DK158676B - METHOD OF ANALOGUE FOR THE PREPARATION OF POLYMERS OR OLIGOMER AMINO ACID DERIVATIVES OR PHYSIOLOGICALLY ACCEPTABLE SALTS. - Google Patents

Description

DK 158676 B

The present invention relates to an analogous process for the preparation of novel polymeric or oligomeric amino acid derivatives having valuable biological and pharmaceutical effects and the general formula I of claim 12, wherein R, R, R, R, Y, B, n, t and r have the same meanings stated.

The process according to the invention is characterized by the characterizing part of the claim.

The compounds of this invention are peptides and are degraded like other peptides in the organism to monomers (amino acid derivatives), and these amino acid derivatives have a broad therapeutic and preventive spectrum of action against morbid changes that can be traced back to damage by "AGAS" (the aerobiosphere genetic adaptation). -15 system).

To illustrate the concept of "AGAS", the following are the main tissues and organs that form this system.

(a) all biological interfaces which are in contact with the external air as a biosphere (skin and skin formation, eye cornea and conjunctiva, oral and pharyngeal cavities, respiratory tract and lung); b) skeleton and joints (tubular and spongy bones, ball joints, synovial membranes, skeletal muscle); C) those for the regulation of the ionic household and participating organs (transepithelial transport system: intestinal tract and renal duct); (d) the teat needed for comminuting the food (rooted in the tandaveolans with roots); (E) hearing, odor and voice organs.

Thus, the compounds of this invention exert a favorable biological or therapeutic effect on the organs or tissues of the AGAS system enumerated herein.

The compounds prepared by the process according to the invention also act on the following functions which are related to the AGAS system: radiation protection, favor of wound healing, general activating 2

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effects on mesenchymal, protection against the ever-growing infection and soiling danger of skin and mucous membranes (the lysozyme production of the moist mucosa, activation of mucosal epithelium in the respiratory tract, etc.), increased protection against the infections caused by viruses and fungi.

The compounds of the present invention are effective against the ever-increasing stress effects associated with mainland life (e.g., meteorological influence, large differences between day and night temperature, increased risk of injury), they stabilize the adaptation syndrome and at the same time avert the peripheral tissue damage of the glucocorticoids (such as damage to the connective tissue, injury to bone-matrix components, etc.). Development of immune homeostasis (increasing body recognition ability of which cells are capable and which are not).

The compounds prepared by the process according to the invention exert their effect, both directly and partly through the regulation of vitamin A metabolism, in the production of vitamin A metabolites of a stronger polar nature. This effect is comparable to the effect of the parathormone on the 25-hydroxycholecalciferol-1α-hydroxylase enzyme in the renal tract. By this information, the broad pharmacological, chemical and therapeutic effect of the compounds of the invention is made understandable. The directions of action of the compounds are as follows: A) Vitamin A grade action a) Pharmacological and biochemical effects: increasing effect on chondroitin sulfate synthesis; beneficial effect on the wound healing or on the experimentally impaired wound healing in rats and dogs by cortisone administration; potentiating effect on vitamin A's effect in rats and chickens in experimentally induced hypo- or hyper-vitaminosis; attenuating effects on the ulceration-dependent stress effects in rats; beneficial effect on the degranulation of mastocytes; increasing effect on the production of lysozyme; effect on the traceability household 3

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(silicon, zinc, copper, manganese, fluorine); promoting effect on epithelial formation; promoting effect on the alkaline phosphatase activity; the effect on the granuloma bag formation 5 (Granulomsackbildung) induced by local action of vitamin A; the extremely flat course of the dose-effect curve or the change of effect sign at large doses; activating effect on the Golgi apparatus; beneficial effect on the formation of mucus or goblet cells; increasing effect on the concentration of vitamin A.

