Note: Descriptions are shown in the official language in which they were submitted.
CA 02416647 2003-O1-20
Method of discovering compounds suitable for the treatment
and/or prophylaxis of obesity
The present invention relates to a method of discovering
compounds suitable for the treatment and/or prophylaxis of
obesity. The invention further relates to the use of
compounds which are capable of inhibiting de novo lipogenesis
in mammals, and which are substantially free of effects
directed towards the central nervous system (= CNS), for the
preparation of drugs for the treatment and/or prophylaxis of
obesity.
Today, especially in the developed industrial nations,
obesity is an increasingly serious problem for the health of
the population, being caused predominantly by unbalanced and
excessively high-fat nutrition. The increase in the
percentage of overweight people in the population is being
accompanied by an increase in the consequences of obesity,
which range from personal discontentment to cardiovascular
disease or certain forms of diabetes. There are therefore
already a number of therapeutic procedures aimed at the
treatment or prophylaxis of obesity. One example which may be
mentioned is lipase-inhibitory compounds, which reduce
lipolysis in the intestinal tract and thereby cut down the
energy yield from the food intake. Thus, in this therapeutic
procedure, at least part of the alimentary fats is excreted
undecomposed. It is however desirable to have other novel
therapeutic procedures for the treatment and/or prophylaxis
of obesity which can complement the previously known forms of
therapy.
It has now been found, surprisingly, that compounds
which are capable of inhibiting de novo lipogenesis in
mammals, especially man, are advantageously suitable for the
effective treatment and/or prophylaxis of obesity.
Particularly good results are achieved by administering the
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above-mentioned compounds over prolonged periods, for example
for periods of several weeks.
The subject of the invention is a method of discovering
compounds suitable for the treatment and/or prophylaxis of
obesity, wherein those compounds are selected which are
capable of inhibiting de novo lipogenesis in mammals,
especially man. Furthermore, a subject of the invention is
the use of compounds which are capable of inhibiting de novo
lipogenesis in mammals, and which are in particular
substantially free of effects directed towards the CNS, such
as anticonvulsant effects, for the preparation of drugs for
the treatment and/or prophylaxis of obesity.
De novo lipogenesis (abbreviated hereafter to DNL) is
understood as meaning the synthesis of endogenous fatty acids
from carbohydrates in the mammalian organism. This synthetic
reaction takes place in the cytosol of body cells and is
based on the so-called citric acid cycle or Krebs-Martius
cycle. In this cycle, in an endogenous biochemical reaction,
citrate is ultimately synthesized from the two components
pyruvate, which originates from carbohydrates, and
bicarbonate via different intermediates - including maleate,
fumarate and a-ketoglutarate. If citrate is synthesized in
excess, it can be converted via the intermediate acetyl-
coenzyme A to free fatty acids (= lipidic subsequent
products), which form fats and can then be stored in fat
cells (= adipocytes). The excessive storage of fats formed
from fatty acids in body cells can quite generally lead to
obesity. A variety of enzymes participate in the citric acid
cycle. The turnover of the citrate cycle depends
substantially on the amount of bicarbonate available. The
amount of bicarbonate available in turn depends on the rate
at which it can be formed from carbon dioxide. This
bicarbonate-yielding equilibrium reaction is catalyzed by so-
called carboanhydrases. Of the known carboanhydrases and
isozymes thereof, predominantly carboanhydrase isozymes of
i
subtypes II (= CA II) and V (= CA V) participate, in mammals,
in the catalysis of reactions which provide bicarbonate for
the citric acid cycle. Carboanhydrases of subtype V, being
present in the mitochondria; have a particular role to play.
The citric acid cycle also takes,place in the mitochondria.
There are various conceivable possibilities for
inhibiting DNL in mammalian cells, all of which aim to reduce
the turnover of the citric acid cycle. This makes it
possible to cut down the concentration of citrate produced in
excess, which is available for the synthesis of fatty acids.
According to the present invention, DNL can preferably be
inhibited by inhibiting the carboanhydrases which catalyze
the reactions yielding bicarbonate for the citric acid cycle.
