DE2517028A1 - METHOD OF ISOLATING GAMMA-L-GLUTAMYLTAURINE - Google Patents

Description

April 16, 1975

Chinoin GySgyszer es Vegyeszeti Termekek Gyara EiD Budapest IV, Τδ utca 1 - 5, Hungary

VJBRVAHRBN FOR INSULATING YON / -L-GLUTAMYLTAURIN

The invention relates to a method for isolating ^ -L-GlutamyItaurine from extracts of the parathyroid glands.

In the registration of the official file number

^ £ ^ f? ^ * :! is the production of a hitherto unknown »from the parathyroid gland isolable concentrate or mixture deposited. That has very valuable biological properties The concentrate or mixture available was called Litoralon (see DE-OS No. 2 234 832) i

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I follow up tI _O r

In further research it was found that the active ingredient of the concentrate called Litoralon is a previously unknown micropeptide Λ, daa ^ -L-glutamyltaurine * '.

The subject of the present invention is - as a further development of the file number above in the Baaia application protected process - a process for the isolation of this new active ingredient in pure form from the Parathyroid extract.

According to that described in the parent application above Procedure is the Nebenachilddräae of mammals respectively their tissue or cell culture, preferably in the form of a powder, with water or an inorganic ion containing aqueous solution extracted, the extract filtered and then treated with a protein precipitant »The precipitate is subjected to gel filtration. The concentrate obtained in this way is used.

According to the present invention, this concentrate becomes the second by spectroscopic determination Fraction showing maximum (fraction B) selected, separated off and purified by means of ion exchange chromatography. The received The concentrate is then subjected to electrophoresis, which is expediently carried out at a pH of about 1.8 and is carried out with relatively high voltages; Finally the product becomes pure by means of chromatography isolated;

For ion exchange chromatography, it is

moderately daa synthetic resin Dowex 50 (Dow Ghent * ι USA) or a. other synthetic resin of the same iCyps used. ·· '■■■■.

As experience shows, the - ^ -L-glutamyl- 509847/1189 migrates

taurine in the direction of the anode during the electrophoresis used according to the invention.

The final step of the isolation process of the invention Representative chromatographic separation is expedient as paper chromatography or column chromatography carried out on cellulose. In this phase of the procedure, the determination of what is to be separated takes place Amino acid derivative expediently with ninhydrin, the pure active ingredient can in the manner described in one concentration obtained that is ten to one hundred thousand times its concentration in the dried parathyroid gland, Any fraction of the cleaning series can in principle also be used for therapeutic purposes will.

The results of the elemental analysis and the determination of the molecular weight are in good agreement with the gross formula Cr ₇ H ^ N ₂ O ₆ S and the resulting composition *
C = 33.07 % H » 5t55% N = 11.02 % 0 = 37.75 % 0 = 0.2.61 II _s 254.27

The assignation of the absorption bands in the infrared spectrum (recorded in KBr) allow with respect to the Structure following keys

SO ;, "and NH, ⁺ zwitterion structurej the free carboxyl group is undissociated,

Characteristic absorption bands i • J (OH), (NH,) at 3600-240Ό cm " ¹

at 3315 cm "" ¹

Valena vibration of the

Carboxyl group at 175C o ™

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-T (CO) amidoarbonyl (l) at 1662 cdT ¹

) at 1576 cm "" ¹

₃ ") at 1296-1031 crn"" ¹

The Nfffi values of the / ~ L ~ Glutarny Itaurine: Chemical shifts (^) t 4.15 (t, 1, ² CH) | 3.65 (m, 2, ² CH) ₅ 3.12 (m, 2, ¹ CH) | 2.7-2.1 (m, 4 » ⁵ CH ₂ ⁴ CIJ ₂ ). Protons Chemical Shift Relative Intensity CH - NH ₂ 4.15 (t) 1

%> - SO ^ H 3.65 (m) 2

CH - NH 3.12 (m) 2

O ^ C-CI ^ -CHg-OH 2.7-2.1 (ta) 4

The 80 percent by volume of ethanol contained-

the ethanol-water mixture shows recrystallized compound the following crystallographic data:

Dimensions of the elementary cell (determined according to Weissenberg's photographic method): a a 6.63 Å, b = 7.87 Ä, c a 11.38 £, ß S3 101 °. The crystals belong to the monoclinic crystal system and in class P2 ^. Density measured »1.55 g / cm3 density calculated: 1.552 g / cnr.

fjaue recording (crystal surface of the index OkO): systematic Extinctions at k »2n + l.

