DE2052131A1 - preparation - Google Patents

Description

Dr. Ing. Α. γση derWerfh

Dr. Franz Lederer

PATENT Attorney I

2052131 23.0Rt.197Q

RAN 4121/10

F. Hofl & nann-La Roche & Co. Aktiengesellschaft, Basel / Switzerland

preparation

The present invention relates to a pharmaceutical preparation which is used for the inhibition of fatty acid synthesis in biological systems is suitable, and a method for its production.

The stereoisomers of hydroxycitric acid and its derivatives are structurally related to citric acid in which one of the four methylene hydrogen atoms of citric acid is replaced by a hydroxyl group. There are therefore four possible stereoisomers of hydroxycitric acid. It has now been found that one of these four stereoisomers, namely (-) - hydroxycitric acid, hereinafter referred to as garcinic acid, and its derivatives, considerably inhibit fatty acid synthesis in biological systems. Garcinic acid can be obtained by isolating it from the fruit of Garcinia cambogia using known methods. Isolation can be carried out, for example, according to the method described by Lewis in “Methods in Enzymology” Vol. 1J>, page 61-5

ah / k.) ο.γα 109819/2067 bad ORIGINAL

«O _M

(American Press, New York, 19Ö9) will.

The pharmaceutical preparation according to the invention is characterized in that it is garcinic acid or a derivative contains thereof together with a pharmaceutically acceptable carrier material.

The inventive method for the production of the preparation consists in that one Garciniasäure or a Derivative thereof using solid or liquid carriers in one suitable for medical administration Brings shape.

Garcinic acid is usually in the form of its lactone isolated. The free acid can be obtained from the lactone by basic hydrolysis, for example by means of sodium hydroxide or Potassium hydroxide, preferably with heating, and subsequent acidification can be obtained in a manner known per se.

The term "derivatives" used in connection with garcinic acid also includes the lactone of garcinic acid Derivatives of one or more carboxylic acid functions of garcinic acid, For example, mono-, di- or tri-esters of garcinic acid or mono- or diesters of lactone of garcinic acid, as well as non-toxic, pharmaceutically acceptable basic salts of garcinic acid, or of lactone or an ester of that.

Ester derivatives which can be used in the context of the present invention of garcinic acid are, for example, lower-alkyl esters, Aryl esters and aryl lower alkyl esters. Under lower ones Alkyl esters are to be understood as meaning esters whose alkyl group is straight-chain or branched and preferably contains 1-7 carbon atoms. Preferred lower alkyl esters of garcinic acid

109819/2067

are the methyl ester, the ethyl ester, the isopropyl ester. and the butyl ester. Examples of aryl esters are the phenyl ester and substituted phenyl ester, where the phenyl radical with halogen, lower-alkyl, lower-alkoxy or nitro can be substituted. The benzyl ester is a preferred aralkyl ester The esters mentioned can be obtained by esterifying garcinic acid with the alcohol in question in the presence of excess mineral acid, for example sulfuric acid, bromine water acid, or the like. Obtained. Suitable alcohols are, for example, lower alkanols, phenol and benzyl alcohol. The esterification can be carried out by methods known per se. Alkyl and aralkyl esters can also by Reaction with diazoalkylenes, for example with diazomethane, diazoethane or phenyldiazomethane, in a manner known per se getting produced.

Garcinic acid can also be used in the form of its pharmaceutically acceptable non-toxic basic salts will. The alkali metal salts, for example the sodium or potassium salt, are preferably used for this purpose Alkaline earth metal salts, for example the calcium salt, or complex salts such as for example ammonium or substituted Ammonium salts, such as the mono-, di- or tri-alkylammonium salt, or a mono-, di- or tri-hydroxyalkylammonium salt is used will.

It is believed that the inhibition of fatty acid synthesis in biological systems by garcinic acid and its derivatives from the inhibition of the citrate cleavage enzyme contained in such systems resulting from these connections. The citrate cleavage is carried out by the citrate cleavage enzyme according to catalyzed by the following formula.

Citrate + CoA (coenzyme Λ) + ATP (adenosine tr! Phosphate) - » Acetyl-CoA + oxaloacetate + ADP (adenosine diphosphate) + P.

109819/2067

In the conversion of carbohydrates and various amino acids into fats in non-ruminating mammals and in humans, the citrate is the main source of the acetyl group of acetyl-coenzyme A, which is used for fatty acid synthesis. The citrate is formed in the mitochondria by the citrate synthesis reaction. It is then metabolized via the citric acid cycle. If the energy consumption exceeds the energy requirement, part of the citrate is directed into the cell space outside the mitochondria and used there for fatty acid synthesis, ie for energy storage.

