Benzodiazepines in complex biological matrices: Recent updates on pretreatment and detection methods

Benzodiazepines(BDZs)are used in clinics for anxiolysis,anticonvulsants,sedative hypnosis,and muscle relaxation.They have high consumptions worldwide because of their easy availability and potential addiction.They are often used for suicide or criminal practices such as abduction and drug-facilitated sexual assault.The pharmacological effects of using small doses of BDZs and their detections from complex biological matrices are challenging.Efficient pretreatment methods followed by accurate and sensitive detections are necessary.Herein,pretreatment methods for the extraction,enrichment,and preconcentration of BDZs as well as the strategies for their screening,identification,and quantitation developed in the past five years have been reviewed.Moreover,recent advances in various methods are summarized.Characteristics and advantages of each method are encompassed.Future directions of the pretreatment and detection methods for BDZs are also reviewed.

The FDA Mandate to Reassess Benzodiazepines: Alprazolam Induces a Positive Conditioned Place-Preference in Male Rats

On September 23, 2020, in order “To address the serious risks of abuse, addiction, physical dependence, and withdrawal reactions, the U.S. Food and Drug Administration (FDA) is requiring the Boxed Warning be updated for all benzodiazepine medicines”. With this announcement, the FDA proclaimed that much more needs to be known about the initiation, continuation, and discontinuation of these widely-used drugs. Unfortunately, relevant information is lacking, since for many years, there has been a notable sparsity in the funding and conduct of basic and clinical research on these drugs. In order to begin to fill the void, it is valuable to (re)examine animal models. We here describe a model of conditioned place-preference (CPP) for rats and for the first time, to our knowledge, show that the representative benzodiazepine alprazolam induces positive place-preference in male rats.

Fast Determination of Benzodiazepines in Human Urine via Liquid-Liquid Extraction with Low Temperature Partitioning and LC-HRMS

A simple and high-throughput method to simultaneously determine selected benzodiazepines (i.e., diazepam, lorazepam, clonazepam, and bromazepam) in urine was developed and validated. The entire methodology consisted of the application of an innovative extraction/cleanup procedure, namely liquid-liquid extraction with low-temperature partitioning (LLE-LTP), and analysis by liquid chromatography combined with high-resolution mass spectrometry (LC-HRMS). The LLE-LTP procedure was optimized via factorial design and by evaluating crucial variables, specifically the freezing mode (either slow or fast), the urine/acetonitrile volume ratio, and the sample ionic strength. The benzodiazepines were quantified using matrix-matched calibration curves where the following parameters were assessed by validation protocol: in general, linearity range of 17 - 200 μg?L–1 (r > 0.9957);limits of detection lower than 5 μg?L–1;relative standard deviations (RSD) lower than 12.5%;and accuracy ranging from 72.3 to 117%. To test this procedure’s performance, the method was applied to determine the content of diazepam in actual urine samples. The validation results obtained for the method demonstrated that the present methodology could be potentially applied in proficient laboratories as a routine approach for determining benzodiazepines compounds content in urine.

Psychoactive Chemical Constituents of Carissa edulis (Forssk.) Vahl Roots and Its Safety Limits in Folk Treatment: Benzodiazepines Analogous

Carissa edulis Vahl is well known in Sudanese herbal medicine,commonly used for treatment of epilepsy,headache,chest pains,rheumatism,skin lesions,mania and other psychoactive diseases.The investigations of the safety use for psychoactive purposes in Sudanese healing traditions and identifying secondary metabolites of the plant extracts are the key steps towards determination of appropriate medicinal doses.Therefore,one of the chemical constituents was isolated and structurally identified by 1H-NMR and LC-MS.With the aim of evaluating Carissa edulis folk random uses,the isolated compound was compared with reference artificial drugs Lormetazepam,a potentially toxic compound.Structure investigations confirm that the isolated product was benzodiazepines analogous 7-chloro 1,4-benzodiazepine-2-ones.It is important to know the potential toxicity of certain plant in order to assess the therapeutic effect of it,as these are slight distinctions between the medicinal and toxic doses.In general the results obtained justify the use of the roots of Carissa edulis in traditional medicine for the treatment of some psychiatric diseases.