The acid amide bond is made by carboxypeptidase not hydrolysiertι this also proves the presence of the ^ Peptide bond.

The electrophoretic migration speed agrees well with the calculated value. When using cysteic acid as the standard substance, it is 0.74 (pH a 6.5, voltage a 1500V, time = 90 minutes, load a 0, Ö3, μM / cni, paper: Whatman 3 times). Optical rotation *! «] | = + 14 ° (water, c -. I _s C.:l;.; _{/ T} decomposition point: 223-226 ⁰ C (corrected)

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Awake hydrolysis can be done in hydrolyzate with an amino acid analyzer Glutamic acid and taurine can be detected by thin-layer chromatographic determination.

For amounts in the order of milligrams, content determinations can be made alkalometrically by determining the sulfo group, be carried out photometrically by the van Slyke method or with ninhydrin. To determine smaller quantities are the chromatographic method with densitometric quantity determination and the detection by the electrophoretically ^ migration speed suitable.

The biological methods of determination will be discussed later.

The stability of the pure product isolated from the parathyroid gland is satisfactory for pharmaceutical purposes. ^{On a sample left in a thermostat at 70 °} C. for 8 days, no decomposition could be detected after examination of the melting point, the optical rotatability and the thin-layer chromatographic and paper electrophoretic fourth.

The isolated active ingredient has a broad spectrum of therapeutic and preventive effects to direct or indirect damage to the "AGAS" (aerobiosphere-genetic Adaptation system) related pathological changes (the development of the parathyroid gland and the formation of life on the mainland coincide closely in evolutionary terms.)

To explain the term ¹¹ AGAS ", the most important tissues and organs that make up this system should be listed here!

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a) All biological interfaces that are in contact with the outside air as a biosphere (skin and skin structures ₉ cornea and conjunctiva, mouth and pharynx, airways and lungs);

b) skeleton and limbs (long bones and sponge bones, Ball joints, synovial membrane, skeletal muscles) 5

c) that of mainland organisms in the regulation of the ion balance participating organs (transepithelial transport system, such as villi and kidney canals);

d) the counter necessary for chopping solid food «. donte (attached by a tooth socket and root) dentition

e) hearing, olfactory and vocal organs of the mainland organisms,

The compound according to the invention has a beneficial biological effect on the organs and tissues of the system mentioned or therapeutic effect.

The compound of the invention also acts on the the following functions related to the AGAS system: radiation protection effect, wound healing promoting effect, the mesenchyme generally activating effect, Schute against the constantly increasing risk of infection and contamination of the skin and mucous membranes (lysozyme generation of the moist th mucous membranes, activation of the ciliated epithelia in the Respiratory tract, etc.), increased protection against infections caused by viruses and fungi;

Against the smch constantly and to a great extent increasing Stress effects (e.g. meteorological influences, strong differences between day and night temperature, increased risk of injury) of life on the mainland is the one according to the invention Effective connection by stabilizing the adaptation syndrome and at the same time reducing the peripheral tissue damage of the glucosus

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fends off chorticoids (e.g. damage to connective tissue, damage to the bone matrix, etc.), development The Immune HomoeostaGe Gso increased knowledge of the body, which cells are the body's own and which not).

