Diana G. Wilkins

4-Methylmethcathinone (Mephedrone): Neuropharmacological Effects of a Designer Stimulant of Abuse

The designer stimulant 4-methylmethcathinone (mephedrone) is among the most popular of the derivatives of the naturally occurring psychostimulant cathinone. Mephedrone has been readily available for legal purchase both online and in some stores and has been promoted by aggressive Web-based marketing. Its abuse in many countries, including the United States, is a serious public health concern. Owing largely to its recent emergence, there are no formal pharmacodynamic or pharmacokinetic studies of mephedrone. Accordingly, the purpose of this study was to evaluate effects of this agent in a rat model. Results revealed that, similar to methylenedioxymethamphetamine, methamphetamine, and methcathinone, repeated mephedrone injections (4× 10 or 25 mg/kg s.c. per injection, 2-h intervals, administered in a pattern used frequently to mimic psychostimulant “binge” treatment) cause a rapid decrease in striatal dopamine (DA) and hippocampal serotonin (5-hydroxytryptamine; 5HT) transporter function. Mephedrone also inhibited both synaptosomal DA and 5HT uptake. Like methylenedioxymethamphetamine, but unlike methamphetamine or methcathinone, repeated mephedrone administrations also caused persistent serotonergic, but not dopaminergic, deficits. However, mephedrone caused DA release from a striatal suspension approaching that of methamphetamine and was self-administered by rodents. A method was developed to assess mephedrone concentrations in rat brain and plasma, and mephedrone levels were determined 1 h after a binge treatment. These data demonstrate that mephedrone has a unique pharmacological profile with both abuse liability and neurotoxic potential.

Higher Binding of the Dopamine D3 Receptor-Preferring Ligand [11C]-(+)-Propyl-Hexahydro-Naphtho-Oxazin in Methamphetamine Polydrug Users: A Positron Emission Tomography Study

Positron emission tomography (PET) findings suggesting lower D2-type dopamine receptors and dopamine concentration in brains of stimulant users have prompted speculation that increasing dopamine signaling might help in drug treatment. However, this strategy needs to consider the possibility, based on animal and postmortem human data, that dopaminergic activity at the related D3 receptor might, in contrast, be elevated and thereby contribute to drug-taking behavior. We tested the hypothesis that D3 receptor binding is above normal in methamphetamine (MA) polydrug users, using PET and the D3-preferring ligand [11C]-(+)-propyl-hexahydro-naphtho-oxazin ([11C]-(+)-PHNO). Sixteen control subjects and 16 polydrug users reporting MA as their primary drug of abuse underwent PET scanning after [11C]-(+)-PHNO. Compared with control subjects, drug users had higher [11C]-(+)-PHNO binding in the D3-rich midbrain substantia nigra (SN; +46%; p < 0.02) and in the globus pallidus (+9%; p = 0.06) and ventral pallidum (+11%; p = 0.1), whereas binding was slightly lower in the D2-rich dorsal striatum (approximately −4%, NS; −12% in heavy users, p = 0.01) and related to drug-use severity. The [11C]-(+)-PHNO binding ratio in D3-rich SN versus D2-rich dorsal striatum was 55% higher in MA users (p = 0.004), with heavy but not moderate users having ratios significantly different from controls. [11C]-(+)-PHNO binding in SN was related to self-reported “drug wanting.” We conclude that the dopamine D3 receptor, unlike the D2 receptor, might be upregulated in brains of MA polydrug users, although lower dopamine levels in MA users could have contributed to the finding. Pharmacological studies are needed to establish whether normalization of D3 receptor function could reduce vulnerability to relapse in stimulant abuse.

Plasma and liver acetaminophen-protein adduct levels in mice after acetaminophen treatment: dose-response, mechanisms, and clinical implications.

At therapeutic doses, acetaminophen (APAP) is a safe and effective analgesic. However, overdose of APAP is the principal cause of acute liver failure in the West. Binding of the reactive metabolite of APAP (NAPQI) to proteins is thought to be the initiating event in the mechanism of hepatotoxicity. Early work suggested that APAP-protein binding could not occur without glutathione (GSH) depletion, and likely only at toxic doses. Moreover, it was found that protein-derived APAP-cysteine could only be detected in serum after the onset of liver injury. On this basis, it was recently proposed that serum APAP-cysteine could be used as diagnostic marker of APAP overdose. However, comprehensive dose-response and time course studies have not yet been done. Furthermore, the effects of co-morbidities on this parameter have not been investigated. We treated groups of mice with APAP at multiple doses and measured liver GSH and both liver and plasma APAP-protein adducts at various timepoints. Our results show that protein binding can occur without much loss of GSH. Importantly, the data confirm earlier work that showed that protein-derived APAP-cysteine can appear in plasma without liver injury. Experiments performed in vitro suggest that this may involve multiple mechanisms, including secretion of adducted proteins and diffusion of NAPQI directly into plasma. Induction of liver necrosis through ischemia-reperfusion significantly increased the plasma concentration of protein-derived APAP-cysteine after a subtoxic dose of APAP. While our data generally support the measurement of serum APAP-protein adducts in the clinic, caution is suggested in the interpretation of this parameter.

