Abstract ID 92838Poster Board 303Objectives: About 1.6 million people have methamphetamine (METH) use disorder (MUD) in the US. There are no direct antagonists approved to treat MUD and small molecule treatments have not produced clinically significant improvements. Behavioral interventions, such as cognitive-behavioral therapies and contingency management are effective in patients with MUD, but few individuals with MUD begin these therapies and retention rates are poor. InterveXion Therapeutics and our research team at UAMS have studied the effects anti-METH antibodies in reducing METH CNS effects as a model for treating MUD for many years. We are now developing a long-acting monoclonal antibody (mAb) that binds to METH with high affinity. Our research objective in this study was to identify the best candidate among a panel of humanized anti-METH binding variable regions by screening mAbs for in vitro specificity and efficacy in a METH-induced locomotor activity model.Methods: A panel of 8 mAbs (IS12-IS19) was chosen for these studies based on their affinity for METH and onset (ka) and offset (kd) rates of binding to METH. Cross-reactivity of the chosen mAbs was tested with several abused drugs, prescription medications, over-the-counter medications, and neurotransmitters. To determine the capacities of mAbs to reduce METH-elicited locomotor stimulation, 48 male Sprague-Dawley rats first received two injections of 0.56 mg/kg METH (sc) and their locomotor activity was video recorded for 4 h post-METH administration using Noldus EthoVision XT. Next, rats were assigned to receive one of eight mAbs (282 mg/kg; iv) and their locomotor response to 0.56 mg/kg METH was assessed 24 h and 96 h after mAb administration. The primary dependent measure in the locomotor activity experiment was the horizontal distance traveled (cm). We also quantified mAb levels in blood serum at <96 h and <264 h after mAb administration in the same sample of rats.Results: Cross-reactivity: mAbs have nanomolar affinity (kd or ki) for (+)-METH (range: 7.78 to 32.36 nM), (+)-amphetamine (170.91 to 1048.93 nM), (+)-3,4-methylenedioxymethamphetamine (9.91 to 65.54 nM), and selegiline (258.81 to 642.80 nM). Affinities for all other test compounds were >1 microM.Locomotor Activity: The mAb with the greatest affinity for METH (IS12) reduced 0.56 mg/kg METH-elicited locomotor stimulation (pre-mAb administration) by 39.44% when tested 24 h after mAb administration and by 18.17% when tested 96 h after mAb administration. Full data sets on cross-reactivity, locomotor stimulant effects, and blood titers of mAbs are forthcoming.Discussion: In this study, we identified IS12, which has the same variable region as the parent antibody, mAb7F9, as the mAb with the greatest affinity for METH and capacity to reduce METH-elicited locomotor stimulation. Our next step is to characterize the pharmacokinetic profile and duration of effect in reducing METH-elicited locomotor stimulation of IS12 produced as a pair of IgGs using two constant domain sequences that increase half-life.Funded by the NIH (NIDA: U01DA056240, PI: Misty Stevens, PhD). Test compounds were provided by the NIDA Drug Supply Program.

Prenatal opioid exposure (POE) and postnatal adverse experiences are early life adversities (ELA) that often cooccur and increase problematic alcohol (EtOH) drinking during adolescence. We investigated the relationship between POE, postnatal adversity, and adolescent EtOH drinking in rats. We also sought to determine whether ELAs affect alpha-adrenoceptor density in the brain because the noradrenergic system is involved in problematic alcohol drinking and its treatment. We hypothesized that the combination of POE and postnatal adversity will increase alcohol drinking in rats compared to rats with exposure to either adversity alone or to control. We also predicted that POE and postnatal adversity would increase alpha 1-adrenoceptor density and decrease alpha 2-adrenoceptor density in brain to confer a stress-responsive phenotype. Pregnant rats received morphine (15 mg/kg/day) or saline via subcutaneous minipumps from gestational day 9 until birth. Limited bedding and nesting (LBN) procedures were introduced from postnatal day (PD) 3-11 to mimic early life adversity-scarcity. Offspring rats (PD 31-33) were given opportunities to drink EtOH (20 %, v/v) using intermittent-access, two-bottle choice (with water) procedures. Rats given access to EtOH were assigned into sub-groups that were injected with either yohimbine (1 mg/kg, ip) or vehicle (2 % DMSO, ip) 30 min prior to each EtOH access session to determine the effects of alpha 2-adrenoceptor inhibition on alcohol drinking. We harvested cortices, brainstems, and hypothalami from EtOH-na & iuml;ve littermates on either PD 30 or PD 70 and conducted radioligand receptor binding assays to quantify alpha 1- and alpha 2-adrenoceptor densities. Contrary to our hypothesis, only LBN alone increased EtOH intake in female adolescent rats compared to female rats with POE. Neither POE nor LBN affected alpha 1- or alpha 2-adrenoceptor densities in the cortex, brainstem, or hypothalamus of early- or late-aged adolescent rats. These results suggest a complex interaction between ELA type and sex on alcohol drinking.

