The neuroprotective effect of exogen melatonin upon fetal hippocampus damage caused by high-dose caffeine administration in pregnant rats.

OBJECTIVE: The aim of this study is to evaluate whether exogenous melatonin (MEL) mitigates the deleterious effects of high-dose caffeine (CAF) administration in pregnant rats upon the fetal hippocampus. MATERIALS AND METHODS: A total of 32 adult Wistar albino female rats were divided into four groups after conception (n = 8). At 9-20 days of pregnancy, intraperitoneal (i.p.) MEL was administered at a dose of 10 mg/kg/day in the MEL group, while i.p. CAF was administered at a dose of 60 mg/kg/day in the CAF group. In the CAF plus MEL group, i.p. CAF and MEL were administered at a dose of 60 and 10 mg/kg/day, respectively, at the same period. Following extraction of the brains of the fetuses sacrificed on the 21st day of pregnancy, their hippocampal regions were analyzed by hematoxylin and eosin and Cresyl Echt Violet, anti-GFAP, and antisynaptophysin staining methods. RESULTS: While there was a decrease in fetal and brain weights in the CAF group, it was found that the CAF plus MEL group had a closer weight average to that of the control group. Histologically, it was observed that the pyramidal cell layer consisted of 8-10 layers of cells due to the delay in migration in hippocampal neurons in the CAF group, while the MEL group showed similar characteristics with the control group. It was found that these findings decreased in the CAF plus MEL group. CONCLUSION: It is concluded that high-dose CAF administration causes a delay in neurogenesis of the fetal hippocampus, and exogenous MEL is able to mitigate its deleterious effects.

Can exposure to lisdexamfetamine dimesylate from juvenile period to peripubertal compromise male reproductive parameters in adult rats?

Lisdexamfetamine (LDX) is a d-amphetamine prodrug used to treat attention deficit and hyperactivity disorder, a common neurodevelopmental disorder in children and adolescents. Due to its action mediated by elevated levels of catecholamines, mainly dopamine and noradrenaline, which influence hormonal regulation and directly affect the gonads, this drug may potentially disrupt reproductive performance. This study evaluated the effects of exposure to LDX from the juvenile to peripubertal period (critical stages of development) on systemic and reproductive toxicity parameters in male rats. Male Wistar rats (23 days old) were treated with 0; 5.2; 8.6 or 12.1 mg/kg/day of LDX from post-natal day (PND) 23 to 53, by gavage. LDX treatment led to reduced daily food and water consumption, as well as a decrease in social behaviors. The day of preputial separation remained unaltered, although the treated animals exhibited reduced weight. At PND 54, the treated animals presented signs of systemic toxicity, evidenced by a reduction in body weight gain, increase in the relative weight of the liver, spleen, and seminal gland, reduction in erythrocyte and leukocyte counts, reduced total protein levels, and disruptions in oxidative parameters. In adulthood, there was an increase in immobile sperm, reduced sperm count, morphometric changes in the testis, and altered oxidative parameters, without compromising male sexual behavior and fertility. These findings showed that LDX-treatment during the juvenile and peripubertal periods induced immediate systemic toxicity and adversely influenced reproductive function in adult life, indicating that caution is necessary when prescribing this drug during the peripubertal phase.

Paternal methamphetamine exposure differentially affects first and second generations in mice.

Amphetamine-type stimulants are abused worldwide, and methamphetamine (METH) accounts for a large majority of seized abused drug cases. Recently, the paternal origin of health and disease theory has been proposed as a concept wherein paternal factors influence descendants. Although METH abuse is more common among males, its effects on their descendants were not examined. Therefore, we investigated the effects of paternal METH exposure on F1 and F2 levels in a mouse model. Sires were administered METH for 21 days and mated with female mice to obtain F1 mice. Growth evaluations (number of births, survival rate, body weight, righting reflex, cliff avoidance tests, and wire-hanging maneuver) were performed on F1 mice. Upon reaching six weeks of age, the mice were subjected to spontaneous locomotion, elevated plus-maze, acute METH treatment, and passive avoidance tests. Additionally, RNA-seq was performed on the striatum of male mice. Male F1 mice were mated with female mice to obtain F2 mice. They were subjected to the same tests as the F1 mice. Paternal METH exposure resulted in delayed growth and decreased memory function in F1 mice, overweight in F2 mice, decreased METH sensitivity, and reduced anxiety-related behaviors in female F2 mice. Enrichment analysis revealed significant enrichment of terms related to behavior in F1 and protein folding in F2. These results indicated that the effects of paternal METH exposure vary across generations. The effects of paternal factors need to be examined not only in F1, but also in F2 and beyond.