Misclassification of Maternal Smoking Status and its Effects on an Epidemiologic Study of Pregnancy Outcomes

Reliance on self-reported smoking status among pregnant women can result in exposure misclassification. We used data from the Calcium for Preeclampsia Prevention trial, a randomized study of nulliparous women conducted from 1992 to 1995, to characterize tobacco exposure misclassification among women who reported at study enrollment that they had quit smoking. Urinary cotinine concentration was used to validate quit status, and factors associated with exposure misclassification and the effects of misclassification on associations between smoking and pregnancy outcomes were evaluated using logistic regression. Of 4,289 women enrolled, 508 were self-reported smokers and 771 were self-reported quitters. Of 737 self-reported quitters with a valid cotinine measurement, 21.6% had evidence of active smoking and were reclassified as smokers. Women who reported having quit smoking during pregnancy were more likely to be reclassified than women who reported quitting before pregnancy (p<.001). Among smokers, factors independently associated with misclassification of smoking status included fewer cigarettes smoked per day and fewer years smoked. After reclassification the odds ratio for a small-for-gestational-age birth among smokers decreased by 14%, and the smoking-related reduction in birth weight decreased by 15%. Effects of misclassification on the association with hypertensive disorders of pregnancy were present but less dramatic. In conclusion, use of self-reported smoking status collected at the time of study enrollment resulted in the introduction of bias into our study of smoking and pregnancy outcomes. The potential for this type of bias should be considered when conducting and interpreting epidemiologic studies of smoking and pregnancy outcomes.

Increased Vesicular Monoamine Transporter Binding during Early Abstinence In Human Methamphetamine Users: Is VMAT2 a Stable Dopamine Neuron Biomarker?

Animal data indicate that methamphetamine can damage striatal dopamine terminals. Efforts to document dopamine neuron damage in living brain of methamphetamine users have focused on the binding of [11C]dihydrotetrabenazine (DTBZ), a vesicular monoamine transporter (VMAT2) positron emission tomography (PET) radioligand, as a stable dopamine neuron biomarker. Previous PET data report a slight decrease in striatal [11C]DTBZ binding in human methamphetamine users after prolonged (mean, 3 years) abstinence, suggesting that the reduction would likely be substantial in early abstinence. We measured striatal VMAT2 binding in 16 recently withdrawn (mean, 19 d; range, 1–90 d) methamphetamine users and in 14 healthy matched-control subjects during a PET scan with (+)[11C]DTBZ. Unexpectedly, striatal (+)[11C]DTBZ binding was increased in methamphetamine users relative to controls (+22%, caudate; +12%, putamen; +11%, ventral striatum). Increased (+)[11C]DTBZ binding in caudate was most marked in methamphetamine users abstinent for 1–3 d (+41%), relative to the 7–21 d (+15%) and >21 d (+9%) groups. Above-normal VMAT2 binding in some drug users suggests that any toxic effect of methamphetamine on dopamine neurons might be masked by an increased (+)[11C]DTBZ binding and that VMAT2 radioligand binding might not be, as is generally assumed, a “stable” index of dopamine neuron integrity in vivo. One potential explanation for increased (+)[11C]DTBZ binding is that VMAT2 binding is sensitive to changes in vesicular dopamine storage levels, presumably low in drug users. If correct, (+)[11C]DTBZ might be a useful imaging probe to correlate changes in brain dopamine stores and behavior in users of methamphetamine.

Age-dependent differential responses of monoaminergic systems to high doses of methamphetamine

Abstract: Abuse of methamphetamine (METH) by adolescents is a major public health issue in the U.S.A. Because of the neurotoxic potential of METH, we examined the response of CNS monoaminergic systems in young (adolescent) animals [postnatal day (PND) 40] to high‐dose treatments (10 mg/kg, four injections, 2‐h intervals) of this drug and contrasted these effects to those seen in older (young adult) rats (PND 90). Consistent with previous reports, we observed that PND 40 animals did not manifest the long‐term (7‐day) deficits in extrapyramidal dopamine (DA) parameters observed in PND 90 rats. In contrast, METH‐induced rapid (1‐h) reduction in the activity of striatal DA transporters occurred in both age groups. In addition, both persistent (7‐day) and rapid (1‐h) deficits in serotonergic systems (measured as reductions in tryptophan hydroxylase activity) were observed in PND 40 and 90 rats. Age‐related differences in METH‐induced hyperthermia did not appear to be a principal cause for our observations; however, age‐dependent pharmacokinetics of this drug might have contributed to the differential METH monoaminergic responses by PND 40 and 90 animals.