Abstract ID 90377Poster Board 078During the 12-month period ending in April 2021, opioid-related deaths, primarily from fentanyl, reached an all-time high in the US, affecting up to 100,000 people annually. People undergoing medication-assisted treatment for opioid use disorder (OUD), including pregnant women, often relapse to fentanyl use. The gold standard treatment for OUD during pregnancy is buprenorphine (BUP), which improves maternal-fetal outcomes but causes neonatal opioid withdrawal syndrome (NOWS) in approximately 50% of newborns during their first weeks of life. We used a rat model to examine the effect of a novel drug, deuterated buprenorphine (BUP-D2), on fentanyl-induced adverse effects in mothers and their neonates. We hypothesized that BUP-D2 would reduce fentanyl-related adverse effects in dams and neonates. Our preclinical model simulated the clinical scenario of fentanyl relapse while undergoing medication-assisted treatment with BUP or BUP-D2. Osmotic minipumps that continuously delivered vehicle (veh; 2% DMSO; 0.120 uL/day), 0.1 mg/kg/day BUP, or 0.1 mg/kg/day BUP-D2 for up to two weeks were subcutaneously implanted in pregnant Sprague-Dawley rats (dams) on gestational day (GD) 9. On GD 13-22, dams received daily s.c. fentanyl (100 μg/kg) or saline injections (1 mL/kg). We assessed post-injection oxygen saturation (O2 sat.), catalepsy, and rectal temperature (temp.). Neonatal opioid withdrawal was precipitated by an i.p injection of naltrexone (NTX; 1 mg/kg) or saline 3-12 hours after delivery. Movement duration, a validated metric of NOWS, was determined using Noldus EthoVision XT. On GD 14, veh+fentanyl maximally induced catalepsy (i.e., to 30 sec cutoff) in all dams 15 min post-injection (p < .0001, vs veh+saline). Fentanyl-induced catalepsy was only partially blocked by BUP (21.25 ± 12.47, p = .0322), but fully blocked by BUP-D2 (10.95 ± 10.96, p = .2046). Veh+fentanyl decreased O2 sat. by 28 ± 25.82% 15 min post-injection relative to pre-injection (p = .0035 vs veh+saline). BUP and BUP-D2 fully blocked the fentanyl-induced decrease in O2 sat (102.0 ± 6.152 % and 100.7 ± 0.6140 % of pre-injection, respectively). Veh+fentanyl decreased temp. relative to veh+saline (35.84 ± 0.56 vs 36.58 ± 0.31 p = .0172) at 15 min post-fentanyl injection. Temp was not affected by BUP+ fentanyl (35.98 ± 1.15) or BUP-D2+fentanyl (36.20 ± 0.59). Lastly, NTX precipitated withdrawals in neonates that were prenatally exposed to BUP+fentanyl (females: 219.61 ± 71.54, p = 0.0229; males: 172.72 ± 89.32, p = 0.0046), but not neonates that were exposed to BUP-D2 + fentanyl (females: 91.46 ±59.7, p=0.8740; males: 102.67 ± 58.16, p = 0.9428). This study is the first to assess effects of fentanyl combined with BUP or BUP-D2 in pregnancy. Although BUP and BUP-D2 equally blocked fentanyl-induced respiratory depression in dams, our data suggest that BUP-D2 more effectively mitigated fentanyl-induced catalepsy and NOWS. This underscores the potential application of BUP-D2 as an improved OUD treatment during pregnancy.Funded by the UAMS Provost Innovator Award and by the NIH (NCATS: TL1 TR003109 and UL1 TR003107; NIDA: T32 DA022981). Fentanyl, BUP, and norbuprenorphine (used to synthesize BUP-D2) were provided by the NIDA Drug Supply Program.

