Note: Descriptions are shown in the official language in which they were submitted.
CA 02360124 2002-O1-14
WO 00/45832 PCT/GB00/00281
I
REGULATION OF ANAESTHESIA
The present invention relates to the regulation of anaesthesia and also a
method of evaluating the anaesthetic needs of a subject.
The metabolic activity of the brain changes in various clinical situations.
For
example the metabolic activity of the brain is increased during an epileptic
fit and
during rapid eye movement sleep. In contrast the metabolic activity of the
brain is
reduced during hibernation and during the administration of a general
anaesthetic.
Anaesthesia may be defined as a loss of feeling or insensibility to external
stimuli. Anaesthesia may be local (the loss of sensation in a specific tissue)
or general
(when it is generally associated with a lack of consciousness). Studies have
shown
that a reduction in brain metabolism of some 47% is associated with a state of
general
anaesthesia. Administration of excessive doses of anaesthetic compounds leads
to a
reduction in metabolic activity in excess of this level and a depth of
anaesthesia that is
excessive and associated with an increased risk of side-effects. It is
therefore
particularly important for a clinician to be able to reliably and sensitively
regulate
brain activity to allow the induction of controlled anaesthesia.
A state of anaesthesia is physiologically different to sleep. For instance, a
subject who is asleep may be easily roused and therefore remains sensitive to
external
stimuli whereas a subject under a general anaesthetic may not be roused to
consciousness by external stimuli. Furthermore sleep is not necessarily
associated
with reduced brain activity (e.g. during Rapid Eye Movement sleep, brain
activity is
normally high) whereas general anaesthetic is associated with reduced
activity. Given
the differences between anaesthesia and sleep it is not surprising that
anaesthetic
compounds do not necessarily act as hypnotics and vice versa.
Small, volatile molecules which induce anaesthesia (e.g. alcohols, halothane,
ether etc) have been known for many years and are, or have been, commonly used
to
CA 02360124 2002-O1-14
WO 00/45832 PCT/GB00/00281
2
induce and maintain anaesthesia prior to, and during, elective surgery etc.
However
many conventional anaesthetics have various disadvantages. These include:
(1) narrow concentration range over which the agent is effective (too little
and the subject regains sensitivity to external stimuli whereas too much
results in coma or death);
(2) slow recovery following anaesthesia;
(3) common side effects such as respiratory depression, cardiovascular
instability and vomiting; and
(4) uncommon but life threatening side-effects such as malignant
hyperpyrexia.
Therefore there is a need to provide compounds which may be used as, or with
anaesthetics, which obviate or mitigate disadvantages associated with the
prior art.
According to a first aspect of the present invention, there is provided the
use
of a compound which modulates Delta-Sleep Inducing Peptide activity for the
manufacture of a medicament for regulating anaesthesia.
DSIP is a nonapeptide (which can exist in linear or cyclic form) with the
amino acid sequence:
Trp-Ala-Gly-Gly-Asp-Ala-Ser-Gly-Glu
DSIP was discovered in the 1970's and has been proposed for sleep induction
(for which it has had only limited success) and for treating drug addicts
during drug
withdrawal. However it has not previously been associated with anaesthesia and
we
have found that compounds which modulate DSIP activity are able to regulate
anaesthesia.
DSIP may cause a reduction in brain metabolism which may be associated
with a changed level of consciousness. However, the inventors have established
that
the reduction in brain metabolism seen with anaesthesia leads to a change in
consciousness which is not typical of normal sleep. In fact, following DSIP
treatment
CA 02360124 2002-O1-14
WO 00/45832 PCT/GBOO100281
3
there is a decrease in the amount of Rapid Eye Movement sleep and an increase
in
delta wave activity. The inventors have correlated these changes with the
anaesthetised state and have therefore established that compounds which
modulate
DSIP activity may be used according to the first aspect of the invention. The
inventors
further believe that DSIP may be important in the induction of hibernation and
the
reduction of brain metabolic activity during hibernation and similar states.
The inventors believe that DSIP is an endogenous "anaesthetic-like" substance
which modulates neurotransmission and brain activity. This belief is founded
upon
observations made whilst conducting studies using PET to assess metabolic
activity
changes that occur in various areas of the brain during anaesthesia with
conventional
anaesthetic agents. The invention arose from the realisation that the areas of
the brain
in which there were changes in metabolic activity in response to a
conventional
anaesthetic agent were the same areas where DSIP has been shown to be located
using
immunohistology techniques.