Melatonin enhances the restoration of neurological impairments and cognitive deficits during drug withdrawal in methamphetamine-induced toxicity and endoplasmic reticulum stress in rats.

Methamphetamine (METH) is a psychostimulant with a very high addiction rate. Prolonged use of METH has been observed as one of the root causes of neurotoxicity. Melatonin (Mel) has been found to have a significant role in METH-induced neurotoxicity. This study aimed to investigate the restorative effect of Mel on behavioral flexibility in METH-induced cognitive deficits. Male Sprague-Dawley rats were randomly assigned to be intraperitoneally injected with saline (control) or Meth at 5 mg/kg for 7 consecutive days. Then, METH injection was withdrawn and rats in each group were subcutaneously injected with saline or Mel at 10 mg/kg for 14 consecutive days. The stereotypic behavioral test and attentional set-shifting task (ASST) were used to evaluate neurological functions and cognitive flexibility, respectively. Rats developed abnormal features of stereotyped behaviors and deficits in cognitive flexibility after 7 days of METH administration. However, post-treatment with Mel for 14 days after METH withdrawal dramatically ameliorated the neurological and cognitive deficits in METH-treated rats. Blood biomarkers indicated METH-induced systemic low-grade inflammation. Moreover, METH-induced endoplasmic reticulum (ER) stress in the prefrontal cortex was diminished by melatonin supplementation. These findings might reveal the therapeutic potential of Mel in METH toxicity-induced neurological and cognitive deficits.

Dichotomous effect of methylphenidate on microglia and astrocytes: Insights from in vitro and animal studies.

Methylphenidate (MPH) has been used for decades to treat attention-deficit/hyperactivity disorder (ADHD) and narcolepsy. Moreover, several studies have shown that it is subject to misuse, particularly among college students and adolescents, for cognitive enhancement or as a recreational drug. This phenomenon causes concern, and it is critical to clarify better how MPH impacts brain cells. In fact, data has suggested that MPH could result in neuroinflammation and neurodegeneration across several brain regions; however, little is known about the effect of MPH on glial cells. To address this, we used microglia N9 cell line and primary cultures of cortical astrocytes that were exposed to MPH (0.01 - 2 mM), as well as Wistar Kyoto rats (WKY) chronically administered with MPH (1.5 mg/kg/day). Several parameters were analyzed, and we concluded that MPH has no significant direct effect on microglial cells, apart from cell migration impairment. On the contrary, MPH promotes astrogliosis, oxidative/nitrosative stress, and increases proinflammatory cytokine TNF levels by astrocytes, which was concordant with the results obtained in the hippocampus of WKY rats. Overall, the present results suggest that brain cells respond differently to MPH, with a more prominent direct effect on astrocytes when compared to microglia.

Overview of blood-brain barrier dysfunction in methamphetamine abuse.

Methamphetamine (METH) is one of the psychostimulants most widely abused in the world. METH abuse can lead to severe neurotoxicity. The blood-brain barrier (BBB) is a natural barrier separating the central nervous system (CNS) from the peripheral blood circulation, which can limit or regulate the exchange of toxic substances, molecules, ions, etc., to maintain the homeostasis of CNS. Long-term or high dose abuse of METH can cause structural or functional abnormalities of the BBB and increase the risk of neurodegenerative diseases. In this review, we discussed the mechanisms of METH-induced BBB dysfunction, summarized the risk factors that could exacerbate METH-induced BBB dysfunction, and introduced some potential therapeutic agents. It would provide an important basis and direction for the prevention and treatment of BBB dysfunction induced by METH.

Synthetic Cathinones and Neurotoxicity Risks: A Systematic Review.

