Comparative Neuropharmacology and Pharmacokinetics of Methamphetamine and Its Thiophene Analog Methiopropamine in Rodents.

Methiopropamine is a novel psychoactive substance (NPS) that is associated with several cases of clinical toxicity, yet little information is available regarding its neuropharmacological properties. Here, we employed in vitro and in vivo methods to compare the pharmacokinetics and neurobiological effects of methiopropamine and its structural analog methamphetamine. Methiopropamine was rapidly distributed to the blood and brain after injection in C57BL/6 mice, with a pharmacokinetic profile similar to that of methamphetamine. Methiopropamine induced psychomotor activity, but higher doses were needed (Emax 12.5 mg/kg; i.p.) compared to methamphetamine (Emax 3.75 mg/kg; i.p.). A steep increase in locomotor activity was seen after a modest increase in the methiopropamine dose from 10 to 12.5 mg/kg, suggesting that a small increase in dosage may engender unexpectedly strong effects and heighten the risk of unintended overdose in NPS users. In vitro studies revealed that methiopropamine mediates its effects through inhibition of norepinephrine and dopamine uptake into presynaptic nerve terminals (IC50 = 0.47 and 0.74 µM, respectively), while the plasmalemmal serotonin uptake and vesicular uptake are affected only at high concentrations (IC50 > 25 µM). In summary, methiopropamine closely resembles methamphetamine with regard to its pharmacokinetics, pharmacodynamic effects and mechanism of action, with a potency that is approximately five times lower than that of methamphetamine.

Mechanistic PBPK Modeling of Urine pH Effect on Renal and Systemic Disposition of Methamphetamine and Amphetamine.

The effect of urine pH on renal excretion and systemic disposition has been observed for many drugs and metabolites. When urine pH is altered, tubular ionization, passive reabsorption, renal clearance, and systemic exposure of drugs and metabolites may all change dramatically, raising clinically significant concerns. Surprisingly, the urine pH effect on drug disposition is not routinely explored in humans, and regulatory agencies have neither developed guidance on this issue nor required industry to conduct pertinent human trials. In this study, we hypothesized that physiologically based pharmacokinetic (PBPK) modeling could be used as a cost-effective method to examine potential urine pH effect on drug and metabolite disposition. Our previously developed and verified mechanistic kidney model was integrated with a full-body PBPK model to simulate renal clearance and area under the plasma concentration-time curve (AUC) with varying urine pH statuses using methamphetamine and amphetamine as model compounds. We first developed and verified drug models for methamphetamine and amphetamine under normal urine pH condition [absolute average fold error (AAFE) < 1.25 at study level]. Then, acidic and alkaline urine scenarios were simulated. Our simulation results show that the renal excretion and plasma concentration-time profiles for methamphetamine and amphetamine could be recapitulated under different urine pH (AAFE < 2 at individual level). The methamphetamine-amphetamine parent-metabolite full-body PBPK model also successfully simulated amphetamine plasma concentration-time profiles (AAFE < 1.25 at study level) and amphetamine/methamphetamine urinary concentration ratios (AAFE < 2 at individual level) after dosing methamphetamine. This demonstrates that our mechanistic PBPK model can predict urine pH effect on systemic and urinary disposition of drugs and metabolites. SIGNIFICANCE STATEMENT: Our study shows that integrating mechanistic kidney model with full-body physiologically based pharmacokinetic model can predict the magnitude of alteration in renal excretion and area under the plasma concentration-time curve (AUC) of drugs and metabolites when urine pH is changed. This provides a cost-effective method to evaluate the likelihood of renal and systemic disposition changes due to varying urine pH. This is important because multiple drugs and diseases can alter urine pH, leading to quantitatively and clinically significant changes in drug and metabolite disposition that may require adjustment of therapy.

Enantioselectivity on the absorption of methylone and pentedrone using Caco-2 cell line: Development and validation of an UHPLC method for cathinones quantification.

