Central Respiratory Effects
Of Benzodiazepines

John Loadsman - 13/2/96

PART ONE

• Transmission in the CNS
• The GABA ergic system
• The Benzodiazepine Receptor

TRANSMISSION IN THE CNS

STRUCTURE

• innumerable neurones
• enormously complex intermingling and cross-talk

FUNCTION

• process and integrate incoming information, physical and psychic needs, emotions and mental processes
• produce automatic and intentional somatic and mental outputs
• retrieve old information
• store new information

MECHANISM

• Interneuronal signalling via neurotransmitters and neuromodulators
• transmembrane ion transport and permeation

Drugs may effect the function of the CNS therefore in 2 ways generally:

1. Interaction with interneuronal signalling
   • Biosynthesis (enzymes and precursors)
   • Storage
   • Release (e.g. modulation by nerve terminal receptors)
   • Inactivation (cellular uptake, enzymatic degradation)
   • Receptors (signal binding sites, allosteric modulatory sites)
   • Post-receptor mechanisms (transduction, 2nd and 3rd messengers)
2. Interaction with ion transport
   • Ion Pumps
   • Ionophores
   • Fixed Channels (K+)
   • Voltage- operated channels (Na+, K+, Ca++)
   • Receptor- operated channels (Na+, K+ Ca++, Cl-)
   • Receptor modulated voltage- operated channels
   • Channels operated/modulated by intracellular messengers e.g., ions, cyclic nucleotides, protein kinases

NOTE: Drugs falling into Group 1 may have a very specific mechanism of action (e.g., Benzodiozepines) but their consequences are frequently diverse c.f. agents having specific group 2 effects which can have very specific consequences.

Benzodiazepine Receptor (BZR) ligands, especially the classic Benzodiazepines (BZs), are among the most specific centrally acting drugs falling into the first group with respect to

1. Site - an allosteric modulatory site on the GABAA receptor
2. Mechanism - interaction with signal recognition and transduction by that receptor

THE GABAergic SYSTEM

GABA

• used in about one third of all brain synapses
• most abundant and important inhibitory neurotransmitter

GABAergic neurones

• mostly interneurons
• a few (e.g., cerebellar cortical Purkinge neurones) are principal output neurones with long axons to distant regions. (other examples:
  • strio-nigral neurons)
  • nigro-thalamic neurons)
  • nigro-tectal neurons)

GABA receptors

• GABAA
  • most abundant
  • on membranes of soma and dendrites of target neurones
  • on membranes of terminals of primary sensory neurones
• GABAB
  • autoreceptors on GABA ergic cell bodies and nerve terminals

* BZs act only on GABAA receptors *

	GABAA	GABAB
Common agonist	GABA	GABA
Selective agonist	Muscimol	Baclofen
Selective antagonist	Bicucculine	Delta-Amino-valeric acid
Effector	Cl- channel	Ca++ channel (inhib) ACh (inhib) K+ channel (stim)
Allosteric modulation	Benzodiazepines Barbiturates Some convulsants

The GABAA receptor

• Part of a membrane chloride channel complex.
• Occupation of the receptor by GABA results in opening of the channel and increased gCl-
• Depending on the transmembrane [Cl-] gradient and the membrane potential this can have 3 effects:
  1. Cl- flux inward causing hyperpolarisation (most common).
  2. Cl- flux outward causing depolarisation.
  3. No Cl- flux causing no change in membrane potential.
• All 3, however, reduce neuronal excitabilty in 2 possible ways :
  1. By impeding the membrane from reaching the threshold potential for A.P. generation or
  2. By short-circuiting the rapid Na+ influx thus slowing or blocking A.P. propagation

* This forms the basis of the inhibitory function of the GABAergic system. *



Some examples of how GABAergic inhibition is thought to work:

• Postsynaptic inhibition
  • (a) } forward inhibition
  • (b) } ' '
  • (d) feed back inhibition
• Presynaptic inhibition (c)

The significance of GABAergic inhibition can be demonstrated by blocking it e.g. with GABA receptor blocking agents :

• generalised decrease (even partial) causes:
  • excitation
  • anxiety
  • convulsions
  • death
• localised decreases causes:
  • focal, uncontrolled epileptiform neuronal activity.

