Download Unusual interactions of benzodiazepine receptor antagonists David

Seminar of Prof. D. Nutt MSc Affective Neuroscience Florence 2004
Unusual interactions of
benzodiazepine receptor
antagonists
David J. Nutt & Philip J. Cowen
MRC Unit and University
Dpt. Of Clinical Pharmacology Radcliffe Infirmary Oxford
Hilary J. Little
Dpt. of Pharmacology Oxford
University
Nature 295 4 Feb 1982
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Excerpt by G.Treviranus
Seminar of Prof. D. Nutt MSc Affective Neuroscience Florence 2004
„Two compounds have recently
been described which act as
potent BDZ antagonists in vivo
and which, in vitro, show high
affinity and selectivity for the
BDZ receptor of the mammalian
CNS.“
(...)
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Excerpt by G.Treviranus
(...)
Seminar of Prof. D. Nutt MSc Affective Neuroscience Florence 2004
„One, ethyl b-carboline-3-carboxylate
(b-CCE) may be related to an
endogenous ligand for the BDZ
receptor1-3.“
(...)
1-3 Baestrup, C., Nielsen, M. et al. 1980
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Excerpt by G.Treviranus
Seminar of Prof. D. Nutt MSc Affective Neuroscience Florence 2004
b-CCE„reverses the effects of
BDZs in vivo4,5 and in vivo6“...
4 Tenen, S., Hirsch, J. 1980 5 Oakley, N., Jones, B. 1980
6 Mitchell, R., Martin, I. 1980
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Excerpt by G.Treviranus
Seminar of Prof. D. Nutt MSc Affective Neuroscience Florence 2004
... „but b-CCE also has intrinsic
activity, as it lowers the seizure
treshold to drugs antagonistic to
(...) GABA5,7“
5 Oakley, N., Jones, B. 1980
7 Cowen, P.J., Green, A., Nutt, D. & Martin, I. 1981
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Seminar of Prof. D. Nutt MSc Affective Neuroscience Florence 2004
„The other compound (Ro 15-1788)
Flumazepil which is also a potent
and specific antagonist of BDZ
binding in vivo and in vitro8, ...
(...)
(...)
8 Möhler, A., Burkard, W.P., Keller, A.,
Richards, J., Haefely, W. 1981
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Excerpt by G.Treviranus
Seminar of Prof. D. Nutt MSc Affective Neuroscience Florence 2004
Flumazenil (Anexate®)... „blocked the
sedative, hypnotic and
anticonvulsant actions of
conventional BDZs, without any
intrinsic activity.9,10“
(...)
9 Hunkeler, W. 1981 10 Darrough, A. 1981
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Excerpt by G.Treviranus
Seminar of Prof. D. Nutt MSc Affective Neuroscience Florence 2004
„Because of the different profiles
of action of these two BDZ
´antagonists´, we have here
investigated their interactions in
two systems for assessing BDZ
activity:“...
(...)
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Excerpt by G.Treviranus
Seminar of Prof. D. Nutt MSc Affective Neuroscience Florence 2004
... „seizure tresholds in vivo11 and
the action of GABA on the
cervical sympathetic ganglia in
vitro12. “
11 Randall, L., Kappell, B. 1973 12 Bowery, N., Dray, A. 1978
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Excerpt by G.Treviranus
Seminar of Prof. D. Nutt MSc Affective Neuroscience Florence 2004
„We find that“ Flumazenil „not only
opposes the actions of BDZs but
also is an effective antagonist of
b-CCE in both systems“...
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Excerpt by G.Treviranus
Seminar of Prof. D. Nutt MSc Affective Neuroscience Florence 2004
... „At high doses“ Flumazenil „has
BDZ-like activity, suggesting that
it may be a partial agonist at the
BDZ receptor site.“
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Excerpt by G.Treviranus
Seminar of Prof. D. Nutt MSc Affective Neuroscience Florence 2004
Seizure tresholds in rats
infused with
epileptogenic drugs
GABA-antagonistic: PTZ & bicuculline
glycine-antagonistic: strychnine
manipulated with Flumazenil
= system 1a
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Excerpt by G.Treviranus
Seminar of Prof. D. Nutt MSc Affective Neuroscience Florence 2004
Seizure tresholds (mg per Kg)
Flumazenil
intra perit.