Synthetic cathinones, such as methylone and pentedrone, are psychoactive derivatives of cathinone, sold in the internet as "plant food" or "bath salts". However, the level at which these compounds and their enantiomers cross the intestinal barrier has not been yet determined. Thus, the present study aimed to analyze the enantioselectivity on the permeability of these drugs through the intestinal barrier by using the Caco-2 cell line, a widely used in vitro model for drug permeability studies. To achieve this goal, an UHPLC-UV method was developed and validated to quantify both synthetic cathinones. The developed UHPLC-UV method revealed high selectivity and a linearity from 1 to 500 µM with correlation coefficients always higher than 0.999. The method has an accuracy that ranged between 89 and 107%, inter-day and intra-day precisions with coefficients of variation below 10%, limits of detection and quantification of 0.31 µM and 0.93 µM for methylone and 0.17 µM and 0.52 µM for pentedrone, respectively. In Caco-2 cells, a differentiated passage of the enantiomers across monolayer was observed for both cathinones. For pentedrone, the difference was observed after the first hour, being R-(-)-pentedrone the most permeable compound. Regarding methylone, the difference was noted after one hour and 30 min, with S-(-)-methylone being the most absorbed enantiomer. In conclusion, a fully validated method was successfully applied for studying the permeability of methylone and pentedrone enantiomers in an in vitro model of human intestine, which allowed to discover, for the first time, the enantioselectivity in drug permeability of this class of drugs.

Acute changes in the retina and central retinal artery with methamphetamine.

Methamphetamine (METH), an addictive stimulant of neurotransmitters, is associated with cardiovascular and neurological diseases. METH-induced ophthalmic complications are also present but have been insufficiently investigated. The purpose of this study is to investigate the retinal effects of METH. C57BL/6 mice were administrated progressively increasing doses of METH (0-6 mg/kg) by repetitive intraperitoneal injections for 5 days (4 times per day). Retinal degeneration was examined by morphological changes and terminal deoxynucleotidyl transferase deoxyuridine triphosphate nick end labeling (TUNEL) assay. Norepinephrine levels were measured by ELISA, protein expression levels were determined by immunoblot and immunostaining, and gelatinase activity was examined by zymography. The thickness of the retina and the number of nuclei in the inner and outer nuclear layers were decreased by METH. Retinal cell death and astrocyte activation by METH treatment were confirmed by TUNEL assay and glial fibrillary acidic protein expression, respectively. Increased tumor necrosis factor-α protein in the retina and elevated norepinephrine levels in plasma were found in METH-treated mice. Platelet endothelial cell adhesion molecule-1 (PECAM-1) protein expression level was decreased in the retina and central retinal artery (CRA) by METH treatment, along with the endothelial proteoglycans glypican-1 and syndecan-1. Moreover, a regulator of the extracellular matrix, matrix metalloproteinase-14 (MMP-14) in the retina, and MMP-2 and MMP-9 in plasma, were increased by METH treatment. In conclusion, METH administration is involved in retinal degeneration with a vascular loss of PECAM-1 and the glycocalyx in the CRA and retina, and an increase of MMPs.

The Role of Biogenic Amine Transporters in Trace Amine-Associated Receptor 1 Regulation of Methamphetamine-Induced Neurotoxicity.

Methamphetamine (MA) impairs vesicular monoamine transporter 2 (VMAT2) and dopamine transporter (DAT) function and expression, increasing intracellular DA levels that lead to neurotoxicity. The trace amine-associated receptor 1 (TAAR1) is activated by MA, but when the receptor is not activated, MA-induced neurotoxicity is increased. To investigate interactions among TAAR1, VMAT2, and DAT, transporter function and expression were measured in transgenic Taar1 knockout (KO) and wild-type (WT) mice 24 hours following a binge-like regimen (four intraperitoneal injections, 2 hours apart) of MA (5 mg/kg) or the same schedule of saline treatment. Striatal synaptosomes were separated by fractionation to examine the function and expression of VMAT2 localized to cytosolic and membrane-associated vesicles. DAT was measured in whole synaptosomes. VMAT2-mediated [3H]DA uptake inhibition was increased in Taar1 KO mice in synaptosomal and vesicular fractions, but not the membrane-associated fraction, compared with Taar1 WT mice. There was no difference in [3H]dihydrotetrabenazine binding to the VMAT2 or [125I]RTI-55 binding to the DAT between genotypes, indicating activation of TAAR1 does not affect VMAT2 or DAT expression. There was also no difference between Taar1 WT and KO mice in DAT-mediated [3H]DA uptake inhibition following in vitro treatment with MA. These findings provide the first evidence of a TAAR1-VMAT2 interaction, as activation of TAAR1 mitigated MA-induced impairment of VMAT2 function, independently of change in VMAT2 expression. Additionally, the interaction is localized to intracellular VMAT2 on cytosolic vesicles and did not affect expression or function of DAT in synaptosomes or VMAT2 at the plasmalemmal surface, i.e., on membrane-associated vesicles. SIGNIFICANCE STATEMENT: Methamphetamine stimulates the G protein-coupled receptor TAAR1 to affect dopaminergic function and neurotoxicity. Here we demonstrate that a functional TAAR1 protects a specific subcellular fraction of VMAT2, but not the dopamine transporter, from methamphetamine-induced effects, suggesting new directions in pharmacotherapeutic development for neurodegenerative disorders.