Garcinic acid and its derivatives can be used to treat obesity and to correct abnormalities in lipid metabolism be used. For this purpose, these compounds can be converted into pharmaceuticals in a customary manner Usable forms are brought by mixing with pharmaceutical, organic or inorganic inert substances suitable for enteral or parenteral administration Carrier materials such as water, gelatin, lactose, starch, magnesium stearate, talc, vegetable oils, polyalkylene glycol, Vaseline etc. The pharmaceutical preparations can be in solid form e.g. as tablets, coated tablets, suppositories, Capsules, or in liquid form, e.g. as suspensions or emulsions. If necessary, they are sterilized and / or or contain auxiliaries such as preservatives, stabilizers, Wetting or emulsifying agents, salts to change the osmotic pressure or buffers. You can have others too Contain therapeutically valuable substances.

A suitable pharmaceutical use form can be from about 15 to about 600 mg of garcinic acid per dosage unit or contain a derivative thereof. Suitable parenteral dosages in mammals and humans are about 1 mg / kg to about 25 rng / kg per day. The specific dosage

109819/2067 bad ORIGINAL

however, should be done according to the respective requirements.

Except in mammals, especially non-ruminants In mammals and humans, fatty acid synthesis is inhibited by garcinic acid or by a Derivative thereof in other biological systems, for example in cell-free enzyme preparations which use the citrate cleavage enzyme (which is also referred to as ATP: citrate oxaloacetate lyase), citrate, coenzyme A, ATP (or systems, which Produce ATP), TPNH (or systems that produce TPNH), and in tissue homogenates, tissue sections and perfused Organs.

example 1

Citrate-cleaving enzyme was isolated from rat liver; the rats starved for two days beforehand and then became fed on a high-glucose diet for three days. The enzyme was prepared according to the method of Inoue et al., J. Biochem. (Japan) 60, 5 ^ 3 (1966). The individual steps this process include ammonium sulfate precipitation a 0-30 ^ saturation and a DEAE column chromatography Separation.

The enzyme activity was measured in the following manner. The reaction mixture contained 20 mM citrate, 20 mM magnesium chloride, 70 mM Tris-HCl buffer (pH 8.0), 200 mM hydroxylamine, 10 mM dithiothreitol, 10 mM adenosine triphosphate (ATP), 0.6 mM Coenzyme A (CoA) and citrate-splitting enzyme. The final volume was 1.0 ml and the temperature 37 ° C. The was started Reaction by the addition of ATP, it was stopped after 20 minutes and the hydroxamate color was v / urde, as in Inoue et al. (quoted above) Written, developed. The formation of hydroxamate ran linearly with time for at least ^ O minutes.

109819/2067

The above method of determination has been used for a number of tricarboxylic acids. These acids were found in the The amounts given in Table I were added to the respective reaction mixture. The reaction mixture also contained 65 micrograms of protein. For the calculation of the educated For the amount of hydroxamate, a millimolar extinction coefficient of 0.8 at 520 millimicrons was used.

Table I.

Citrate concentration in mM Added substance 0 1 10

mM Entangende
Rydroxamate (mjuMol / mg / fain .) No 25 mM 0 210 405 Homocitrate, 25 25 mM 6th 52 310 Homoisocitrate, 1 mm 12th 190 401 Homoaconitat, 12th 198 385 Garcinic acid, 0 2 101

Table I shows that garcinic acid is a powerful inhibitor of the citrate-cleaving enzyme, which is what it does the effect that in the presence of this substance less hydroxamate is formed. The other citric acid analogs tested produce significantly less inhibition, despite being 25-fold larger amount than the garcinic acid were used.

Example 2

This example shows the stereospecific nature of the inhibition of the citric acid-cleaving enzyme by the Garcinic acid. In this experiment the method described in Example 1 was used, with the exception that Garcinic acid and the stereoisomeric (+) - allo-hydroxycitronic acid in the amounts as shown in the table below.

109819/2067

are given, have been added.

Table II

Citrate concentration (in mM)

Added substance 0.5 10

split citrate (ΐημΜοΐ / min.)

165

144 96

162 140

No 69 Garcinic acid 10 μΜ 25th 100 μΜ 6th (+) - allohydroxy citric acid 100 μΜ 68 1000 μΜ 33

From the results summarized in Table II it can be seen that garcinic acid is a considerably stronger: The citrate-cleaving enzyme inhibitor is structurally related Stereoisomeric (+) - allo-hydroxycitric acid.