ID 24021 Poster Board 215 Alcohol (EtOH) use significantly contributes to the three leading causes of death among adolescents: unintentional injuries, suicide, and homicide. Adolescents consume more EtOH per drinking occurrence than adults. Early life adversities (ELA), including prenatal opioid exposure (POE) and postnatal adverse experiences, increase the risk of problematic EtOH use among adolescents. POE is any exposure to opioids during gestation and postnatal adverse experiences include abuse, neglect, or household dysfunction, such as parental substance use disorder. Because they often co-occur and are risk factors for early EtOH use, we investigated the relationship between POE, postnatal adversity, and adolescent EtOH. We also aimed to determine how α-adrenoceptor activity is affected by these ELAs to in turn affect adolescent EtOH intake. To model POE, pregnant rats received 15 mg/kg/day morphine or vehicle via subcutaneous minipumps from gestational day 9 to birth. To model postnatal adversity, we used limited bedding and nesting (LBN) procedures in which dams and litters were restricted to 20% of normal bedding and nesting materials from postnatal day (PD) 3 to 11. LBN increases maternal stress and decreases maternal care quality. Starting on PD 31 or 33, rats were given access to bottles containing either a 20% EtOH (v/v) solution or water under an intermittent-access, two-bottle choice procedures for 24-hour sessions, three days per week for four weeks (12 sessions). Rats received either yohimbine (1 mg/kg; ip) or vehicle 30-min prior to each EtOH access session to determine if pharmacologically-induced sympathomimetic activity enhances the acquisition of EtOH in adolescent rats and to assess for altered α2-adrenoceptor activity in rats that experienced POE and/or LBN. Cortices and brainstems from littermates without EtOH exposure were harvested on either PD 30 or PD 70 and used in radioligand receptor binding assays to quantify α1- and α2-adrenoceptor density. We hypothesized that adolescents with a history of combined POE and LBN (“two-hit” group) will consume more EtOH, have higher brain densities of α1-adrenoceptor, and have lower brain densities of α2-adrenoceptor than adolescents with either condition alone. In general, female rats drank more EtOH than male rats, and EtOH consumption was more affected by treatment condition in females than males. For example, female rats, more so than male rats, in the two-hit treatment group drank more EtOH after administration of yohimbine. Also, in the two-hit group, there was a decrease in EtOH consumption compared to the LBN and control groups within each sex. In our adrenoceptor density study, there was no difference in α1-adrenoceptor expression between males and females and across treatment groups. However, in the female cortex, there was high variability and a main effect of sex in α2-adrenoceptor expression with male expression being greater. Together, these results indicate a complex interaction between ELA, sex, and α-adrenoceptor activity that may support a stress inoculation hypothesis. Support/Funding Information: NIDA 5T32DA022981 ABI/ACRI Grant Award

OBJECTIVES/GOALS: Buprenorphine (BUP) is used for opioid use disorder during pregnancy but causes neonatal opioid withdrawal syndrome (NOWS). The goal of this study was to determine the contribution of the active metabolite, norbuprenorphine (NorBUP), to the development of NOWS when the parent drug, BUP, is administered during pregnancy. METHODS/STUDY POPULATION: Subcutaneously implanted osmotic minipumps delivered BUP (0, 0.01, 0.1 or 1 mg/kg/day) ± NorBUP (1 mg/kg/day) to pregnant Long-Evans rats from gestation day 9 until after delivery. NOWS was measured between 3 and 12 hours after delivery. Withdrawal was precipitated by an intraperitoneal injection of a mu opioid receptor antagonist naltrexone (NTX; 0, 1 or 10 mg/kg), and movement duration (MD; a validated proxy for NOWS) was measured using Noldus Ethovision. Concentrations of BUP, NorBUP, and their glucuronide conjugates in the brains of neonatal littermates not undergoing withdrawal testing were determined using LC/MS/MS. Two-way ANOVA and multiple linear regression analyses tested for interactions between BUP and NorBUP on MD and related brain concentrations to MD, respectively. RESULTS/ANTICIPATED RESULTS: There was no interaction effect between BUP and NorBUP on MD for either sex or at any dose of naltrexone. In females, but not males, BUP (1 mg/kg/day) significantly increased NorBUP-induced MD by 58% following an injection with 1 mg/kg NTX. A multiple linear regression model that included BUP and NorBUP brain concentrations as predictors of MD was significant and well-fitting [FEMALES: F (2, 40) = 23.97, P < .0001, adj R2 = 0.52; MALES: F (2, 40) = 5.84, P = .0059, adj R2 = 0.19]. There was a differential contribution of NorBUP brain concentrations to MD based on sex. The partial regression coefficient for NorBUP was 51.34 (p < .0001) for females and 19.21 (p = 0.093) for males. The partial regression coefficient for BUP was similar for females and males (FEMALES:βBUP = 10.62, p = .0017; MALES:βBUP = 11.38, p = .009). DISCUSSION/SIGNIFICANCE: We show for the first time a differential contribution of NorBUP to BUP-associated NOWS in each sex, suggesting sex differences in NorBUP susceptibility and implicating that treatment strategies reducing prenatal NorBUP exposure may be more effective for females than males.