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.

Identification of three new phase II metabolites of a designer drug methylone formed in rats by N-demethylation followed by conjugation with dicarboxylic acids.

1. Methylone (3,4-methylenedioxy-N-methylcathinone, MDMC), which appeared on the illicit drug market in 2004, is a frequently abused synthetic cathinone derivative. Known metabolic pathways of MDMC include N-demethylation to normethylone (3,4-methylenedioxycathinone, MDC), aliphatic chain hydroxylation and oxidative demethylenation followed by monomethylation and conjugation with glucuronic acid and/or sulphate. 2. Three new phase II metabolites, amidic conjugates of MDC with succinic, glutaric and adipic acid, were identified in the urine of rats dosed subcutaneously with MDMC.HCl (20 mg/kg body weight) by LC-ESI-HRMS using synthetic reference standards to support identification. 3. The main portion of administered MDMC was excreted unchanged. Normethylone, was a major urinary metabolite, of which a minor part was conjugated with dicarboxylic acids. 4. Previously identified ring-opened metabolites 4-hydroxy-3-methoxymethcathinone (4-OH-3-MeO-MC), 3-hydroxy-4-methoxymeth-cathinone (3-OH-4-MeO-MC) and 3,4-dihydroxymethcathinone (3,4-di-OH-MC) mostly in conjugated form with glucuronic and/or sulphuric acids were also detected. 5. Also, ring-opened metabolites derived from MDC, namely, 4-hydroxy-3-methoxycathinone (4-OH-3-MeO-C), 3-hydroxy-4-methoxycathinone (3-OH-4-MeO-C) and 3,4-dihydroxycathinone (3,4-di-OH-C) were identified for the first time in vivo.

Novel nanoformulation to mitigate co-effects of drugs of abuse and HIV-1 infection: towards the treatment of NeuroAIDS.

Drug abuse (e.g., methamphetamine-Meth or cocaine-Coc) is one of the major risk factors for becoming infected with HIV-1, and studies show that in combination, drug abuse and HIV-1 lead to significantly greater damage to CNS. To overcome these issues, we have developed a novel nanoformulation (NF) for drug-abusing population infected with HIV-1. In this work, a novel approach was developed for the co-encapsulation of Nelfinavir (Nel) and Rimcazole (Rico) using layer-by-layer (LbL) assembled magnetic nanoformulation for the cure of neuroAIDS. Developed NF was evaluated for blood-brain barrier (BBB) transmigration, cell uptake, cytotoxicity and efficacy (p24 assay) in HIV-1 infected primary astrocyte (HA) in presence or absence of Coc and Meth. Developed magnetic nanoformulation (NF) fabricated using the LbL approach exhibited higher amounts of drug loading (Nel and Rico) with 100% release of both the therapeutic agents in a sustained manner for 8 days. NF efficacy studies indicated a dose-dependent decrease in p24 levels in HIV-1-infected HA (~55%) compared to Coc + Meth treated (~50%). The results showed that Rico significantly subdued the effect of drugs of abuse on HIV infectivity. NF successfully transmigrated (38.8 ± 6.5%) across in vitro BBB model on the application of an external magnetic field and showed >90% of cell viability with efficient cell uptake. In conclusion, our proof of concept study revealed that sustained and concurrent release of sigma σ1 antagonist and anti-HIV drug from the developed novel sustained release NF can overcome the exacerbated effects of drugs of abuse in HIV infection and may solve the issue of medication adherence in the drug-abusing HIV-1 infected population.