B) Clinical-therapeutic effects: keratoconjunctivis sicca; Sjagren's syndrome; rhino-laryngo-pharingitis sicca; ozæna; chronic bronchitis; sinobronchitis; muco-viscidose; constitutional lung disease in young children; 15 periodontosis; skin and mucosal susceptibility to viruses and fungi; cortisone-antagonistic effect; beneficial effect on healing of operative and mucosal wounds; erosio colli; pruritus-like disorders; reducing the sense of smell and taste.

20 B) Vitamin A-grade effects a) Pharmacological and biochemical effects: effects on blood sugar levels with respect to a transient 2 ^ decrease; increasing effect on phosphaturia, lowering effect on serum phosphate level; radiation protective effect; reducing the time required to reach the goal of maze trials in inactivated animals; diminishing effect on experimentally induced fluorine and cadmium toxicosis; increasing effect on the cyclic adenosine monophosphate depletion of the kidneys; attenuating effect on the symptoms of experimentally induced lathyris mice; decrease in histamine sensitivity; enhancing effect on the activity of the liver enzyme tyrosine aminotransferase.

JD

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b) Therapeutic effects: weak radiation damage; vitiligo; muscle hypotonia; psychoenergetic effect; beneficial effect on involutional and gerontological conditions as well as on the mnestic functions; keloid 5 inclinations; spondylosis ankylopoetica; diseases of the movement organs due to wear and tear; sclerotic fundus; amyloidosis; morphea; fibrocytic mastopathy.

When administering the 10 compounds of the invention, the duration of treatment is extremely different. Many diseases (eg rhino-laryngo-parangitis siccaT, by oral administration of 3 x 5 days daily, are already symptom-free after two weeks, for symptomatic improvement of other diseases (eg, paradoxic, Sjogren's syndrome) one to two months are needed. , in yet other diseases (eg, spondylosis ankylopoetica), treatment must be treated in the ethics quarter to six months.

From the compounds of the process according to the invention, 20 desired pharmaceutical, preventive or veterinary medicinal products can be prepared in a simple manner. The compositions may contain a single active substance or a combination of active substances.

The dose of a pure active compound is 50-500 ng / day / kg body weight and is administered in three single doses.

A tablet contains 2-20 µg, preferably 10 µg of active substance in addition to biologically inactive carriers.

In the case of injection preparations, the appropriate dose per ampoule 5-10 µg. The active compounds may also be administered as an infusion. Suppositories contain from 2 to 30, preferably approx. 10 µg of active substance and was prepared from cocoa butter or other synthetic waxes or fats suitable for this purpose. The content of the active compound in ointments for healing the skin is 0.1-1 µg / g. The ointment base may be hydrophilic or hydrophobic and contains the usual components. In addition, the active compounds can be formulated as aerosol preparations in which the content is also 0.1-1 µg / g. In formulation 5

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as perlingual tablets, a tablet contains 10 pg of effective compound, the decomposition time of the tablet is half and one hour. For example, polymers for retarded release of the active substance may be prepared as a suspension and contain 1-5 µg of active compound / g polymer.

In veterinary medicine, the compounds of the invention have similar fields of application as in human medicine, ie. for example, skin damage (peeling), wound healing and bone fractures.

The process according to the invention is illustrated in the following by means of some examples.

Example 1 0.52 g (4 mval) of α-poly-L-glutamic acid (degree of polymerization 80) (Acta Chim. Acad. Sci. Hung. 5, 267, 1955) are dissolved in 10 ml of dimethylformamide and the solution is cooled with ice water. With stirring, 1.39 g (10 mmol) of p-2Q nitrophenol and 0.82 g (4 mmol) of dicyclohexylcarbodiimide are added. After ten minutes, dicyclohexylurea is separated. The mixture is stirred for one day at room temperature after which the dicyclohexyl urea is filtered off, with the addition of 0.3 ml of glacial acetic acid, the non-converted di-cyclohexylcarbodiimide is also converted to dicyclohexyl urea and this is also filtered. The solution is poured into a mixture of 100 ml ether, 100 ml petroleum ether, 20 ml ethyl acetate and 2 ml glacial acetic acid. The precipitated product is centrifuged, washed several times, with ether and then dried under vacuum over sulfuric acid. 0.80 g of α-poly-L-gluamic acid active ester 1 is obtained, the content of p-nitrophenolate of 2.6 mval / g.

