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 × 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.

In Utero Exposure to Norbuprenorphine, a Major Metabolite of Buprenorphine, Induces Fetal Opioid Dependence and Leads to Neonatal Opioid Withdrawal Syndrome.

Buprenorphine is the preferred treatment of opioid use disorder during pregnancy but can cause fetal opioid dependence and neonatal opioid withdrawal syndrome (NOWS). Notably, withdrawal severity is independent of maternal buprenorphine dose, suggesting that interindividual variance in pharmacokinetics may influence risk and severity of NOWS. Using a rat model of NOWS, we tested the hypothesis that clinically relevant doses of the active metabolite norbuprenorphine (NorBUP) can induce in utero opioid dependence, manifested as naltrexone-precipitated withdrawal signs in the neonate. Pregnant Long-Evans rats were implanted with 14-day osmotic minipumps containing vehicle, morphine (positive control), or NorBUP (0.3-10 mg/kg per day) on gestation day 9. By 12 hours post-delivery, an intraperitoneal injection of the opioid antagonist naltrexone (1 or 10 mg/kg) or saline was administered to pups. Precipitated withdrawal signs were graded by raters blinded to treatment conditions. In a separate group, NorBUP concentrations in maternal and fetal blood and brain on gestation day 20 were determined by liquid chromatography-tandem mass spectrometry. Steady-state maternal blood concentrations of NorBUP in dams infused with 1 or 3 mg/kg per day were comparable to values reported in pregnant humans treated with buprenorphine (1.0 and 9.6 ng/ml, respectively), suggesting a clinically relevant dosing regimen. At these doses, NorBUP increased withdrawal severity in the neonate as shown by an evaluation of 10 withdrawal indicators. These findings support the possibility that NorBUP contributes to fetal opioid dependence and NOWS following maternal buprenorphine treatment during pregnancy.

Effect of Bile Duct Ligation-induced Liver Dysfunction on Methamphetamine Pharmacokinetics and Locomotor Activity in Rats.

PURPOSE: Methamphetamine (METH) abuse is associated with hepatic dysfunction related comorbidities such as HIV, hepatitis C, and polysubstance abuse with acetaminophen-containing opioid formulations. We aimed to develop a bile duct ligation (BDL)-induced hepatic dysfunction model for studying both METH and experimental treatments for METH abuse in this comorbidity. METHODS: Sham or BDL surgery was performed in male Wistar rats on day 0. Liver function was measured throughout the study. On days 7 and 19, serum pharmacokinetics studies were performed with 1 mg/kg subcutaneous (sc) METH. On day 21, this dose was repeated to determine 2 h post-METH brain concentrations. METH-induced open field behaviors were measured every other day (days 12 - 16) with ascending sc doses (0.3 - 3 mg/kg). RESULTS: BDL transiently increased alanine aminotransferase levels and altered liver structure, which resulted in significantly greater METH serum and brain exposure. In the BDL compared to sham group, there was a longer duration of METH-induced locomotor activity (after 1 and 3 mg/kg) and stereotypy (after 3 mg/kg). CONCLUSIONS: In rats, liver dysfunction reduced METH clearance, increased brain METH concentrations, and enhanced METH effects on locomotor activity in a dose dependent manner. In addition, this model could be further developed to simulate the associated hepatic dysfunction of key METH abuse comorbidities for preclinical testing of novel pharmacotherapies for effectiveness and/or toxicity in vulnerable populations.

PEGylation of a High-Affinity Anti-(+)Methamphetamine Single Chain Antibody Fragment Extends Functional Half-Life by Reducing Clearance.

PURPOSE: 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. METHODS: 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). RESULTS: 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. CONCLUSIONS: PEGylation of scFv7F9Cys significantly increase the functional half-life of scFv7F9Cys, suggesting it may be a long-lasting pharmacological treatment option for METH 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 (14)C-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 (r(2) = 0.98, y = 0.98x + 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.

Treatment with a monoclonal antibody against methamphetamine and amphetamine reduces maternal and fetal rat brain concentrations in late pregnancy.

