Note: Descriptions are shown in the official language in which they were submitted.
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1
Use of sodium channel blockers and their analogues for the treatment of
nicotine dependency
Field of the invention
The present invention refers to the use of a sodium channel blocker such as
tetrodotoxin or saxitoxin, their analogues and derivatives as well as their
physiologically
acceptable salts, in medicinal products for human therapeutics and/or animal
research
for the treatment of nicotine dependency.
Background of the invention
Nicotine is a natural product that can be isolated from the leaves of the
tobacco plant.
Tobacco has been consumed for centuries in Mezo- and South America, valued for
its
effects after smoking. It is now consumed worldwide. Even though its effect on
the
body are not as devastating as that of other drugs which produce, dependency
it
nevertheless has developed into an enormous health care problem with lung
cancer
being one of the main causes for death.
For the moment any de-addiction treatment from nicotine mainly involves
behavioral
therapy sometimes helped by medication. Accordingly there is a high need for
an
effective and successful de-addiction therapy especially for useful agents in
the
pharmacotherapy of addiction from nicotine.
Therefore the subject of this invention is the use of a sodium channel
blocker, and/or
one of its derivatives for the production of a drug for treatment of addiction
to nicotine,
to treat dependency upon nicotine or ameliorate the effects of a de-addiction
treatment
from nicotine. The sodium. channel blocker is optionally used in the form of
its
racemate, pure stereoisomers, especially enantiomers or diastereomers or in
the form
of mixtures of stereoisomers, especially enantiomers or diastereomers,
preferably in
any suitable ratio; in neutral form, in the form of an acid or base or in form
of a salt,
especially a physiologically acceptable salt, or in form of a solvate,
especially a
hydrate.
So it is now established that a sodium channel blocker, such as tetrodotoxin
(TTX) or
saxitoxin (STX), could help in de-addiction treatment of nicotine.
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2
The term "sodium channel blocker" mentioned in this application is defined as
a
compound that specifically binds to and specifically inhibits sodium channels,
which
are classified as either TTX-resistant or TTX-sensitive. The term TTX-
resistant" and
TTX-sensitive refers to a difference in the tightness of TTX binding, with the
TTX
resistant channel having a binding constant as mentioned in Hunter et
al.,Current
Opinion in CPNS Investigational Drugs 1(1), 1999 as well as Clare et al. DDT,
5(11),
2000, 506-520 included here by reference and the TTX sensitive channel having
a
binding constant as mentioned in Hunter et al., Current Opinion in CPNS
Investigational Drugs 1(1), 1999 as well as Clare et al. DDT, 5(11), 2000, 506-
520. A
preferred sodium channel blocker thus binds to a sodium channel with a IC50 of
less
than 200 M, preferably less than 100 M or with an IC50 of 2 M. Said
inhibition refers
to suppression or modification of any downstream effect caused by activation
of said
sodium channels. More preferably, the term "sodium channel blocker" mentioned
in
this invention refers to compounds binding to an alpha subunit of sodium
channels,
especially TTX-resistant or TTX-sensitive sodium channels. More preferably,
the term
"sodium channel blocker" mentioned in this invention refers to compounds
binding to
either a SS1 or SS2 region of an alpha subunit of sodium channels, especially
TTX-
resistant or TTX-sensitive sodium channels. Preferred sodium channel blockers
for
use in this invention are tetrodotoxin and saxitoxin which both specifically
inhibit said
sodium channels.
The term "analogues" as used in this application is defined here as meaning a
chemical compound that is a derivative of a compound which has similar
biochemical
activity to that compound. "Analogues" of TTX and STX bind to the same site on
the
alpha subunit of sodium channels as does TTX and STX.
The term "derivatives" as used in this application is defined here as meaning
a
chemical compound having undergone a chemical derivation such as substitution
or
addition of a further chemical group to change (for pharmaceutical use) any of
its
physico-chemical properties, such as solubility or bioavailability.
Derivatives include so-
called prodrugs, e.g. ester and ether derivatives of an active compound that
yield the
active compound per se after administration to a subject.
Examples of well known methods of producing a prodrug of a given acting
compound
are known to those skilled in the art and can be found e.g. in Krogsgaard-
Larsen et al.,
Textbook of Drugdesign and Discovery, Taylor & Francis (April 2002).