Psychomotor effect differences between l-methamphetamine and d-methamphetamine are independent of murine plasma and brain pharmacokinetics profiles.

l-Methamphetamine has been occasionally referred to as a stimulant similar to d-methamphetamine, probably owing to insufficient comparative studies. Here, we directly compared psychomotor efficacies and pharmacokinetics of methamphetamine enantiomers in mice. Only d-methamphetamine, but not l-methamphetamine, induced stereotypy and sensitization at 1-10 mg/kg. However, plasma pharmacokinetic parameters of 10 mg/kg l-methamphetamine were ≥tenfold those of 1 mg/kg d-methamphetamine. These results clearly indicate that differential psychomotor efficacies of methamphetamine enantiomers are independent of their pharmacokinetic profiles.

Medico-legal assessment of methamphetamine and amphetamine serum concentrations-what can we learn from survived intoxications?

Medico-legal experts are increasingly enlisted to assess the methamphetamine and amphetamine serum concentrations after a criminal offense. However, since criminal users rarely provide useful information to medico-legal experts regarding the substances abused, when the substance(s) was/were used, dose of ingestion tools are needed to interpret the analytical data, which can be used as objective evidence in such cases. A comparative series of methamphetamine and amphetamine serum concentrations were used to analyze the frequency of concentrations, to determine methamphetamine/amphetamine concentration ratios, and prove them as a tool to distinguish pure methamphetamine from mixed amphetamine/methamphetamine ingestion. Additionally, two cases of survived accidental methamphetamine intoxication, resulting from ingestion smuggling which was longitudinally monitored, and pharmacokinetic parameters were assessed. In a series of 628 samples where the most frequent concentration of methamphetamine exceeded the therapeutic level, there was a strong correlation suggesting pure methamphetamine consumption, when the ratios of methamphetamine/amphetamine concentrations were within the range between 3 and 10. In the two cases of methamphetamine bodypacking, the relevant serum concentrations of methamphetamine and amphetamine, which could be measured up to 9 days after ingestion, indicated a decrease of the methamphetamine/amphetamine ratios in an exponential manner. However, the ratios were not always within the range between 3 and 10. Lastly, the course of the serum concentrations suggested an increase of the apparent elimination half-life of methamphetamine. In terms of the objective evidence required in criminal law, calculating methamphetamine/amphetamine concentration ratio is not a suitable to means to distinguish pure methamphetamine intake and that of mixed amphetamine/methamphetamine abuse in an individual case. Instead, methamphetamine high serum concentrations and the possible increase in apparent elimination half-life suggest that an extended detection period may be used to distinguish between "illicit use" as compared to "therapeutic use" of methamphetamine.

Trans-generational neurochemical modulation of methamphetamine in the adult brain of the Wistar rat.

Chronic methamphetamine (METH) abuse has been shown to elicit strong neurotoxic effects. Yet, with an increasing number of children born to METH abusing mothers maturing into adulthood, one important question is how far do the neurotoxic effects of METH alter various neurotransmitter systems in the adult METH-exposed offspring. The purpose of this study was to investigate long-term trans-generational neurochemical changes, following prenatal METH exposure, in the adult Wistar rat brain. METH or saline (SAL-control animals) was administered to pregnant dams throughout the entire gestation period (G0-G22). At postnatal day 90, dopamine, serotonin, glutamate and GABA were measured in the adult brain before (baseline) and after a METH re-administration using in vivo microdialysis and liquid chromatography/mass spectrometry. The results show that METH-exposure increased basal levels of monoamines and glutamate, but decreased GABA levels in all measured brain regions. Acute challenge with METH injection in the METH-exposed group induced a lower increase in the monoamine system relative to the increase in the GABAergic and glutamatergic system. The data show that prenatal METH exposure has strong effects on the monoaminergic, GABAergic and glutamatergic system even when exposure to METH was limited to the prenatal phase. Toxicological effects of METH have therefore longer lasting effects as currently considered and seem to affect the excitatory-inhibitory balance in the brain having strong implications for cognitive and behavioral functioning.

Safety of Intravenous Methamphetamine Administration During Ibudilast Treatment.