We hypothesized that treatment of pregnant rat dams with a dual reactive monoclonal antibody (mAb4G9) against (+)-methamphetamine [METH; equilibrium dissociation rate constant (KD) = 16 nM] and (+)-amphetamine (AMP; KD = 102 nM) could confer maternal and fetal protection from brain accumulation of both drugs of abuse. To test this hypothesis, pregnant Sprague-Dawley rats (on gestational day 21) received a 1 mg/kg i.v. METH dose, followed 30 minutes later by vehicle or mAb4G9 treatment. The mAb4G9 dose was 0.56 mole-equivalent in binding sites to the METH body burden. Pharmacokinetic analysis showed baseline METH and AMP elimination half-lives were congruent in dams and fetuses, but the METH volume of distribution in dams was nearly double the fetal values. The METH and AMP area under the serum concentration-versus-time curves from 40 minutes to 5 hours after mAb4G9 treatment increased >7000% and 2000%, respectively, in dams. Fetal METH serum did not change, but AMP decreased 23%. The increased METH and AMP concentrations in maternal serum resulted from significant increases in mAb4G9 binding. Protein binding changed from ∼15% to > 90% for METH and AMP. Fetal serum protein binding appeared to gradually increase, but the absolute fraction bound was trivial compared with the dams. mAb4G9 treatment significantly reduced METH and AMP brain values by 66% and 45% in dams and 44% and 46% in fetuses (P < 0.05), respectively. These results show anti-METH/AMP mAb4G9 therapy in dams can offer maternal and fetal brain protection from the potentially harmful effects of METH and AMP.

The fate and function of therapeutic antiaddiction monoclonal antibodies across the reproductive cycle of rats.

During preclinical development of neuroprotective antiaddiction therapeutic monoclonal antibodies (mAbs) against phencyclidine (PCP) and (+)-methamphetamine, we discovered novel, gestation stage-specific changes in mAb disposition spanning the entire reproductive cycle of female rats. Each pharmacological change was independent of mAb dose and antigen target but was precisely coincident with transitions between the gestational trimesters, parturition, and lactation periods of the female reproductive cycle. Whereas anti-PCP mAb6B5 terminal elimination half-life (t(1/2λz)) in nonpregnant females was 6.6 ± 1.6 days, the mAb6B5 t(1/2λz) significantly changed to 3.7 ± 0.4 days, then 1.4 ± 0.1 days, then 3.0 ± 0.4 days in the second trimester, third trimester, and postpartum periods, respectively (p < 0.05 for each change). Initially, these evolving changes in mAb6B5 clearance (3.3-fold), distribution volume (1.8-fold), and elimination half-life (4.7-fold) affected our ability to sustain sufficient mAb6B5 levels to sequester PCP in the bloodstream. However, understanding the mechanisms underlying each transition allowed development of an adaptive mAb-dosing paradigm, which substantially reduced PCP levels in dam brains and fetuses throughout pregnancy. These mAb functional studies also revealed that antidrug mAbs readily cross the placenta before syncytiotrophoblast barrier maturation, demonstrating the dynamic nature of mAb pharmacokinetics in pregnancy and the importance of maintaining maternal mAb levels. These studies provide the first preclinical pregnancy model in any species for chronic mAb dosing and could have important implications for the use of antibody therapies involving blood organ barriers (such as addiction) or other chronic diseases in women of childbearing age (e.g., irritable bowel diseases, multiple sclerosis, breast cancer, rheumatoid arthritis).

Gestation time-dependent pharmacokinetics of intravenous (+)-methamphetamine in rats.

We tested the hypothesis that differences in (+)-methamphetamine (METH) disposition during late rat pregnancy could lead to increased vulnerability to acute METH effects. The disposition of a single 1 mg/kg i.v. METH dose was studied during early (gestation day 7, GD7) and late (GD21) gestation. Results showed gestation time-dependent pharmacokinetics, characterized by a significantly higher area under the METH serum concentration versus time curve and a lower clearance on GD21 (p < 0.05; total, renal, and nonrenal clearance). The terminal elimination half-life (t(1/2λz)) of METH and (+)-amphetamine (AMP; a pharmacologically active metabolite of METH) were not different on GD7, but by GD21, AMP t(1/2λz) was 37% longer than METH t(1/2λz) (p < 0.05). To identify the mechanism for AMP metabolite changes, intravenous AMP pharmacokinetics on GD21 were compared with AMP metabolite pharmacokinetics after intravenous METH. The intravenous AMP t(1/2λz) was significantly shorter than metabolite AMP t(1/2λz) (p < 0.05), which suggested AMP metabolite formation (not elimination) was the rate-limiting process. To understand the medical consequence of METH use during late-stage pregnancy, timed-pregnant rats received an intravenous dose of saline or METH (1, 3, or 5.6 mg/kg) on GD21, 0 to 2 days antepartum. Although one rat died and another had stillbirths at term after the 5.6-mg/kg dose, the pharmacokinetic values for all of the other animals were not significantly different. In conclusion, late-gestational clearance reductions lengthen METH exposure time, possibly increasing susceptibility to adverse effects, including death.