Intranasal Delivery of Two Benzodiazepines, Midazolam and Diazepam, by a Microemulsion System

Nasal application of benzodiazepines might be an alternative to intravenous administration in acute clinical situations such as seizures emergencies. However, irritation and pain as well as symptoms like teary eyes, dizziness, discomfort, nasal drainage and bad taste usually accompany subject received midazolam and diazepam via the nasal route. The purpose of this study was to evaluate the use of a new alcohol-free microemulsion system as a carrier for diazepam or midazolam given intranasally. Midazolam (base) or diazepam was solubilized in the microemulsion to obtain a high drug concentration of 25 mg/g (2.5% by weight), to provide 2.5 mg drug in 100 μl spray (d ≈ 1.00 g/ml). The nasal absorption of both drugs from the same microemulsion formulation (containing 20% aqueous phase) was found to be fairly rapid after administration of 0.4 mg/kg to rabbits. The absolute bioavailability of diazepam after intranasal administration using this formulation was 33.45% ± 12.36% and the tmax was 18.33 ± 23.09 min, which was twice longer than the tmax obtained after midazolam administration, 9.25 ± 6.75 min. The pharmacokinetic parameters of midazolam in W/O (20% water) microemulsion and their comparison with midazolam in O/W (50% water) microemulsion have shown that both formulations resulted in a relatively short time to reach the peak plasma level (tmax), that is, 9.25 ± 6.75 min and 6.75 ± 5.67 min, respectively. However, the peak plasma levels (Cmax) and the absolute bioavailability (FA) of midazolam were significantly higher after administration of the W/O formulation than those obtained after application of O/W formulation, i.e., 46.62 ± 17.38 μg/ml vs. 15.44 ± 4.00 μg/ml, and 35.19% ± 11.83% vs. 19.83% ± 16.32%, respectively. Our results suggest that the new microemulsion system may be useful for getting rapid-onset of midazolam and diazepam following intranasal administration, resulting in reasonable peak plasma levels and bioavailability, but most importantly, providing a high measure of tolerability and comfort.

Original and Simple Protocol for Withdrawal of Benzodiazepines to Achieve Sustained Remission

Benzodiazepines are psychotropic medications for effective management of anxiety. However, after over 50 years of unrestrained prescription use, prescribers realize the severity of side effects in select patients. The most challenging of these side effects is the emergence of a physiological dependence that explains virtual impossibility of successful withdrawal that can ensure sustained abstinence. We propose a method for successful withdrawal using substitution and innovative downward tapering of diazepam oral solution for all benzodiazepines to attain sustained remission. Our protocol reported here allowed not only successful withdrawal for all of 20 patients included in this pilot study but also helped in totally weaning them off of benzodiazepines demonstrating absence of relapse even 2 years after the total discontinuance.

Research progress on benzodiazepines to treat alcohol withdrawal syndrome

Alcohol withdrawal syndrome(AWS)refers to a series of symptoms and signs that chronic al-coholics experience when they suddenly stop drinking or reduce their drinking,usually 12 to 24 h later.These in-clude tremors,fatigue,sweating,hyperreflexia,and gas-trointestinal symptoms.This article will analyze the drug treatment of this disease and make a brief review.

Emergency department management of acute agitation in the reproductive age female and pregnancy

BACKGROUND:Agitation is a common presentation within emergent departments(EDs).Agitation during pregnancy should be treated as an obstetric emergency,as the distress may jeopardize both the patient and fetus.The safety of psychotropic medications in the reproductive age female has not been well established.This review aimed to explore a summary of general agitation recommendations with an emphasis on ED management of agitation during pregnancy.METHODS:A literature review was conducted to explore the pathophysiology of acute agitation and devise a preferred treatment plan for ED management of acute agitation in the reproductive age or pregnant female.RESULTS:While nonpharmacological management is preferred,ED visits for agitation often require medical management.Medication should be selected based on the etiology of agitation and the clinical setting to avoid major adverse effects.Adverse effects are common in pregnant females.For mild to moderate agitation in pregnancy,diphenhydramine is an effective sedating agent with minimal adverse effects.In moderate to severe agitation,high-potency typical psychotropics are preferred due to their neutral effects on hemodynamics.Haloperidol has become the most frequently utilized psychotropic for agitation during pregnancy.Second generation psychotropics are often utilized as second-line therapy,including risperidone.Benzodiazepines and ketamine have demonstrated adverse fetal outcomes.CONCLUSION:While randomized control studies cannot be ethically conducted on pregnant patients requiring sedation,animal models and epidemiologic studies have demonstrated the effects of psychotropic medication exposure in utero.As the fetal risk associated with multiple doses of psychotropic medications remains unknown,weighing the risks and benefits of each agent,while utilizing the lowest effective dose remains critical in the treatment of acute agitation within the EDs.