The connection according to the invention exerts its effect on Partly directly, partly through the control of the vitamin A metabolism, through the production of vitamin A metabolites more polar in character. This effect is that of the parathyroid hormone on the 25-hydroxy ~ cholecalciferol-1-cl ~ hydroxylase ~ enzyme comparable to the kidney canals. Through this explanation the broad pharmacological, biochemical and understandable therapeutic effect of the compound according to the invention. The directions of action are as follows: A) Effects with vitamin A character

a) Pharmacological and biochemical effects «Labeled sulfates are to an increased extent in the cartilage of the rat or the lens of the eye, the liver and lying tissue of the chicken embryo incorporatedj radioactive marking * Increased levels of phosphorus are built into the cartilage of the rat § increasing the synthesis of chondroitin sulfate Effect * favorable effect on wound healing or on the administration of cortisone experimentally impaired wound healing in dogs and rats; potentiating the effect of vitamin A. Effect on rats and dogs experimentally demonstrated » called hypo- or hypervitaminosesj die uleus-related stress effect reducing effect Rats; effect favoring the degranulation of mastocytes; increasing effect on the production of

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NACHQERgIOHT I - 8 -

Lyaocyin; Effect on the BUDGET _h of the trace elements (silicon, zinc, copper, manganese, fluorine); promoting effect on epithelial formation; increasing effect on alkaline phosphatase activity the effect exerted on the granuloma sac formation caused by the local action of vitamin A; the very flat course of the dose-effect curve or II

the change in sign for large doses. b) Clinical-therapeutic effects ent keratooonjunctivis sicca; SJogren's syndrome; rhino-laryngo-pharyngitis sioca; ozaena; chronic bronchitis, sinobronchitis; Cystic fibrosis; constitutional lung diseases in young children; Periodontal disease; Skin susceptibility to infection and the mucous membranes for viruses and fungi; cliortisone antagonistic Effect, beneficial effect on the healing process in surgical wounds and mucous membrane wounds; erosio colli; pruritus-like; Reduction of the odor and Sense of taste »activating effect on the Golgi

Apparatus. B) Effects without vitamin A character

a) Pharmacological and biochemical effects: effect on the blood sugar level in the sense of a temporary lowering; increasing effect on phosphaturia * decreasing effect on the phosphorus level in the blood serum; radioprotective effect; in the case of labyrinth experiments with inactive animals, the effect of reducing the time required to achieve the goal; reducing effect on experimentally induced fluorine and cadmium toxicosis; the cyclic adenosine monophosphate deflation of the kidneys increasing effecti

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- O, M

. increasing effectiveness the activity of the liver enzyme tyrosine aminotransferasej

b) Therapeutic effects * weak radiation damage j Vitiligoj muscle hypotonia j p3ychoener.getising Effect; favorable effect on involutional and gerontological conditions as well as on mystical functions; cheloid tendencies; Ankylopoietic spondylosis; diseases of the locomotor organs due to wear and tear § sclerotic fundus; Amyloidosis morphea, fibrooycti " see mastopathy.

When the compound according to the invention is administered, the duration of the treatment varies greatly. Some diseases (e.g. rhinolaryngo-pharyn- ^: gitis aicca) become symptom-free after two weeks with oral administration of 3x5 micrograms daily, for symptomatic improvement of other diseases (e.g. paradentosis, SJögren's syndrome) one to two months are required, other diseases (for example spondylosis ankylo * · poetica) have to be treated for a quarter to a half year »

From the compound according to the invention, any desired pharmaceutical, preventive, cosmetic or veterinary preparations are produced. The preparations can have a single active ingredient or contain a combination of active ingredients. The dose of pure action " substance is JPO-500 nanograms per day and kilo of body weight and is applied in three individual doses.

One tablet contains 2-20 micrograms, preferably 10 micrograms, of active ingredient and also biologically inert carriers (e.g. milk sugar, starch) and the usual tabbing aids (granulating and lubricating agents »

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such as polyvinylpyrrolidone, gelatin, talc, magnesium stearate, Aeroeil etc.) With the extremely low dose, it is advisable to use the active ingredient before the Add granulation in the form of a solution to the tablet mass and homogenize with a kneader. To this Way, the active ingredient is evenly distributed, the low active ingredient content enables the production on a scale large laboratory. The active ingredient is stable, the tablets can therefore be consumed in the without specifying a deadline Trade to be brought. The active ingredient content of delayed effective tablets or spansular capsules can be 10-20 micrograms, for injection preparations is the appropriate dose per ampoule 5-10 micrograins, the parenteral It can be used intramuscularly, subcutaneously or intravenously. Damages in the specified concentration the active ingredient neither tissue nor vascular walls. The active ingredient can therefore also be administered as an infusion.