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3
Tetrodotoxin (alternatively in the context of this application abbreviated
TTX), also
known as Ti Qu Duo Xin, is an alkaloid found in puffer fish (Tetradontiae).
The
chemical name is Octahydro-12-(Hydroxymethyl)-2-imino-5, 9, 7, lOa-dimethano-
10aH-[1,3]dioxocino[6,5-d]pyrimidine-4,7,10,11,12-pentol with a molecular
formula
CIIH17N308 and a Molecular weight of 319.27. It is a potent non-protein
neurotoxin and
an indispensable tool for the study of neurobiology and physiology.
Tetrodotoxin (TTX)
is a marine organic toxin which is mainly found in testicies, ovaries, eggs,
livers,
spleens, eyeballs, and blood of puffer fish as well as in diverse animal
species,
including goby fish, newt, frogs and the blue ringed octopus and even in
marine alga.
Several processes for producing TTX are known. Usually TTX is extracted from
marine
organisms (e.g. JP 270719 Goto and Takahashi) but besides numerous others
methods of synthesis are also described (and used for the preparation of
tetrodotoxin
in connection to this invention) in US 6,552,191, US6,478,966, US 6,562,968 or
2002/0086997, all of which are included here by reference. Tetrodotoxin is a
well
known compound described for example in W002/22129 as systemically acting as
analgesic. For one of the many descriptions of TTX it is recommended turn to
e.g. Tu,
Anthony (Ed.) Handbook of Natural Toxins, Vol. 3: Marine Toxins and Venoms,
1988,
185-210 as well as Kao (1966), Pharmacol. Rev. 18:997 - 1049 and others.
The usefulness of TTX in treatment of addiction to alkaloids and synthetic non-
amino
acid nitrogen-containing compounds is already claimed in EP 0 750 909 and also
described in CN 1145225 and CN 1227102. WO 03/099301 describes aerosol, spray
or gasoloid formulation for addiction treatment in regards to alkaloids with
the majority
being opioids. Even so, this art does not touch the current invention because
nicotine
dependency from which millions of smoker's suffer never is summarized under
drug
addiction. Therefore, it came as a surprise when during our experiments it was
discovered that TTX couid help in treating nicotine addiction.
In the scientific literature however, the main use of TTX (and to a lesser
extent)
saxitoxin has been its use as pharmacological research tools in neurobiology,
pharmacology and electrophysiology. Respective exemplary experiments referring
to
TTX were performed in the context of the actions of heroin (Rita et al, 2002),
cocaine
(Erb et al., 2001; McLaughlin et al., 2001), ethanol (Yan et al., 2003)
morphine (De
Rover et al., 2005) and nicotine (Benwell et al., 1993).
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4
A recent study performed by Corrigal et al (Corrigal et al., 2002) TTX was
used as a
research tool in the context of nicotine dependence. In this study TTX was
infused
intracranially into the pedunculopontine tegmental nucleus, thereby provoking
a
temporary functional lesion of said area with the aim to block cholinergic
neuron
signalling. The amount of TTX used in this neurophysiological study is
extremely high
compared to a therapeutic dosage when one considers the weight of this
respective
lesioned brain area. This study shows only that TTX-mediated functional
deletion of a
special cell type such as cholinergic neurons in a cerebral nucleus leads to a
modification of behavior of drug-dependent rats. Consequently, a person
skilled in the
art would consider the study performed by Corrigal et al. as being out of the
scope of
the present invention, both in the sense of mechanism as well as in the sense
of TTX
usage. In addition we consider especially that the prior art as a whole does
not
anticipate use of TTX, or any other sodium channel blocker, in treatment of
subjects
having an established addiction to nicotine.
The phrase "its (tetrodoxin's) derivatives" according to this invention is
defined - using
the definition of US 6,030,974 (included here by reference) - as meaning amino
perhydroquinazoline compounds having the molecular formula C11H17N308.
