SK612003A3 - Method for locating compounds which are suitable for the treatment and/or prophylaxis of obesity - Google Patents

Abstract

The invention relates to a method for locating compounds which are suitable for the treatment and/or prophylaxis of obesity, whereby the ability of the examined compounds to inhibit de novo lipogenase in mammals and/or humans is determined. Also disclosed is the use of compounds which are capable of inhibiting de novo lipogenase in mammals in the production of medicaments for the treatment and/or phophylaxis of obesity.

Description

A method for determining suitable compounds for the treatment or prevention of obesity

Technical field;

The invention relates to a method for determining suitable compounds for the treatment or prevention of obesity. In particular, the invention relates to the use of compounds which have the ability to inhibit de-lipo-lipogenesis in mammals and which do not substantially affect the central nervous system (Zentrales Nervensystem - ZNS) for the preparation of medicaments for the treatment or prevention of obesity.

BACKGROUND OF THE INVENTION

Obesity is currently becoming a problem for the health of the population, especially in developed countries, as a result of an unbalanced and fat-rich diet. As the proportion of the population with excessive body weight increases, so do the consequences of obesity, ranging from personal dissatisfaction to heart disease and circulation and / or certain forms of diabetes. There are already some treatments for treating or preventing obesity. An example of such a procedure is the use of lipase suppressants that reduce fat breakdown in the digestive tract and in this way also the energy effect of the ingested food. In this therapy, the fats are at least partially non-degradable and are excreted. Additional means for treating and preventing obesity need to be developed to complement existing therapies.

SUMMARY OF THE INVENTION

It has been found that compounds that can inhibit de novo-lipogenesis in mammals, and especially in humans, are useful for the effective treatment or prevention of obesity. Good results are obtained when these compounds are administered over a prolonged period of time, for example for several weeks.

It is an object of the present invention to provide compounds which are useful in the treatment or prevention of obesity, wherein such compounds are capable of inhibiting de-lipo-lipogenesis in mammals, especially humans. It is also an object of the present invention to use compounds having the ability to inhibit de-lipo-lipogenesis in mammals and which have substantially no effect on the central nervous system, such as anticonvulsant action, in the preparation of medicaments for treating or preventing obesity.

The term de-novo-lipogenesis (hereinafter abbreviated as DNL) means the synthesis of the body's own fatty acids from carbohydrates in a mammalian organism. This synthesis occurs in the cytosol of body cells, and is based on the so-called. the citric acid cycle or the Krebs-Martio cycle. In a biochemical reaction taking place in the body from both essential constituents, which are carbohydrate-derived pyruvate, and bicarbonate, via various intermediate steps such as e.g. maleate, fumarate and α-ketoglutarate, eventually citrate is formed. If an excess of citrate is synthesized, this excess can be converted to the fatty acids (= lipidic downstream products) that form fats through the intermediate stage, which is acetyl coenzyme A, and these fats are stored in adipocytes. Inadequate deposition of fatty acid fats generally leads to obesity.

Various enzymes are involved in the citric acid cycle.

The metabolism taking place in the citrate cycle depends essentially on the amount of bicarbonate available.

The amount of bicarbonate available depends on the rate at which the oxide can form bicarbonate, by carboanhydrases. On the catalysis of bicarbonate for the cycle of known carbonic anhydrases and their carbon dioxide. This reaction, which catalyzes the so-called.

The carboanhydrase isozymes of subtypes II (= CA II) and V present in mitochondria are predominantly involved in the reactions provided by citric acid among mammalian isozymes. In mitochondria (= CA V). Carboanhydrases of subtype V are important because they are also in the citric acid cycle.

