Anxiolytics: Pharmacology and Toxicology

• The document discusses anxiolytics, their pharmacology, toxicology, and analytical methods for detection.

Introduction to Anxiolytics

• Anxiety exists as a normal human emotion that can manifest in varying degrees, from simple worry to terror.
• Pathological anxiety is abnormal anxiety.
• It often involves phobias, obsessive-compulsive disorders, panic attacks, post-traumatic stress, and generalized anxiety.

Anxiolytics Defined

• Anxiolytics are psychotropic medications used to treat the psychic and somatic manifestations of pathological anxiety.
• They belong to the psycholeptic group, decreasing mental activity.
• They often have a sedative effect, historically known as "tranquilizers".
• Benzodiazepines are the most used.

Benzodiazepines (BZDs)

• Sternbach discovered benzodiazepines, with chlordiazepoxide synthesized in 1957.
• Diazepam was introduced in 1963.
• Over 3,000 benzodiazepines were synthesized between 1960 and 2002.
• Roughly 35 of these are commercially available worldwide.
• There were rising concerns about dependence risks by 1980.

Physicochemical Properties

• BDZs are crystalline powders that are white or slightly yellowish, odorless, and have a bitter taste.
• They behave as weak bases.
• They are not dissociated at physiological pH.
• BDZs are slightly soluble in water but soluble in ethanol, chloroform, and ether.

Structure of BDZs

• Contain a benzene ring and a diazepine nucleus with 7 atoms (5 carbon and 2 nitrogen).
• There are three categories of BDZs, separated by the positioning of nitrogen atoms.

Structure-Activity Relationship

• The nature and position of substituents are responsible for the activity of the molecule.
• Substitution at the 7th position increases activity.
• NO2 at this position results in hypnotic action (e.g., Flunitrazepam).
• X at this position results in anxiolytic action (e.g., Diazepam).
• Alkylation at the 1st position increases activity.
• The -COOH group at the 3rd position acts as a prodrug needing activation to yield an active metabolite.
• Suppression of the benzene ring at the 5th position leads to a loss of activity.

Mechanism of Action

• The GABA A receptor: transmembrane protein.
• It has 6 binding sites on its extracellular face.
• 1 primary site binds the neurotransmitter GABA
• 5 secondary sites bind GABA effect modulating molecules, regulating chloride permeability.
• BZDs bind to a secondary site on the GABAA receptor, allosterically modifying the receptor.
• The receptor's affinity for GABA increases.
• Without GABA at the primary site, regulatory molecules do not impact chlorine permeability.
• Ligands that increase chloride permeability will increase the anxiolytic effect.
• Ligands that decrease chloride permeability cause an anxiogenic effect.

Ligands Binding to Modulatory Sites

• Agonists increase chlorine permeability, leading to an increased GABA effect.
• These agonists can be full or partial.
• Inverse agonists: These decrease chlorine permeability.
• The agonists reduce the effectiveness of GABA, yielding an anxiogenic effect.
• Antagonists block the secondary site without affecting GABA's efficacy.
• Flumazenil is a neutral ligand, preventing agonists and antagonists from acting.

Pharmacological Properties

• Anxiolytic effects
• Hypnotic effects
• Anticonvulsant effects
• Myorelaxant effects

Absorption

• When taken orally, BDZs which are weak bases, are rapidly and completely absorbed in the intestinal tract.
• Bioavailability ranges from 60% to 100%, depending on the specific BZD.
• Peak plasma concentration occurs between 0.5 to 3 hours after ingestion.
• It is delayed when overdosed.
• Intramuscular absorption is erratic.
• It is affected by other medications that influence transit.
• Diazepam and chlordiazepoxide have irregular and slower V.O. absorption.
• Satisfactory V.R. can convulse children, so intravenous V. IV use is not recommended.

Distribution

• Binding to plasma proteins ranges from 75% to 95%.
• BDZs are lipophilic.
• They penetrate tissues well.
• They cross the placental and blood-brain barriers.