Although we do not wish to be bound by any hypothesis, we believe that
compounds which modulate DSIP activity are effective because they regulate
binding
of ligands with a neuromodulatory binding site on neuroreceptors which have
been
linked to the regulation of anaesthesia (e.g. the site described by Mihic et
al. (1997)
Nature 389 p385-389 on GABAA receptors and glycine receptors). We believe
binding of DSIP to these receptors modulates signalling from these receptors
and
thereby regulates the level of brain metabolism and the level of anaesthesia.
Our hypothesis that DSIP acts as an anaesthetic was confirmed by experiments
which established that administration of DSIP induces anaesthesia and also
prolongs
anaesthesia induced by other anaesthetic agents. For instance, anaesthesia
following a
7mg/kg iv bolus of propofol was approximately 28% longer in animals pretreated
with DSIP (lmg/kg IP, 15 mins prior to the propofol bolus) compared to animals
treated with propofol alone. Further experimental data illustrating the
efficacy of
DS1P, and related compounds, is presented in the Example below.
CA 02360124 2002-O1-14
WO 00/45832 PCT/G800/00281
4
According to a first embodiment of the first aspect of the invention, we have
found that compounds which increase DSIP activity may be administered alone,
or
preferably in combination with certain other anaesthetic agents, to induce or
maintain
anaesthesia. When used as part of a regime to induce anaesthesia, compounds
which
increase DSIP activity may be administered at the time of induction or at an
earlier
time as part of a regimen of pre-medication.
Several classes of compound which are capable of increasing DSIP activity
may be used according to the invention. Such compounds include agonists or
partial
agonists of DSIP neuromodulatory binding sites, agents which enhance the
release of
endogenous agonists of DSIP neuromodulatory binding sites, agents which
enhance
the synthesis of endogenous agonists of DSIP neuromodulatory binding sites,
agents
which attenuate the breakdown (or removal/sequestration) of endogenous DSIP
agonists, agents which increase DSIP expression or activity and agents which
enhance
the mechanisms involved in signal transduction between the Iigand bound DSIP
binding site and effector systems.
Preferred compounds which increase DSIP activity are DSIP agonists and
include DSIP per se and derivatives and/or pharamaceutically acceptable salts
thereof.
Preferred DSIP agonists which may be used according to the first embodiment
of the first aspect of the invention include the phosphorylated nanapeptides
disclosed
in British Patent No. 2 000 511. (which are incorporated herein by reference).
Biologically active fragments of DSIP, biologically active DSIP derivatives
and larger peptides comprising the nonapeptide (or biologically active
fragments and
derivatives thereof) are also preferred compounds for use according to the
first
embodiment of the first aspect of the invention. For example a preferred
derivative of
DSIP is Cyclo(-GLY-DSIP) which is described by Nekrasov et al. (Biochem. Mol.
Biol. Int. 1996:38 p739-745). This derivative is more lipophilic than DSIP and
crosses
the blood brain barrier more readily. Cyclo (-GLY-DSIP) is particularly useful
for
rapid induction of anaesthesia.
CA 02360124 2002-O1-14
WO 00145832 PCT/GB00/00281
It will be appreciated that non-peptide compounds which mimic peptide DSIP
agonist activity (which may be isolated tcom nature or rationally designed)
may also
be used.
Compounds which modulate DSIP activity may be used in a method of
inducing anaesthesia comprising administering to a patient to be anaesthetised
an
effective amount of a compound which promotes DSIP activity to induce at least
part
of the desired level of anaesthesia.
We believe that DSIP (and functional analogues thereof induce or maintain
anaesthesia according to the first embodiment of the first aspect of the
invention for
the following reasons:
( 1 ) It is a neuromodulator, not necessarily a neurotransmitter, which we
believe influences a transmembrane binding site on the GABAA, glycine and
possibly
other receptors in a manner consistent with a modulator working via the same
site as
the ethanol site and/or the enflurane anaesthetic site.
(2) It is an anticonvulsant.
(3) 1t has analgesic properties. We believe DSIP acts as an analgesic because
it promotes the release of met-enkephalin.