"Tetrodoxin's derivatives" according to this invention encompasses compounds
described in US 5,846,975 (included here by reference) as amino hydrogenated
quinazolines and derivatives including the substances set forth from column 3
line 40
to column 6 line 40. Specifically exemplified "derivatives of tetrodotoxin"
according to
this invention are including but are not limited to anhydro-tetrodotoxin,
tetrodaminotoxin, methoxytetrodotoxin, ethoxytetrodotoxin, deoxytetrodotoxin
and
tetrodonic acid, 6 epi-tetrodotoxin, 11-deoxytetrodotoxin as well as the
hemilactal type
TTX derivatives (e.g. 4-epi-TTX, 6-epi-TTX, 11-deoxy-TTX, 4-epi- 11 -deoxy-
TTX, TTX-
8-O-hemisuccinate, chiriquitoxin, 11-nor-TTX-6(S)-ol, 11-nor-TTX-6(R)-oI, 11-
nor-TTX-
6,6-diol, 11-oxo-TTX and TTX-11-carboxylic acid), the lactone type TTX
derivatives
(e.g. 6-epi-TTX (lactone), 11-deoxy-TTX (lactone), 11-nor-TTX-6(S)-ol
(lactone), 11-
nor-TTX-6(R)-ol (lactone), 11-nor-TTX-6,6-diol (lactone), 5-deoxy-TTX, 5,11-
dideoxy-
TTX, 4-epi-5,11-didroxy-TTX, 1-hydroxy-5,11-dideoxy-TTX, 5,6,11-trideoxy-TTX
and
4-epi-5,6,11-trideoxy-TTX) and the 4,9-anhydro type TTX analogs (e.g. 4,9-
anhydro-
TTX, 4,9-anhydro-6-epi-TTX, 4,9-anhydro-11-deoxy-TTX, 4,9-anhydro-TTX-8-O-
hemisuccinate, 4,9-anhydro-TTX-11-O-hemisuccinate). The typical derivatives of
TTX
possess only 1/8 to 1/40 of the toxicity of TTX in mice, based upon bioassay
in mice. It
has been observed that these derivatives produce joint action, and do not
interact
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adversely. Examples of TTX derivatives inciude novel TTX derivatives isolated
from
various organisms, as well as those that are partially or totally chemically
synthesized
(see e.g., Yotsu, M. et al. Agric. Biol. Chem., 53(3):893-895 (1989)).
5 According to U. S. Patent No. 6,030,974,"saxitoxin" or "STX" refers to a
compound
comprising a tetrahydropurine moiety composed of two guanidine units fused
together
in a stable azaketal linkage, having a molecular formula CIoHI7N704 (mol. wt.
299.30)
and to derivatives thereof, including but not limited to hydroxysaxitoxins and
neosaxitoxin. Bower et al., Nonprotein Neurotoxins, Clin. Toxicol. 18 (7): 813-
863
(1981).
In connection with this invention "neutral form" refers either to a non-ionic
form or to a
neutrally net charged form, for example a Zwitter-lon at its isoelectric
point.
The term "salt" according to this invention is to be understood as meaning any
form of
the active compound according to the invention in which this compound assumes
an
ionic form or is charged and - if applicable - is also coupled with a counter-
ion (a
cation or anion). By this are also to be understood complexes of the active
compound
with other molecules and ions, in particular complexes which are complexed via
ionic
interactions. As preferred examples of salts this includes the acetate, mono-
trifluoracetate, acetate ester salt, citrate, formate, picrate, hydrobromide,
monohydrobromide, monohydrochloride or hydrochloride.
The term "physiologically acceptable salt" in the context of this invention is
understood
as meaning a"salt" (as defined above) of at least one of the compounds
according to
the invention which are physiologically tolerated by humans and/or mammais.
The term "solvate" according to this invention is to be understood as meaning
any form
of the active compound according to the invention which has another moiecule
(most
likely a polar solvent) attached to it via non-covaient bonding. Examples of
solvates
include hydrates and alcoholates, e.g. methanolate.
The term "effects of a de-addiction treatment" in the context of this
invention is
understood as including any side effect coming with any de-addiction
treatment,
especially any kind of withdrawal syndrome.
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The term "ameliorate" in the context of this invention is understood as
meaning any
improvement on the situation of the patient treated - either subjectively
(feeling of or on
the patient) or objectively (measured parameters).
The term "treatment" or "to treat" in the context of this specification means
administration of a compound or formulation according to the invention to
prevent,
ameliorate or eliminate one or more symptoms associated with de-addiction from
nicotine. Such symptoms can arise from withdrawal, or can be associated with
relapse
behavior, such as a craving of a subject for nicotine. "Treatment" also
encompasses
preventing, ameliorating or eliminating the physiological sequelae of removal
of
nicotine from an addicted subject that may cause the exhibited symptoms
perceived by
the subject. "Treatment" also encompasses a reduction in the amount of
nicotine that
is consumed by a subject.