To suppress DNLs in mammalian cells, they are intended to be suppressed in all cases in the citric acid cycle.

the concentration available to the invention may vary.

metabolism,

This can be reduced by taking for the citric acid cycle. advantageous suppression of carbonic anhydrase carbonic anhydrase CA V.

which may non-specifically suppress carboanhydrases supplying

For the purposes of subtype II, excess citrate produced is for the synthesis of fatty acids. Accordingly, the production of DNL is preferably suppressed by suppressing the carbonic anhydrases that catalyze the reaction of the bicarbonate of the invention is and / or V, preferably

Compounds (= non-specific or classical CA-inhibitors) used for a long time as medicaments, especially as in ophthalmology. An overview of the use of these are known and diuretics, classic ones are already or

CA-inhibitors disclose e.g. C. T. Supuran in Expert Opinion on Therapeutic Patents, 10 (5), 575-600, (2000). The phrase "specific CA-inhibitors" refers to those compounds that predominantly suppress only one CA subtype (e.g., CA V subtype) or a particular group of CA subtypes. The use of classical C'A-inhibitors for the targeted treatment and / or prevention of obesity is not yet known.

It is generally believed that most of the known cases of obesity are due to an excessive proportion of fat in foods. In developed countries such as the United States of America, fats account for up to 30% of obese people 's food intake. According to current knowledge, fat deposition and consequently obesity are predominantly due to fat intake in food, therefore the suppression of DNL affecting carbohydrate metabolism has no effect on this process.

Although suppression of DNL results in a reduction in fatty acid deposition in adiposytes, the contribution of DNL to this effect, reducing fatty acid deposition in body cells, is still considered insufficient for the treatment and / or prevention of human obesity. A summary of expert opinions on this issue can be found eg. in M. K. Hellerstein, European Journal of Clinical Nutrition 53 (1) 53-65, (1999). This view, prevailing in the professional world, has hitherto been in the way of the targeted search and development of drugs for the treatment and / or prevention of obesity based on the principle of DNL suppression.

It has been surprisingly found in the present invention that compounds having the ability to inhibit DNL in mammals, particularly humans, can be used more effectively for the treatment and / or prevention of obesity, particularly where these compounds can be used by patients for a prolonged period of time. , e.g. over a period of more than six weeks. These obese persons can achieve significant weight loss during this longer period by suppressing DNL, although DNL has only a relatively low effect on adipocyte body fat. By suppressing DNL over an extended period of time, this low impact can accumulate so that it can cause a significant weight loss overall.

As an example of Topiramate, it can be shown that the method according to the invention is indeed suitable for the targeted search for compounds suitable for the treatment and / or prevention of obesity. Topiramate is an antiepileptic drug described in EP 0 138 441 A2. . . Topiramate is known to have a multifactorial pharmacological action. Can block sodium channels voltage dependent _and thus stabilize the membrane potential of cells, activates GABAReceptory and thereby amplifies the inhibition caused by GABA, acts as a carbonic anhydrase inhibitor and finally is able to suppress the kainate / AMPA receptors, which are one type of glutamate receptors, thereby reducing occurrence of AMPA-induced currents. Topiramate also shows a pronounced effect on ZNS. It is not known whether the antiepileptic properties of Topiramate are related to the pharmacologically relevant factors mentioned above.

It is known from WO 98/00130 that Topiramate also has pharmacological properties which indicate that it could be useful in the treatment of obesity. These properties have been found to be concomitant in long-term trials of its therapeutic effects on epilepsy. It is not yet known which pharmacological properties of Topiramate cause a decrease in body weight of patients suffering from epilepsy. Topiramate belongs to the group of anticonvulsants for the treatment of obesity of the general formula I described in WO 98/00130.

It has now been shown that, using the method of the invention, Topiramate is a potent CA-inhibitor, is a potent inhibitor in particular of the carboanhydrases of subtypes II and V occurring in mammals, and that it effectively suppresses DNL in mammalian cells. According to the method of the present invention, it has been shown that the hitherto unexplained effects of weight loss in patients suffering from epilepsy are side effects of Topiramat, essentially consisting in its ability to effectively suppress DNL in mammals.