Background: Hippocampal and cerebellar neuropathology occurs in individuals with alcohol use disorders (AUD), resulting in impaired cognitive and motor function.Objectives: Evaluate the effects of ethanol on the expression of pro- and anti-inflammatory molecules, as well as the effects of the anti-inflammatory PPAR-γ agonist pioglitazone in suppressing ethanol-induced neuroinflammation.Methods: Adult male and female mice were treated chronically with ethanol for just under a month followed by a single acute binge dose of ethanol. Animals were provided liquid diet in the absence of ethanol (Control; n = 18, 9 M/9F), liquid diet containing ethanol (ethanol; n = 22, 11 M/11F), or liquid diet containing ethanol plus gavage administration of 30.0 mg/kg pioglitazone (ethanol + pioglitazone; n = 20, 10 M/10F). The hippocampus and cerebellum were isolated 24 h following the binge dose of ethanol, mRNA was isolated, and pro- and anti-inflammatory molecules were quantified by qRT-PCR.Results: Ethanol significantly (p < .05) increased the expression of pro-inflammatory molecules IL-1β, TNF-α, CCL2, and COX2; increased the expression of inflammasome-related molecules NLRP3 and Casp1 but decreased IL-18; and altered the expression of anti-inflammatory molecules including TGFβR1 in the hippocampus and cerebellum, though some differences were observed between males and females and the two brain regions. The anti-inflammatory pioglitazone inhibited ethanol-induced alterations in the expression of most, but not all, inflammation-related molecules.Conclusion: Chronic plus binge administration of ethanol induced the expression of inflammatory molecules in adult mice and pioglitazone suppressed ethanol-induced neuroinflammation.

Diagnoses of maternal opioid use disorder (MOUD) at delivery increased more than 500% between 1999 and 2017 in the United States. Today, cases of in utero opioid exposure due to MOUD exceed cases of the most common birth defects, including cleft lip, cleft palate, clubfoot, and Down syndrome, combined. Children exposed to opioids in utero are at increased risk of adverse childhood experiences (ACEs) due to parental care being compromised by SUDs. These ACEs include abuse, neglect, loss of a parent from death or incarceration, parental divorce, and household domestic violence. Altered sensitivity to opioid analgesia later in life is one potential outcome shared by in utero opioid exposure and ACEs that may contribute to poorly controlled pain and increased risk of opioid addiction. Studies using rat models of prenatal opioid exposure (POE) or early life adversity (ELA) have shown that each decreases opioid-induced antinociception long after exposure has ceased. However, the contributions of POE and ELA, alone and in combination, to opioid analgesic response in humans remains unknown. In this study, we used a rodent model of combined POE and ELA to disambiguate their long-term effects more quickly and with greater experimental control than human studies. We hypothesized that combined POE and ELA will decrease morphine-induced antinociception in adolescence relative to POE or ELA alone. We used a two-by-two between-subjects factorial design in which timed-pregnant Long-Evans rats were exposed to morphine (15 mg/kg/day; "POE" group) or saline ("vehicle" group) via subcutaneous osmotic minipump from gestation day 9 until delivery. Litters were fostered to untreated dams then randomly assigned to normal housing or ELA conditions using a limited bedding and nesting procedure from postnatal days 3-11. Morphine-induced antinociception was measured between postnatal days 30 and 54 using a warm-water tail withdrawal (WWTW) assay. Cumulative dose-response of morphine-induced antinociception was determined on test day 0 (1.0-30 mg/kg morphine, s.c.) and test day 13 (3.0-100 mg/kg, s.c.). On test days 1-12, rats were dosed with 18 mg/kg, s.c., every 12 hours, and WWTW was conducted on odd test days. Interesting, preliminary results of this ongoing study indicate that morphine-induced antinociception was enhanced on test day 0 in POE + normal-housed males (n = 2). These rats exhibited 100% maximum possible effect (MPE) and significantly higher potency (mean ED50=5.96 mg/kg; 95% CI = -12.12, 24.05), while mean MPE for the other groups (n=2-3) was less than 70% and mean ED50>17 mg/kg. Males exhibited partial tolerance throughout the 12-day chronic morphine treatment period. In the POE + normal-housed males, MPE decreased from 100% on test day 1 to 58% on test day 11. For the remaining groups, MPE decreased from 44-57% on test day 1 to 12-23% on test day 11. Morphine was more potent in the POE + normal-housed males on test day 13 (mean ED50=66.21 mg/kg; 95% CI = -281.6, 414) than in other males (mean ED50>104.4 mg/kg). No clear group effect was observed in females. These unexpected results suggest a complex sex-dependent interaction between POE and ELA on opioid antinociception.