For the purpose of the invention, it is possible to inhibit
preferably carboanhydrases of subtypes II and/or V and
particularly preferably CA V.
Compounds which are capable of inhibiting
carboanhydrases non-specifically (= non-specific or
conventional CA inhibitors) are known per se and have been
used for a relatively long time in various therapeutic
fields, mainly as diuretics or in ophthalmology. A survey of
fields of use of such conventional CA inhibitors can be found
e.g. in C.T. Supuran, Expert Opinion on Therapeutic Patents,
(5) (2000) 575 - 600. "Specific CA inhibitors", on the
other hand, is to be understood to mean compounds which
largely inhibit only one CA subtype (e. g. CA V) or a defined
group of CA subtypes. The use of conventional CA inhibitors
for the specific treatment and/or prophylaxis of obesity is
not known.
It is generally accepted that the overwhelming majority
of known cases of obesity are attributable to the
comparatively excessive proportion of exogenous fats in the
food intake. In highly developed countries like the USA, up
to 30s of the food consumed by obese people is in the form of
CA 02416647 2003-O1-20
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fats. According to current knowledge, fatty acid deposits
leading to obesity accordingly originate predominantly from
an excessive intake of alimentary fats. These cannot be
influenced by the inhibition of DNL, which targets
carbohydrate metabolism.
Thus, although a reduction in the storage of fatty acids
in adipocytes is also achieved by DNL inhibition, the latter
has hitherto been regarded as an unsuitable starting point
for the treatment and/or prophylaxis of obesity in man
because of its inherently small contribution to the storage
of fatty acids in body cells. A summary of the opinions
expressed by experts in this subject can be found for example
in M.K. Hellerstein, European Journal of Clinical Nutrition
53 (1) (1999) 53 -65. This view prevailing among experts has
so far stood in the way of the specific search for drugs for
the treatment and/or prophylaxis of obesity which are based
on the principle of DNL inhibition, or of their development.
Within the framework of the present invention, it has
now been found, surprisingly, that compounds which are
capable of inhibiting DNL in mammals, especially man, can be
used effectively for the treatment and/or prophylaxis of
obesity, particularly if these compounds are administered to
the patients in question over prolonged periods of e.g. more
than six weeks. Accordingly, over prolonged periods,
significant reductions in the body weight of obese persons
can be achieved by DNL inhibition even though DNL per se
makes only a relatively small contribution to the body fat
stored in adipocytes. Thus, by means of DNL inhibition over
prolonged periods, this inherently small effect can
accumulate in such a way that it makes a significant overall
contribution to the reduction in body weight.
Using the compound topiramate as an example, it can be
shown that the method according to the invention is indeed
suitable for specifically discovering compounds suitable for
CA 02416647 2003-O1-20
the treatment and/or prophylaxis of obesity. Topiramate is
an antiepileptic known from EP 0 138 441 A2. Topiramate is
also known to have a multifactorial pharmacological spectrum
of action. Thus topiramate can block the tension-dependent
sodium channels and hence stabilize the membrane potential of
cells, it activates the GABA receptors and thereby enhances
GABA-mediated inhibition, it acts as a carboanhydrase
inhibitor and, lastly, it is capable of inhibiting the
AMPA/kainate receptors, a subtype of the glutamate receptors,
thereby inhibiting the appearance of AMPA-induced flows.
Accordingly, topiramate exhibits pronounced effects on the
CNS. It is not known whether the antiepileptic properties of
topiramate are attributable to the pharmacologically relevant
factors mentioned above.
It is known from WO 98/00130 that topiramate also
possesses pharmacological properties which make it appear
suitable for the treatment of obesity. These properties were
discovered by chance as side effects in long-term studies on
epileptic patients. It is not yet known which
pharmacological properties of topiramate are responsible for
the weight loss observed in epileptic patients. Topiramate
is one of a group of anticonvulsant compounds of a general
formula I indicated in WO 98/00130 as suitable for the
treatment of obesity.