Example 3

This example shows the inhibition of fat formation (lipogenesis), which is achieved by treatment with garcinic acid in isolated rat liver sections. In these experiments, female Charles River rats were left with one Weight 150-175 6 starve for two days, followed by three days with a diet consisting of TOJo Dextrose, 25 / & Casein, $ 5 Phillips and hard salt mixture were fed ad libitum.

The rats were decapitated, their livers quickly excised, immediately for 30 seconds Ice was placed and pieces weighing

109819/2067

100-150 mg cut using a modified Staty Riggs tissue cutting device. The sections (apart from the serous sections, which were discarded) were placed in 50 ml beakers standing in ice, which contained a Hanks solution (10 times the amount of dissolved tissue) in 5 mM Tris buffer pH 7.4-7, 6 contained a fatty acid precursor in the form of alanine together with the transaminase acceptor α-ketoglutaric acid in a molar ratio of 2: 3 at a pH of 7 Λ-Ί.6 . Garcinic acid was also added either in the form of the lactone or in the form of the free acid, in the concentration given in the tables below. The incubation was carried out at a temperature of 37 ° C. and under a 100% oxygen atmosphere in an Eberbach water bath shaking device, in each case for 60 minutes, unless otherwise stated. The incubation period began with the addition of

C-alanine (specific activity = 107.2 mc / mM) until a final concentration of 10 μο / g tissue was reached. Before adding C-alanine, ¹¹ O "time control samples were mixed with 2 ml of a 5N sodium hydroxide solution and corresponding corrections were made to the preliminary test samples.

The sections were taken out with tweezers, cooled directly on ice, placed in 10 ml homogenizing tubes made of glass containing 2 ml of water and homogenized with 5 strokes of a Teflon pestle. The homogenates were introduced into tubes which contained 2 ml of a 5N sodium hydroxide solution, and were saponified ^{there at 90 ° C. for 3 hours.} The samples were then acidified with 2.5 ml of 5N sulfuric acid and extracted twice with 5 ml of petroleum ether (boiling point 40 ° -60 ° C.) each time. The supernatant liquid was poured directly into glass counting vials, evaporated to dryness and mixed with 10 ml of a toluene-PPO-POPOP scintillation liquid. The samples were checked for radioactivity in a Packard Tri-Carb scintillation counter. the

109819/2067

Results were in ΐημΜ incorporated C-alanine / gram of tissue / 60 minutes printed out.

In the first group of experiments, liver sections were incubated for 60 minutes with 10 mM C-alanine and 15 mM oc-ketoglutarate. Table III shows the lipogenesis inhibition by garcinic acid under these conditions.

Tabel additive III ^ Inhibition sample control
5 μΜ garcinic acid *
50 μΜ garcinic acid *
500 μΜ garcinic acid * m / z mol / g / 60 ¹ 5.0
29.0
51.7 1
2
3
4th 167.5
159.2
118.9
80.9

* neutralized to pH 7.4

Table IV shows the fatty acid inhibition by

Garcinoic acid and garcinoic acid lactone in liver slices,

14th
which for 60 minutes with 5 mM C-alanine and 7.5 mM oc-keto

glutaric acid were incubated. These data show that the physiological apparent inhibition constant (K.) of

Garciniasäure 350 μΜ and the K. of Garciniasaurelacton
30 μΜ.

Table IV

Sample addition n ^ mol / g / ÖO * % inhibition

1 control 144.1

2 4 μΜ garcinic acid * 128.8 10.6

3 50 μΜ garcinic acid * 109.9 23.7

4,500 μΜ garcinic acid * 52.0 63.9

5 5000 μΜ garcinic acid * 76.5 46.9

6 5 μΜ garcinias aurelactone 89.0 38.2

7 50 μΜ garcinias aurelactone 57.9 59.8

8 500 µΜ garcinias aurelactone 62.7 56.5

9 5OOO μΜ Garciniasaurelacton 39i5 72.6

109819/2067

* neutralized to pH 7.4.

Table V shows the rapid time kinetics of the inhibition by Garcinic acid under the same experimental conditions as in Table IV.