Accordingly, it is expected that by using the process of the present invention it will be possible in the future to determine compounds suitable for the treatment and / or prevention of obesity. The method according to the invention makes it possible for the first time to determine the pharmacologically interesting compounds relatively quickly and easily on the basis of their ability to inhibit DNL. Using the methods of the invention, on the basis of this principle, substances potentially suitable for the treatment and prevention of obesity can be selected by first guideline without having to carry out long and expensive in vivo experiments, such as those in which the effect of feeding on experimental animals is investigated.

This finding has produced surprising results since, in a previous investigation of pharmacological properties, its effect as a CA-inhibitor has always been assessed as insignificant or only minor and therapeutically as insignificant (see, e.g., BEMaryanoff et al., (Journal of Medicinal Chemistry 30, 880-). 887 (1987); BEMaryanoff et al., Journal of Medicinal Chemistry 41 1315-1343 (1998); SJDodgson et al., Epilepsy 41 (1), 35-39 (2000)). Inhibitory effect of Topiramate used always test solutions containing CA of different mammals, which additionally contained different body organs such as blood or different tissues.

According to a first embodiment of the invention, compounds suitable for the treatment and / or prevention of obesity may be found by selecting those compounds which have the ability to inhibit the activity of carbonic anhydrase in at least some mammals. Experimentally, at least one test compound may be contacted with a single carbonic anhydrase and subsequently those compounds that suppress the activity of at least one carbonic anhydrase can be identified in a known manner. Preferably, carboanhydrases of mammals such as humans or rodents are used in this embodiment, e.g. rats, mice or guinea pigs, in particular carbonic anhydrases of subtypes II and / or V. Human carbonic anhydrases are preferred. Suitable carbonic anhydrases may be e.g. from mammals isolated and optionally purified, or may conveniently be prepared by known chemical or biotechnological techniques.

In a preferred variation of this first embodiment, the change in carbonic anhydrase activity can be measured by the test compounds using a known in vitro enzymatic activity assay using isolated and at least largely purified carbonic anhydrase. Preferably, the carbonic anhydrases used are prepared by a chemical or biotechnological process, as these are available for use in an extremely pure form. Assays for determining the in vitro activity of carbonic anhydrases are known. In vitro in vitro enzymatic activity assays performed in the methods of the invention, in which the change in carbonic anhydrase activity is measured, e.g. using an assay to measure pH change due to carbonic anhydrase (see KM Wilbur, NGAndersen, Journal of Biological Chemistry 176 147-154 (1948); G.Sanyal, THMären, Journal of Biological Chemistry 256, 608-612 (1981) , the measurement is performed using the stop-flow method (see RGKhalifah, Journal of Biological Chemistry 246, 2561-2573 (1971)) or used

4-nitrophenyl acetate esterase method (see Y.Pocker, J.T. Stone, Journal of the American Chemical Society 87 5497-5498 (1965)). In the latter test, the rate of hydrolysis of 4-nitrophenyl acetate is measured by the carbonic anhydrase under test, utilizing the ability of the carbonic anhydrase to act simultaneously as esterases.

In this regard, the method of determining the CA-inhibitory effect of the compounds described in CTSupuran et al., European Journal of Medicinal Chemistry 33, 577-594 (1998) (see, in particular, page 592; cited as "Supuran et al.") or in A.Scozzafava et al., Journal of Medicinal Chemistry 42 3690-3700 (1999) (see especially p. 3697; hereinafter cited as "Scozzafava et al.). The description of the invention, which is f

the subject matter of the present invention explicitly relates to the above methods of determination according to Supuran and co-workers and according to Scozzafava and co-workers. Compounds which, according to one of these in vitro assays for Supuran and coworkers or Scozzafava and coworkers, exhibit IR ₅ values of 10 gmol / l or lower (i.e., have higher potency) may be selected as suitable compounds according to the invention. CA-inhibitory effects (= CA-inhibitors). If the activity of the human CA-subtypes is to be determined, other methods than the 4-nitrophenylacetate esterase-based method may be more appropriate. Particularly suitable may be methods which also make it possible to observe the relatively rapid reaction progress.