THE BENZODIAZEPINE RECEPTOR

1975 - First suggestion that classic BZs (agonists) acted by enhancing GABAergic inhibition.

Since shown that BZ agonist facilitation of GABAergic transmission occurs in all areas of the CNS where GABAA receptors have been found.

BZR distribution has been shown to coincide with GABA receptor distribution by autoradiograms of animal brain sections after treatment with 3H-diazepam and more recently by P.E.T. scanning of live animal and human brains after injection with 11C-Flumazenil.

Radioisotope and Flourescent BZR bigands have also been used to demonstrate their high affinity and high specificity for the BZR and also the fact that these sites are saturable.

Studies of the action of BZR agonists on the GABAergic system have shown the following :

• A dose-response relationship between GABA and gCl-.
• A dose dependent left shift of the GABA/gCl- curve by BZR agonists.
• An increase in GABA to GABAA receptor binding in the presence of BZR agonists.
• An increase in BZ binding to BZRs in the presence of GABA.

In 1985 the BZR was isolated using monoclonal antibodies and it was found that as well as BZs it bound GABA, leading to the conclusion that the GABAA receptor and the BZR were both part of one complex, the GABAA - BZR - Cl- channel complex.

This was comfirmed in 1987 when DNA for alpha and beta subunits of the complex were cloned and injected into cells not normally having GABAA receptors, resulting in the synthesis and membrane inclusion of fully functional GABAA - BZR - Cl- channel complexes. The gCl- of the cells varied in the predicted manner in response to GABA before and after addition of BZs .(No response was seen with addition of BZs alone). This confirmed the theory that the BZR is an allosteric modulatory site on the GABAA receptor and that GABA and BZs allosterically modulate each other's binding to their respective receptors.

It was thought in 1987 that the receptor complex contained 2 a subunits (containing the BZRs) and 2 b subunits (containing GABAA receptors) but by late 1990 subunits of 4 classes (alpha ,beta , gamma , delta) had been identified and coded with even further subdivision within the classes. Diverse structural and pharmacological heterogeneity of the complex has now been demonstrated.

It has also been shown in the 2nd half of the 1980's that the GABAA receptor complex occurs in virtually every neuron in the brain, on glia, and also exists in the periphery.

PART TWO: THE VARIOUS LIGANDS OF THE BZR

BASIC STRUCTURE OF BZR LIGANDS

The core of all BZR ligands is a benzene ring joined to a 7-membered 1,4-diazepine ring:

The various substituents determine the ligand's pharmacological properties:

• its AFFINITY to bind the receptor
• its INTRINSIC EFFICACY or capability to produce a functional change in the receptor. Ligands capable of activating the receptor (AGONISTS) are said to have intrinsic efficacy. ANTAGONISTS which bind the site but are unable to activate the receptor have zero intrinsic efficacy ideally while ligands which bind the site but produce the opposite effects to AGONISTS are called INVERSE AGONISTS and are said to have negative intrinsic efficacy.
• other properties such as lipophilicity, water solubility etc.

THE BZR AGONISTS (The Classic Benzodiazepines)

• 1st produced in 1961 ( chlordiazepoxide )
• about 25 in clinical use
• all important ones have a 5-aryl substituent

DIAZEPAM

LORAZEPAM

MIDAZOLAM

all produce the same effects ( to varying degrees )

1. Anticonvulsant (at 20-25% occupancy)
2. Anxiolytic (20-30%)
3. Sedative / Hypnotic (25-30%/60-90%)
4. Muscle relaxant (centrally mediated)
5. Amnesic

• effects also depend on receptor occupancy (see above)
• were also thought to have a glycine-mimetic effect in spinal cord and brainstem (glycine is another inhibitory neurotransmitter) but this is not mentioned in current literature.
• most are water insoluble except for Midazolam whose solubility is due to the fused imidazole ring.