vehicle
PentyleneT Bicuculline Strychnine
etraZol  GABA  glycine
30 ±4
0.24 ±0.02
2.2 ±0.4
1
-----------
0.25 ±0.02
-----------
10
29 ±2
0.26 ±0.02
-----------
50
100
2.2 ±0.4 no
0.29 
----------- proP<0.01 40  P<0.05----------- P<0.05
36 ±5 
tection
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Excerpt by G.Treviranus
Seminar of Prof. D. Nutt MSc Affective Neuroscience Florence 2004
Seizure tresholds in rats pretreated
with anticonvulsants
Diazepam
Phenobarbital VALPROATE Progabide
& proconvulsant b-CCE
infused with the GABA-antagonistic
epileptogenic drug  PTZ
manipulated by Flumazenil intra peritoneum
 system
1b
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Excerpt by G.Treviranus
Seminar of Prof. D. Nutt MSc Affective Neuroscience Florence 2004
Drug

Flumazenil
pretreatment
Vehicle
i. perit.
Seizure treshold to
P value
 PTZ (mg per Kg)
Saline
34 ±02
b-CCE
26 ±03  35 ±3 
< 0.001
Diazepam
62 ±14  39 ±4 
52 ±05  56 ±9
< 0.001
Phenobarb.
35 ±3
VALPROATE
57 ±14  53 ±10
Progabide 50 ±06  55 ±11
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N/S
N/S
N/S
N/S
Excerpt by G.Treviranus
Seminar of Prof. D. Nutt MSc Affective Neuroscience Florence 2004
... Flumazenil „antagonized both the
anticonvulsant effects of diazepam
& the proconvulsant effects of bCCE without having any detectable
effect of ist own. (..;it) seems to be
selective for (...) BDZ receptor.“
(...)
(...)
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Excerpt by G.Treviranus
Seminar of Prof. D. Nutt MSc Affective Neuroscience Florence 2004
Perfused isolated sympathetic
cervical ganglion of rat
(system 2)
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Excerpt by G.Treviranus
Seminar of Prof. D. Nutt MSc Affective Neuroscience Florence 2004
{ GABA perf. of rat cervical ggl.
depolariz [Cl -] } ~ in CNS
Ø (GABA) bicuculline
{GABA   depolariz [Cl -] }
 chlordiazepoxide & bicuculline
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Excerpt by G.Treviranus
Seminar of Prof. D. Nutt MSc Affective Neuroscience Florence 2004
{ GABA perf. of rat cervical ggl.
depolariz [Cl -] } ~ in CNS
Ø (GABA) bicuculline
-] }
i{GABA   depolariz
[Cl

 chlordiazepoxide & bicuculline
 Flumazenil 0.8 mM  blocks CDP
effect neutralizing bicuculline
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Excerpt by G.Treviranus
Seminar of Prof. D. Nutt MSc Affective Neuroscience Florence 2004
{ GABA perf. of rat cervical ggl.
depolariz [Cl -] } ~ in CNS
Ø (GABA) bicuculline
ii{GABA   depolariz [Cl -] }
 chlordiazepoxide & bicuculline
 Flumazenil > 167 mM  effect
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Excerpt by G.Treviranus
Seminar of Prof. D. Nutt MSc Affective Neuroscience Florence 2004
{ GABA perf. of rat cervical ggl.
depolariz [Cl -] }
iii {GABA   depolariz [Cl -] }
 barbiturate
 Flumazenil : no effect
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Excerpt by G.Treviranus
Seminar of Prof. D. Nutt MSc Affective Neuroscience Florence 2004
„It thus seems that in two experimental systems in which
specific BDZ activity ma be assessed  b-CCE
has
the opposite effect to BDZs,“
Flumazenil „seems to be a selective &
potent antagonist of both compounds
in both systems having any detectable effect of its own. (...; it) seems to
be selective for (...) BDZ receptor.“
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Excerpt by G.Treviranus
Seminar of Prof. D. Nutt MSc Affective Neuroscience Florence 2004
Drug Tmt.
Flumazenil
Vehicle
i. perit.
P value
Seizure treshold to PTZ (mg per Kg)
Saline
34 ±02
b-CCE
26 ±03  35 ±3 
< 0.001
Diazepam
62 ±14  39 ±4 
52 ±05  56 ±9
< 0.001
Phenobarb.
35 ±3
VALPROATE
57 ±14  53 ±10
Progabide 50 ±06  55 ±11
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N/S
N/S
N/S
N/S
Excerpt by G.Treviranus
Seminar of Prof. D. Nutt MSc Affective Neuroscience Florence 2004
Drug Tmt.
„We suggest 3 possible explanations
to lthese findings...