Table V

Time (in ιημΜοί / % sample addition minutes) g / t inhibition

1 control 60 110.3 24, 1 2 500 μΜ garcinic acid * 60 83.7 3 control 90 239.6 42, 6th 4th 500 μΜ garcinic acid * 90 137.4 5 control 120 737.4 66 3 6th 500 μΜ garcinic acid * 120 248.1

* neutralized to pH 7.4

Example 4

This example demonstrates the in vivo activity of synthetic garcinic acid and its lactone. Individual groups of female Charles River rats weighing 150-175 ß were fasted for two days and then fed from 9-12 o'clock in the morning with a putter consisting of $ 70 dextrose, 2.% casein and 5% Philipps - and hard salt mixture

On the last day of putter administration, about 5 hours After feeding, the animals were treated with 2,2-dichloro-l, ldifluoroethyl methyl ether lightly anesthetized and the following mixture was injected into the tail vein: 12.6 mg of alanine as a fatty acid precursor, 30.6 mg of α-ketoglutarate

14th

as transaminase acceptor and 5 μο C-alanine (specific Activity = 156 mc / mM) dissolved in a total volume of 0.25 ml saline solution with a pH of 7.4-7.6.

'109819/2067

The rats were beheaded 30 minutes later, their livers quickly excised, weighed, in 30 ml Beakers with 15 ml of water are introduced and roughly cut. The liver samples were then placed in glass homogenization tubes introduced and homogenized by 5 blows with a Teflon pestle. There were 3 ml of each of these Liver homogenates placed in tubes containing 2.1 ml a 5N sodium hydroxide solution, and with 2.6 ml of a 5N Sulfuric acid saponified and twice with 5 ml petroleum ether each time (Boiling point 4o-6o ° C) extracted. The supernatant liquid was introduced directly into glass viscous vials for Evaporated dry and with 10 ml of a toluene-PPO-POPOP Scintillation fluid added. The samples were in a Packard Tri-Carb scintillation counter for their absolute Activity examined. The results obtained were in ΐημΜοΙ C-alanine introduced / gram of tissue / 30 minutes expressed.

Fourteen rats were fasted for two days and then fed as indicated above for three days. On the last day of feeding, a group of rats received 10 mg of garcinic acid in the form of lactone, dissolved in a total volume of 0.25 nil saline solution with a pH of 7.4-7.6. The lactone was administered by injection into the tail vein 60 minutes prior to the administration of the ¹ C-alanine. An additional amount of 5 mg of Garcinic Acid Lactone was added

14th

administered together with the C-alanine. The control animals

14th

received the C-alanine injection only. The one found Inhibition of lipogenesis by the Garciniasaureleton is shown in Table VI, with those with the numbers 1-7 rats designated were control animals, while the rats designated 8-14 were administered garcinic acid lactone became.

109819/2067 _ba d

Table VI mmol / g / 30 ' 218.1 Rat No. (controls) 445.0 ' 1 187.3 2 228.7 3 388.8 4th 180.7 5 729.7, 6th 339.8 -75.7 7th Average 83.5 63.4 8 (garcinic acid lactone) 104.8 9 34.9 10 65.4 11 49.2 12th 66.5 13th 66.8-8.5 14th Average Escapement = $ 80

The other group of rats was fed each morning for ten days from 9-12 pm with Purina laboratory chow, after which the animals munched two days fasting Hess and then for 5 days with a putter high dextrose.

On the last day of putter administration, four rats received 10 rr.g Garcinic acid lactone 60 minutes before the C-alanine administration and an additional amount of 4.6 mg of garcinic acid lactone together

14th
with the C-alanine injection.

Table VII shows the inhibition in the animals treated with garcinic acid lactone 5-8 compared to FIG the control animals 1-4.

109819/2067

Table VII _{m (iMol / g / 30} , Rat. No. (Control) 1018.9
1554.4
836.2
782.4 1
2
3
4th 1048-176.3
386.1
249.1
290.9
257.8 5 (garcinic acid lactone)
6th
7th
8th Average 296-31.4 Inhibition = 12% Average
Example 5

A solution of (+) - garcinia lactone (10 g) in tetrahydrofuran (100 ml) is treated with a solution of diazomethane in ether until the yellow color persists. The solution is then left to stand for one hour at room temperature and the solvent is then evaporated off under reduced pressure and the residue is crystallized from a mixture of ether and hexane, 8 g of (-t -) - Garciniasäurelacton-dimethylester with a melting point of 70 -72 ⁰ C. Another 2.4 g of this substance, having a melting point of 65-70 ⁰ C, obtained from the mother liquors. Crystallization from ether gives an analytical sample with a melting point of 72-73 ° C; [a) ^ ^J = + 85.65 ° (c = 1.0; CHCl3); IR (CHCl,) 3550, 1810 and 1755 cm " ¹ , NMR (CDCl-) 4.91 (singlet, IH, CH), 4.14 (singlet, IH, OH), 4.91 (singlet, 3H, - OCH ₃ ), 3.78 (singlet, 3H, -OCH) and 2.99 (quartet, 2H, CH ₀ ).