BACKGROUND: Methamphetamine dependence is a significant public health concern without any approved medications for treatment. We evaluated ibudilast, a nonselective phosphodiesterase inhibitor, to assess the safety and tolerability during intravenous methamphetamine administration. We conducted a randomized, double-blind, placebo-controlled, within-subjects crossover clinical trial. METHODS: Participants received ibudilast (20 mg twice daily followed by 50 mg twice daily) and placebo, with order determined by randomization, and then underwent intravenous methamphetamine challenges (15 and 30 mg). We monitored cardiovascular effects, methamphetamine pharmacokinetics, and reported adverse events. RESULTS: Ibudilast treatment had similar rates of adverse events compared with placebo, and there was no significant augmentation of cardiovascular effects of methamphetamine. Pharmacokinetic analysis revealed no clinically significant change in maximum concentration or half-life of methamphetamine with ibudilast. CONCLUSIONS: Methamphetamine administration during ibudilast treatment was well tolerated without additive cardiovascular effects or serious adverse events, providing initial safety data to pursue ibudilast's effectiveness for the treatment of methamphetamine dependence.

Extended Detection of Amphetamine and Methamphetamine in Oral Fluid.

BACKGROUND: Amphetamine and methamphetamine are popular drugs of abuse worldwide and are important components of drug monitoring programs. Windows of detection for amphetamine and methamphetamine in oral fluid after high doses have not been investigated. Repeated high-dose ingestions are likely to cause positive samples for extended periods. Common routes of administration of amphetamine/methamphetamine in Norway are oral intake or injection. The aim of this study was to investigate windows of detection for amphetamine and methamphetamine in oral fluid from drug addicts under sustained abstinence during detoxification. METHODS: Twenty-five patients admitted to a closed detoxification unit were included in this study. Oral fluid samples were collected daily in the morning and evening, and urine every morning for 10 days. A blood sample was drawn during the first 5 days after admission if the patient consented. Oral fluid results were compared with urine results to determine whether a new ingestion occurred. Oral fluid was collected with the Intercept oral fluid collection device. In-house cutoff concentrations for amphetamine and methamphetamine were 6.8 and 7.5 mcg/L, respectively, in oral fluid, and 135 and 149 mcg/L, respectively, in urine. RESULTS: Amphetamines were detected in 11 oral fluid, 5 urine, and 2 blood specimens from 25 patients. Patients self-reported amphetamines intake of up to 0.5-2 g daily. Windows of detection for amphetamine and methamphetamine in oral fluid were up to 8 days, longer than in urine at the applied cutoff values. CONCLUSIONS: These data confirm that oral fluid is a viable alternative to urine for monitoring amphetamine abuse, and that these substances might be detected in oral fluid for at least 1 week after ingestion of high doses. Such long detection times were, as far as we are aware, never reported previously for oral fluid amphetamines.

Methamphetamine absorption by skin lipids: accumulated mass, partition coefficients, and the influence of fatty acids.

Occupants of former methamphetamine laboratories, often residences, may experience increased exposure through the accumulation of the methamphetamine in the organic films that coat skin and indoor surfaces. The objectives of this study were to determine equilibrium partition coefficients of vapor-phase methamphetamine with artificial sebum (AS-1), artificial sebum without fatty acids (AS-2), and real skin surface films, herein called skin oils. Sebum and skin oil-coated filters were exposed to vapor-phase methamphetamine at concentrations ranging from 8 to 159 ppb, and samples were analyzed for exposure time periods from 2 h to 60 days. For a low vapor-phase methamphetamine concentration range of ~8-22 ppb, the equilibrium partition coefficient for AS-1 was 1500 ± 195 µg/g/ppb. For a high concentration range of 98-112 ppb, the partition coefficient was lower, 459 ± 80 µg/g/ppb, suggesting saturation of the available absorption capacity. The low partition coefficient for AS-2 (33 ± 6 µg/g/ppb) suggests that the fatty acids in AS-1 and skin oil are responsible for much high partition coefficients. We predict that the methamphetamine concentration in skin lipids coating indoor surfaces can exceed recommended surface remediation standards even for air concentrations well below 1 ppb.

A critical role of striatal A2A R-mGlu5 R interactions in modulating the psychomotor and drug-seeking effects of methamphetamine.