Active vaccination reduces reinforcing effects of MDPV in male Sprague-Dawley rats trained to self-administer cocaine.

RATIONALE: 3,4-Methylenedioxypyrovalerone (MDPV) is a synthetic cathinone abused for its cocaine-like psychostimulant effects in "bath salts" products. While there are currently no pharmacotherapies for MDPV abuse, rodent studies suggest immunotherapy may offer a feasible treatment option. OBJECTIVES: These studies tested the capacity of active vaccination to reduce the reinforcing effects of MDPV in Sprague-Dawley rats. METHODS: Rats acquired cocaine self-administration (0.32 mg/kg/inf) on an FR1 schedule. Dose-effect functions for cocaine (0.032-1.0 mg/kg/inf) and MDPV (0.001-0.32 mg/kg/inf) were determined under an FR5 schedule. Rats in the vaccine group were immunized during cocaine self-administration. All rats transitioned to a progressive-ratio (PR) schedule to establish breakpoints for cocaine (0.1-1.0 mg/kg/inf) and MDPV (0.01-0.32 mg/kg/inf). Responding was extinguished, and cue-induced and MDPV-primed reinstatement (0.56 mg/kg, IP) were evaluated. RESULTS: No endpoints of cocaine self-administration differed between groups, but the ED50 for MDPV self-administration was significantly lower in control relative to vaccinated rats. Under the PR schedule, MDPV was ~ 2.5-fold more potent in maintaining responding in control than vaccinated rats, but Emax was not different between groups. Vaccination did not reduce MDPV-primed reinstatement, perhaps due to a decrease in antibody titer. CONCLUSIONS: Vaccination did not alter acquisition of cocaine self-administration, demonstrating pharmacological selectivity and suggesting that the vaccine did not affect learning or motivation, while effectively reducing the potency of MDPV as a reinforcer. The protective effects of the vaccine were surmounted by large unit doses of MDPV, suggesting maximal efficacy of drug-conjugate vaccines in substance abuse disorders will likely require concurrent behavior modification therapy.

Effects of a methamphetamine vaccine, IXT-v100, on methamphetamine-related behaviors.

RATIONALE: Vaccines have been developed as a potential treatment for methamphetamine (meth) use disorder (MUD). Immunization with the meth vaccine IXT-v100 has previously been shown to elicit antibodies with high affinity for meth and thus may be an effective treatment for MUD. OBJECTIVES: These studies were designed to determine the efficacy of IXT-v100 on meth-taking and meth-seeking behaviors in rats. METHODS: In the acquisition and maintenance study, male and female rats were trained to self-administer meth (0.06 mg/kg/infusion) over an 8-week period following vaccination. In the last 4 weeks, the dose of meth was increased or decreased each week. To assess meth-seeking behavior, the meth-primed reactivity model was used. Rats were trained to self-administer meth for 5 weeks, followed by a 5-week or 11-week forced abstinence period during which the animals were vaccinated. Rats were then placed back into the self-administration chamber immediately after being injected with meth (1 mg/kg, i.p.) but did not receive meth during the session. Responses were recorded and used as a measure of meth seeking. RESULTS: Results from the acquisition and maintenance study in Wistar rats show that vaccination with IXT-v100 adjuvanted with glucopyranosyl lipid A stable emulsion decreases the percentage of animals that will self-administer a moderate level of meth. In the meth-primed reactivity studies, results from males showed that vaccination significantly attenuates meth-seeking behavior. CONCLUSION: Together, these results suggest vaccination with IXT-v100 may be effective at decreasing meth-taking and meth-seeking behaviors in humans suffering with MUD.

Development of active and passive human vaccines to treat methamphetamine addiction.

Methamphetamine (METH) abuse is a major worldwide epidemic, with no specific medications for treatment of chronic or acute effects. Anti-METH antibodies have the potential to save lives and reduce the crippling effects of METH abuse. While they are not expected to be the magic bullet to immediately cure addiction, immunotherapy could provide a breakthrough medication to continuously block or attenuate METH effects during a comprehensive addiction recovery plan. A unique challenge for METH antibody antagonists is the need to protect the brain from the complex direct and indirect adverse effects of long-term METH use. To meet this challenge, a new generation of passive monoclonal antibodies and active immunization therapies are at an advanced stage of preclinical development. Both of these vaccines could play an essential role in a well planned recovery program from human METH addiction by providing long-lasting protection from the rewarding and reinforcing effect of METH.