The infrared spectrum shows the following maxima for the characteristic groups: NH 3300 cm C = 0 (COONP)

__ 1765 cm S C = 0 (amide I) 1660 cm C = 0 (amide II) 1550 cm S

35 in NO2 1530 and 1360 cm.

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Dissolve 0.25 g of α-poly-L-glutamic acid-γ-p-nitrophenyl ester-1 in 7 ml of pyridine. With stirring, 0.125 g (1 mmol) of taurine is added in 1 ml of water and 0.28 ml (2 mmol) in ten portions in such a way that the reaction proceeds continuously in clear solution. An additional 1 ml of water is required. The reaction mixture is left at room temperature for three days, then evaporated under vacuum and the residue dried. After thorough leaching with ether, the residue is torn to a powder. After dissolving in water and subsequent lyophilization, 0.20 g of α-poly-Y-glutamyltaurine-1 is obtained. The chromatogram shows a contamination of 0.4% taurine.

Analysis: S 10.1%.

The infrared spectrum exhibits the following maxima for the characteristic groups: OH 3100 cm (wide); 15 C = 0 (amide I) 1650 cm 1: C = 0 (amide II) 1550 cm 1; S = 0 (SO 2 OH) 1220 , 1040 and 600 cm "1.

Example 2 2Q 0.26 g (2 mval) of α-poly-L-glutamic acid (degree of polymerization 580) (J.A.C.S. 80, 4631, 1958) is swollen and dissolved in 15 ml of dimethylformamide. To the solution, 0.69 g (5 mmol) of p-nitrophenol and 0.41 g (2 mmol) of dicyclohexylcarbodiimide are added with stirring. The reaction mixture is stirred for two days at room temperature. 0.30 g of α-poly-L-glutamic acid active ester-2 is obtained.

The infrared spectrum exhibits the following maxima for the characteristic groups: C = 0 (COOH) 1720 cm. The other bands are the same as those described in Example 2Q 1 but they are wider. From the obtained α-poly-L-gluta-minic acid-Y-β-nitrophenyl ester-2, 0.27 g of lyophilized α-poly-γ-L-glutamyltaurine-2 is obtained in the manner described in Example 1. According to chromatographic study, the contamination of the product with taurine is less than 0.4%.

Analysis: S 7.3%.

The infrared spectrum is identical to that described above.

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Example 3 0.26 g (2 mval) of α-L polyglutamic acid (degree of polymerization 580) is dissolved in 8 ml of dimethylformamide. The solution is cooled to -10 ° C in a cold mixture of ice and boiling salt. After the addition of 0.28 ml (2 mmol) of triethylamine, the solution gels and even with the addition of a further 8 ml of dimethylformamide and intensive stirring, it does not become liquid again. Now 0.28 ml (2 mmol) of hydrochloric acid isobutyl ester and 0.16 ml of cysteamine in 2 ml of dimethylformamide are added after 30 minutes of activation time. The reaction mixture is stirred at -5 ° C for two hours and then at room temperature for four hours, then poured into a mixture of 50 ml of chloroform and 50 ml of petroleum ether. The precipitated white precipitate is centrifuged, washed several times with a mixture of chloroform and petroleum ether, sometimes swelled in alcohol and then precipitated with ether.

0.33 g of α-poly-γ-L-glutamyl cysteamine is obtained.

Analysis: S 14.7%.

0.16 g of α-poly-γ-L-glutamylcysteamine is suspended in 5 ml of glacial acetic acid and then 1 ml of 30% hydrogen peroxide is added. The reaction mixture is left at room temperature for three days, slowly dissolving the solution. It is diluted with water and then lyophilized. 0.20 g of white α-poly-γ-L-glutamyltaurine are obtained whose chromatographically determined taurine contamination amounts to 0.5%.

Analysis: S 11.9%.

The infrared spectrum matches what has already been described.