By the method according to the invention, it was now
possible to show that topiramate is a potent CA inhibitor,
especially a potent inhibitor of carboanhydrases of subtypes
II and V occurring in mammals, and that topiramate is capable
of effectively inhibiting DNL in mammalian cells. Thus the
method according to the invention made it possible for the
first time to prove that the hitherto inexplicable
pharmacological side effects of topiramate leading to weight
loss in epileptic patients are based substantially on its
ability effectively to inhibit DNL in mammals. It is
accordingly to be expected that the method according to the
CA 02416647 2003-O1-20
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invention will make it possible in the future to discover
compounds suitable for the treatment and/or prophylaxis of
obesity. The method according to the invention thus opens up
for the first time the possibility of discovering, with
comparative speed and ease, pharmacologically potent
compounds which act according to the principle of DNL
inhibition. By virtue of the present invention, compounds
acting according to the above-mentioned principle and
suitable for the treatment and/or prophylaxis of obesity can
now be selected, at least in a first preliminary selection
process, without lengthy and expensive in vivo tests such as
test-animal feeding experiments.
These results are surprising as earlier investigations
into the pharmacological properties of topiramate indicated
that its CA-inhibitory activity was always unexceptional or
even rather small and of no therapeutic importance (cf. e.g.
B.E. Maryanoff et al., Journal of Medicinal Chemistry 30
(1987) 880 - 887; B.E. Maryanoff et al., Journal of Medicinal
Chemistry 41 (1998) 1315 - 1343: S.J. Dodgson et al.,
Epilepsia 41 (1) (2000) p. 35 - p. 39). In the earlier
investigations mentioned above, the CA-inhibitory activity of
topiramate was always determined using test solutions
containing CA from different mammals, which still contained
various body constituents such as blood or organ tissue.
In a first embodiment of the method according to the
invention, compounds suitable for the treatment and/or
prophylaxis of obesity can be discovered by selecting as
suitable those compounds which are capable of inhibiting the
activity of at least one carboanhydrase occurring in mammals.
For example, at least one test compound can be brought into
contact with at least one carboanhydrase and those compounds
which inhibit the activity of at least one carboanhydrase can
then be identified in a manner known per se. In this
embodiment it is preferable to use carboanhydrases which
occur in mammals such as man or rodents, for example rats,
CA 02416647 2003-O1-20
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mice or guinea-pigs, especially carboanhydrases of subtypes
II and/or V. It is particularly preferable to use
carboanhydrases which occur in man. For example, suitable
carboanhydrases can be isolated from the above-mentioned
mammals and purified if desired, or they can preferably be
prepared by chemical or biotechnological methods known per
se.
In a preferred variant of this first embodiment, the
change in activity of the carboanhydrases under the influence
of the test compounds can be determined in an in vitro enzyme
activity test known per se, the carboanhydrases being present
as isolated enzymes which have been at least substantially
freed of impurities. It is preferable to use carboanhydrases
which have been prepared by chemical or biotechnological
methods as these can be used in especially pure form. In
vitro activity tests for determining the activity of
carboanhydrases are known per se. The in vitro enzyme
activity tests which are suitable within the scope of the
present invention to determine changes in the activity of
carboanhydrases are for example the measurement of the change
in pH value under the influence of carboanhydrases, (cf.
K. M. Wilbur, N. G. Andersen, Journal of Biological Chemistry
176 (1948) 147 - 154; G. Sanyal, T. H. Maren, Journal of
Biological Chemistry 256 (1981) 608 - 612), the stop-flow-
measurement method (cf. R. G. Khalifah, Journal of Biological
Chemistry 246 (1971) 2561 - 2573) or the
4-nitrophenylacetate-esterase method (cf. Y. Pocker,
J. T. Stone, Journal of the American Chemical Society 87
(1965) 5497 - 5498). In the latter test, the rate of
hydrolysis of 4-nitrophenylacetate under the influence of the
carboanhydrases to be investigated is determined, in which
the property of carboanhydrases also to act as esterases is
utilised.