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.

The objectives of this study were to determine alcohol consumption after administration of (R)(-)-2,5-dimethoxy-4-iodoamphetamine (DOI) or naltrexone in Long-Evans rats, and to assess the effectiveness of these treatments based on individual differences in alcohol consumption. Adult male Long-Evans rats (N = 16) were given opportunities to orally self-administer a 20% (v/v) ethanol (EtOH) solution using an intermittent access, two-bottle (vs. tap water) choice procedure in their home cages. EtOH consumption and preference, total fluid consumption and food intake were measured. Last, we assessed the effects of naltrexone (1 mg/kg; subcutaneous) and (R)(-)-DOI (0.1-1 mg/kg; subcutaneous) on EtOH intake and preference using a quartile analysis. Rats showed stable EtOH (20%) intake and preference after 15 EtOH access sessions. Naltrexone produced a transient decrease in EtOH intake, but an inconsistent effect on EtOH preference, whereas DOI dose-dependently reduced EtOH intake and preference for at least 24 h. Subsequent quartile analyses revealed that rats with the highest EtOH intake during the first 60 min of access to EtOH showed greater reductions in EtOH intake and preference after DOI treatment. This is the first report to show that DOI-elicited reductions in EtOH intake and preference in rats depend on baseline EtOH intake, perhaps supporting a 'baseline dependency' hypothesis of effectiveness with phenethylamine psychedelics on EtOH consumption. If so, individuals with greater potential to develop severe AUDs may be particularly responsive to the positive motivational changes produced by treatment with psychedelics that target the 5-HT2 receptor family.

RATIONALE:There is a renewed interest in the use of 3,4-methylenedioxymethamphetamine (MDMA) for treating psychiatric conditions. Although MDMA has entered phase II clinical trials and shows promise as an adjunct treatment, there is an extensive literature detailing the potential neurotoxicity and adverse neurobehavioral effects associated with MDMA use. Previous research indicates that the adverse effects of MDMA may be due to its metabolism into reactive catechols that can enter the brain and serve directly as neurotoxicants. One approach to mitigate MDMA's potential for adverse effects is to reduce O-demethylation by deuterating the methylenedioxy ring of MDMA. There are no studies that have evaluated the effects of deuterating MDMA on behavioral outcomes.OBJECTIVES:The purpose of the present study was to assess the motor-stimulant effects of deuterated MDMA (d2-MDMA) and compare them to MDMA in male mice.METHODS:Two experiments were performed to quantify mouse locomotor activity and to vary the drug administration regimen (single bolus administration or cumulative administration).RESULTS:The results of Experiments 1 and 2 indicate that d2-MDMA is less effective at eliciting horizontal locomotion than MDMA; however, the differences between the compounds diminish as the number of cumulative administrations increase. Both d2-MDMA and MDMA can elicit sensitized responses, and these effects cross-sensitize to the prototypical drug of abuse methamphetamine. Thus, d2-MDMA functions as a locomotor stimulant similar to MDMA, but, depending on the dosing regimen, may be less susceptible to inducing sensitization to stereotyped movements.CONCLUSIONS:These findings indicate that d2-MDMA is behaviorally active and produces locomotor effects that are similar to MDMA, which warrant additional assessments of d2-MDMA's behavioral and physiological effects to determine the conditions under which this compound may serve as a relatively safer alternative to MDMA for clinical use.

Invertebrate animal studies of methamphetamine (METH) could allow for high throughput, inexpensive, and high-animal number pharmacology and toxicology studies. We hypothesized that in Periplaneta americana cockroaches, METH would increase locomotion compared to saline and produce lethality. Lethal dose, 50% (LD50) was determined with 0-1,780 mu g/g (mg/kg) METH (n = 15-16/group) using logit analysis. Locomotor activity after METH (0-560 mg/kg, intra-abdominal, n = 8 per group) administration and spontaneous locomotor activity in surviving cockroaches in an open field 24 h after LD50 study doses was measured with Noldus Ethovision. The LD50 of METH was 823.1 mg/kg (more than 10-fold greater than the value in rats). There were significant decreases in spontaneous locomotor activity in surviving cockroaches after administration of 650 and 750 mg/kg METH (P < 0.05). While 100 mg/kg METH did not significantly increase METH locomotor activity relative to saline, 300 mg/kg METH significantly increased locomotor activity compared to saline (P < 0.05), and 560 mg/kg METH resulted in most of the cockroaches slowly moving around the open field in the supine position for most of the trial. In conclusion, METH produces pharmacological and toxicological effects in P. americana. The high availability, low cost, and relative ease of use of these animals makes them a potential, very accessible option for studying METH use disorder.