Antibody production and pharmacokinetics of METH in rats following vaccination with the METH vaccine, IXT-v100, adjuvanted with GLA-SE.

BACKGROUND: Methamphetamine use disorder continues to be inadequately treated, but improvements are being made in the field of immunotherapeutics, including vaccines, which could provide new options for treatment. Cocaine and nicotine vaccines have been tested clinically, but have yet to elicit the necessary antibody concentrations required to be effective. Methamphetamine vaccines have been tested in multiple nonclinical models and appear promising. Improved adjuvants have the potential to further stimulate the immune system to reach effective levels of antibodies. Previously, the methamphetamine vaccine IXT-v100 was administered with GLA-SE, a toll-like receptor 4 agonist, in mice to produce higher levels of antibodies than when it was administered with two other widely used adjuvants, Alhydrogel and Sigma Adjuvant System. METHODS: The purpose of this research was to evaluate IXT-v100, given in combination with the adjuvant GLA-SE, to determine its efficacy in antagonizing methamphetamine disposition in a rat pharmacokinetic study. Additional rat studies were conducted to compare the ability of IXT-v100 manufactured with greater hapten densities to elicit higher antibody levels. RESULTS: As expected based on prior studies with anti-methamphetamine monoclonal antibodies, the antibodies resulting from vaccination with IXT-v100 altered methamphetamine pharmacokinetics by increasing serum concentrations and extending the half-life. Furthermore, intentional variations in the ratio of components during manufacturing led to production of vaccines with higher hapten densities. The higher hapten densities resulted in production of antibodies that maintained the ability to bind methamphetamine with high affinity. CONCLUSIONS: The results support continued development of IXT-v100 for the treatment of methamphetamine use disorder.

Cardiovascular effects of 3,4-methylenedioxypyrovalerone (MDPV) in male and female Sprague-Dawley rats.

BACKGROUND: 3,4-methylenedioxypyrovalerone (MDPV) toxicity includes intense neurological and cardiovascular events. We examined MDPV-induced cardiovascular, temperature, and locomotor effects following escalating and repeated MDPV administration in adult male and female Sprague-Dawley rats and compared these effects to cocaine in male rats. METHODS: Telemetry devices were surgically implanted to allow continuous measurement of cardiovascular, temperature, and locomotor activity over a 22 h period after dosing. Rats were administered increasing intraperitoneal (IP) MDPV doses (1-5.6 mg/kg) every other day, followed two days later by a binge regimen of four injections of 3 mg/kg MDPV at 2 h intervals. MDPV serum concentrations were measured by LC-MS/MS. Cocaine (3-30 mg/kg) and four injections of 30 mg/kg IP were administered to male rats for comparison with male MDPV data. RESULTS: The duration of MDPV cardiovascular effects was significantly greater (p < 0.05) in male rats than female rats at 3-5.6 mg/kg. The ED50 for MDPV-induced locomotor was significantly lower in males (2.4 ± 0.3) than females (3.4 ± 0.2). Males showed significantly greater variability in MDPV serum concentrations than females after binge dosing. MDPV produced five-fold more potent cardiovascular effects than cocaine in male rats. MDPV did not alter thermoregulation in either sex, but cocaine binge administration decreased temperature. CONCLUSION: Effects of MDPV on temperature were not significantly different between sexes. MDPV-induced cardiovascular and locomotor effects in males lasted significantly longer and were more potent than in females. These differences appeared to be related to pharmacokinetic factors leading to greater variance in MDPV serum concentrations in males.

Development and preclinical testing of a high-affinity single-chain antibody against (+)-methamphetamine.