Furthermore, the terms "to treat" or "treatment" according to this invention
include the
treatment of symptoms of de-addiction, nicotine dependency or nicotine
withdrawal,
especially certain subtypes of de-addiction, drug dependency or nicotine
withdrawal,
the treatment of the consequences causing the symptoms, the prevention or the
prophylaxis of the symptoms of de-addiction, or nicotine withdrawal,
especially certain
subtypes of de-addiction, or nicotine withdrawal, the prevention or
prophylaxis causing
the symptoms of de-addiction, or nicotine withdrawal, as well as the
prevention or the
prophylaxis of the consequences causing the symptoms.
The wording "nicotine replacement therapy" used in this application refers to
any form
of nicotine replacement that can help lessen the urge to smoke. "Nicotine
replacement
therapy" also includes the utilization of nicotine patches, gums, nasal sprays
and any
other form of nicotine application, either oral or parenteral.
For treatment of human subjects, the dose administered is normally between 5
and
4000 g/day when tetrodotoxin, or a derivative or analog thereof is given.
When TTX is
administered the dosage is usually from 5 to 4000 g/day. Administration is
typically
twice per day and on such a schedule each administered dose will typically
contain
from 2.5 to 2000 g of TTX or of a derivative or analog thereof. Preferably,
from 2.5 to
500 ug is administered per dose, more preferably from 50 to 500 g per dose,
or from
50 to 400 g per dose. In some administrations, from 2.5 to 30 g per dose is
given.
The dosage will vary with the route of administration, with intravenous
administration
typically utilizing a lower dosage than oral administration.
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A substance named as an "active ingredient" will have a purity of at least
97%. For
example, a formulation said to have "500 g of TTX as the active ingredient"
may
contain as much as 15 g of anhydrotetrodotoxin as an impurity. On the other
hand, a
formulation said to have "500 g of TTX and 500 .g of anhydrotetrodotoxin as
active
ingredients" will contain at least 485 ug of TTX and 485 g of
anhydrotetrodotoxin, but
may contain as much as 30 g of other substances as impurities of the active
ingredients. Of course, substances named as other components of a formulation
are
not included when the purity of the active ingredient is considered.
In a preferred embodiment of the invention the use according to the invention
is
restricted to treat dependency upon nicotine or ameliorate the effects of a
treatment for
de-addiction from nicotine.
In a preferred embodiment of the invention the use according to the invention
is
restricted to tetrodotoxin as the active ingredient. The TTX is optionally
provided in the
form of its racemate, pure stereoisomers, especially enantiomers or
diastereomers or
in the form of mixtures of stereoisomers, especially enantiomers or
diastereomers,
preferably in a suitable ratio; in neutral form, in the form of an acid or
base or in form of
a salt, especially a physiologically acceptable salt, or in form of a solvate,
especially a
hydrate.
In a preferred embodiment of the invention the use according to the invention
is
restricted to tetrodotoxin as the active ingredient, preferably in any
suitable ratio in
neutral form or as a salt, especially a physiologically acceptable salt.
In a preferred embodiment of the invention tetrodotoxin, any of its
derivatives and/or
one of its analogues is used as the active ingredient in an amount between 10
g/day
and 4 mg/day.
In a preferred embodiment of the invention tetrodotoxin, its derivative or its
analogue is
isolated from a biological source, preferably from fish, especially puffer
fish.
In a preferred embodiment of the invention tetrodotoxin, its derivative or its
analogue is
synthetic.
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In a preferred embodiment of the invention, nicotine in any type of
formulation as
mentioned in this application, is administered in combination with
tetrodotoxin or
saxitoxin, their derivatives or analogues. The amount of nicotine is typically
chosen
according to the needs of nicotine of the patient (as weli-known in the art
for de-
addiction using only nicotine) and preferably decreases over time. It is
desirable to
ultimately administer no nicotine.
In a preferred embodiment of the invention, nicotine is administered in
combination
with tetrodotoxin or saxitoxin, their derivatives or analogues, either in form
of a patch, a
gum, a nasal spray, an inhaler or any other form of topical administration.
The term "in combination" includes but does not exclusively mean the
concomitant
administration of compounds, but also the sequential administration of each of
the
compounds, as well as administration of one compound, followed by
administration of
the other compound, whereas the time interval between the two administrations
can be
between 1 min and 1 week. Additionally, "in combination" refers to a
formulation
containing all compounds in its respective carrier such as a tablet, capsule,
a pill or a
gum. Most preferably, "in combination" means a fixed combination of compounds
in a
single dosage form such as a tablet, a pill , a capsule or a gum.