Addiction to psychostimulants is a major public health problem with no available treatment. Adenosine A2A receptors (A2A R) co-localize with metabotropic glutamate 5 receptors (mGlu5 R) in the striatum and functionally interact to modulate behaviours induced by addictive substances, such as alcohol. Using genetic and pharmacological antagonism of A2A R in mice, we investigated whether A2A R-mGlu5 R interaction can regulate the locomotor, stereotypic and drug-seeking effect of methamphetamine and cocaine, two drugs that exhibit distinct mechanism of action. Genetic deletion of A2A R, as well as combined administration of sub-threshold doses of the selective A2A R antagonist (SCH 58261, 0.01 mg/kg, i.p.) with the mGlu5 R antagonist, 3-((2-methyl-4-thiazolyl)ethynyl)pyridine (0.01 mg/kg, i.p.), prevented methamphetamine- but not cocaine-induced hyperactivity and stereotypic rearing behaviour. This drug combination also prevented methamphetamine-rewarding effects in a conditioned-place preference paradigm. Moreover, mGlu5 R binding was reduced in the nucleus accumbens core of A2A R knockout (KO) mice supporting an interaction between these receptors in a brain region crucial in mediating addiction processes. Chronic methamphetamine, but not cocaine administration, resulted in a significant increase in striatal mGlu5 R binding in wild-type mice, which was absent in the A2A R KO mice. These data are in support of a critical role of striatal A2A R-mGlu5 R functional interaction in mediating the ambulatory, stereotypic and reinforcing effects of methamphetamine but not cocaine-induced hyperlocomotion or stereotypy. The present study highlights a distinct and selective mechanistic role for this receptor interaction in regulating methamphetamine-induced behaviours and suggests that combined antagonism of A2A R and mGlu5 R may represent a novel therapy for methamphetamine addiction.

Methamphetamine induces a rapid increase of intracellular Ca(++) levels in neurons overexpressing GCaMP5.

In this study, methamphetamine (Meth)- and glutamate (Glu)-mediated intracellular Ca(++) (Ca(++) i) signals were examined in real time in primary cortical neurons overexpressing an intracellular Ca(++) probe, GCaMP5, by adeno-associated viral (AAV) serotype 1. Binding of Ca(++) to GCaMP increased green fluorescence intensity in cells. Both Meth and Glu induced a rapid increase in Ca(++) i, which was blocked by MK801, suggesting that Meth enhanced Ca(++) i through Glu receptor in neurons. The Meth-mediated Ca(++) signal was also blocked by Mg(++) , low Ca(++) or the L-type Ca(++) channel inhibitor nifedipine. The ryanodine receptor inhibitor dantrolene did not alter the initial Ca(++) influx but partially reduced the peak of Ca(++) i. These data suggest that Meth enhanced Ca(++) influx through membrane Ca(++) channels, which then triggered the release of Ca(++) from the endoplasmic reticulum in the cytosol. AAV-GCaMP5 was also injected to the parietal cortex of adult rats. Administration of Meth enhanced fluorescence in the ipsilateral cortex. Using immunohistochemistry, Meth-induced green fluorescence was found in the NeuN-containing cells in the cortex, suggesting that Meth increased Ca(++) in neurons in vivo. In conclusion, we have used in vitro and in vivo techniques to demonstrate a rapid increase of Ca(++) i by Meth in cortical neurons through overexpression of GCaMP5. As Meth induces behavioral responses and neurotoxicity through Ca(++) i, modulation of Ca(++) i may be useful to reduce Meth-related reactions.

Chronic Nicotine Exposure Attenuates Methamphetamine-Induced Dopaminergic Deficits.

Repeated methamphetamine (METH) administrations cause persistent dopaminergic deficits resembling aspects of Parkinson's disease. Many METH abusers smoke cigarettes and thus self-administer nicotine; yet few studies have investigated the effects of nicotine on METH-induced dopaminergic deficits. This interaction is of interest because preclinical studies demonstrate that nicotine can be neuroprotective, perhaps owing to effects involving α4ß2 and α6ß2 nicotinic acetylcholine receptors (nAChRs). This study revealed that oral nicotine exposure beginning in adolescence [postnatal day (PND) 40] through adulthood [PND 96] attenuated METH-induced striatal dopaminergic deficits when METH was administered at PND 89. This protection did not appear to be due to nicotine-induced alterations in METH pharmacokinetics. Short-term (i.e., 21-day) high-dose nicotine exposure also protected when administered from PND 40 to PND 61 (with METH at PND 54), but this protective effect did not persist. Short-term (i.e., 21-day) high-dose nicotine exposure did not protect when administered postadolescence (i.e., beginning at PND 61, with METH at PND 75). However, protection was engendered if the duration of nicotine exposure was extended to 39 days (with METH at PND 93). Autoradiographic analysis revealed that nicotine increased striatal α4ß2 expression, as assessed using [(125)I]epibatidine. Both METH and nicotine decreased striatal α6ß2 expression, as assessed using [(125)I]α-conotoxin MII. These findings indicate that nicotine protects against METH-induced striatal dopaminergic deficits, perhaps by affecting α4ß2 and/or α6ß2 expression, and that both age of onset and duration of nicotine exposure affect this protection.