(4) Studies to investigate a possible action of DSIP in sleep promotion have
shown that it does not induce normal sleep stages but promotes delta wave
activity on
the electroencephalograph as do many anaesthetics. During anaesthesia the
electroencephalograph shows a complex pattern which may include a delta wave
component but this pattern is distinct from that seen during natural sleep
stages.
(S) It may regulate excitation and inhibition within the brain. It may
modulate
thermoregulation, as do general anaesthetics.
The analgesic properties of the compounds (3 above) represents a particular
advantage of compounds used according to the first embodiment of the first
aspect of
the invention. Under certain circumstances the analgesic activity of a
compound may
outlast the anaesthetic action. This is of particular benefit as it will
promote pain relief
CA 02360124 2002-O1-14
WO 00/45832 PCT/GB00/00281
6
during a recovery period following surgery etc. Furthermore it will be
appreciated that
the analegesia promoted by the compounds is not associated with respiratory
depression (a common side-effect of many known analgesics e.g. morphine).
The inventors have found that compounds which increase DSIP activity are
particularly useful for treating patients who require long term ventilation in
the
intensive care setting. A problem associated with such patients relates to the
long
term maintenance of an adequate state of anaesthesia such that the patient is
maintained pain free and can be ventilated. Extensive clinical experience has
shown
that increasing doses of anaesthetic agents are required. In general, gaseous
agents
are not used because of a number of major drawbacks including pollution of the
local
environment. Continuous intravenous anaesthesia using propofol is often used.
However, accumulation of elements of the propofol formulation results in
undesirable
effects. Another major problem is that tolerance to the anaesthetic effects of
propofol
develops, in some cases rapidly, such that ever larger doses are required to
maintain
the patient. Finally, when the time comes to wean patients off the anaesthetic
in order
to wean the patient off the ventilator, the respiratory depression caused by
conventional general anaesthetics is a major problem. The use of compounds
that
increase DSIP activity in this clinical setting has particular advantages
because
increased DSIP activity does not cause respiratory depression. Furthermore
tolerance
has not been observed to the effects of the naturally occurring hormone. In
addition,
DSIP has activities that will confer additional benefits over many
conventional
anaesthetics as follows:
1) DSIP has been shown to have analgesic activity of its own, possibly through
the
release of met enkcphalin; (pain is frequently a prominent problem in the long
term ventilated patient); and
2) DSIP has been shown to have a beneficial effect on the adaptive responses
to
stress (the intensive care setting is extremely stressful).
We have found that compounds which increase DSIP activity are also
particularly useful as adjuncts to other anaesthetics. When given in
conjunction with
other anaesthetics, compounds that increase DSIP activity prolong the duration
of
CA 02360124 2002-O1-14
WO 00!45832 PCT/GB00/00281
7
anaesthesia. Equally, when a compound according to the first embodiment of the
first
aspect of invention is used as an adjunct, a satisfactory depth of anaesthesia
may be
achieved at a reduced level of the other anaesthetic (compared to use of other
anaesthetics alone). This has the advantage of reducing the risk of side
effects andlor
the discomfort associated with recovery from the use of higher amounts of
anaesthetic
compounds. For instance, known anaesthetics can be associated with respiratory
depression whereby patients stop spontaneous breathing. DSIP is not associated
with
respiratory depression. Therefore, administration of DSIP with a reduced level
of
known anaesthetic results in an acceptable level of anaesthesia without
respiratory
depression.
The use of DSIP and other compounds according to the first embodiment of
the first aspect of the invention has the advantage that there is less risk of
cardiovascular instability. Other advantages of using the compounds include:
(i) the kinetics of DSIP in vivo is non-saturable (metabolism is by plasma
and other non-specific esterases);
(ii) peptide compounds such as DSIP are not toxic and need not be used as
a gas. Therefore there is less environmental pollution during manufacture, use
and
disposal; and
(iii) compounds which promote DSIP activity also allow for instantaneous
reversal, or at least quicker reversal, of general anaesthesia thereby further
improving
or eliminating anaesthetic recovery times and improving anaesthetic safety
(e.g. the
use of DSIP as an anaesthetic cofactor in combination with propofol helps
smooth out
propofol induced anaesthesia and allows fewer intraoperative side effects)
DSIP is degraded by a number of non-specific peptidases including
Angiotensin Converting Enzyme (ACE). Therefore it is preferred for some
applications that compounds according to the first embodiment of the first
aspect of
the invention are formulated with (or co-administered with) ACE inhibitors in
order
that DSIP activity may be potentiated. This is preferred when DSIP needs to be
used
for relatively long periods of time (e.g. anaesthesia and analgesia during
intensive
care).
