity was measured for 90 minutes. METH was administered consecutively with a two‐day washout period in between doses. This regimen was also performed on separate mice 7–8 months after administration of AAV‐7F9‐Fc therapy to test duration of protection. Our results show that by incorporating an IgG Fc moiety into the anti‐METH constructs, the average expression increased by over 30 fold compared to a our first generation scFvs to a peak serum concentration of 5.227.7 μg/ml, and mean of 2,116.1 μg/ml, and persisted over 239 days. The AAV‐7F9‐Fc mice showed significantly less locomotor activity (p < 0.05) than empty AAV8 treated mice at the 1.0 and 3.0 mg/kg METH doses. Furthermore, the AAV‐7F9‐Fc treated mice showed no significant activity differences from baseline at any of the METH doses administered. In a separate, long‐term study, 7–8 months after AAV‐7F9‐Fc treatment, mice showed significantly lower levels of locomotor activity at 0.56 and 1.0 mg/kg sc METH doses than the sham treated group. These data suggest our AAV‐7F9‐Fc can protect against stimulant effects of METH, and that the duration of action is at least 8 months in mice. Overall, these data suggest that 1) the addition of the IgG Fc region into the AAV‐scFv constructs resulted in dramatically higher anti‐METH antibody circulating concentrations than our first generation anti‐METH scFvs and 2) this higher antibody titer was able to provide protection against METH stimulant effects at low to moderate doses for at least 8 months in mice.Support or Funding InformationSupported by NIDA R01 DA036600 & NIGMS T32 GM106999This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

The heavy chain IGHV1-69 germline gene exhibits a high level of polymorphism and shows biased use in protective antibody (Ab) responses to infections and vaccines. It is also highly expressed in several B cell malignancies and autoimmune diseases. G6 is an anti-idiotypic monoclonal Ab that selectively binds to IGHV1-69 heavy chain germline gene 51p1 alleles that have been implicated in these Ab responses and disease processes. Here, we determine the co-crystal structure of humanized G6 (hG6.3) in complex with anti-influenza hemagglutinin stem-directed broadly neutralizing Ab D80. The core of the hG6.3 idiotope is a continuous string of CDR-H2 residues starting with M53 and ending with N58. G6 binding studies demonstrate the remarkable breadth of binding to 51p1 IGHV1-69 Abs with diverse CDR-H3, light chain, and antigen binding specificities. These studies detail the broad expression of the G6 cross-reactive idiotype (CRI) that further define its potential role in precision medicine.

Methamphetamine (METH) abuse is a worldwide drug problem, yet no FDA-approved pharmacological treatments are available for METH abuse. Therefore, we produced an anti-METH single chain antibody fragment (scFv7F9Cys) as a pharmacological treatment for METH abuse. ScFv's have a short half-life due to their small size, limiting their clinical use. Thus, we examined the pharmacokinetic effects of conjugating poly(ethylene) glycol (-PEG) to scFv7F9Cys to extend its functional half-life.The affinity of scFv7F9Cys and PEG conjugates to METH was determined in vitro via equilibrium dialysis saturation binding. Pharmacokinetic and parameters of scFv7F9Cys and scFv7F9Cys-PEG20K (30 mg/kg i.v. each) and their ability to bind METH in vivo were determined in male Sprague-Dawley rats receiving a subcutaneous infusion of METH (3.2 mg/kg/day).Of three PEGylated conjugates, scFv7F9Cys-PEG20K was determined the most viable therapeutic candidate. PEGylation of scFv7F9Cys did not alter METH binding functionality in vitro, and produced a 27-fold increase in the in vivo half-life of the antibody fragment. Furthermore, total METH serum concentrations increased following scFv7F9Cys or scFv7F9Cys-PEG20K administration, with scFv7F9Cys-PEG20K producing significantly longer changes in METH distribution than scFv7F9Cys.PEGylation of scFv7F9Cys significantly increase the functional half-life of scFv7F9Cys, suggesting it may be a long-lasting pharmacological treatment option for METH abuse.

Methamphetamine (METH) is considered one of the top drug problems in the United States today, yet there are no FDA approved pharmacological treatments available for METH abuse. To this end, we have designed and produced an anti-METH single chain antibody fragment (scFv7F9Cys) and a PEGylated scFv (scFv7F9Cys-PEG) as anti-METH pharmacological treatments. However, the effects of these pharmacokinetic antagonists on methamphetamine neurotoxicity markers have not been previously tested. Therefore, the purpose of this study was to determine the effects of chronic administration of METH and treatment with an anti-METH scFv on three makers in 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. Rats received 3.2 mg/kg/day of METH from an osmotic pump (s.c.) for a total of 144 hours. Approximately 48 hours after pump implantation, scFv7F9Cys or scF7F9Cys-PEG was administered (30 mg/kg, i.v.). Brain samples were harvested 144 hours post-pump implantation, 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 (DAT, TH, VMAT2, NR2A, GluR2, PSD-95). qRT-PCR results showed a significant reduction in TH, NMDA, AMPA receptors and PSD-95 mRNA levels in the scFv7F9Cys and scFv7F9Cys-PEG treatment groups. However, Western blotting revealed that only TH protein levels were significantly reduced in the scFv7F9Cys-PEG group. These unexpected findings will be the basis for further study of the relationship of anti-METH scFvs and neuronal markers of toxicity.