30

As mentioned, the polymeric and oligomeric amino acid derivatives prepared by the process according to the invention are broken down into the corresponding monomeric amino acid derivatives, and thus, they exert the pharmacological action in the organism.

It will be elucidated in the following by two biological test reports, A and B. These reports show the bio- 8

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logical effect of four different types of monomeric amino acid derivatives formed in the organism by degradation of the oligo or polymeric compounds with the general formula I.

5 Biological Test Report A.

Examination of γ-L-glutamyltaurine, which in this report is referred to as litoralon._ In the following Tables 1-6, test results 10 are shown as mean values + standard deviation with the number of parentheses determined. Determination of. whether there is a significant difference between control experiments and experiments on treated animals was done with Student's t-test.

Table 1

Effect of Litoralon on vitamin A concentration in serum.

Group of Litoralone Vitamin A Concentration in Serum _ug / day_ 20 I. Control - 28.3 + 0.7 (20) II. 0.1 41.9 + 1.0 * (20) III. 0.3 32.9 + 1.1 ** (20) IV. 1.0 27.4 + 0.6 (20) 25 x P <0.001 xx P <0.01 20 Sprague Pawley rats (10 males and 10 females) weighing 180-200 g were orally treated with the daily doses listed in Table 1 -30 sees litoralon in the form of an aqueous solution for a period of 8 days. On the 9th test day, the animals were sacrificed by decapitation and the blood was collected. The vitamin A concentration in the serum was determined by Neeld and Pearson's method.

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Table 2

The effect of litoralone and vitamin A on granuloma formation induced by implanted cotton balls_ 5 Group Dose Dry weight of

Vitamin Ax litoralon granuloma. local local oral "_mg _yg_yg / day_ I. Control - - - 52 + 1.0 (24) 1Q II.Check +

Solvent - - - 54 + 3.1 (8) III. 2 - - 64 + 2.5 (8) IV. 2 0.1 - 65 + 2.7 (8) V. - 0.1 73 + 2.9 (8) 15 VI. 2 - 0.1 90 + 4.1 (8) x Hoffmann La Roche

The difference is significant; between groups II and III at 20 P <0.05, between II and V at P <0.001, and between V and VI at P <0.01. Granule formation was determined according to Lee et al with male Sprague-Dawley rats weighing 110-120 g. The tampons were removed from the dorsolateral subcutaneous implants after 10 days and weighed after drying to constant weight at 65 ° C.

25 30 35

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Table 5

The effect of Litoralon on the metamorphosis of Rana dalmatina

Group Body length Tail length 5 _mm_mm_

Control 45.9 ± 0.2 / 31.4 ± 0.2 (60)

Treated animals 40.2 + 0.2X 26.7 + 0.2X (60) x Significance: P <0.001 10 The experiment was conducted with larvae of Rana dalmatia with a age of 30 days, a body length of 20-25 mm, and with already visible projecting hind legs. Over a 30-day period, the test animals were placed in tap water containing 0.5 µg / ml litoralone for two hours each day. The control group was placed in 15 tap water without the addition of litoralone. The nipples were measured on the 3rd day.

Table 6 2q Litoralone's effect on blood sugar levels

Group I. Study II. Study _mg% ___ mg% _

Control 94 + 3.6 (10) 94 + 4.09 (10)

Treated animals 82.4 +3.8 (10) 81 + 1.32 (10) 25

Significance at I. study: P <0.05 Significance at II. study: P <0.01

Male CGY white body rats 160-180 g were used for the experiment. The animals were kept on a standard diet. Blood samples were taken on the 5th day of testing after 18 hours of fasting. The blood glucose assays were performed according to the method of E. Hultman. Litoralon was given orally at daily doses of 1 µg / kg.

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Biological Test Report B.