Assay for enzymatic activity by the method of Scozzafava et al. (see above), which is based on the reaction of 4-nitrophenylacetate esterase (originally described in Y.Pocker and JTStone, Biochemistry 6 668-678 (1967)), it has been shown in the present invention that Topiramat exhibits a significant inhibitory action on human carboanhydrase of subtype II, obtained by biotechnology (IC50 = 5 nmol / l), and that this inhibitory action is significantly more potent than that of the classical CA-inhibitors of acetazolamide or metazolamide used as comparators. II was obtained as described in Scozzofava et al.

By the same assay, Topiramate also showed a significant inhibitory effect on carboanhydrase of the mouse subtype Va (= mCA Va) (IC50 = 74 nmol / l) obtained by biotechnology. In this case, the enzyme mCA Va at a concentration of 120 nM was used differently from the protocol of Scozzafava et al. Carboanhydrase of mCA Va was obtained according to the method described by H. R. Heck et al., Journal of Biological Chemistry 269, 24742-24746, following a substantially known method. The BL 21 (DE3) strain and plasmid-coordinator of the mCA Va sequence were used, under the control of the isopropyl-β---thiogalactopyranoside-induced T7 promoter (IPTG). The bacterial culture was inoculated at 37 ° C with stirring in Luria-Bertani liquid medium containing ampicillin (100 µg / ml) and its growth was monitored spectrophotometrically at

600 nm.

When the bacterial culture entered the exponential growth phase, it was added

IPTG at a final concentration of 1 nmol / L.

After a three hour incubation period (37 ° C, with stirring), the bacterial culture was centrifuged at 7000 g for 15 minutes and the upper layer was removed. Redistilled water and lysozyme (1.00 µg / ml) were added to the obtained pellet. Cells were lysed by ultrasound by treating 10 ml of bacterial suspensions in ultra-sonicated containers, intermittently four times for 3 minutes. After each 3-minute ultrasound treatment, the absorption of the samples was determined at 600 nm, each time 100 μΐ of the sample was diluted with 900 μΐ of redistilled water. The final state was reached when the absorption value of the sample at 600 nm was about 1/10 of the initial value. After the cell digestion was complete, CaCl ₂ -binding buffer (manufactured by Stratagene) was added, and the cell lysate obtained was subjected to purification on a column packed with a calmodulin sorption resin. This purification is based on the high affinity of the resin-bound calmodulin domain in the terminal group capable of binding to that nitrogen-containing domain of the obtained mCA Va protein.

Purification is carried out in a manner known per se (see the Affinity LIC Cloning and Protein Purification Kit Manual of Stratagene).

According to a second embodiment of the method according to the invention, it is possible to find substances suitable for the treatment and / or prevention in that the citric acid-lowering products are selected such that the metabolism is selected in which they reduce the amount of mammalian acid cycles. citric acid. Acid-soluble metabolic products, such as citrate, maleate, fumarate and / or α /, are suitable as the metabolic products of the citric acid cycle or cycle, the amount of which is measurably reduced due to the test compounds ^. α-ketoglutarate, preferably citrate. Lipidic subsequent products of the citric acid cycle, such as free fatty acids, are further suitable as metabolic products. In a second embodiment, the ability of the compounds to inhibit the activity of at least one mammalian carbonic anhydrase is also examined; the test model used is only otherwise arranged as in the previous embodiment. The principle of this second method is essentially the known transfer of radioactivity from ¹⁴ C-isotope-labeled citric acid cycle substrates to metabolic intermediates or citric acid isolated on subsequent products of the acid cycle from living mammalian cells and comparing the observed conditions results but with those obtained in the presence of compounds. the same they have

CA-inhibitory effects.

isolated live or guinea pigs may be used in this embodiment. Preferably used rodent cells, hepatocytes. Preferably, human cells are used, rodent cells such as those used by human can be used can be successfully rat, mouse cells. When adipocytes or adipocytes are rodents.

these may be adipocytes or hepatocytes. The use of human hepatocytes is preferred.