According to the EU Early Warning System (EWS), synthetic cathinones (SCs) are the second largest new psychoactive substances (NPS) class, with 162 synthetic cathinones monitored by the EU EWS. They have a similar structure to cathinone, principally found in Catha Edulis; they have a phenethylamine related structure but also exhibit amphetamine-like stimulant effects. Illegal laboratories regularly develop new substances and place them on the market. For this reason, during the last decade this class of substances has presented a great challenge for public health and forensic toxicologists. Acting on different systems and with various mechanisms of action, the spectrum of side effects caused by the intake of these drugs of abuse is very broad. To date, most studies have focused on the substances' cardiac effects, and very few on their associated neurotoxicity. Specifically, synthetic cathinones appear to be involved in different neurological events, including increased alertness, mild agitation, severe psychosis, hyperthermia and death. A systematic literature search in PubMed and Scopus databases according to PRISMA guidelines was performed. A total of 515 studies published from 2005 to 2022 (350 articles from PubMed and 165 from Scopus) were initially screened for eligibility. The papers excluded, according to the criteria described in the Method Section (n = 401) and after full text analyses (n = 82), were 483 in total. The remaining 76 were included in the present review, as they met fully the inclusion criteria. The present work provides a comprehensive review on neurotoxic mechanisms of synthetic cathinones highlighting intoxication cases and fatalities in humans, as well as the toxic effects on animals (in particular rats, mice and zebrafish larvae). The reviewed studies showed brain-related adverse effects, including encephalopathy, coma and convulsions, and sympathomimetic and hallucinogenic toxidromes, together with the risk of developing excited/agitated delirium syndrome and serotonin syndrome.

Cyanidin prevents MDPV withdrawal-induced anxiety-like effects and dysregulation of cytokine systems in rats.

Psychostimulant exposure and withdrawal cause neuroimmune dysregulation and anxiety that contributes to dependence and relapse. Here, we tested the hypothesis that withdrawal from the synthetic cathinone MDPV (methylenedioxypyrovalerone) produces anxiety-like effects and enhanced levels of mesocorticolimbic cytokines that are inhibited by cyanidin, an anti-inflammatory flavonoid and nonselective blocker of IL-17A signaling. For comparison, we tested effects on glutamate transporter systems that are also dysregulated during psychostimulant free period. Rats injected for 9 d with MDPV (1 mg/kg, IP) or saline were pretreated daily with cyanidin (0.5 mg/kg, IP) or saline, followed by behavioral testing on the elevated zero maze (EZM) 72 h after the last MDPV injection. MDPV withdrawal caused a reduction in time spent on the open arm of the EZM that was prevented by cyanidin. Cyanidin itself did not affect locomotor activity or time spent on the open arm, or cause aversive or rewarding effects in place preference experiments. MDPV withdrawal caused enhancement of cytokine levels (IL-17A, IL-1ß, IL-6, TNF=α, IL-10, and CCL2) in the ventral tegmental area, but not amygdala, nucleus accumbens, or prefrontal cortex, that was prevented by cyanidin. During MDPV withdrawal, mRNA levels of glutamate aspartate transporter (GLAST) and glutamate transporter subtype 1 (GLT-1) in the amygdala were also elevated but normalized by cyanidin treatment. These results show that MDPV withdrawal induced anxiety, and brain-region specific dysregulation of cytokine and glutamate systems, that are both prevented by cyanidin, thus identifying cyanidin for further investigation in the context of psychostimulant dependence and relapse.

Amphetamines abuse and depression: Focus on TRPC channels.

Amphetamines, such as amphetamine (AMPH), methamphetamine (METH) and 3,4-methylenedioxymethamphetamine (MDMA), are the psychotropic substances widely abused in the world. Amphetamines abuse can damage dopaminergic and serotonin neurons and cause neuroinflammation and neurotoxicity. Neuropsychiatric disorders induced by amphetamines abuse include depression, anxiety, auditory hallucinations, mania, and cognitive disorders, of which depression has a higher incidence. Transient receptor potential (TRP) channels can regulate the inflow and outflow of Ca2+. In TRP family, transient receptor potential canonical (TRPC) channels are closely associated with the development of some neurological diseases, such as Parkinson's disease and Alzheimer's disease. However, the correlation between TRPC channels and depression and the specific mechanism of TRPC channels in depression still haven't been fully clarified. This review elaborates the pathophysiological mechanisms of depression induced by amphetamines abuse, the functions of TRPC channels in the nervous system, and the possible correlation between TRPC channels and depression induced by amphetamines abuse, which would provide the theoretical basis for the development of the novel and effective therapeutic drugs for amphetamines abuse-induced depression.