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The invention is illustrated by the following examples explained in more detail without, however, remaining limited to the examples,

example 1

Obtaining Ak-glutamyltaurine from natural substances IO s lyophilized and ground minor acyroid gland powder are degreased according to the well-known Auerbach method in the following way: The glandular powder is thoroughly mixed with 25 ml acetone at room temperature and then the suspension is _{filtered through a glass filter (G 4} Schott). Well sucked off glandular powder is rubbed thoroughly with chloroform, also at room temperature, and the suspension is filtered again through a glass filter. This operation is repeated once more. The extracted substance is then suspended again in 25 ml of acetone and thoroughly sucked off through a glass filter. The gland powder purified in this way is dried in vacuo at room temperature. About 7-9 g of dry, fat-free gland powder are obtained; this weight depends on the fat content of the starting material.

10 g of the defatted dry powder glands are extracted at 50 ⁰ C three times with 100 ml of distilled water ·. Each extraction takes one hour * After each extraction, the aqueous solution is separated from the powder by means of a high-speed laboratory centrifuge. Until further use the aqueous extracts under nitrogen bf> i temperatures are kept for 3-5 ⁰ G in the refrigerator. The aqueous extract shows a positive reaction to hinhydrin, biuret and trichloreasiga acid,

The aqueous extracts are combined before the next operation. The reSnig ^ en ^ xtratb is under G.

ORIGINAL INSPECTED

Shake drop by drop enough 50 vol .-% aqueous trichloroacetic acid solution added so that the final concentration of trichloroacetic acid is 15 % .

The aqueous solution containing the precipitate ^{is left to stand for 2 hours at 3-5 °} C. in the refrigerator and the precipitate formed by the action of the trichloroacetic acid is then centrifuged off. The clear solution gives no further precipitate on addition of erichloroacetic acid.

The excess of l'richloroacetic acid is replaced by a serial extraction at room temperature removed, ether is used for extraction. To the entire trichlores To remove excess acid, it is necessary to extract 17 to 18 times, in some cases even? 20 to 25 one-time extraction. After the last extraction, the aqueous phase should have a pH value between 4-5 and 5.

It

To remove the ether, a stream of nitrogen is passed through the aqueous phase at a temperature of below 30 ^{° C. by means of a water jet pump}

The entire Athör is hours away, the operation is over associated with a loss of volume, since at the reduced pressure part of the water evaporates as well as the ether;

It

They checked by smelling _t if all ether removed is.

The aqueous solution is then lyophilized, and the substance, since it is hygroscopic, is stored in a well-closable vessel.The yield of substance is 10-15 % based on the gland powder that has not yet been defatted, which naturally depends on the fat content of the expansion ;; ydrüGenpulver .

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The lyophilized substance obtained is on Sephadex G-15 (Pharmacia, Sweden, üpsala | particle size 90-120 microns) gel-filtered using the descending method.

The elution diagram is determined by measuring the absorbance at 280 nm. A total of 7 maxima (peaks) are obtained (AG),
Characteristic parameters of gel filtration »

approach

Column diameter Column height Flow rate Time duration Fraction size maxima (peak)

5.1 g of lyophilized substance dissolved in 10 ml of distilled water 4.85 cm 149 cm 0.78 ml / min 10 min 7.8 ml / tube No. of tubes

77-103 104-128 129-134 135-162 163-189 I9O-217 218-227.

The active substance is in the fraction labeled B, up to tube no. 104 the volume of the eluate is 814 ml «The total volume of fraction B is 188 ml ·

Fraction B is then lyophilized. With 5 g of starting material, 0.43-0 * 53 g of Lyophilir.at is obtained.

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Identification during the <ielfi'i.tr <itioDS

SUBMITTED

If the gel filtration is carried out under the conditions given above, the active substance appears at the volume (0.67 ± 0.02) χ V ₀ , where V _{0 is} that eluate volume at which the inorganic ions appear in the eluate (positive chloride reaction, with Silver nitrate tested).

The lyophilized fraction B is subjected to ion exchange chromatography subject.