CA 02360124 2002-O1-14
WO 00/45832 PCT/GB00/00281
8
According to a second embodiment of the first aspect of the invention
compounds may be used which decrease DSIP activity.
Compounds according to the second embodiment of the first aspect of the
invention may be used for increasing brain activity for inducing recovery from
anaesthesia.
Several classes of compound which are capable of decreasing DSIP activity
may be used according to the second embodiment of the first aspect of the
invention.
Such compounds include antagonists or partial agonists of DSIP neuromodulatory
binding sites, agents which inhibit the release of endogenous agonists of DSIP
neuromodulatory binding sites, agents which inhibit the synthesis of
endogenous
agonists of DSIP neuromodulatory binding sites, agents which promote the
breakdown (or removal/sequestration) of endogenous DSIP agonists, agents which
decrease DSIP expression or activity and agents which inhibit the mechanisms
involved in signal transduction between the ligand bound DSIP binding site and
effector systems.
Preferred compounds which decrease DSIP activity are DSIP anatagonists and
include rnelatonin, dalargin and neokyotorphin.
A preferred use of compounds which decrease DSIP activity is to promote
recovery from anaesthesia. Thus, immediately before an operation, compounds
according to the first embodiment of the first aspect of the invention may be
used
(alone or in conjunction with another anaesthetic) to anaesthetise a subject
and then,
once the procedure has been completed, compounds according to the second
embodiment of the first aspect of the invention may be used to expedite
recovery from
anaesthesia.
Brain activity may be regulated with compounds which modulate DSIP
activity according to either embodiment of the first aspect of the invention
as a
CA 02360124 2002-O1-14
WO 00/45832 PCT/GB00/00281
9
monotherapy or in combination with other agents. For instance, anaesthesia may
be
induced with compounds according to the first embodiment of the first aspect
of the
invention alone (a monotherapy) or in combination with other known anaesthetic
agents {e.g. combination therapy with a DSIP agonist as an anaesthetic
cofactor for
propofol or with a gaseous agent to reduce MAC. MAC being the Minimum Alveolar
Concentration of anaesthesia necessary to achieve loss of movement to a
noxious
stimulus in 50% of subjects).
When the compounds are used in combination with other agents, a lower dose
of that agent may be required. This will reduce the incidence and severity of
side-
effects known to be caused by such agents. The dose requirements are typically
reduced by 20 - 50% depending upon the specific combination used.
The compounds used according to the first aspect of the invention may take a
number of different forms depending, in particular on the manner in which the
composition is to be used. Thus, far example, the composition may be in the
form of
a powder, tablet, capsule, liquid, ointment, cream, gel, hydrogel, aerosol,
spray,
micelle, liposome or any other suitable form that may be administered to a
person or
animal. It will be appreciated that the vehicle of the composition of the
invention
should be one which is well tolerated by the subject to whom it is given and
enables
delivery of the compounds to the target tissue.
Preferred formulations include sterile, isotonic solutions for injection and
micronised powders with excipients for oral inhalation.
The compounds may be used in a number of ways. For instance, systemic
administration may be required in which case the compound may be contained
within
a composition which may for example be administered by injection into the
blood
stream. Injections may be intravenous (bolus or infusion} or subcutaneous
(bolus or
infusion). The compounds may also so be administered by inhalation.
Alternatively
the compound may be ingested orally in the form of a tablet, capsule or
liquid.
CA 02360124 2002-O1-14
WO 00/45832 PCT/GB00/00281
Compounds modulating DSIP activity may be administered centrally by
means of intracerebral, intracerebroventricular, or intrathecal delivery.
It will be appreciated that the amount of a compound required is determined
by biological activity and bioavailability which in turn depends on the mode
of
administration, the physicochemical properties of the compound employed and
whether the compound is being used as a monotherapy or in a combined therapy.
The
frequency and/or rate of administration will also be influenced by the above
mentioned factors and particularly the half life of the compound within the
subject
being treated. It will be appreciated that an anaesthetist will need to
monitor the depth
of anaesthesia of a subject during anaesthesia and adjust the required dose of
the
compound as required.