Flumazeni
 the partial antagonist effect of Flumazenil
may be sufficient to account for its reversal of the
34 ±02
35 ±3
N/S
Saline
actions of b-CCE. This seems unlikely
Vehicle
i. perit.
P value
because the concentration of Flumazenil
that antagonizes b-CCE had no
antagonist action when tested alone.“
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Excerpt by G.Treviranus
Seminar of Prof. D. Nutt MSc Affective Neuroscience Florence 2004
„We suggest 3 possible explanations to these findings...
 the functional inter-action of ligands
with the BDZ receptor may be unusual in
that specific high-affinity ligands may
produce opposite pharmacological effects
and yet have a common antagonist. (...)
predicted from receptor theory. The
allosteric model ...
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Excerpt by G.Treviranus
Seminar of Prof. D. Nutt MSc Affective Neuroscience Florence 2004
The allosteric model ...
„The allosteric models of receptor
behaviour suggest that receptors
exist in equilibrium between two
states (which may be termed
„active“ and „inactive“) and that
this equilibrium is altered by ligand
binding.“
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Excerpt by G.Treviranus
Seminar of Prof. D. Nutt MSc Affective Neuroscience Florence 2004
the binding of A and B to an allosteric protein are
coupled - influencing each other - and this kind of
conformational coupling is known as allostery
The allosteric model ...
Jacques
Monod
Nobel laureate Medicine1965 1920 - 1976
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Excerpt by G.Treviranus
Seminar of Prof. D. Nutt MSc Affective Neuroscience Florence 2004
effector
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Excerpt by G.Treviranus
Seminar of Prof. D. Nutt MSc Affective Neuroscience Florence 2004
effector
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Excerpt by G.Treviranus
Seminar of Prof. D. Nutt MSc Affective Neuroscience Florence 2004
1. KoH = KAH - No allosteric interactions - there
is no effect of binding of one ligand on the binding
of the 2nd ligand.
2. KoH > KAH - A is an allosteric inhibitor
- when A is bound to R it inhibits the binding
of H to R.
≡ negative cooperativity
3. KoH < KAH - A is an allosteric activator
- when A is bound to R it promotes (or activates) the binding of H to R.
≡ positive cooperativity
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Excerpt by G.Treviranus
Seminar of Prof. D. Nutt MSc Affective Neuroscience Florence 2004
„If we apply this theory to our findings, BDZ
would be considered to be agonists, binding to
the active state, whereas Flumazenil
would be a conventional antagonist, binding to
both forms without altering the euqilibrium. If
b-CCE bound preferably to the receptors in the
inactive state it would shift the equlibrium in the
opposite direction. This form of antagonism has
been predicted but not so far demonstrated.“
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Excerpt by G.Treviranus
Seminar of Prof. D. Nutt MSc Affective Neuroscience Florence 2004
same
site
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Excerpt by G.Treviranus
Seminar of Prof. D. Nutt MSc Affective Neuroscience Florence 2004
„We suggest 3 possible explanations to these findings...
 „binding studies12 suggest (...)
subclasses of BDZ receptors“ w/
„identical affinities to BDZ including
Flumazenil, but differ(ing) in affinities for
b-carbolines“
12 Bowery, N., Dray, A. 1978
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Excerpt by G.Treviranus
Seminar of Prof. D. Nutt MSc Affective Neuroscience Florence 2004
Which explanation was
supported by further
subclasses of BDZ receptors“ w/
„identical affinities to BDZ including
Flumazenil, but differ(ing) in affinities for
b-carbolines“
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Excerpt by G.Treviranus
Seminar of Prof. D. Nutt MSc Affective Neuroscience Florence 2004
update 2004 (1)
update 2004 (1)
The crystallization of a ACh-BindingProtein of a snail Lymnea stagnalis (Smits,
Titia Sixma, Amsterdam) by known
homologies common to the pentameric
ligand-gated ion channels superfamily
(nACh, GABA A & C, glycine rec, 5-HT3)
allowed to define the aromatic-cage pocket
between subunit interfaces into which bCEE, flumazenil and also Ro-15-1788 bind.