According to the invention, a test method for
determination of the CA-inhibitory properties of compounds
CA 02416647 2003-O1-20
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which is described by C.T. Supuran et al., European Journal
of Medicinal Chemistry 33 (1998) 577 - 594 (cf. p. 592 in
particular; cited hereafter as "Supuran et al.") or by
A. Scozzafava et al., Journal of Medicinal Chemistry 42
(1999) 3690 - 3700 (cf. p. 3697 in particular; cited
hereafter as "Scozzafava et al.") is preferentially suitable
in this context. Within the framework of the disclosure of
the present invention, express reference is hereby made to
said test methods described by Supuran et al. and Scozzafava
et al. Compounds which have IC5p values of at least
umol/1 or below (= higher activity) in one of the above-
mentioned in vitro standard activity tests according to
Supuran et al. or Scozzafava et al. can be selected as
suitable CA-inhibitory compounds (= CA inhibitors) in terms
of the present invention. If the activity of human CA
subtypes is to be determined, methods other than the 4-
nitrophenylacetate-esterase method may be better suited. In
particular, methods which make it possible also to have
relatively rapid reactions may be suitable.
In an enzyme activity test (originally described by Y.
Pocker and J. T. Stone, Biochemistry 6 (1967) 668-678)
operating according to the 4-nitrophenylacetate-esterase
method described by Scozzafava et al. (vide supra) in terms
of the present invention it was demonstrated that topiramate
has a pronounced inhibitory action on human carboanhydrase of
subtype II (ICSp = 5 nmol/1) obtained by biotechnological
methods, and that this inhibitory action is considerably
stronger than the inhibitory action caused by the
conventional CA inhibitors acetazolamide or methazolamide
measured as reference substances. The human carboanhydrase of
subtype II was obtained by the method described by Scozzafava
et al.
In the same test, it was demonstrated that topiramate
also has a pronounced inhibitory action on carboanhydrase of
the subtype Va of mice (= mCA Va) which is obtained by
CA 02416647 2003-O1-20
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biotechnological processes (ICSo = 74 nmol/1). In a departure
from the test procedure described by Scozzafava et al, in
this case the enzyme mCA Va was used in a concentration of
120 nM. The mCA Va was obtained in known manner by the method
described by H.R. Heck et al., Journal of Biological
Chemistry 269 (1994) 24742 - 24746. To this end, the strain
of bacteria Escherichia coli BL 21 (DE3) was used, which was
used with a plasmid vector which contained the sequence
coding for mCA Va under the control of the T7 promoter which
can be induced by isopropyl-a-D-thiogalactopyranoside (IPTG).
The bacterial culture was inoculated at 37°C with stirring
into a Luria-Bertani liquid medium containing ampicillin
(100 ug/ml) and its growth was monitored by spectrophotometry
at 600 nm. Once the bacterial culture had entered the
exponential growth phase, IPTG was added in a final
concentration of 1 mmol/1. After 3 hours' incubation time
(37°C with stirring), the bacterial culture was centrifuged
at 7000 x g for 15 min and the supernatant was discarded. The
resulting pellet was taken up in 0.1 vol twice-distilled
water, and lysozyme (100 ug/ml) was added thereto. The cell
lysis took place under ultrasound treatment. To this end,
aliquotes of 10 ml of the resulting bacterial suspension were
poured into a glass vessel which was open at the top, and
each of these samples was treated with ultrasound 4 times for
3 min. After each ultrasound pulse, the absorption of the
samples was determined at 600 nm, for which each 100 u1 of a
sample was diluted with 900 u1 twice-distilled water. The end
point was reached once the value of the 600 nm absorption of
a sample was about 1/10 of the initial value. Once cell lysis
had taken place, CaCl2-binding buffer (Stratagene) was added,
and the resulting cell lysate was poured on to a calmodulin
Affinity resin column for purification. The purification is
based on the high affinity of the calmodulin domain which is
bound to the resin to the calmodulin binding peptide tag
which is present at the N-terminal end of the expressed mCA
Va protein. The purification was effected in known manner
CA 02416647 2003-O1-20
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(cf. manual "Affinity LIC Cloning and Protein Purification
Kit Manual" from Stratagene).