Metabolism

• Hepatic biotransformation is key for BZD clearance.
• Only a small percentage of the administered dose gets eliminated unchanged.
• The main reactions are microsomal oxidation reactions.
• Metabolites undergo glucuroconjugation.
• They are the eliminated in the urine.

Phase I Reactions

• N1 oxidative dealkylation
• C3 hydroxylation
• Reduction of nitro groups into amino groups
• Diazepine ring opening

Phase II Reactions

• Glucuronoconjugation to increase hydrosolubility leads to a loss of activity.

Elimination

• BDZs are mainly eliminated through the renal system in the form of glucuroconjugated metabolites.
• A small fraction is eliminated in bile.
• BDZs can also be found in breast milk.
• BDZs are classified into three groups based on half-life.
• Short: less than 10 hours (e.g., triazolam, midazolam)
• Intermediate: 10 to 38 hours (e.g., bromazepam, oxazepam, clobazam, alprazolam, tetrazepam)
• Long: 47 to 200 hours (e.g., chlordiazepoxide, diazepam, clonazepam)

Acute Toxicity

• Toxic dose is >50 mg in children and >500 mg in adults.
• It brings neurological effects, such as behavioral disturbances (drunkenness, agitation, aggression).
• It augment sedative effects on the central nervous system (CNS) with drowsiness.
• Can evolve into a hypotonic coma, possibly a shallow one.
• Creates respiratory depression (generally moderate).
• Causes cardiovascular issues, like hypotension and bradycardia.
• Induces digestive problems, such as nausea and vomiting.
• Causes renal issues.
• Causes urinary retention
• Anterograde amnesia often occurs.
• Prognosis: generally favorable, and death is rare.

Special Attention Should be Given

• Massive ingestion of a highly sedative molecule (e.g., triazolam, flunitrazepam).
• Pre-existing conditions (chronic renal failure, hepatic insufficiency, respiratory failure).
• Combined use with other central nervous system depressants and/or alcohol.

Chronic Toxicity

• Prolonged treatment can lead to drug addiction.
• Alters the alertness.
• Alters physical performance.
• Can cause confusion, muscle hypotonia.
• Reduces concentration and anterograde amnesia.
• Can result in a depressive state.
• Can create paradoxical effects.
• Hostility and insomnia
• Tolerance develops in 2 to 4 weeks of use.
• Rebound effect
• Serious dermatological effects.

Tolerance + Psychological and Physical Dependence

• Abrupt cessation of BDZ treatment leads to withdrawal syndrome.
• Anxiety, insomnia
• Headaches, muscle spasms, tremors
• Anorexia, nausea, vomiting, paresthesia
• Photophobia
• Convulsions

Interactions

• Combination with lithium, cimetidine, erythromycin, or acute ethyl alcohol intoxication increases sedative effects through pharmacokinetic interaction and CYP inhibition.
• Combination with morphine increases respiratory depression through pharmacodynamic interactions.
• Combination with phenobarbital, rifampicin, or chronic alcoholism decreases activity through CYP activation and enzyme induction.
• Concomitant use of other benzodiazepines decreases activity due to competitive antagonism.

Treatment

• Treatment is mainly symptomatic.
• Tracheal intubation and assisted ventilation are required in cases of coma.
• Provide hydro-electrolytes.
• Correct hemodynamic disturbances with hypotension.
• Identify and treat co-intoxications.
• Activated charcoal is administered early.
• Implement a neutral osmotic diuresis.
• Flumazenil has a stronger affinity for BZD receptors to displace the BZDs.

Flumazenil Administration

• Administered through injection.
• A rapid and marked effect in 30 seconds to 3 minutes.
• Administer repeated injections of 0.2 to 0.3mg IV.
• Lift respiratory depression in the patient.
• Follow by continuous perfusion to prevent patient from falling back asleep.