Known procedures, such as those conventionally employed by the
pharmaceutical industry (e.g. in vivo experimentation, clinical trials etc),
may be used
to establish specific formulations of compositions and precise therapeutic
regimes.
Generally, a dose of between 0.01 hglkg of body weight and 1.0 gJkg of body
weight of a compound which modulates DSIP activity may be used for the
regulation
of brain activity depending upon which specific compound is used and the
reason for
regulating activity. For instance, a suitable dose of a DSIP agonist will be
in the range
of between 1.0 p,g/kg and I.0 mg/kg (preferably 20 - 400pg/kg). Purely by way
of
example a suitable dose of DSIP for use in combination with propofol (e.g.
7mg/kg
LV. bolus) for inducing anaesthesia is between O.Olmg and 100 mg/kg and
preferably
between 0.02 mg/kg and 10 mglkg.
Administration may be required frequently or continuously depending upon
the requirements of an anaesthetist. By way of example between 1 p,glkg/hr and
1 g/kg/hr, and preferably between l Opg/kg/hr and 100mg/kg/hr of DSIP may be
required to maintain anaesthesia.
CA 02360124 2002-O1-14
WO 00/45832 PCTIGB00/00281
11
According to a second aspect of the present invention, there is provided a
method of regulating anaesthesia comprising administering to a subject in need
of
treatment a compound which modulates Delta-Sleep Inducing Peptide activity.
The abovementioned compounds which modulate DSIP activity according to
the first aspect of the invention may be used according to the method of the
second
aspect of the invention.
According to a third aspect of the present invention there is provided a
method
of evaluating the anaesthetic needs of a subject to be anaesthetised
comprising
assaying a sample taken from the subject for the presence of Delta-Sleep
Inducing
Peptide.
By "anaesthetic needs" we mean an assessment of the dose of an anaesthetic
required to induce or maintain a desired level of anaesthesia.
We have found that anaesthetic dose requirements are directly related to
endogenous levels of DSIP. Thus a pre-operative assay of DSIP levels in a
subject
(e.g. a simple urine or blood test screening for DSIP) provides an anaesthetic
dosage
guide for predicting anaesthetic requirements. Higher than average endogenous
levels
of Delta-Sleep Inducing Peptide assayed from the sample indicate the subject
will
have lower than average anaesthetic requirements. Lower than average
endogenous
levels of Delta-Sleep Inducing Peptide assayed from the sample indicate the
subject
will have higher than average anaesthetic requirements.
It will be appreciated that the normal range for endogenous DSIP will depend
upon the assay employed and the population studied. Purely by way of example
DSIP
levels may be assessed using the assay described by Seifritz et al. (Peptides
1995; 16
(8); p1475 - 1481). Using this assay the range of DSIP in blood is
approximately 0.1
- 11 ng/ml. 'Therefore subjects with DSIP levels greater than about 5.0 ng/ml
are
likely to need less anaesthetic than normal whereas subjects with DSIP levels
less
than about 5.0 ng/ml are likely to require more anaesthetic than normal.
CA 02360124 2002-O1-14
WO 00!45832 PCT/GB00/00281
12
A suitable assay for measuring DSIP levels in a sample is a quantitative
immunoassay utilising antibodies raised against DSIP. For instance, the enzyme
immunoassay described by Kato et al. (Neuroendocrinology 1984;39:p39-44) may
be
adapted for use as a pre-operative test to evaluate anaesthetic requirements.
An
alternative assay which may be used according to the third aspect of the
invention is a
radioimmunoassay (e.g. as described by Seifritz et al. Supra). It is preferred
that the
assay mediates a colourmetric change which may be interpreted by eye or
spectrophotometrically.
The sample is most suitably a blood or urine sample.
Such a method may be used pre-operatively to evaluate the anaesthetic needs
of elective surgical patients.
According to one embodiment of the third aspect of the invention, an
anaesthetist, nurse or theatre technician may test a blood or urine sample
from a subject a
short while (approximately 30 minutes or less) before anaesthesia to evaluate
the
anaesthetic needs of the subject. This test may be by means of inserting into
the sample
a dip-stick which undergoes a colour change (depending upon the DSIP levels in
the
sample). An anaesthetist can then interpret the measured levels and adapt the
anaesthetic
regime accordingly.