THE OTHER LIGANDS

• A full range of Benzodiazepines from complete inverse agonists through antagonists to complete agonists have been produced (see examples below)
• All are competetive binders of the BZR i.e. antagonists will block/displace both agonists and inverse agonists.
• Unlike the agonists whose binding is enhanced by GABA, inverse agonists are inhibited from binding by GABA while antagonists' binding is not affected at all.
• Partial agonists may prove very useful due to selective effects e.g. Anxiolysis or anticonvulsant without sedation or muscle relaxation. Other potential properties are reduced tolerance, reduced ethanol potentiation and reduced physical dependence.

MIDAZOLAM: an agonist - intrinsic efficacy +1.0

Ro 16-6028: a partial agonist - intrinsic efficacy +0.5

FLUMAZENIL: an antagonist - intrinsic efficacy 0

Ro 15-4513: a partial inverse agonist - intrinsic efficacy -0.5

Ro 19-4603: an inverse agonist - intrinsic efficacy -1.0




NON BENZODIAZEPINE BZR LIGANDS

• It is thought that inosine and hypoxanthine have some agonist activity at the BZR.
• There are beta - carboline derivatives (e.g. DMCM) with strong inverse agonist activity.

THE QUESTION OF ENDOGENOUS LIGANDS

• ?mechanism of anxiety states, epilepsy etc i.e. lack of endogenous agonist or presence of endogenous inverse agonist.
• some putative ones found but none behave as predicted.
• extremely low concentrations of BZs such as diazepam and nordiazepam were discovered in animal and human brain in 1987 but it is thought that these come from plants (found naturally in potatoes and cereals and other plant nutrients).

PART THREE: CENTRAL RESPIRATORY EFFECTS

The first studies of the respiratory effects of diazepam were carried out in the late '60s. They examined CO2 responses only and most concluded there was no effect.

In 1971, Catchlove & Kafer used Read's rebreathing technique for the first time to examine the effect of 0.14mg/kg (10mg/70kg) i.v. diazepam on CO2 responses and steady-state gas exchange. VT decreased 20%, PaCO2 increased 19%, and VD/VT increased 34%. The slope of the CO2 response was depressed in 46%, unchanged in 23% and increased in the rest. 84% showed a rightward shift.

Forster et al (1980) studied deltaVE/deltaPETCO2 and deltaP0.1/deltaPETCO2 after 0.3mg/kg diazepam and 0.15mg/kg midazolam i.v.. Both drugs depressed both variables to a similar degree. No rightward shift could be demonstrated.

Gross et al (1982) showed the CO2 response to be depressed significantly for at least 25 minutes after 0.4mg/kg i.v. diazepam. The degree of depression correlated well with the level of sedation. Again no right shift demonstrated.

Power et al (1983) claimed neither 0.15mg/kg diazepam nor 0.075mg/kg midazolam i.v. caused depression of the CO2 response despite the mean slopes falling to 0.72 and 0.60 of their control values respectively. Main problem was n=7 (i.e. Power had no power! :-)

Alexander and Gross (1988) demonstrated a 53% decrease in the slope of the isohypercapnic (PETCO2 50mmHg) hypoxic response (deltaVE/-deltaSaO2) after 0.1mg/kg midazolam i.v. It also obliterated the haemodynamic response to hypoxia.