Analysis calculated for C ₀ H ₁ -O ₇ : C = 44.04; H *. X 4.62; 10981 9/2067

found: C = 44.35; H = 4.87-

Example 6

10 ml of dry, liquid ammonia are mixed with 1 g of (+) - garcinia lactone, whereupon the mixture is stirred for a long time until the solid has dissolved. The ammonia is then allowed to evaporate, the residue is dissolved in 5 ml of water and the solution is passed through a cation exchange column filled with 10 ml of Amberlit IR 120. The acidic liquid which has passed through is evaporated to dryness, a colorless solid being obtained. Crystallization from a mixture of methanol and ethanol gives 800 mg of (+) - Garciniasäurelacton-monoammonium salt with a melting point of 2 ^ 1 ° (decomposition); [aJ ^ + 92.9 ° (c = 1.0; H ₂ O); IR (KBr) 3460-2500 (broad), 1800, 1770 and 1β25 (broad) cm ^"1; NMR (DMSO) 2.71 (quartet, 2H, CH ₃₎ and 4,6θ (singlet, IH, CH).

Neutralization equivalent: 207

Analysis: Calculated for C ₆ H ₉ NO ₇ : C = 34.79; H = 4.38; N = 6.76 Found: C = 34.65; H = 4.48; N = 6.72.

Example 7

3 ml of acetyl chloride are added to 50 ml of absolute ethanol and after a few minutes 5 g of (+) - garcinic acid lactone, after which the solution is refluxed for three hours. That Solvent is then evaporated off under reduced pressure and 7 * 5 g of a pale yellow oil are obtained, which one Purified by distillation under reduced pressure, 5.2 g of a colorless viscous liquid having a boiling point obtained from 136-140 ° / 0.15 mm; IR (CHCI3) - 36ΟΟ, 18IO and 1745 cm ". The NMR analysis indicates that a Geir.iscn of 2 parts (+ J-Garciniasäurelacton-Diethylester and 1 part Triethyl garcinate is present. The triester is converted into the diester by repeated distillation and you can

109819/2067

BATH ORIGINAL

in this way pure diester lactone is obtained.

Example 8

Capsules with the following content are produced in the usual way:

per capsule

Garcinic Acid Lactone Lactose Sugar Corn Starch Talc

Total weight 210 mg

Example 9

Capsules with the following content are produced in the usual way.

10 mg 165 mg 30th mg 5 mg

Per capsule mg 50 mg 125 mg 30th mg 5

Garcinic Acid Lactone Milk Sugar Corn Starch Talk

Total weight 210 mg

Example 10

Standard white tablets are used to produce the following composition:

109819/2067

) ro tablet

Garciniasaurelacton 25 * 00 mg

Dicalcium phosphate dihydrate 175 »00 mg

Corn starch 24.00 mg

Magnesium stearate 1.00 mg

Total weight 225.00 mg

Example 11

The following tablets are prepared in the usual way
Composition ago:

Garciniasaurelacton lactose corn starch Prehydrolyzed corn starch calcium stearate

Total weight 410 mg

per tablet mg 100 mg 202 mg 80 mg 20th mg 8th

1 0981 9/2067

Claims (10)

Claims 1. A process for the production of a pharmaceutical preparation, characterized in that one garcinic acid or a derivative thereof using solid or liquid carriers in one for medical administration brings suitable shape. 2. The method according to claim 1, characterized in that that as a derivative of garcinic acid, the lactone of this Acid used. >. Method according to claim 1, characterized in that that an ester of this acid is used as a derivative of arcinic acid. 4. The method according to claim 3 *, characterized in that that a lower alkyl ester is used, 5. The method according to any one of claims 1-4, characterized characterized in that one has about 15 to about 600 mg of the active substance used per dosage unit. 10981'3 / 206? 6. Pharmaceutical preparation, characterized in that that it contains garcinic acid or a derivative thereof together with a pharmaceutically acceptable carrier material. 7. Preparation according to claim 6, characterized in that that the derivative of garcinic acid is its lactone. 8. Preparation according to claim 6, characterized in that the derivative of garcinic acid is an ester of this acid, 9. Preparation according to claim 8, characterized in that the ester is a lower alkyl ester. 10. Preparation according to one of claims 6-9, characterized in that it contains about 15 to about 600 mg of active substance per dosage unit contains. 109819/2067