BACKGROUND:Several disease states commonly associated with methamphetamine (METH) use produce liver dysfunction, and in the bile duct ligation (BDL) model of hepatic dysfunction, rats with liver injury are more sensitive to METH effects. Additionally, both female rats and humans are known to be more sensitive to METH than males. In consideration of known sex-dependent differences in METH pharmacokinetics, this study sought to determine the potential interaction between sex and liver dysfunction variables on METH pharmacokinetics.METHODS:Sham or BDL surgery was performed on male and female rats on day 0. Serum biomarker and pharmacokinetics studies with 3 mg/kg subcutaneous (SC) METH were performed on day 7. METH-induced weight loss was measured on day 8. Liver histology evaluation and brain METH concentration measurements were performed on day 9.RESULTS:While BDL surgery produced significantly elevated alanine aminotransferase and bile duct proliferation in male compared to female rats, there were no significant interactions between sex and liver function in the pharmacokinetic parameters. Both liver dysfunction and female sex, however, were associated with significantly slower METH serum clearance and significantly higher brain METH concentrations (p < .05).CONCLUSIONS:BDL-induced hepatic dysfunction produces substantial reductions in METH clearance and increased brain METH concentrations in both male and female rats, despite less liver injury in females. This preclinical model may be useful to identify and correct potential liver dysfunction comorbidity-related problems with future pharmacotherapy for stimulant use disorder with METH prior to expensive clinical trials.

BACKGROUND:Recent clinical studies support the use of 3,4-methylenedioxymethamphetamine (MDMA) as an adjunct treatment for posttraumatic stress disorder (PTSD). Despite these promising findings, MDMA administration in controlled settings can increase blood pressure, heart rate, and body temperature. Previous studies indicate thatO-demethylated metabolites of MDMA contribute to its adverse effects. As such, limiting the conversion of MDMA to reactive metabolites may mitigate some of its adverse effects and potentially improve its safety profile for therapeutic use.METHODS:We compared the interoceptive and hyperthermic effects of a deuterium-substituted form of MDMA (d2-MDMA) to MDMA using rodent drug discrimination and biotelemetry procedures, respectively.RESULTS:Compared to MDMA, d2-MDMA produced full substitution for a 1.5 mg/kg MDMA training stimulus with equal potency and effectiveness in the drug discrimination experiment. In addition, d2-MDMA produced increases in body temperature that were shorter-lasting and of lower magnitude compared to equivalent doses of MDMA. Last, d2-MDMA and MDMA were equally effective in reversing the hypothermic effects of the selective 5-HT2A/2C antagonist ketanserin.CONCLUSION:These findings indicate that deuterium substitution of hydrogen at the methylenedioxy ring moiety does not impact MDMA's interoceptive effects, and compared to MDMA, d2-MDMA has less potential for producing hyperthermic effects and likely has similar pharmacodynamic properties. Given that d2-MDMA produces less adverse effects than MDMA, but retains similar desirable effects that are thought to relate to the effective treatment of PTSD, additional investigations into its effects on cardiovascular functioning and pharmacokinetic properties are warranted.

Disease states such as hepatitis C and HIV are both associated with methamphetamine (METH) use and are known to produce liver dysfunction. Indeed, bile duct ligation (BDL)-induced liver dysfunction enhances METH effects in rats. While female rats are more sensitive to METH, male rats are more prone to BDL-induced liver damage. The objective of this study was to determine if BDL disproportionately affects METH pharmacokinetics in male compared to female rats. On day 0, sham or BDL surgery was performed on male and female rats. On day 7, serum biomarker samples were collected prior to pharmacokinetics studies with 3 mg/kg subcutaneous METH. METH-induced weight loss was measured on day 8. On day 9, samples were collected for liver histology and brain METH concentration measurement. While BDL surgery produced significantly elevated alanine aminotransferase and bile duct proliferation in male versus female rats, there were no significant interactions between sex and liver function in the pharmacokinetic parameters. Both liver dysfunction and female sex, however, were associated with significantly slower serum METH clearance and higher brain METH concentrations (p<.05). While there were no sex-dependent differences, there was a significant reduction in post-METH weight in BDL compared to sham animals (p<.05). BDL-induced hepatic dysfunction produces substantial elevations in serum and brain METH exposure in both male and female rats. This inexpensive model could potentially be used to find and correct liver dysfunction-related issues with future METH use disorder medications prior to expensive clinical trials. Funding: Marshall University School of Pharmacy Faculty Research Seed Grant.

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.