Altered methamphetamine place conditioning in mice vaccinated with a succinyl-methamphetamine-tetanus-toxoid vaccine.

BACKGROUND AND OBJECTIVES: We previously reported that an anti-methamphetamine (MA) vaccine attenuated drug-conditioned effects in mice, but it used a carrier protein and adjuvant not available for clinical use. Here we produced a vaccine with the same hapten (succinyl-methamphetamine, SMA) but attached to tetanus toxoid (SMA-TT) and adsorbed to aluminum hydroxide, components approved for use in humans. We then assessed the vaccine's ability to generate anti-MA antibodies, alter acquisition and reinstatement of MA place conditioning, and prevent MA brain penetration. METHODS: Mice were administered SMA-TT at weeks 0 and 3 and non-vaccinated mice received saline. Anti-MA antibody concentrations were determined at 8 and 12 weeks. Place conditioning began during week 9 in which vaccinated and non-vaccinated mice were divided into groups and conditioned with .5, or 2.0 mg/kg MA. Following acquisition training, mice were extinguished and then a reinstatement test was performed in which mice were administered their original training dose of MA. Separate groups of non-vaccinated and vaccinated mice were administered .5 and 2.0 mg/kg MA and brain MA levels determined. RESULTS AND CONCLUSIONS: Anti-MA antibody levels were elevated at week 8 and remained so through week 12. The SMA-TT vaccine attenuated acquisition and reinstatement of MA place conditioning. Significantly greater proportions of vaccinated mice during acquisition and reinstatement tests showed conditioned place aversion. Moreover, MA brain levels were decreased in vaccinated mice following administration of both doses of MA. SCIENTIFIC SIGNIFICANCE: Results support further development of anti-MA vaccines using components approved for use in humans.

Nonlinear pharmacokinetics of (+/-)3,4-methylenedioxymethamphetamine (MDMA) and its pharmacodynamic consequences in the rat.

3,4-Methylenedioxymethamphetamine (MDMA) is a widely abused illicit drug that can cause severe and even fatal adverse effects. However, interest remains for its possible clinical applications in posttraumatic stress disorder and anxiety treatment. Preclinical studies to determine MDMA's safety are needed. We evaluated MDMA's pharmacokinetics and metabolism in male rats receiving 2.5, 5, and 10 mg/kg s.c. MDMA, and the associated pharmacodynamic consequences. Blood was collected via jugular catheter at 0, 0.5, 1, 2, 4, 6, 8, 16, and 24 hours, with simultaneous serotonin (5-HT) behavioral syndrome and core temperature monitoring. Plasma specimens were analyzed for MDMA and the metabolites (±)-3,4-dihydroxymethamphetamine (HHMA), (±)-4-hydroxy-3-methoxymethamphetamine (HMMA), and (±)-3,4-methylenedioxyamphetamine (MDA) by liquid chromatography-tandem mass spectrometry. After 2.5 mg/kg MDMA, mean MDMA Cmax was 164 ± 47.1 ng/ml, HHMA and HMMA were major metabolites, and <20% of MDMA was metabolized to MDA. After 5- and 10-mg/kg doses, MDMA areas under the curve (AUCs) were 3- and 10-fold greater than those after 2.5 mg/kg; HHMA and HMMA AUC values were relatively constant across doses; and MDA AUC values were greater than dose-proportional. Our data provide decisive in vivo evidence that MDMA and MDA display nonlinear accumulation via metabolic autoinhibition in the rat. Importantly, 5-HT syndrome severity correlated with MDMA concentrations (r = 0.8083; P < 0.0001) and core temperature correlated with MDA concentrations (r = 0.7595; P < 0.0001), suggesting that MDMA's behavioral and hyperthermic effects may involve distinct mechanisms. Given key similarities between MDMA pharmacokinetics in rats and humans, data from rats can be useful when provided at clinically relevant doses.

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.