Catatonia:A deep dive into its unfathomable depths

This editorial addresses catatonia,a complex neuropsychiatric syndrome characterised by a spectrum of psychomotor disturbances.The editorial seeks to clarify the ambiguous aspects of catatonia,integrating recent research findings,including global studies and diagnostic advancements.It discusses catatonia’s clinical manifestations,prevalence,and associated psychiatric and medical conditions,with particular emphasis on its frequent co-occurrence with schizophrenia and mood disorders.The prevalence of catatonia,which varies across psychiatric populations,is illustrated by a significant study conducted in Nelson Mandela Bay,South Africa.This study provides valuable insights into the effectiveness of the Bush-Francis Screening Instrument compared to the Diagnostic and Statistical Manual 5 criteria in diagnosing catatonia.The editorial evaluates treatment approaches,primarily focusing on benzodiazepines and electroconvulsive therapy,and discusses emerging therapeutic strategies.It underscores the importance of robust diagnostic frameworks and early intervention in managing catatonia,as recommended by the latest evidence-based consensus guideline.Furthermore,it suggests future research directions,particularly in exploring the neurobiological and genetic factors of catatonia,to enhance our understanding and improve treatment outcomes.This editorial succinctly aims to demystify catatonia and provide valuable insights for clinicians and researchers in mental health care.

In（OTf）3-Catalyzed Synthesis of Functionalized 1,5-Benzodiazepines from o-Phenylenediamine and Alkyl Propiolates under Solvent-Free Reaction Conditions

A simple, environmental-friendly, and practical method for the synthesis of benzodiazepine derivatives through a reaction of substituted o-phenylenediamines with alkyl propiolates has been developed. The reactions generated the 1,5-benzodiazepines in good to excellent yields in the presence of catalytic amount of In（OTf）3 under sol- vent-free reaction conditions.

Cycloaddition of benzoheteroazepine Ⅲ Reaction of 2,3-dihydro-lH-l,5-benzodiazepines with dichlorocarbene and stereo-structures of products

Reaction of 2,3-dihydro-1H-l,5-benzodiazepines with dichlorocarbene generated in situ using benzyltriethylammonium chloride (TEBA) as a phase transfer catalyst in chloroform-aqueous sodium hydroxide mixture gave mainly 1,2-cycloadducts, as and trans-la,3-disubstituted-1, 1-dichloro-1a,2,3,4-tetrahydro-1H-azirino[l,2-a][l,5]benzodiazepines (2,3), and formylated 1,2-cycloadducts, trans-la,3-disubstituted-l, 1-dichloro-4-formyl-1a,2,3,4-tetrahydro-lH-azirino[l,2-a][l,5]benzodiazepines (4). The stereo-structures of cycloadducts and the mechanism are also discussed.

Mass spectrometric studies of cis- and trans-1a,3-disubstituted-1, 1-dichloro-4-formyl-1a ,2,3,4-tetrahydro-1H-azirino [ 1,2-a] [1,5] benzodiazepines

The mass spectrometric behaviour of four cis- and trans-1a, 3-disuhsdtuted-1,1-dichloro-4-formyl-1a,2,3,4-tetrahydro-1H-azirino[1, 2-a] [1, 5]benzodiazepines has been studied with the aid of mass-analysed ion kinetic energy spectrometry and exact mass measurements under electron impact ionization. All compounds show a tendency to eliminate a chlorine atom from the aziridine ring, and then eliminate a neutral propene or styrene from the diazepine ring to yield azirino[1, 2b][1,3] benzimidazole ions. These azirino[1,2-a][1,5]benzodiazepines can also eliminate HCl, or Cl plus HCl simultaneously to undergo a ring enlargement rearrangement to yield 1,6-benzodiazocine ions, which further lose small molecular fragments, propyne or phenylacetylene, with rearrangement to give quinoxaline ions.