Chronic or excessive (+)-methamphetamine (METH) use often leads to addiction and toxicity to critical organs like the brain. With medical treatment as a goal, a novel single-chain variable fragment (scFv) against METH was engineered from anti-METH monoclonal antibody mAb6H4 (IgG, kappa light chain, K(d) = 11 nM) and found to have similar ligand affinity (K(d) = 10 nM) and specificity as mAb6H4. The anti-METH scFv (scFv6H4) was cloned, expressed in yeast, purified, and formulated as a naturally occurring mixture of monomer ( approximately 75%) and dimer ( approximately 25%). To test the in vivo efficacy of the scFv6H4, male Sprague-Dawley rats (n = 5) were implanted with 3-day s.c. osmotic pumps delivering 3.2 mg/kg/day METH. After reaching steady-state METH concentrations, an i.v. dose of scFv6H4 (36.5 mg/kg, equimolar to the METH body burden) was administered along with a [(3)H]scFv6H4 tracer. Serum pharmacokinetic analysis of METH and [(3)H]scFv6H4 showed that the scFv6H4 caused an immediate 65-fold increase in the METH concentrations and a 12-fold increase in the serum METH area under the concentration-time curve from 0 to 480 min after scFv6H4 administration. The scFv6H4 monomer was quickly cleared or converted to multivalent forms with an apparent t(1/2lambdaz) of 5.8 min. In contrast, the larger scFv6H4 multivalent forms (dimers, trimers, etc.) showed a much longer t(1/2lambdaz) (228 min), and the significantly increased METH serum molar concentrations correlated directly with scFv6H4 serum molar concentrations. Considered together, these data suggested that the scFv6H4 multimers (and not the monomer) were responsible for the prolonged redistribution of METH into the serum.

Maternal high fat diet during the perinatal period alters mesocorticolimbic dopamine in the adult rat offspring: reduction in the behavioral responses to repeated amphetamine administration.

RATIONALE: Early environment can shape the development and function of the mesocorticolimbic dopamine (DA) system and represents a possible risk factor for adult pathologies. One critical variable in the early environment is nutrition, and exposure to high fat (HF) in adulthood is known to change this DA system. OBJECTIVES: We tested whether perinatal HF intake in rats could have long-term effects on DA function and behavior in adult offspring. MATERIALS AND METHODS: Rat dams were fed either a control (5% fat, CD) or high fat (30% fat, HF) diet during the last week of gestation and lactation, and adult offspring were tested (PND 56-90) after weaning on CD. Locomotor responses to acute and repeated doses of D: -amphetamine (AMP, 0.75 mg/kg bw) were determined as were indices of DA function in the ventral tegmental area (VTA), nucleus accumbens (NAc), and the prefrontal cortex (PFC). RESULTS: Adult HF offspring displayed increased tyrosine hydroxylase expression in the VTA and NAc and significant increases in DA and DOPAC content in the NAc, suggesting an elevated DA tone in this target field. In the NAc, there were no significant changes in D1, D2 receptors, or DA transporter (DAT) levels between diet groups. Perinatal HF feeding reduced AMP-induced locomotion and behavioral sensitization to AMP, suggesting that early diet might have caused long-lasting desensitization of postsynaptic receptor mechanisms in the NAc. CONCLUSIONS: Our results demonstrate that both synthetic activity in VTA neurons and the responsiveness of accumbens DA neurons is altered by maternal nutrition. These effects subside long after termination of exposure to the HF diet.

Engineering and characterization of a mouse/human chimeric anti-phencyclidine monoclonal antibody.

Previously, our laboratory produced a high affinity, anti-phencyclidine (PCP) murine monoclonal antibody (mAb6B5) that also binds other PCP-like arylcyclohexylamines. In this project, mAb6B5 is engineered into a mouse/human chimera (ch-mAb6B5) to assess the feasibility of developing it into a medication for PCP and PCP-like drug abuse. To create ch-mAb6B5, the light and heavy chain constant regions of mAb6B5 were replaced with human kappa and IgG(2) constant regions in order to decrease its potential immunogenicity in humans. To be an effective anti-PCP medication, ch-mAb6B5 must retain the critical immunochemical binding properties of mAb6B5. Expression vectors containing ch-mAb6B5 light chain and heavy chain cDNA were constructed and expressed in the murine myeloma cell line P3X63-Ag8.653. Immunoassays confirm that ch-mAb6B5 is indeed a chimera, composed of mAb6B5's PCP-binding variable domains and human kappa and IgG constant regions. Radioimmunoassays show that ch-mAb6B5 has the same drug-binding profile as mAb6B5. Ch-mAb6B5 and mAb6B5 bind PCP with a K(D) of 0.67 nM and 1.17 nM (respectively) and bind PCP-like arylcyclohexylamines 1-[1-(2-thienyl)cyclohexyl]piperidine and N-ethyl-1-phenylcyclohexylamine with similar specificity. Additionally, ch-mAb6B5 and mAb6B5 have the same calculated isoelectric points and molecular weights, critical properties in antigen-antibody interactions. These data demonstrate that mouse/human ch-mAb6B5, a "more human" version of murine mAb6B5, retains mAb6B5's unique drug-binding properties. This work supports our continued efforts to develop ch-mAb6B5 into a medication for PCP and PCP-like drug abuse - introducing the intriguing possibility of using a single therapeutic mAb for treating a class of abused drugs.