Molecular mechanisms and treatment strategies for methamphetamine­induced neurodegeneration, inflammation and neurotoxicity.

Methamphetamine (METH) is a highly addictive psychostimulant known for its profound impact on the nervous system. Chronic METH use leads to neurotoxicity characterized by various molecular and structural alterations in the brain. This review article primarily aims to elucidate the mechanisms underlying METH­induced neurotoxicity. METH's mechanism of action involves the inhibition of dopamine, serotonin, and norepinephrine reuptake, resulting in altered synaptic function. Prolonged METH exposure triggers oxidative stress, endoplasmic reticulum stress, mitochondrial dysfunction, impaired axonal transport, autophagy, and programmed cell death, ultimately contributing to neurotoxicity. These neurotoxic effects manifest as increased neuronal firing rate, disruptions in intracellular ion balance (Ca2+ and Na+), energy production imbalances, and excessive reactive oxygen species production. The blood­brain barrier is compromised, leading to structural, functional, and neurochemical alterations, particularly in the fronto­striatal circuit. While our comprehensive review addresses these intricate molecular and structural changes induced by METH, we also examined the latest therapeutic strategies designed to mitigate neurotoxicity. Our investigation sheds light on the critical need to comprehend the complex pathways underlying METH­induced neurotoxicity and develop effective treatment approaches.

Methamphetamine induced neurotoxic diseases, molecular mechanism, and current treatment strategies.

Methamphetamine (MA) is a extremely addictive psychostimulant drug with a significant abuse potential. Long-term MA exposure can induce neurotoxic effects through oxidative stress, mitochondrial functional impairment, endoplasmic reticulum stress, the activation of astrocytes and microglial cells, axonal transport barriers, autophagy, and apoptosis. However, the molecular and cellular mechanisms underlying MA-induced neurotoxicity remain unclear. MA abuse increases the chances of developing neurotoxic conditions such as Parkinson's disease (PD), Alzheimer's disease (AD) and other neurotoxic diseases. MA increases the risk of PD by increasing the expression of alpha-synuclein (ASYN). Furthermore, MA abuse is linked to high chances of developing AD and subsequent neurodegeneration due to biological variations in the brain region or genetic and epigenetic variations. To date, there is no Food and Drug Administration (FDA)-approved therapy for MA-induced neurotoxicity, although many studies are being conducted to develop effective therapeutic strategies. Most current studies are now focused on developing therapies to diminish the neurotoxic effects of MA, based on the underlying mechanism of neurotoxicity. This review article highlights current research on several therapeutic techniques targeting multiple pathways to reduce the neurotoxic effects of MA in the brain, as well as the putative mechanism of MA-induced neurotoxicity.

Increased sperm deoxyribonucleic acid damage leads to poor embryo quality and subfertility of male rats treated with methylphenidate hydrochloride in adolescence.

BACKGROUND: Methylphenidate hydrochloride (MPH) is a psychostimulant widely used in the treatment of attention-deficit hyperactive disorder (ADHD), as well as a performance enhancer, for at least 60 years. Despite the notable effectiveness as a psychostimulant, ADHD is a chronic disorder and has a two-third chance of accompanying the individual throughout life. Long-term use of MPH has been associated not only with an increase in the development of neurodegenerative diseases, but it also causes side effects on male fertility in experimental animals. OBJECTIVES: To investigate whether methylphenidate poses a risk to sperm DNA structure and to the quality of embryos conceived after treatment during adolescence in rats. MATERIALS AND METHODS: Wistar rats at 38 days of age were treated either with 5 mg/kg body weight of MPH, in a single daily dose for 30 days, via gavage or with distilled water-only protocol. Levels of oxidative stress in testicular and epididymal tissues were evaluated. Sperm chromatin quality and acrosome integrity was assessed under flow cytometry. From 107 days of age, animals were mated with untreated females. The effects of the paternal contribution at two different embryo development moments-cleavage stage (2.5 days post coitum) and late gestation (20 days post coitum) -were analyzed. RESULTS: MPH caused high levels of sperm DNA damage, which was reflected in 40% of decrease in early embryo quality and a lower number of live pups at 20 dpc. DISCUSSION: The high level of fragmentation seen in the embryos sired from the MPH group is consistent with the poor chromatin structure of the sperm and does not seem to be a result of oxidative stress in the reproductive tissues. CONCLUSIONS: The results presented here suggest that the subchronic use of MPH during male prepubertal phase may cause long-term subfertility and compromise embryo survival.