301 g of the lyophilized fraction B are dissolved in a formic acid-acetic acid mixture of pH 1.8 to give a 30 # solution. The solution is chromatographed on a column of size 132 χ 1 cm, which is adjusted to pH 1.8 with a formic acid-acetic acid mixture and is filled with Dowex 50x2 (Dow Chemical, USA) at a flow rate of 13 ml per hour, with fractions of 4 ml are collected. »These individual fractions are combined to form fractions AI on the basis of their extinction maxima measured at 280 nm!

description of
fraction Tube number Weight (mg)
(lyophilized) A. 9-12. 8.4 B. 13-17 18.4 C. 18-23 64.4 D. 24-29 5.2 E * 30-36 2.2 3? 37-42 ■ 0.5 G 43-50 3.1 H 51-57 7.3 I. 58-80 6.8

116.3 (38.6 %) ₍

*) (86 ml glacial acetic acid and 25 ml formic acid / 88 vol. 96 / with dist. Water diluted to 1000 ml.)

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BATH ORIGINAL

The active substance is in fractions D, B and F. By lyophilizing the combined eluates of these three fractions, 7.9 mg of substance is obtained, which corresponds to 2.62% of the gel-filtered starting fraction B.

The fractions D, B and F obtained by ion exchange chromatography and then lyophilized are ' the böi a pH value of 1.8 on a 22 cm wide paper (Whatman 3 MM) at a voltage of 1500 V for 90 minutes subjected to electrophoresis.

The active substance shows positive ninhydrin reaction and moves uniformly from the starting line in the direction of the positive pole, namely at 1500 V voltage ..

After electrophoresis, the one against ninhydrin positive strips containing the substance possessing a corresponding migration speed, cut out and in the way on a new paper chromatographic sheet sewn on so that the direction of development is opposite to the direction of electrophoretic migration further purification is done by descending paper chromatography. A butanol-pyridine-glacial acetic acid -Water mixture used in the ratio 15 * 10 * 3 * 12. The at Paper chromatography performed at room temperature on Whatman 3MM paper lasts 60 hours.

In the specified superplasticizer, the pure compound has an R _f value of 0.19. The relative mobility (based on cysteinic acid) determined by electrophoresis at pH 6.5 paper is 0.74. After hydrolysis of the product with hydrochloric acid, the hydrolyzate is generally glutamic acid and

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Taurine detectable. The isolated substance is not hydrolyzed by carboxypeptidase. Formolt C ₇ H ₁₄ N ₂ O ₆ S, M = 254.27.

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Claims (1)

Puαent anaprü cho l.yv / suppurative development of the i-lr. yf protected process to a process for the isolation of ^ -L-glutamyltaurine from the extract of the parathyroid gland, characterized in that - after the parathyroid gland or its tissue or cell culture, preferably in powdered form, with water or an inorganic Aqueous solution containing ions extracted, the filtered extract freed from iüiweiß and lyophilized - the substance dissolved in water and the solution subjected to a gel filtration, the fraction containing the active ingredient, preferably by spectrographic determination, selected and lyophilized, the lyophilisate dissolved in water and the aqueous solution is purified by means of ion exchange chromatography, the active ingredient concentrate obtained is subjected to electrophoresis and finally the ^ -L-glutamyltaurine is isolated by chromatography, 2. The method according to claim 1, characterized « that for the purifying ion exchange chromatography that fraction of the gel filtration is selected which appears in the course of the bleeding at a volume of V (0.67 + 0.02), wherein ο - V means the volume at which inorganic ions * appear in the fi lerate . 3, method according to claim 1, characterized in that, for further purification, that bluate of gel filtration is selected as the starting material, the extinction of which, measured at 280 nm, has the second maximum. 4.Verfahren according to claim 1, characterized in that the electrophoresis at pH «* l, 8 and using holier Tension is performed. 509847/1189 λ * 5 · Method according to claims 1 and 2, characterized characterized in that the chromatography see purification as Paper chromatography or cellulose column chromatography is performed. 6, medicinal product preparation, characterized in that it contains -L-glutamylitaurine isolated from the parathyroid as an active ingredient. 509847/1189