Gillis et al (1988) performed an elegant study where they applied various BZR ligands to the intermediate area of the ventral surface of the medula of cats anaesthetised with a-chloralose. Both midazolam (0.75-250mg/side) and chlordiazepoxide (100-1000mg/side) caused dose related depression of VE, VT, heart rate and blood pressure, although some tachyphylaxis was demonstrated. Respiratory frequency did not change. The effect was evident within 1 minute and lasted 30-60 minutes. Application of the agents to the rostral area caused neither effect and application to the caudal area caused hypotension without depressing respiration. Only the largest of these doses given i.v. had any effect but increasing i.v. doses (up to 1.0mg/kg midazolam) produced the same dose related response as the smaller topically applied ones, with the addition of depressed resiratory rate. Topically applied flumazenil (250mg/side) and bicuculline (a selective GABAA antagonist - 10mg/side) had no effect on their own but both prevented (if applied before) and reversed (if applied after) the depression caused by midazolam whether it was given topically or i.v. Ethyl-beta-carboline-3-carboxylate (a BZR inverse agonist - 12.5-30mg/side) applied topically to the medulla caused the opposite effects to midazolam and was also reversed by flumazenil.

Also in 1988, Skatrud et al examined the effects of up to 3 times the normal oral dose of triazolam. They claimed no effect on VE (VT decreased and f increased). However, they had a similar result with 0.15mg/kg i.v. morphine (a fairly big dose) and their intersubject variability was very large.

In 1990, Wettstein, Teeple and Morse examined the effects of a variety of BZR ligands and other agents (given i.v.) on the respiration of awake rhesus monkeys. The BZ agonists alprazolam (0.01-1.0mg/kg), lorazepam (0.3-10.0mg/kg) and quazepam (1.0-5.6mg/kg) all depressed VT and VE during both 5%CO2 and air inhalation. Pentobarbital (3.0-30mg/kg) also depressed frequency and it's dose/effect relationship on VT was steeper. Two beta-carbolines (beta-CCE (0.3-5.6mg/kg) and FG 7142 10mg/kg)) increased frequency and VE. Both flumazenil and CGS 8216 (a weak inverse agonist)(both 1.0mg/kg) reversed the effects of the agonists. Alprazolam reversed the stimulation of FG 7142.

Dahan and Ward claimed in 1991 that midazolam (0.025mg/kg followed by infusion at 1.0mg/70kg/h - a piddling dose!) did not reduce the acute ventilatory response to isohypercapnic hypoxia. In their discussion however they state there was a reduction but it failed to reach significance, hardly surprising when there were only five subjects and the PETCO2 was actually higher in the midazolam experiments! The midazolam also caused a greater hypoxic ventilary decline.

Gross, Weller and Conard reversed midazolam (0.13mg/kg) with flumazenil (1.0mg/kg) and found that the sedation and right shift of the CO2 response were rapidly reversed but the depression of the slope of the CO2 response was not.

Blouin et al (1993) confirmed midazolam's (0.12mg/kg) depression of the isohypercapnic hypoxic ventilatory response (deltaVE/deltaSpO2 reduced to 59%). This was fully reversed with flumazenil (1.0mg/kg).

SUMMARY

BZR agonists almost certainly cause dose-related centrally mediated (medullary) respiratory depression. VT seems to be affected more than frequency and the sites of these actions may be different. Inverse agonists cause the opposite effect and pure antagonists reverse the effect. Flumazenil may not be a pure antagonist given the variability in it's reversal of the effects of agonists on ventilatory responses. Alternatively, the level of receptor occupancy may play a role in these differences.

BIBLIOGRAPHY

1. Wood & Wood: Drugs and Anaesthesia. 2nd Edition, 1990.
2. Amrein et al: Clinical pharmacology of Dormicum (Midazolam) and Anexate (Flumazenil). Resuscitation, 16, Suppl (1988) S5-S27.
3. W.Haefely: Pharmacology of the Benzodiazepine receptor. European archives of Psychiatry and Neurological Sciences, (1989) 238: 294-301.
4. S.Ymer et al : Structural and functional characterisation of the g1, subunit of GABAA/benzodiazepine receptors. The EMBO Journal, Vol 9; No 10 (1990) : 3261-3267.