Study of γ-aspartyl taurine and its derivatives 1. β-aspartyl-N-methyltaurine 5 a) Effect on blood sugar level Control 107 mg%

Treated animals 93 mg%

Significance: P <0/05

20-20 rats were used for the samples. Measurements were carried out -10 after 18 hours of fasting. The dose used was 1 μgAg body weight for 4 days in the form of a solution by oral administration.

b) Effect to increase serum vitamin A levels

Oral dose Induced vitamin A yg% 15 µg / 200 g body weight 0 8.5 5 11, 0 0 11.5 0.3 12.5 20 0.1 16.1

0.05 14, S

0.01 12.5 0.005 10.5

Significance: P <0.01

For the experiments 20-20 Wistar male body weights 200-220 g were used.

Trial period: 6 days.

c) Effect on blood silicon level:

Silicon (mg / g blood) _0 hours_20 days_40 days_

Control group 0.110 + 0.004 0.120 + 0.010 0.154 + 0.015

Treatment group I 5 µg / day 0.100 + 0.005 0.315 + 0.014XX 0.345 + 0.015XX · 35

treatment Group

II 10 µg / day 0.107 + 0.009 0.370 + 0.1B'XX 0.360 + 0.017XX

xx Significance: P <0.001

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14

The results are significant from the 13th day at the significance level P <0.01 and from the 20th day at the level P <0.001. The experiments were performed on inbred male rabbits of body weight 2.5-3 kg. The active ingredient was administered orally in the 5 daily doses shown in the table. The determination of silicon was performed according to Gaubatz's method (Gaubatz E., Clin. Wschrft. 14, 1753, 1935) in 5 ml of blood samples taken from the animals' ears.

d) The effect of β-aspartyl-N-methyl-taurine and vitamin A on the 10 granulomatous effects caused by the implantation of cotton wool:

Group Dose Weight of dry

Vitamin Ax β-aspartyl-N-Sranuloma methyl'taurine local local oral mg yg yg / day 15--

Control I. - - - 54 + 1.7

Control II. + solvent - - - 55 + 3.4

Treatment Group III. 2 - - 66 + 12.5

Treatment Group IV. 2 0.1 - 67 + 2.6 ^ Treatment group V. - - 0.1 79 + 2.8

Treatment Group VI. 2 - 0.1 96 + 4.4 x Hoffmann la Roche

The differences are significant as follows: 25 Between groups II and III P <0.05, between groups II and V P <0.001 and between groups V and VI P <0.01.

The determination of granuloma was performed on male Sprague-Dawley body weights 110-120 g by the method of Lee et al (Lee K.H., Fu Ch.Ch., Spencer M.R. Tong T.G. and Poon R.J., Pharm.

30 Sci., 62, 895, 1973). The dorsolaterally subcutaneously implanted tampons were removed after 10 days and measured after drying at 65 ° C to constant weight.

2. β-Asparphyl homotaurin.

35 a) Effect on blood sugar level

Control group 105 mg%

Treated group 94 mg% 15

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Significance / number of test animals and test method were as indicated in section. 1 a) above.

5 b) Increasing effect on serum vitamin A level

Oral dose Induced vitamin A

yg / 200 g body weight _ugl_ 0 -8/6 5 12/0 10 1 13/5 0.3 * 14.0 '0/1 15/8 0.05 15.0 · 0.01 12.0 15 _0, 005_10,0_

Significance, number of test animals and test method were as indicated above under section. Ib).

c) Effect on blood silicon level: 20 -

Silicon (mg / g blood) _ 0 hours_20 days_40 days_

Control group 0.104 + 0.009 0.134 + 0.015 0.157 + 0.020

Treatment group xv vv I. 5yg / day 0.094 + 0.007 0.309 + 0.01¾ ^ 0.340 + 0.014 25

Treatment Group II, 10 µg / day 0.109 + 0.010 0.372 + 0.120 0.363 + 0.018

Significance, number of test animals, arrangement and method of determination as indicated above under cl. 1 c).

30 35 16

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(d) The effect of β-aspartyl homotaurine and vitamin A on the granulomatous effect of cotton implantation;

Dose Weight of dried

Vitamin Ax-β-aspartyl-9-celluar 5 local homotaurin mg local oral _y g yg / day_.