Alcohol use disorder (AUD) is a prevalent (>15 million Americans) chronic condition characterized by compulsive alcohol use and dependency. Less than 10% of people with an AUD seek treatment, and less than 20% of people with an alcohol disorder who receive treatment will remain abstinent for a full year. As such, it is important to identify adjunct treatment strategies for AUDs and methods for promoting long‐term alcohol abstinence. The goals of the present study were to establish and validate a model of ethanol consumption in laboratory rats, in order to evaluate the effectiveness of psychedelic drugs in persistently reducing ethanol consumption. Adult male Long‐Evans rats (N=16) were trained to orally self‐administer 20% ethanol using an intermittent access two‐bottle choice procedure in their home cages. Rats were given opportunities to drink from a bottle containing 20% ethanol or a bottle containing water in three 24‐hour sessions per week; on intervening days, rats were given two bottles of water. Ethanol consumption and preference, total fluid consumption, and food intake were quantified during acquisition, until rats showed stable ethanol intake and preference with no apparent trend (considered at 17 ethanol access sessions). During the final three sessions of acquisition, ethanol intake was 4.87 g/kg/24 hours and preference was ~50%. Following acquisition, rats were divided into high consumers (n=8) and low consumers (n=8) using a median split. During the final three ethanol acquisition sessions, high consumers showed significantly greater levels of intake (7.33 g/kg/24 hours) and preference (72%) compared to the intake (2.5 g/kg/24 hours) and preference (26%) of low consumers. After the acquisition phase, all rats were given intermittent, 24‐hour access to varying concentrations of ethanol (5, 10, 20, 40% v/v) to generate ethanol concentration effect curves (ECEC). In the complete sample of rats, peak intake occurred at 20% ethanol, however preference was greater at 5% (86% preference) and 10% (75% preference) ethanol concentrations. High consumers and low consumers all showed peak intake at 20% ethanol, but the ECEC for ethanol preference in high consumers was upwardly shifted compared to low consumers. Furthermore, a regression analysis revealed that the point of indifference in ethanol preference (i.e., a 50% preference) occurred at 31.7% ethanol for high consumers, but at 13.97% ethanol in low consumers. Thus, these results indicate that rats' ethanol consumption patterns during the acquisition phase of the experiment are predictive of their intake and preference when given access to varying ethanol concentrations. Following the initial ECEC determination, the effects of naltrexone pretreatment on ethanol intake and preference will be assessed for model validation. Thereafter, the capacity of the psychedelic serotonin 5‐HT2A agonist 2,5‐dimethoxy‐4‐iodoamphetamine (DOI) to reduce ethanol intake and preference in rats will be assessed. It is hypothesized that naltrexone will produce a global reduction in consummatory behaviors in rats, while DOI is expected to specifically decrease ethanol consumption, but have no effect on water or food consumption.Support or Funding InformationNIDA T32DA022981This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Recreational abuse of illicit synthetic cathinones is an ongoing public health concern. Recent studies indicate that the methcathinone derivative 4-methylmethcathinone (4-MMC) produces behavioral and neurochemical effects similar to the entactogen 3,4-methylenedioxymethamphetamine (MDMA). Whereas polysubstance abuse is common, most preclinical studies of drug abuse liability only evaluate the effects of single drugs. Utilizing the locomotor sensitization paradigm, the present study assessed the combined locomotor stimulant effects of 4-MMC and MDMA for induction of sensitization following repeated administration and for expression of sensitization to a challenge dose of either substance alone after a 10-day period of drug abstinence. Male Sprague-Dawley rats received once daily intraperitoneal injections of saline, 4-MMC (1.0 mg/kg or 5.0 mg/kg), MDMA (3.0 mg/kg), or a mixture containing 4-MMC (1.0 mg/kg or 5.0 mg/kg) + MDMA (3.0 mg/kg) for 7 consecutive days. Following a 10-day drug-free period, rats were given a single intraperitoneal injection of either saline, 4-MMC (1.0 or 5.0 mg/kg), or 3.0 mg/kg MDMA. Activity was recorded for 1 h immediately before and 1 h immediately after injections on days 1, 7, and 17. 4-MMC treatment failed to induce locomotor sensitization, but, when combined with MDMA, sensitization was induced to a greater extent than with MDMA alone. Furthermore, the expression of sensitization to a subsequent challenge dose of MDMA was observed only in animals previously exposed to MDMA or a 5.0 mg/kg 4-MMC + MDMA mixture. In consideration of these findings along with the fact that 4-MMC has similar neurochemical actions to MDMA, further research may be warranted to determine the abuse liability of drug mixtures including 4-MMC and MDMA.