Toxicology Analysis

• Specimens for toxicological analysis include: blood, urine, hair.
• When testing samples within a week, refrigerate between 0 and 8°C.
• Otherwise, freeze the sample (< -18°C).

Extraction

• Liquid-liquid extraction occurs.
• Solid phase extraction

Thin Layer Chromatography

• Phosphoric acid reagent is flaujnatre
• Iodoplatinate
• Dragendorff is used as a general developer

Diazocopulation Colorimetric Technique

• Acid hydrolysis of BZDs and their metabolites produces a diazotizable primary aromatic amine.
• It can be characterized and measured spectrophotometrically at 550nm.
• Paracetamol has cross reaction.
• Diazepam and tetrazepam can't be diazocopulated.

UV Spectrum

• The extraction residue's identification can be done with spectrophotometry UV after adding HCL 1N.

Immunological Methods

• They are qualitative techniques for urine.
• Between 100 and 300ng/ml determine positiveness.
• Immunochemical methods are used to screen serum or urinary benzodiazepines.
• The methods depend on competition assays.
• Fluorescence Polarization Immuno Assay.
• Enzyme Multiplied Immunoassay Technique
• Cloned Enzyme Donor Immunoassay
• Kinetic Interaction of Microparticles in Solution
• The tests units are on support solide

Gas Chromatography

• The GC is coupled with

• Electron capture detector

• Nitrogen-phosphorus detector

• Mass spectrometry

• Has 2 advantages:

• Quantification

• Identification

• From 1 to 5 ng/mL in diazepam plasma, clobazam, flunitrazepam, triazolam, midazolam, oxazepam, lorazepam and several demethylized, hydroxylized metabolites.

High-Performance Liquid Chromatography

• HPLC is the chosen method to analyse blood samples.
• Sample preparation includes L-L extraction.
• HPLC, using the HPLC-SM method, performs analysis in 5-10 minutes.
• Vary the detection limit depending on the nature of the benzodiazepine, the possibility of derivatization, the matrix and the material.

Structure of Carbamates

• Salts or esters of carbamic or aminoformic acid.
• Lumirelax
• The risk of coma and death are involved with overdose, it means, it should be related to serious El.

Pharmacological Properties

• Anxiolytic
• Hypnotic
• Muscle relaxants

Action mechanism

• Meprobamate regularizes sleep with sedative features.
• They have similar pharmacodynamic qualities as BZD since both act on GABA.
• Carbamates treat symptoms using the system of excitable limbs.
• Compared to benzodiazepines, it can be considered that carbamates have rather poor efficiency and high toxicity.

Toxicokinetics

• Absorption: TGI with plasmatic features.
• Metabolism: About hydroxylated compounds are made into glucuroconjugated versions during hepatic metabolism.

Distribution

• 20% plasma proteins.
• The volume is 0.7 L/kg.

Elimination

• Slow-acting, urinary.
• 10 % is excreted without being modified following treatment and 65% are glucuroconjugated.
• 10 % are retrieved in the fecal matter.

Toxicity

• Risk causes breathing difficulties and cardiovascular problems.
• The dose is 40g for adults.

Disorders and Intoxication

• neurological effects result in the onset of cardiovascular harm.
• Neurology can involve giddiness.
• Somnolence can result in coma.
• Sedative use and other central nervous system depressants is a central cause.

Secondary Effects

• Tiredness
• Tolerance is an effect.

Treatment

• Supply air
• Fix temperatures.
• Stabilize the circulatory
• Fight fits of seizures
• Stop further harm to cardiovasculars.

Toxicology Analysis

• Bloods should use serum, however urine or other materials are useable.
• Samples should be treated with anti coagulants, should be chilled and only kept up to a month.

Conclusion

• Anxiolytics is the most issued pills around.
• BZD can make a negative impact.
• Serious is rarely the case, mainly in situations with central nervous system or alcohol.
• The market removes a number of anxiolytics.