The mammalian cells used in this embodiment are obtained by conventional methods of cell proliferation and cloning. Natural or biotechnologically modified mammalian cells may be used in the invention. When determining the transfer of radioactivity to acid-cycle intermediates which are a soluble substrate, preferably transfer of the environment is used as ¹⁴ C-lipidic bicarbonate of the subsequent products as ¹⁴ C-labeled substrate of preferably radioactivity into the citric acid cycle using [U- ¹⁴ C] glucose .

second

A preferred variant of the ability of the compounds to inhibit DNL in S.A. Hazen et al., FASEB for performing assays in mammals is disclosed.

Journal 10 (4) (1996) 481a et al. compounds

-490 (hereinafter referred to as "Hazen et al.") The disclosure of the present invention expressly relates to the above Hazen assay method, which, according to this assay of collaborator activities, significantly inhibits transmission

the labeled substances are precursors of the citric acid cycle, e.g. transfer from ¹⁴ C-bicarbonate to citrate, maleate, Hazen and ¹⁴ Cfumarate and / or α-keto-glutarate with an IS ₅ of at least 10 gmol / l or less (= higher efficacy) may be selected as suitable compounds within the meaning of the invention.

In the test model described by Hazen and co-workers of the present invention, Topiramate showed a significant ability to suppress the formation of soluble metabolic products in the acidic environment of the citric acid cycle by adipocytes of the rat 3T3-F442A cell line, obtained by biotechnology. This action of Topiramate (IC 50 = 348 nmol / L) was significantly more potent than that of the classical CA-inhibitor Etoxyolamide, which was measured as a reference. ^!

In a more preferred variation of the method of determining compounds that are useful in the treatment and / or prevention of obesity, those compounds which, in the above-mentioned embodiment of the invention, particularly in said in vitro enzymatic activity assay, have been shown to be suitable for suppressing at least one mammalian carboanhydrase are selected. and which have also been shown to be useful in reducing the i

the amount of metabolic products produced by isolated living mammalian cells in the citric acid cycle. In a first embodiment of the invention, the ability of the individual compounds to inhibit mammalian carboanhydrates can be rapidly and efficiently investigated. The second embodiment of the present invention provides, inter alia, whether the investigated compounds also have the ability to penetrate into the mitochondria of living mammalian cells containing CA V. The first and second embodiments of the present invention may be used to select suitable compounds or may be used in any order.

According to the invention, compounds having the ability to inhibit DNL in mammals are useful in the preparation of medicaments for the treatment and / or prevention of obesity. Compounds having the ability to inhibit at least one mammalian carboanhydrase are selected. The selected compounds must be physiologically tolerable and meet the general requirements for drugs, i. e.g. requirements relating to the safety and compatibility of medicinal products. Preference is given to the use of compounds which inhibit carboanhydrases of mammals of subtype II and / or V. More preferred is the use of compounds which specifically inhibit CA II and / or CA V, in particular CA V.

Compounds having CA-inhibitory action are known in the art which, when used for a long time, cause patients to lose weight, e.g. Topiramate also exhibits a pronounced action on the central nervous system, such as anticonvulsant action. Compounds that are to be used for a long time to treat and / or prevent obesity cannot have central nervous system side effects. Compounds which have been found to be useful in the treatment and / or prevention of obesity as described above may be examined for their action on the central nervous system. E.g. anticonvulsant action can be detected. To monitor anticonvulsant properties, e.g. suitable so-called. Supramaximal-Elektroschocktest (SES-Test), also called the "Maximal-Elektroschocktest (MES-Test), described in G.Chen et al., Proceedings of the Society for Experimental Biology and Medicine, 87 (1954) 334- 339 (hereinafter referred to as "Chen et al.") And JEPToman et al., Journal of Neurophysiology 9 (1946) 231 (hereinafter referred to as "Toman et al.). The description of the invention, which is the subject of this application, expressly relates to the above tests by Chen and co-workers and by Toman and co-workers. Compounds intended to be used for a long time to treat and / or prevent obesity should be shown to be substantially ineffective in the above-mentioned Chen and co-workers and Toman and co-workers tests, and preferably also at doses higher than at least 100 mg / kg po should not show any significant effects according to the usual criteria of this test (ie "Protective Dose PD ₅₀ by G. Chen et al.> 100 mg / kg po; with PD ₅₀ values according to G. Chen et al. Minimal Effective Dosis (MED). Compounds which have been determined to be suitable based on the above compound determination method and which have additionally been found to be substantially ineffective by Chen and co-workers according to the above MES-Test are particularly suitable for the preparation of medicaments for the treatment and / or prevention of obesity. The above-mentioned SES-Test (or MES-Test) are pharmacological standard tests that can be performed as routine tests by service providers in the relevant area (eg Panlabs).