Bioavailability of (+)-methamphetamine in the pigeon following an intramuscular dose.

Pigeons are used frequently as subjects in behavioral pharmacology research. An advantage of the pigeon is an exceedingly vascular breast muscle, which is easily accessible for injections. The purpose of these studies was to provide a profile of the pharmacokinetics of (+)-methamphetamine (METH) and (+)-amphetamine (AMP), a pharmacologically active metabolite, in pigeons (n=6) after intramuscular (i.m.) and intravenous (i.v.) dosing (0.8 mg/kg). LC-MS/MS analysis was used to determine serum concentrations of METH and AMP. A modified crossover design was used to determine the bioavailability, time to maximum concentration, total clearance, the volume of distribution, the maximal concentration, the area under the concentration-time curve (AUC), and terminal elimination half-life for METH. The route of administration did not significantly affect these pharmacokinetic parameters. The time to maximum concentration for METH and AMP following i.m. administration was 0.3 h. Maximum AMP serum concentrations were achieved in 2 h, irrespective of the route of administration, and these concentrations remained essentially constant for an additional 6 h. The metabolism of METH to AMP was not affected by the route of administration, and the molar ratio AMP to METH AUC values were the same (i.v.=0.57; i.m.=0.41). These results show that METH pharmacokinetics after i.m. administration in the pigeon are similar to i.v. administration. Thus i.m. is a reasonable route of administration for METH behavioral assays in the pigeon if sufficient time for absorption is given following the dose, and the behavioral endpoint is not dependent on the rapid input of METH following an i.v. dose.

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.

Sex differences in (+)-amphetamine- and (+)-methamphetamine-induced behavioral response in male and female Sprague-Dawley rats.

(+)-Methamphetamine (METH) and (+)-amphetamine (AMP) are structurally similar drugs that are reported to induce similar pharmacological effects in rats of the same sex. Because pharmacokinetic data suggest female rats should be more affected than males, the current studies sought to test the hypothesis that the behavioral and temporal actions of METH and AMP should be greater in female Sprague-Dawley rats than in males. Using a dosing regimen designed to reduce the possibility of tolerance and sensitization, rats were administered 1.0 and 3.0 mg/kg intravenous drug doses. Distance traveled, rearing events and focal stereotypies (e.g., head weaving, sniffing) were measured. Female rats traveled significantly greater distances and displayed a greater number of rearing events than males after both doses. Analysis of stereotypy ratings after 3.0 mg/kg revealed that focal stereotypies were more pronounced and lasted longer in females. The second study compared the potencies of METH and AMP in inducing locomotor activity and focal stereotypies in each sex. No differences in potency were found when METH and AMP effects were compared within males or females. In summary, these studies showed female rats displayed greater and longer-lasting locomotor activity and more stereotypic behaviors, supporting earlier evidence of significant sexual dimorphism in pharmacokinetics.

Chiral determination of 3,4-methylenedioxypyrovalerone enantiomers in rat serum.

The emerging stimulant drug of abuse (3,4)-methylenedioxypyrovalerone [(R,S)-MDPV] is self-administered as a racemic mixture by intranasal, iv, oral, and smoking routes. The individual enantiomers are known to have widely different pharmacological effects, with (S)-MDPV showing much greater potency than (R)-MDPV in pharmacological testing. The goal of these studies was to develop and validate an analytical method for quantitation of (R)-MDPV, (S)-MDPV and (R,S)-MDPV in small volumes of rat serum using a chiral separation column and liquid chromatography-mass spectrometry. The method was validated for selectivity, precision, accuracy, recovery, sensitivity, and reproducibility. The method was also used to determine the enantiomeric stability of the individual enantiomers during sample cleanup and analysis. The linear dynamic range of the calibration curve was 1 - 1000 ng/ml for each enantiomer. Concentration values for the lower limit of quantitation (1 ng/ml) were within 30% of their nominal value, but all other calibration standards were <20% of their nominal value. With proper storage and handling of samples, the two MDPV enantiomers were shown to remain stable in rat serum without any apparent racemization during the time needed for analysis. Finally, the ruggedness of the method was demonstrated with diluted and undiluted serum samples collected from Sprague Dawley rats in a preliminary pharmacokinetic study at 3 mg/kg of (R,S)-MDPV. In summary, the assay used a simple sample preparation method, reversed-phase chiral chromatography, and tandem mass spectrometry to achieve accurate and selective determinations of MDPV enantiomer concentrations in small volumes of serum.