In a second embodiment of the method according to the
invention, compounds suitable for the treatment and/or
prophylaxis of obesity can be discovered by selecting those
compounds which are capable of reducing the amount of
metabolic products formed in the citric acid cycle of
isolated living mammalian cells or of lipidic subsequent
products of the citric acid cycle. Suitable metabolic
products of the citric acid cycle whose amount is measurably
reduced under the influence of the test compounds are acid-
soluble metabolic products such as citrate, maleate, fumarate
and/or a-ketoglutarate. Citrate is preferred. Furthermore,
lipidic subsequent products of the citric acid cycle, such as
free fatty acids, are suitable as metabolic products. Lastly,
in this second embodiment, the ability of the test compounds
to inhibit the activity of at least one carboanhydrase
occurring in mammals is also determined, although the test
model used is set up differently from the first embodiment
mentioned. The process of the second embodiment is based on
the principle of determining in known manner the uptake of
radioactivity from substrates of the citric acid cycle
labelled with the 14C-isotope into metabolic intermediate
products or subsequent products of the citric acid cycle of
isolated, living mammalian cells and comparing the results
obtained with the results which were obtained under otherwise
identical conditions, but under the influence of CA-
inhibitory compounds. Isolated living cells of rodents such
as rats, mice or guinea-pigs, or of man, are preferably used
in this variant of the method. Human cells are preferred.
If cells of rodents are used, these can be adipocytes or
hepatocytes. Adipocytes of rodents are preferred. If human
cells are used, adipocytes or hepatocytes may be used. Human
hepatocytes are preferred. The mammalian cells used in this
variant of the method can be obtained by conventional culture
and/or cloning processes. Natural or biotechnologically
CA 02416647 2003-O1-20
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modified mammalian cells can be used. If the uptake of
radioactivity in acid-soluble intermediate products of the
citrate cycle is to be determined, preferably 14C-hydrogen
carbonate (= NaH[14C)03) is used as the 14C-labelled
substrate. If the uptake of radioactivity in lipidic
subsequent products of the citrate cycle is to be determined,
preferably [U-14C]glucose is used as the 14C-labelled
substrate.
A preferred variant of this second embodiment for
determining the ability of compounds to inhibit DNL in
mammals is indicated by S.A. Hazen et al. in FASEB Journal 10
(4) (1996) 481 - 490 (cited hereafter as "Hazen et al.").
Within the framework of the disclosure of the present
invention, express reference is hereby made to said test
method described by Hazen et al. Compounds which, in said
test according to Hazen et al., significantly inhibit the
incorporation of radioactivity from 14C-labelled substances
which can serve as precursors in the citric acid cycle, for
example the incorporation of 14C-bicarbonate into citrate,
maleate, fumarate and/or a-ketoglutarate, with an IC5p value
of at least 10 umol/1 or below (= higher activity) can be
selected as suitable compounds in terms of the present
invention.
In a test model corresponding to that described by Hazen
et al., which was carried out within the framework of the
present invention, topiramate exhibited a pronounced
inhibitory effect on the formation of acid-soluble metabolic
products of the citric acid cycle of rat adipocytes of the
3T3-F442A cell line obtained by biotechnological methods.
This inhibitory effect of topiramate (ICSp = 348 nmol/1) was
markedly more pronounced than the effect of the conventional
CA inhibitor ethoxzolamide measured as a reference substance.