Synthetic cathinones (SC) are one of the largest categories of emerging novel psychoactive substances, and are typically abused as ‘bath salts’ preparations. 3,4‐ methylenedioxypyrovalerone (MDPV) is a common SC frequently mentioned in both clinical case reports and calls to poison control centers, and has been implicated in increased risky behaviors. Impulsivity is a major factor in risky behaviors, and psychostimulants like cocaine may modulate baseline impulsivity through actions on dopaminergic systems. Like cocaine, the psychoactive effects of MDPV are elicited by selective and potent reuptake inhibition at catecholamine transporters, and studies from our lab have indicated that long‐term administration of MDPV increases impulsive choice in rats. The behavioral construct of impulsivity comprises multiple components, including impulsive choice and motor impulsivity. In the current study, we characterized the effects of acute MDPV on motor impulsivity using a differential reinforcement of low rate of responding (DRL 20 sec) task. Several endpoints were quantified, including number of reinforcers earned (SR), total number of premature responses (PRE), response efficiency (RE, # of reinforcers earned /total premature and reinforced responses), and perseverative responses (PRSV, # of responses following premature responses or reinforced nose pokes until the next available signaled reinforcer). Eighteen drug‐naïve, adult male Sprague‐ Dawley rats were split into three groups of six: a saline negative control (SAL), a cocaine positive control (COC), and an MDPV test group (MDPV). Animals were matched based on RE for the final five days of training (days 45–50), yielding approximately the same RE for each group (MDPV = 20.22%, COC = 19.64%, and SAL = 21.71%). Following stabilization, a satiety challenge was implemented in which animals were fed their daily allotment of food 1 hour prior to the start of their scheduled session. Satiation increased both RE and SR in all groups, while decreasing PRE and PRSV, indicating decreases in motor impulsivity. After a 5 day restabilization, each animal received an acute saline injection, followed by daily injections of either subcutaneous (SQ) saline (SAL group), 1.0 – 30.0 mg/kg intraperitoneal cocaine (COC group), or 0.1 – 3.0mg/kg SQ MDPV (MDPV group). Acute saline injections had no effect on any measure in any group, and further injections of acute saline in the SAL group had no effects on any DRL endpoints. However, escalating acute injections of both cocaine and MDPV dose dependently increased motor impulsivity. At the highest doses tested, RE was reduced to 8.36% in COC animals and to 0.81% in MDPV rats, though operant behavior was completely suppressed in 2 of the 6 MDPV animals. Moreover, the number of PRE and PRSV were increased in both COC and MDPV groups, though MDPV was more potent and effective than cocaine in this regard. Further experiments investigating the effects of long‐term MDPV administration on motor impulsivity are currently underway.Support or Funding InformationThese studies were supported in part by DA039195 and T32DA022981.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

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.

Substituted amphetamines represent one of the largest and most pharmacologically‐diverse classes of abused drugs. Modifications to the amphetamine base structure are seemingly limitless, and novel psychoactive substituted amphetamines continuously emerge on the illicit market. Amphetamine analogues containing a ketone at the beta carbon are referred to as cathinones, which are typically the active constituents in abused “bath salts” products. Each “generation” of cathinone analogues introduces new structural motifs, most likely in response to regulatory control of previous compounds. One recently identified “bath salts” constituent is 4‐chloroethcathinone (4‐CEC), consisting of the base amphetamine structure with the addition of an ethyl side chain, a chlorine at the 4‐position, and a ketone group at the beta carbon. In these studies, we compared 12 structurally‐analogous substituted amphetamines and cathinones increasing in structural complexity from amphetamine to 4‐CEC in order to assess structure‐activity relationships for locomotor stimulant and toxic effects in mice. Automated photobeam chambers were used to record various aspects of mouse locomotor activity including jump count, time in stereotypy, vertical time, speed, and distance. Full dose‐effect curves were determined for each compound, starting with a dose that produced saline‐like distance traveled, and increasing in half‐log units until distance decreased from that drug's peak, or until lethal effects were observed. For the substituted amphetamines, increasing the length of the side chain increased maximal effectiveness on locomotor endpoints. As compared to analogous unsubstituted compounds, addition of a chlorine at the 4‐position did not substantially impact distance traveled, but increased time spent in stereotypy. These same trends were also observed with the substituted cathinones. Interestingly, 4‐CEC produced lethality in all subjects at doses on the ascending limb of the dose‐effect curve for distance traveled. Further development of structure‐activity relationships for these drugs may determine which moieties of their chemical structures are involved in producing specific pharmacological effects, and may allow predictions about abuse liability or toxic effects of new pharmacological entities with similar structural motifs.Support or Funding InformationThese studies supported by DA022981, R01‐DA039195, and DEA/FDA HHSF223201610079C.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.