Pilot Studies on the Novel Hypothesis that TSPO (Peripheral Benzodiazepine Receptor) Is Involved in Benzodiazepine/“Z-Drug” Physical Dependence and/or Withdrawal

Benzodiazepines and other benzodiazepine receptor agonists, such as the “Z” drugs, are widely prescribed medications mainly used for treating anxiety and seizures, and for inducing sedation. Unfortunately, despite their popularity, benzodiazepine prescribing often exceeds recommendations and the consequences can be severe. On September 23, 2020, the United States FDA announced a new requirement for a Boxed Warning for benzodiazepines prescribing. Along with this announcement, the FDA stated that relevant information regarding the initiation, continuation, and discontinuation of benzodiazepines is lacking. Here, we describe initial pilot studies intended to investigate the questions 1) can animal models be developed that demonstrate benzodiazepine physical dependence and/or withdrawal symptoms, and 2) determine whether translocator protein (TSPO) plays a role in benzodiazepine dependence and/or withdrawal processes. The former was demonstrated, methodological limitations prevented the latter.

Catatonia:Our current understanding of its diagnosis, treatment and pathophysiology

Catatonia is a psychomotor syndrome that has been reported to occur in more than 10% of patients with acute psychiatric illnesses. Two subtypes of the syndrome have been identified. Catatonia of the retarded type is characterized by immobility, mutism, staring, rigidity, and a host of other clinical signs. Excited catatonia is a less common presentation in which patients develop prolonged periods of psychomotor agitation. Once thought to be a subtype of schizophrenia, catatonia is now recognized to occur with a broad spectrum of medical and psychiatric illnesses, particularly affective disorders. In many cases, the catatonia must be treated before any underlying conditions can be accurately diagnosed. Most patients with the syndrome respond rapidly to low-dose benzodiazepines, but electroconvulsive therapy is occasionally required. Patients with longstanding catatonia or a diagnosis of schizophrenia may be less likely to respond. The pathobiology of catatonia is poorly understood, although abnormalities in gamma-aminobutyric acid and glutamate signaling have been suggested as causative factors. Because catatonia is common, highly treatable, and associated with significant morbidity and mortality if left untreated, physicians should maintain a high level of suspicion for this complex clinical syndrome. Since 1989, we have systematically assessed patients presenting to our psychiatry service with signs of retarded catatonia. In this paper, we present a review of the current literature on catatonia along with findings from the 220 cases we have assessed and treated.

Electroconvulsive therapy in catatonic patients: Efficacy and predictors of response

Recent evidence favors the view of catatonia as an autonomous syndrome, frequently associated with mood disorders, but also observed in neurological, neurodevelop-mental, physical and toxic conditions. From our systematic literature review, electroconvulsive therapy(ECT) results effective in all forms of catatonia, even after pharmacotherapy with benzodiazepines has failed. Response rate ranges from 80% to 100% and results superior to those of any other therapy in psychiatry. ECT should be considered first-line treatment in patients with malignant catatonia, neuroleptic malignant syndrome, delirious mania or severe catatonic excitement, and in general in all catatonic patients that are refractory or partially responsive to benzodiazepines. Early intervention with ECT is encouraged to avoid undue deterioration of the patient's medical condition. Little is known about the long-term treatment outcomes following administration of ECT for catatonia. The presence of a concomitant chronic neurologic disease or extrapyramidal deficit seems to be related to ECT non-response. On the contrary, the presence of acute, severe and psychotic mood disorder is associated with good response. Severe psychotic features in responders may be related with a prominent GABAergic mediated deficit in orbitofrontal cortex, whereas non-responders may be characterized by a prevalent dopaminergic mediated extrapyramidal deficit. These observations are consistent with the hypothesis that ECT is more effective in "top-down" variant of catatonia, in which the psychomotor syndrome may be sustained by a dysregulation of the orbitofrontal cortex, than in "bottom-up" variant, in which an extrapyramidal dysregulation may be prevalent. Future research should focus on ECT response in different subtype of catatonia and on efficacy of maintenance ECT in long-term prevention of recurrent catatonia. Further research on mechanism of action of ECT in catatonia may also contribute to the development of other brain stimulation techniques.