Methamphetamine treatment rapidly inhibits serotonin, but not glutamate, transporters in rat brain

Previous studies have demonstrated that multiple methamphetamine (METH) administrations rapidly and reversibly decrease dopamine transporter activity assessed in striatal synaptosomes. A role for reactive oxygen species was suggested by findings that: (1) METH treatment increases the formation of oxygen radicals in vivo; and (2) oxygen radicals, generated by the enzyme xanthine oxidase, attenuate dopamine uptake in vitro. To test the selectivity of transporter responses, the present study examined effects of METH and xanthine oxidase on [3H]serotonin ([3H]5HT) and [3H]glutamate transport into striatal synaptosomes. Multiple doses of METH, or incubation with xanthine oxidase, rapidly attenuated [3H]5HT transport; an effect attributable to a decrease in Vmax. The METH-induced decrease in transport activity completely recovered by 24 h, but was decreased again 1 week later. In contrast, [3H]glutamate transport was essentially unchanged after METH treatment or incubation with xanthine oxidase. These findings indicate that: (1) METH causes a rapid and reversible decrease in 5HT transporter activity; and (2) glutamate transporters are less susceptible than 5HT transporters to effects of reactive species or METH treatment.

Tolerance to the neurotoxic effects of methamphetamine in young rats

The present study examined whether exposure to methamphetamine during adolescence (as determined in post-natal day 40 rats) might alter its effects when used in young adulthood (as assessed in post-natal day 90 rats). Results confirm that high-dose methamphetamine administration (4x10 mg/kg/injection, s.c., 2-h intervals) decreases striatal dopamine uptake and transporter ligand binding in post-natal day 90 rats; effects that were blocked if animals received six biweekly methamphetamine pretreatments (15 mg/kg; s.c.) beginning at post-natal day 40. This neuroprotection was not likely due to pharmacokinetic tolerance, since brain methamphetamine concentrations did not differ 1 h after the high-dose methamphetamine regimen among treated rats regardless of pretreatment. The methamphetamine biweekly pretreatment attenuated the hyperthermia caused by the neurotoxic methamphetamine regimen; a phenomenon that may have contributed to the neuroprotection.

Bupropion increases striatal vesicular monoamine transport

The vesicular monoamine transporter-2 (VMAT-2) is principally involved in regulating cytoplasmic dopamine (DA) concentrations within terminals by sequestering free DA into synaptic vesicles. This laboratory previously identified a correlation between striatal vesicular DA uptake through VMAT-2 and inhibition of the DA transporter (DAT). For example, administration of methylphenidate (MPD), a DAT inhibitor, increases vesicular DA uptake through VMAT-2 in a purified vesicular preparation; an effect associated with a redistribution of VMAT-2 protein within DA terminals. The purpose of this study was to determine if other DAT inhibitors, including bupropion, similarly affect VMAT-2. Results revealed bupropion rapidly, reversibly, and dose-dependently increased vesicular DA uptake; an effect also associated with VMAT-2 protein redistribution. The bupropion-induced increase in vesicular DA uptake was prevented by pretreatment with eticlopride, a DA D2 receptor antagonist, but not by SCH23390, a DA D1 receptor antagonist. We previously reported that MPD post-treatment prevents persistent DA deficits associated with multiple methamphetamine (METH) administrations. Although bupropion attenuated the METH-induced reduction in VMAT-2 activity acutely, it did not prevent the long-term dopaminergic toxicity or the METH-induced redistribution of VMAT-2 protein. The findings from this study demonstrate similarities and differences in the mechanism by which MPD and bupropion affect striatal dopaminergic nerve terminals.

Serum Caffeine and Paraxanthine as Markers for Reported Caffeine Intake in Pregnancy

PURPOSE Previous studies of maternal caffeine use and pregnancy outcome have relied on self-reported use. Even if these were perfectly accurate, inter-individual differences in caffeine metabolism result in a relatively weak correlation between caffeine intake and serum concentration. The purpose of this study was to determine whether the serum concentration of caffeine or its primary metabolite, paraxanthine, obtained at an unknown time during working hours, is useful to distinguish between pregnant women who report consuming small and large amounts of caffeine. METHODS We selected from the Birmingham fetal growth study 60 women with normal pregnancy outcomes who reported consuming < or = 0.8 mg/kg/day of caffeine in a 24-hour dietary recall, 60 who consumed 0.81-2.5 mg/kg/day, 60 who consumed 2.51-5.0 mg/kg/day and 59 who consumed > or = 5.01 mg/kg/day. These women had serum drawn for storage during regular clinic hours on the same day as the recall interview. Caffeine and paraxanthine were measured in the stored serum using high performance liquid chromatography. RESULTS The weighted kappa coefficient between strata of caffeine intake and quartiles of serum paraxanthine was 0.58 among smokers and 0.53 among nonsmokers, versus 0.44 and 0.51, respectively, for quartiles of serum caffeine. The Pearson correlation coefficient between intake and paraxanthine was 0.50 for smokers and 0.53 for nonsmokers, and 0.37 and 0.51, respectively, for serum caffeine. These values are comparable to the correlation between reported smoking and serum cotinine in pregnancy. CONCLUSIONS The serum concentrations of paraxanthine, and to a lesser degree, caffeine are useful to distinguish between women with varying levels of caffeine intake.