In general a preferred embodiment of the invention is a combination comprising
a substance A selected from the group of sodium channel blockers, and/or one
of its
derivatives, optionally in the form of its racemate, pure stereoisomers,
especially
enantiomers or diastereomers or in the form of mixtures of stereoisomers,
especially
enantiomers or diastereomers; preferably in any suitable ratio; in neutral
form, in the
form of an acid or base or in form of a salt, especially a physiologically
acceptable salt,
or in form of a solvate, especially a hydrate
and
substance B, selected from nicotine as well as its derivatives.
A preferred embodiment of this invention is a combination wherein substance A
selected from the group of sodium channel blockers is tetrodotoxin, optionally
in the
form of its racemate, pure stereoisomers, especially enantiomers or
diastereomers or
in the form of mixtures of stereoisomers, especially enantiomers or
diastereomers;
preferably in any suitable ratio; in neutral form, in the form of an acid or
base or in form
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9
of a salt, especially a physiologically acceptable salt, or in form of a
solvate, especially
a hydrate.
Another preferred embodiment of the invention is a medicament comprising a
substance A selected from the group of sodium channel biockers, and/or one of
its
derivatives, optionally in the form of its racemate, pure stereoisomers,
especially
enantiomers or diastereomers or in the form of mixtures of stereoisomers,
especially
enantiomers or diastereomers; preferably in any suitable ratio; in neutral
form, in the
form of an acid or base or in form of a salt, especially a physiologically
acceptable salt,
or in form of a solvate, especially a hydrate
and
substance B, selected from nicotine as well as its derivatives.
Another preferred embodiment of the invention is the use of an above-mentioned
combination for the production of a drug for the treatment of nicotine
dependence, de-
addiction of nicotine dependence or amelioration of the effects of a de-
addiction
treatment from nicotine.
A preferred embodiment of this invention is a use according to this invention
wherein
the drug is manufactured to be administered parenterally and/or orally.
A preferred embodiment of this invention is a use according to this invention
wherein the drug is manufactured to be administered in form of a patch, a gum,
a
nasal spray, an inhaler or any other form of topical administration.
Another preferred embodiment of the invention is a dosage form of TTX, and/or
of an analog and/or derivative thereof, for the treatment of nicotine
addiction.
Another preferred embodiment of the invention is the use of TTX, and/or of an
analog and/or derivative thereof, to prepare a medicament for treatment of
nicotine
addiction.
Another preferred embodiment of the invention is a kit comprising a drug
comprising a substance A selected from the group of sodium channel blockers,
and/or
one of its derivatives, optionally in the form of its racemate, pure
stereoisomers,
especially enantiomers or diastereomers or in the form of mixtures of
stereoisomers,
especially enantiomers or diastereomers, in any suitable ratio; in neutral
form, in the
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form of an acid or base or in form of a salt, especially a physiologically
acceptable
salt, or in form of a solvate, especially a hydrate
and a drug comprising substance B, selected from nicotine as well as its
derivatives.
5
Any formulation or pharmaceutical composition according to the invention
contains the
active ingredient (nicotine, saxitoxin or TTX, its derivatives and/or its
analogues) as
well as optionally at least one auxiliary material and/or additive.
The auxiliary material and/or additive can be one ore more of conserving
agents,
emulsifiers and/or carriers for parenteral application. The selection of these
auxiliary
materials and/or additives and of the amounts to be used depends upon how the
pharmaceutical composition is to be administered. Examples include especially
parenteral routes like intravenous subcutaneous or intramuscular
administration.
Routes of administration of tetrodotoxin its derivatives and its analogues can
include
intramuscular injection, intraveneous injection, subcutaneous injection,
sublingual,
bucal, patch through skin, oral ingestion, implantable osmotic pump, collagen
implants,
aerosols or suppository.