Propofol vs traditional sedatives for sedation in endoscopy:A systematic review and meta-analysis

BACKGROUND Propofol is commonly used for sedation during endoscopic procedures.Data suggests its superiority to traditional sedatives used in endoscopy including benzodiazepines and opioids with more rapid onset of action and improved postprocedure recovery times for patients.However,Propofol requires administration by trained healthcare providers,has a narrow therapeutic index,lacks an antidote and increases risks of cardio-pulmonary complications.AIM To compare,through a systematic review of the literature and meta-analysis,sedation with propofol to traditional sedatives with or without propofol during endoscopic procedures.METHODS A literature search was performed using MEDLINE,Scopus,EMBASE,the Cochrane Library,Scopus,LILACS,BVS,Cochrane Central Register of Controlled Trials,and The Cumulative Index to Nursing and Allied Health Literature databases.The last search in the literature was performed on March,2019 with no restriction regarding the idiom or the year of publication.Only randomized clinical trials with full texts published were included.We divided sedation therapies to the following groups:(1)Propofol versus benzodiazepines and/or opiate sedatives;(2)Propofol versus Propofol with benzodiazepine and/or opioids;and(3)Propofol with adjunctive benzodiazepine and opioid versus benzodiazepine and opioid.The following outcomes were addressed:Adverse events,patient satisfaction with type of sedation,endoscopists satisfaction with sedation administered,dose of propofol administered and time to recovery post procedure.Meta-analysis was performed using RevMan5 software version 5.39.RESULTS A total of 23 clinical trials were included(n=3854)from the initial search of 6410 articles.For Group I(Propofol vs benzodiazepine and/or opioids):The incidence of bradycardia was not statistically different between both sedation arms(RD:-0.01,95%CI:-0.03–+0.01,I2:22%).In 10 studies,the incidence of hypotension was not statistically difference between sedation arms(RD:0.01,95%CI:-0.02–+0.04,I2:0%).Oxygen desaturation was higher in the propofol group but not statistically different between groups(RD:-0.03,95%CI:-0.06–+0.00,I2:25%).Patients were more satisfied with their sedation in the benzodiazepine+opioid group compared to those with monotherapy propofol sedation(MD:+0.89,95%CI:+0.62–+1.17,I2:39%).The recovery time after the procedure showed high heterogeneity even after outlier withdrawal,there was no statistical difference between both arms(MD:-15.15,95%CI:-31.85–+1.56,I2:99%).For Group II(Propofol vs propofol with benzodiazepine and/or opioids):Bradycardia had a tendency to occur in the Propofol group with benzodiazepine and/or opioidassociated(RD:-0.08,95%CI:-0.13–-0.02,I2:59%).There was no statistical difference in the incidence of bradycardia(RD:-0.00,95%CI:-0.08–+0.08,I2:85%),desaturation(RD:-0.00,95%CI:-0.03–+0.02,I2:44%)or recovery time(MD:-2.04,95%CI:-6.96–+2.88,I2:97%)between sedation arms.The total dose of propofol was higher in the propofol group with benzodiazepine and/or opiates but with high heterogeneity.(MD:70.36,95%CI:+53.11–+87.60,I2:61%).For Group III(Propofol with benzodiazepine and opioid vs benzodiazepine and opioid):Bradycardia and hypotension was not statistically significant between groups(RD:-0.00,95%CI:-0.002–+0.02,I2:3%;RD:0.04,95%CI:-0.05–+0.13,I2:77%).Desaturation was evaluated in two articles and was higher in the propofol+benzodiazepine+opioid group,but with high heterogeneity(RD:0.15,95%CI:0.08–+0.22,I2:95%).CONCLUSION This meta-analysis suggests that the use of propofol alone or in combination with traditional adjunctive sedatives is safe and does not result in an increase in negative outcomes in patients undergoing endoscopic procedures.