Control group I - - - 52 + 1.5

Control group II - - - 53 + 3.2 + solvent

Treatment Group III. 2 - - 64 + 3.2.3

Treatment Group IV. 2 0.1 - 65 + 2.4

Treatment group V. - - 0.1 77 + 2.6

Treatment Group VI. 2 - 0.1 94 + 4.2 x Hoffmann la Roche 15

Significance, number of test animals, arrangement and method of determination were as indicated above under point (ld).

3. γ-Glutamyl-colamine phosphate.

2o (a) Effect on blood sugar level

Control group 104 mg%

Treatment group 93 mg%

Significance, number of test animals, arrangement and dosage were as indicated above under point 1a).

25 b) Increasing effect on serum vitamin A level

Oral dose of Vitamin A action yg / 200 g body weight _yg% _ 0 8.8 30 5 · 12.3 1 13.4 0.3 14.3 0.1 16.0 '0.05 15.5 35 0, 01-11 11 0.005_. _9 ^ 2_ 17

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d) The effect of γ-glutamyl-cholamine phosphate and vitamin A on the granuloma-inducing effect of water implantation

Dose Weight of dried granuloma

Vitamin Ax γ-glutamyl mg 5 Local Colamine Phosphate mg Local Oral _gg / day

Control group I. - - 49 + 1.2

Control group II. + solvent - - - 50 + 2.9 10 Treatment group III. 2 - 61 + 12.0

Treatment Group IV. 2 0.1 - 62 + 2.1

Treatment group V. - - 0.1 74 + 2.3

Treatment group Vi. 2 - 0.1 91 + 3.9. _ x Hoffmann la Roche 15

Significance, number of test animals, arrangement and method of determination were as indicated under 1 d) above.

Significance, number of test animals, arrangement and method of determination were as indicated above under point 1 (b).

C) Effect on blood silicon level:

Silicon (mg / g blood) _0 hours_20 days_40 days_

Control group 0.106 + 0.011 0.136 + 0.017 0.159 + 0.022 Treatment group I 5 µg / day 0.096 + 0.009 0.311 + 0.016 0.340 + 0.017 x

Treatment group VY vv II 10 µg / day 0.111 + 0.011 0.374 + 0.121xx 0.365 + 0.019xx

Significance, number of test animals, arrangement and method of determination were as indicated above under point 1 (c).

35

Claims (4)

5 NH-CH-CO * - (NH-CHR6-CO) -YI r (CHO) II 2 η 1 CO-N-CH- (CH 2) .- B 1 O 2 t RR 10 where R represents hydrogen, alkoxycarbonyl with 1-4 carbon atoms in the alkoxy moiety or phenyl-C 1-4 alkoxycarbonyl optionally substituted with methoxy or nitro, 2 R represents hydrogen or a carboxyl group, R represents hydrogen or methyl, g 15. represents a group of the formula - (CHO) -CO-N -CH - (CHO). - B1 2. i i 2 t R R Y represents hydroxy or C 1-4 alkoxy, B1 represents -SO20H or -OPO (OH2), n represents the number 1 or 2, t represents the number 1 or 2, r denotes an average degree of polymerization up to a molecular weight of not more than 2000, 25 or physiologically acceptable salts thereof, characterized in that a compound of the general formula NH - CH - (CH-). - B1 II I h 2 t 30. R 2 2 1 'wherein R, R and t have the meanings given above and B has one of the meanings given for B or represents -SH, is reacted with an α-polyaminodicarboxylic acid m-activated ester of the general formula 35 R1 158676 B R 1 - NH - CH - CO - fNH - CH - CO] - Y (CT2) \ <CH2) n J III hop1 to to 7 r 5 where R, Y, n and r have the meanings given above and COA denotes an activated ester group to oxidize a won compound containing a group -SH at the site of B and then, if desired, convert the won compound 10 of formula X to a physiologically acceptable salt thereof or convert a won salt to the free one. compound I. 15 "20 25 30 35