In a particularly preferred variant of the method
according~to the invention for discovering compounds suitable
CA 02416647 2003-O1-20
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for the treatment and/or prophylaxis of obesity, those
compounds are selected which, in the above-mentioned first
embodiment of the method, especially in an above-mentioned in
vitro enzyme activity test, have been selected as suitable
for inhibiting at least one carboanhydrase occurring in
mammals, and which additionally, in the above-mentioned
second embodiment of the method, have been selected as
suitable for reducing the amount of metabolic products formed
in the citric acid cycle of isolated living mammalian cells
as well. In the first embodiment of the method, the ability
of compounds to inhibit carboanhydrases occurring in mammals
can be checked rapidly and effectively. The second
embodiment of the method provides, inter alia, clues as to
whether the compounds investigated are also capable of
penetrating mitochondria of living mammalian cells where CA V
is located. Suitable compounds can be selected by carrying
out the first and second above-mentioned embodiments in
parallel or consecutively in either order.
According to the invention, compounds which are capable
of inhibiting DNL in mammals are suitable for the preparation
of drugs for the treatment and/or prophylaxis of obesity.
Compounds selected here are those which are capable of
inhibiting at least one carboanhydrase occurring in mammals.
The selected compounds must of course be physiologically
compatible and meet the demands generally made on
pharmaceutical active substances, e.g. regarding safety and
compatibility. It is preferable to use compounds which are
capable of inhibiting carboanhydrases of subtypes II and/or V
occurring in mammals. Particularly preferred compounds are
those which are capable specifically of inhibiting CA II
and/or CA V, especially CA V.
Previously known compounds with a CA-inhibitory effect
which cause a reduction in patients' body weight after
prolonged administration, for example topiramate, also have
pronounced effects directed towards the CNS, for instance
CA 02416647 2003-O1-20
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anticonvulsant effects. Side effects directed towards the
CNS are often undesirable for compounds which are intended
for long-term administration aimed at the treatment and/or
prophylaxis of obesity. Compounds which have been selected
by the above-mentioned method according to the invention as
suitable for the treatment and/or prophylaxis of obesity can
therefore additionally be tested for effects directed towards
the CNS. For example, the compounds can be tested for any
anticonvulsant properties which may be present. An example
of a suitable method of testing compounds for anticonvulsant
properties is the so-called "supramaximal electroshock test"
(= SES test, occasionally also referred to as the "maximal
electroshock test" or MES test) according to G. Chen et al.,
Proceedings of the Society for Experimental Biology and
Medicine 87 (1954) 334 - 339 (cited hereafter as "Chen et
al.") and J.E.P. Toman et al., Journal of Neurophysiology 9
(1946) 231 (cited hereafter as "Toman et al."). Within the
framework of the disclosure of the present invention, express
reference is hereby made to said test method described by
Chen et al. and Toman et al. Thus compounds which are aimed
at the treatment and/or prophylaxis of obesity by
administration to patients over prolonged periods should be
substantially ineffective in the above-mentioned SES test
according to Chen et al. and Toman et al., and should
preferably have no effects to be regarded as significant
according to the criteria conventionally applied to this
test, even in higher doses of at least 100 mg/kg p.o. (i.e.
protective dose, PDSp, according to G. Chen et al. X100 mg/kg
p.o.; the PD5p values given by G. Chen et al. substantially
correspond here to the more common indication of dose as the
minimum effective dose, MED). Compounds which have been
selected as suitable in the above-mentioned method of
discovering compounds, and which additionally are
substantially ineffective in the above-mentioned MES test
according to Chen et al., are particularly suitable for the
preparation of drugs for the treatment and/or prophylaxis of
obesity. Said SES (or MES) tests are standard
CA 02416647 2003-O1-20
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pharmacological tests and can be performed as routine methods
by appropriate service providers (e.g. "Panlabs").
The compounds discovered by the method according to the
invention as being suitable for the treatment and/or
prophylaxis of obesity can usually be contained as drugs with
conventional pharmaceutical auxiliary substances in galenic
formulations, e.g. tablets, capsules, suppositories or
solutions. These galenic formulations can be prepared by
methods known per se using conventional solid or liquid
excipients, e.g. lactose, starch or talcum or liquid
paraffins, and/or using conventional pharmaceutical auxiliary
substances, for example tablet disintegrants, solubilizers or
preservatives. Pharmaceutical preparations suitable according
to the invention are for example also known from
EP 0 138 441 A2 and from WO 98/00130.