Compounds which have been found to be useful in the treatment and / or prevention of obesity according to the invention may be formulated in various dosage forms, such as e.g. tablets, capsules, suppositories or solutions. These dosage forms can be prepared by known methods using conventional solid or liquid carriers such as milk sugar, starch or talc, optionally liquid paraffins and / or tablet release agents, solubility enhancers or preservatives. Dosage forms suitable for use in the present invention are described e.g. also in EP 0 138 441 A2 and in WO 98/00130.

Claims (17)

What is claimed is: 1. A method for the determination of compounds suitable for the treatment or prevention of obesity, which comprises selecting compounds which have the ability to suppress de novo-lipogenesis in mammals, particularly humans. The method according to claim 1, characterized in that those compounds having the ability to inhibit the activity of at least one carboanhydrase occurring in the mammalian organism are determined to be suitable. The method of claim 2, wherein the at least one compound is contacted with the at least one carbonic anhydrase and subsequently those compounds that suppress the activity of the at least one carbonic anhydrase are identified. Method according to claim 2, characterized in that carboanhydrases occurring in rodent organisms or in the human organism are used. 5th process according to claim 4, characterized by with and by that is use the carbonic anhydrases occurring the in human body. 6th process according to claim 2, characterized in with and by that is use carboanhydrases of subtype II and / or V, occurring in mammalian organisms, in particular in the human organism. The method according to claim 6, characterized in that carboanhydrases of subtype V occurring in mammalian organisms, in particular in the human organism, are used. The method according to claim 3, characterized in that the carbonic anhydrase is an enzyme obtained by a chemical or biotechnological process, and in that the change in activity of the carbonic anhydrase under the influence of said compounds is determined by an in vitro enzyme activity assay. The method according to claim 1, characterized in that those compounds having the ability to reduce the amount of metabolic products resulting from the citric acid cycle of isolated living mammalian cells are determined to be suitable. Method according to claim 9, characterized in that the reduction of citrate, maleate, fumarate, α-ketoglutarate and / or subsequent lipidic products resulting from the citric acid cycle is determined by the action of said compounds. The method of claim 9, wherein said isolated living cells are human or rodent cells. Method according to claim 11, characterized in that adipocytes or hepatocytes are used. Method for the determination of compounds suitable for the treatment and / or prevention of obesity, characterized in that compounds which have been identified as suitable for this purpose by the method of claim 8 and additionally by the method of claim 9 are determined. Method for the determination of compounds suitable for the treatment and / or prevention of obesity, characterized in that compounds which have been identified as suitable for this purpose by the process of claim 1 and which are also a standard test for. determination of anticonvulsant properties identified as essentially ineffective. Use of compounds which are capable of inhibiting de-novo-lipogenesis in mammals for the preparation of a medicament for the treatment and / or prevention of obesity. Use according to claim 15, characterized in that said compounds are compounds having the ability to inhibit at least one carboanhydrase found in mammalian organisms. Use according to claim 15 or 16, characterized in that said compounds are compounds which have the ability to inhibit carboanhydrases of subtypes II and / or V occurring in mammalian organisms. Use according to claim 15, characterized in that said compounds are compounds which have substantially no anticonvulsant properties. Use of the compounds which according to claim 1 have been identified as suitable for the treatment and / or prevention of obesity, for the preparation of a medicament for the treatment and / or prevention of obesity.