Method Development for the Detection of Basic/Weak Basic Drugs in Hair by LCMSMS: Comparison between Methanolic and Alkaline Extraction on Real Samples

Detection of drugs in hair has become popular in recent years. The significantly long drug detection window (months) in hair has allowed the retrospective investigation and measurement of past consumption of drug. As the majority of drugs are basic, an extraction method was developed based on a methanolic solution for detection of basic/weak basic drugs in hair. It was compared with alkaline digestion (NaOH) followed by LLE. A filtration step with filtration vials was added and their materials were compared. After filtration, extracts were injected directly onto a C18 column coupled to Sciex ABI 2000 MSMS. The mobile phase was 50% methanol, 0.1% formic acid and 2 mM ammonium acetate (isocratic). Both methods were compared by applying them to real samples. Results showed that calibration was linear with r2 of 0.991-0.999 for 20 tested analytes. The matrix effect was assessed to be between 91.4%- 110.2% for 18 analytes. PTFE filter material showed better recoveries over the GMF and PVDF based filters. Stability of analytes during extraction in general was better with methanolic incubation rather than alkaline digestion. With regard to real sample recovery, 6 out of 10 analytes recovered better with alkaline digestion. In conclusion, the methanolic method is capable of extracting most basic drugs in hair samples but only part of the total incorporated drug. Therefore, these results suggest that a combination of both methods (methanolic and alkaline extractions) in hair sample processing for general detection of basic and weak basic drugs may produce better results. However, not all basic drugs are stable under alkaline digestion.

An Intentional Poisoning by Lormetazepam in a Patient Treated with Nordiazepam

A case of suspected poisoning by benzodiazepines is presented in this paper. Toxicological analyses were applied on the blood collected 48 hours after the facts. GC-MS and HPLC-DAD have detected only nordiazepam which was prescribed in the treatment. Therefore, LC-MS/MS has detected lormetazepam and lorazepam (major metabolite of lormetazepam) in addition of nordiazepam. The concentrations of lormetazepam, lorazepam and nordiazepam measured in blood were 6.8, 6.3, and 16.2 ng/mL, respectively. The initial concentration of each drug at the relevant time, has been calculated from these residual concentrations, using an extrapolation approach. According to estimates, only lormetazepam (189 ng/mL) and lorazepam (68 ng/mL), were active at the target moment. A poisoning by lormetazepam was then considered. The more technology we have, the greater chance we have to get an outcome that is favorable to justice.

Treating Insomnia in Older Adult Patients: Limiting Benzodiazepine Use

As aging comes, an increased prevalence of medical maladies and chronic pain independently or interactively disrupt sleep, which in turn can exacerbate either one. Furthermore, anxiety about pain can further negatively impact sleep. Fortunately, good quality sleep can improve pain management. Because benzodiazepine receptor agonists (including the “Z” drugs) can reduce anxiety and improve sleep, they seem a convenient choice. However, their use in this population, particularly for more than short-term (guidelines range from 2 to 6 weeks max), is not recommended because of increased likelihood of falls, further disruption of sleep, dependence, and problems with discontinuation (withdrawal). Besides, this population is often likely to take concomitant medication for pain or other central nervous system depressants leading to potentially serious and even life-threatening interactions involving synergistic amplification of respiratory depression (opioids being a particularly dangerous interaction). Therefore, insomnia in older adults should ideally be treated with a non-benzodiazepine receptor agonist;if indicated, they may be used, but should be closely monitored and tapered to avoid long-term adverse problems (direct or from withdrawal). Older adult patients with insomnia may be more optimally treated with sleep aids that do not interact with the GABAA receptor.

The necessity of regional DOHaD centers based on programming/imprinting and embedding-like phenomena

A mini-review is presented for the evidence of growth-inhibitory effects of several psychoneurotropic drugs and glucocorticoids on developing animals and humans, together with our own data obtained in experimental models, as well as in epidemiologic studies confirming female predominance in morbidity caused by affective disorders and in consumption of some psychoneurotropic drugs. The emerging concepts of pharmacotoxicologic programming/imprinting and embedding are discussed, justifying the necessity of regional DOHaD centers.