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Seminar of Prof. D. Nutt MSc Affective Neuroscience Florence 2004
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update 2004 (6)
Excerpt by G.Treviranus
Seminar of Prof. D. Nutt MSc Affective Neuroscience Florence 2004
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update 2004 (6)
Excerpt by G.Treviranus
Seminar of Prof. D. Nutt MSc Affective Neuroscience Florence 2004
update 2004 (4)
Fig. 10. Scheme summarizing the relationship
of the benzodiazepine binding site with the
barbiturate site and the R1 and R2 binding sites
(Sigel, E. et al. Mol Pharmacol. 2001 Jun;59(6):1470-7)
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Excerpt by G.Treviranus
Seminar of Prof. D. Nutt MSc Affective Neuroscience Florence 2004
Intrinsic activity Compounds
Full agonists
update 2004 (6)
(Nutt & Malizia 2001)
BDZ = pos. allosteric modulator
Partial agonists bretazenil, abecarnil, imidazenil
Antagonist
Flumazenil, Ro 15-1788 competitive
Partial inverse
agonist
RU-33965, sarmazenil, FG 7142
Full inverse
agonist
b-CCE neg. allosteric modulator
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Excerpt by G.Treviranus
Seminar of Prof. D. Nutt MSc Affective Neuroscience Florence 2004
Intrinsic activity Compounds
Full agonists
update 2004 (6)
(Faravelli 2003)
BDZ anxiolysis + sedation,
ataxia, memory, dependence
Partial agonists unconvincing separation of
anxiolysis from unwanted side-effects
Antagonist
Blocker w/o intrinsic activity
Partial inverse
agonist
Full inverse
agonist
unconvincing separation of
anxiolysis from unwanted side-effects
anxiogenic, proconvulsant
stimulating, activate memory
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Excerpt by G.Treviranus
Seminar of Prof. D. Nutt MSc Affective Neuroscience Florence 2004
update 2004 (6)
postsynaptic GABAA = fast 50 ms
synaptic inhibition - ? terminated also by
GABA-reuptake (neuron, glia) by 12trans-mb-domain GABA-Transporters
(cerebral presynaptic GAT-1 & GAT -3
also in astrocytes, meningeal GAT-2 &
postsynaptic BGT-1: all cloned).
Catabolism: GABA-shuntKrebs-
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Excerpt by G.Treviranus
Seminar of Prof. D. Nutt MSc Affective Neuroscience Florence 2004
update 2004 (6)
affinity chromatography at BDZ
binding site: 250 kD complex with 5
subunits around central pore 49 – 58 kD
cloning: they belong to ligandgated ion channel superfamily w/
conserved agonist binding site
homologous to BDZ binding site
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Excerpt by G.Treviranus
Seminar of Prof. D. Nutt MSc Affective Neuroscience Florence 2004
update 2004 (6)
17 subunits – clustered around 5 genetic loci – the
subunit chaos
≥80% homology: same greek letter: a-6, b1-3, g1-3, d, e,
g, q, p
a & b subunits combined produce GABA-gated
currents
g subunit is required for BDZ pos. allosteric
modulation
many subunit combinations are non-functional
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update 2004 (6)
Seminar of Prof. D. Nutt MSc Affective Neuroscience Florence 2004
transgenic K.O.-mice:
a1 confers BDZ-sedation
a2 confers BDZ-anxiolysis
a5 confers BDZ-memory 
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Excerpt by G.Treviranus
update 2004 (6)
Seminar of Prof. D. Nutt MSc Affective Neuroscience Florence 2004
a1, b2, g2 major isoform of GABAA:
a1 interface g2 forms w1/BZ-1 site BDZ
a1 interface b2: agonist/ GABA binding sites
a2, b3, g2 isoform of GABAA w/ w2 BZ-2 site
a1, b3, g2 w/ A1 zolpidem / b-CCE site
a2...6, b3, g2 GABAA with A2...3 sites.
http://pharmrev.aspetjournals.org/cgi/reprint/50/2/291.pdf
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Seminar of Prof. D. Nutt MSc Affective Neuroscience Florence 2004
update 2004 (6)
courtesy Prof. Erwin SIgel
as seen
from
the
neuron
towards
the
synaptic
cleft:
BDZ
2 GABA binding sites
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binding
site
Sternbach was born 1908 in the croatian part of the austrohungarian empire from where he came to ETH in Zurich, were
he married the land lady‘s daughter. In Krakow he studied the
hepto-xdiazines and Roche saved him from the germans in
1940, calling him to Switzerland and 1941 to New Jersey. In
1954 he created 40 all inert derivatives, of which he treated
one with methylamine. During a cleanup in 1957 he decided
to send it in. It became chlordiazepoxide. After clouding of
some elderlies tested with CDP Roche wanted to stop its
evaluation, but Sternberg disobeyed....