Included in this invention are especially also methods of treatments of a
patient or a
mammal, including man, suffering from addiction from nicotine, the method
comprising
the administration of tetrodotoxin, of its derivatives and/or of its analogues
optionally in
the form of its racemate, pure stereoisomers, especially enantiomers or
diastereomers
or in the form of mixtures of stereoisomers, especially enantiomers or
diastereomers;
preferably in any suitable ratio; in neutral form, in the form of an acid or
base or in form
of a salt, especially a physiologically acceptable salt, or in form of a
solvate, especially
a hydrate. It is aiso preferred if the method of treatment is restricted to
tetrodotoxin,
optionally in the form of its racemate, pure stereoisomers, especially
enantiomers or
diastereomers or in the form of mixtures of stereoisomers, especially
enantiomers or
diastereomers, preferably in any suitable ratio; in neutral form, in the form
of an acid or
base or in form of a salt, especially a physiologically acceptable salt, or in
form of a
solvate, especially a hydrate. It is also preferred if the method of treatment
is restricted
to tetrodotoxin, in neutral form or as a salt, especially a physiologically
acceptable salt,
whereas preferably tetrodotoxin, its derivative and/or one of its analogues is
used in an
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11
amount between 10 g/day and 4 mg/day, is isolated from a biologicai source,
preferably from fish, especially puffer fish, or is synthesized.
The present invention also includes articles of manufacture that include a
dosage form
of TTX, or of one or more of its derivatives and/or analogs, or a mixture of
TTX and
one or more of its derivatives and /or analogs, packaged together with written
material
that urges the administration of the dosage form for the treatment of nicotine
addiction.
The dosage form provided with the article of manufacture according to the
invention is
preferably one that is appropriate for oral administration or administration
by a"patch"
that comprises substances for carrying the active ingredients through the
skin. The
dosage form included in the' article of manufacture may be one appropriate for
parenteral injection.
Articles of the scientific periodical and patent literature cited herein are
hereby
incorporated by reference in their entirety by such citation.
The examples and figures in the following section describing pharmacological
trials are merely illustrative and the invention cannot be considered in any
way as
being restricted to these applications.
Examples:
Example 1:
Example formulation of an injectable (imliv) solution of TTX
Tetrodotoxin (TTX) (powdered material) 15 mg
0.5% diluted acetic acid 1 ml
Acetic Acid - actetate buffer solution (pH=3-5) 50 ml
Water for injection c.s.p., add to 1000 ml
The TTX is dissolved in the 0.5% acetic acid and then the TTX solution is
further
diluted in the acetate buffer and finally diluted with the water for
injection. The
formulation is sterilized by ultrafiltration and packaged in injection
bottles.
The dosage of TTX for injection is 30 p,g in 2 mi.
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Example 2:
Nicotine dependence and withdrawal
Nicotine dependence was induced by using Aizet osmotic minipumps (Model 2001)
(Alzet", Cupertino, CA, USA). These minipumps, implanted subcutaneously under
brief
ether anaesthesia, contained saline or nicotine solutions and delivered a
constant
subcutaneous flow in a rate of 1 l/h. The concentration of nicotine was
adjusted to
compensate for differences in the body weight of the mice. Thus, the average-
weighed
mice received a dose of approximately 25 mg/kg/day nicotine during six days.
Nicotine
withdrawal syndrome was precipitated six days after minipump implantation by
injection of the nicotinic receptor antagonist, mecamylamine (1 mg/kg, sc).
Tetrodotoxin (3 pg/kg, sc) or vehicle was administered 30 min before
mecamylamine
injection. The somatic signs of withdrawal were evaluated immediately after
mecamylamine injection during a period of 30 min. The number of wet dog
shakes,
front paw tremors, writhes and scratches was counted. Body tremor, ptosis,
teeth
chattering, genital licks and piloerection were scored 1 for appearance or 0
for non-
appearance within each 5 min time. The locomotor activity over 5 min periods
was
rated 0, 1 or 2 (0 for inactivity, 1 for low activity and 2 for normal
activity). A global
withdrawal score was calculated for each animal by giving each individual sign
a
relative weight (Castane et a)., Neuropharmacology 43: 857, 2002).
The effects induced by the administration of tetrodotoxin on the expression of
nicotine
physical dependence were evaluated. Animals were distributed as follows:
Group 1 (n = 10): chronic vehicle + acute vehicle
Group 2 (n = 10): chronic nicotine + acute vehicle
Group 3 (n = 10): chronic vehicle + acute tetrodotoxin
Group 4 (n = 10): chronic nicotine + acute tetrodotoxin
The results shown in figure 1 and 2 clearly demonstrate that administration of
tetrodotoxin
attenuates the somatic expression of nicotine withdrawal syndrome
significantly.