The invention will be further illustrated by the following non-limiting
Example.
CA 02360124 2002-O1-14
WO 00/45832 PCT/GB00/00281
13
EXAMPLE
Experiments were performed in rodents to evaluate the effect of DSIP on
anaesthesia induced by propofol.
Methods
Nine female Sprague-Dawley rats weighing 230 to 287g had free access to water
and rat Purina chow. All animals were maintained, cared for, and handled in
accordance
with IACUC animal utilization policy. Animals were divided into two groups to
test the
interactions between DSIP and the intravenous anaesthetic agent propofol (n=5}
or the
inhalational anaesthetic agent isotlurane (n=4). For the propofol test, rats
were randomly
selected to receive either Delta-sleep inducing peptide (Peninsula Labs, CA) 1
mg/kg i.p.
in 3 ml of sterile water or just 3 ml of sterile water i.p. alone (placebo) 15
minutes prior
to injection of propofol (Trademarks: Diprivan or Rapinoivet) 7 mg/kg i.v.
into a tail
vein over approximately 10 s. Following injection of propofol animals were
tested for
loss of righting reflex. On loss of righting reflex the animals were placed on
their sides
in the center of a large plastic bowl with a flat bottom. Sleep time was
recorded as the
time taken to regain righting with all 4 feet on the ground. The following
week, those
animals that had received DSIP now received placebo pretreatment and those
that had
received placebo now received DSIP pretreatment. Again sleep time was assessed
for
each rat, after giving each rat the identical dose of propofol that it had
been given the
previous week.
For the inhalational test, rats were placed on a rotating rod in the middle of
an
anaesthetizing chamber. The Level of inhalational agent was slowly titrated
upwards in
0.05% increments every 10-15 min until the rats could no longer walk forward
on the
rotating rod. At week one, rats were randomly selected to receive either DSIP
0.1 mg/kg
i.p. 15 min prior to testing, or placebo. The following week rats were crossed
over to the
other treatment arm (i.e. placebo to DSIP and DSIP to placebo).
Data were analyzed with a paired two-tailed t-tests.
CA 02360124 2002-O1-14
WO 00/45832 PCT/GB00/00281
14
Results
Intraperitoneal injection of lmg/kg DSIP did not cause any rat to loose
consciousness. Rats did, however, display a paucity of movement almost
immediately
after i.p. injection of DSIP. The animals did appear to be under the influence
of some
pharmacologic effect following DSIP pretreatment, perhaps best described by
noting that
the rats appeared to have a "vacant" look about them when left undisturbed.
The
animals would, however, move appropriately when approached, but then would
quickly
resume a crouched position when left alone.
Sleep times following propofol iv injection (7mg/kg) for each animal are shown
in Table I.
Table 1
animal Sleep time (Sec) Sleep time (Sec)
DSIP lmg/kg placebo
1 406 242
2 527 446
3 748 577
4 637 581
737 689
Each animal slept longer when pretreated with DSIP. The mean sleep time for
propofol alone was 477 +/- 158 Sec. The mean sleep time for DSIP pretreatment
followed by propofol was 611 +/- 145. This difference was significant at the
P<0.01
level and represents a mean 28% increase in sleep time.
The dose of isoflurane anaesthesia (chamber ISO%) required to prevent each
animal from being able to walk forward on a rotating rod is shown in Table 2.
CA 02360124 2002-O1-14
WO 00145832 PCT1GB00/00281
Table 2
animal Chamber iso % Chamber iso
DSIP O.lmg/kg placebo
ip
1 0.24 0.30
2 0.12 0.20
3 0.26 0.31
4 0.18 0.21
The mean (+/- SD) concentration of isoflurane that prevented animals from
being
able to walk on the rotarod following placebo alone was 0.26 +/- 0.06%. The
DSIP
pretreatment reduced this value 23% to 0.20 +/- 0.06%. This reduction was
statistically
significant at the p = 0.01 level.
These data illustrate that DSIP was particularly effective when used as an
adjunct
to both propofol and isoflurane. Table 1 illustrates that DSIP prolongs the
length of
anaesthesia whereas Table 2 illustrates that DSIP is able to lower the
concentration of
another anaesthetic which is required to induce anaesthesia.