A review of basic to clinical studies of the association between hyperammonemia, methamphetamine.

Methamphetamine (METH), an addictive psychostimulant drug, is the second most widely used type of drug all around the world. METH abusers are more likely to develop a psycho-neurological complication. Hyperammonemia (HAM) causes neuropsychiatric illnesses such as mental state changes and episodes of acute encephalopathy. Recently, there are some shreds of evidence about the relationship between METH complication and HAM. Both METH intoxication and HAM could induce psychosis, agitation, memory impairment, and psycho-neuronal disorders. They also have similar mechanisms of neuronal damages, such as excitotoxicity, oxidative stress, mitochondrial impairments, and inflammation responses, which can subsequently increase the glutamate level of the brain. Hence, the basic to clinical studies of the association between HAM and METH are reviewed by monitoring six case studies and a good body of animal studies literature. All instances of METH-associated HAM had changes in mental state and some level of confusion that were improved when the ammonia serum level returned to the normal level. Furthermore, most of them had typical vital signs. Several studies suggested some sources for METH-associated HAM, including METH-induced liver and renal damages, muscular hyperactivity, gut bacterial overgrowth, co-abuse of other substances, and using some forms of NH3 in METH cooking. In conclusion, it seems that mental status changes in METH abusers may be related to ammonia intoxication or HAM; therefore, it is important to assess the serum level of ammonia in METH intoxicated patients and resolve it.

The relationship between serum brain-derived neurotrophic level and neurocognitive functions in chronic methamphetamine users.

Methamphetamine (METH) is a highly addictive psychostimulant with serious neurotoxic effects. Given evidence indicating that brain-derived neurotrophic factor (BDNF) is associated with addictive behaviors, this study aimed to investigate the serum level of BDNF and cognitive functions in chronic METH users and healthy participants. Thirty-seven chronic METH users and 37 healthy participants were recruited in this study. Cognitive functioning, including executive functions and working memory, were assessed using the Wisconsin Card Sorting Test (WCST) and Wechsler Memory Scale (WMS), respectively. The levels of serum BDNF were also examined using an enzyme-linked immunosorbent assay kit. METH users showed significant impairment in executive function and working memory compared to healthy participants. The serum BDNF concentrations of METH users were significantly higher than healthy participants (42 ± 13.34 ng/ml vs. 24 ± 7 ng/ml). BDNF concentration was significantly correlated with duration (r = 0.37, p = 0.02) and dose of METH use (r = 0.35, p = 0.02). Besides, the BDNF level was not associated with any subscales of WCST and WMS. These results provide further evidence regarding the role of BDNF in the pathophysiology of METH addiction. Besides, these findings suggest that increased BDNF level is not related to cognitive impairments in METH users.

The Crosstalk Between Neurons and Glia in Methamphetamine-Induced Neuroinflammation.

Methamphetamine (METH), an illicit psycho-stimulant, is widely known as an addictive drug that may cause neurotoxic effects. Previous researches on METH abuse have mainly focused on neurotransmitters, such as dopamine and glutamate. However, there is growing evidence that neuroinflammation also plays an important role in the etiology and pathophysiology of brain dysfunction induced by METH abuse. This has cast a spotlight on the research of microglia and astrocyte, which are critical mediators of neuroimmune pathology in recent years. In the central nervous system (CNS) immunity, abnormalities of the microglia and astrocytes have been observed in METH abusers from both postmortem and preclinical studies. The bidirectional communication between neurons and glia is essential for the homeostasis and biological function of the CNS while activation of glia induces the release of cytokines and chemokines during pathological conditions, which will affect the neuron-glia interactions and lead to adverse behavioral consequences. However, the underlying mechanisms of interaction between neurons and glia in METH-induced neuroinflammation remain elusive. Notably, discovering and further understanding glial activity and functions, as well as the crosstalk between neurons and glia may help to explain the pathogenesis of METH abuse and behavioral changes in abusers. In this review, we will discuss the current understanding of the crosstalk between neurons and glia in METH-induced neuroinflammation. We also review the existing microglia-astrocyte interaction under METH exposure. We hope the present review will lead the way for more studies on the development of new therapeutic strategies for METH abuse in the near future.

The effects of minocycline in improving of methamphetamine withdrawal syndrome in male mice.

Methamphetamine (METH) is a potent psychostimulant drug with an increasing rate of abuse over recent years. Depressive-like behaviors are one of the major symptoms patients in the METH withdrawal period experience. There is limited evidence regarding the METH withdrawal treatment, and conventional managements are not completely effective. Furthermore, extensive promising literature supports minocycline, a well-known antibiotic with anti-oxidant, anti-inflammatory properties, to treat depressive-like behaviors. Therefore, we hypothesized that administration of minocycline might mitigate the methamphetamine (METH) induced depression in male mice. Administration of METH (2 mg/kg) to mice two times a day for 14 constitutive days was done to induce the METH-induced withdrawal syndrome model. Animals were divided into 10 groups (n = 10 in each group), and three doses of minocycline (2.5, 5 and 10 mg/kg) were daily administered to male albino mice for 10 days. Following the behavioral test, the animals were scarified, their hippocampus were dissected to measure oxidative stress parameters. Our data revealed that chronic administration of minocycline provoked antidepressant effects in behavioral tests, such as forced swim test (FST), tail suspension test (TST) and splash test. Additionally, minocycline was able to improve oxidative stresses and neuronal damage in the hippocampus and restore the body's antioxidant system by increasing glutathione (GSH) and the cellular energy (ATP) and reducing the malondialdehyde (MDA) level. According to our promising results of minocycline on targeting mitochondria and its performance, we suggest minocycline as a new therapeutic option in clinical trials of depression treatment.

Behavioural and neurochemical effects after repeated administration of N-ethylpentylone (ephylone) in mice.

N-ethyl-pentylone (NEP), also known as 'ephylone' and N-ethylnorpentylone, has been identified as one of the most recent novel psychostimulants to emerge into the illicit drug market and it has been associated with some intoxications and even fatalities. However, little is known about the consequences of its repeated consumption as well as the role of the monoaminergic system in such consequences. Thus, the aim of our study was to investigate the neurochemical profile and the behavioural effects after both acute and repeated NEP exposure. Male OF1 mice were acutely (1, 3, 10 mg/kg, i.p.) or repeatedly (1, 3, 10 mg/kg, i.p., 5 days, twice/day) exposed to NEP, and anxiety-like behaviour, aggressiveness, social interaction, depressive-like symptoms, body temperature, changes in monoaminergic enzymes and neurotransmitters levels as well as ΔFosB in striatum and prefrontal cortex (PFC) from post-mortem tissue were analysed short after drug-exposure or during drug-withdrawal. Acute administration of NEP induced anxiolytic effects but also an aggressive behaviour and social exploration deficits in mice, which persist during NEP-withdrawal. Moreover, NEP induced hyperthermia as well as depressive-like symptoms after repeated administrations that may be related to the decrease in serotonin and noradrenaline levels observed in striatum and PFC. Finally, the long-term increase in ΔFosB levels in striatum after NEP chronic exposure points to a high risk of dependence. Altogether indicates that NEP consumption induces different neurological and neuropsychiatric disorders accompanied by changes in the monoaminergic system, posing a threat to public health.

Effect of repeated sodium valproate and topiramate administration on mania­like behaviors induced by methylphenidate in mice.

Novel and effective treatments for mania are needed, and well­validated animal models are important to reach this goal. The psychostimulant­induced hyperactivity is the most frequently animal model of mania used. Although this model is validated pharmacologically using mood stabilizers, data about its predictive validity with negative controls (i.e., drugs that are clinically ineffective in treating mania) are lacking. The present study evaluated the effects of the repeated administration of a clinically effective drug (sodium valproate) and clinically ineffective drug (topiramate) on methylphenidate (MPH)­induced manic­like behaviors in Swiss mice in the behavioral pattern monitor (BPM). Methylphenidate increased locomotor activity and center activity in the BPM. Valproate attenuated the effect of MPH on locomotor and general activity, with no effect on center activity. Topiramate did not affect any MPH­induced manic­like behaviors. Methylphenidate did not change exploratory activity (rearing or nose poking). These results support the predictive validity of MPH­induced hyperactivity for screening antimanic­like drugs.