DE3427618A1 - HOMOCARNOSINE AND ITS SALTS FOR USE AS THERAPEUTIC ACTIVE SUBSTANCES - Google Patents

Description

The invention relates to homocarnosine and its salts, in particular its pharmacologically acceptable salts, for use as therapeutic agents, in particular to promote the healing of wounds, ulcerations and inflammatory processes.
10

The age of medicinal substances with anti-inflammatory, anti-edematous, antigranular and analgesic effects began with the discovery of the clinical effects of cortisone 194-9 · Today these drugs are only used in limited numbers Scope applied.

There are numerous inflammatory processes with the usual ones Antiphlogistics cannot be treated, for example decubitus, corneal ulcer, cervical erosion and ulcer cruris on the lower legs. The natural or spontaneous healing of inflammatory diseases would be ideal. However, it is the experience that spontaneous healings are very rare.

25th

The invention has for its object to be effective drugs for the treatment of various types of inflammatory Develop processes. The solution to this problem is based on the surprising finding that homocarnosine and its salts, in particular its pharmacologically acceptable salts, are suitable for use as therapeutic agents, especially to promote the healing of wounds, ulcerations and inflammatory processes.

35

Experimental testing for anti-inflammatory activity

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with honocarnosine resulted in an increase in the formation of granulation tissue in the granuloma test.

Homocarnosine is a dipeptide, namely L-histidinyl-'f-aminobutyric acid. The compound was isolated in 1961 by Pisano and colleagues from the brain extract of cows. The compound is present in the brain in an amount of about $ 0.007. Since its discovery, the compound's physiological and pharmacological significance has remained unknown.
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Homocarnosine has the following formula:

HC ===== C - CH ₀ CHCOOH

I I ι

, 5 HN _x ^ N ₂₂₂₂

It is a white crystalline powder with a melting point of 24-2-24-3 ⁰ C and a rotation value ß * J ^ = + 23.2 °. Homocarnosine turns into L-histidine and Y- aminobutyric acid in vivo

. split. Y- aminobutyric acid is a commercial product.

Various methods of producing homocarnosine are known. According to the method described in The Journal of Biological Chemistry, Vol. 236, No. 2 (1961), pp 499-502, ^{triethylamine is added to a suspension of carbobenzyloxy- 1} JT-aminobutyric acid in methylene chloride. After the resulting solution has cooled to -5 ° C., ethyl chloroformate is added and the mixture is kept at this temperature for 10 minutes. A solution of L-histidine methyl ester is then quickly added to the solution. The ester was prepared by adding triethylamine to a suspension of L-histidine methyl ester dihydrochloride in methylene chloride at 0 ^° C. The reaction mixture obtained is left to stand at 25 ° C. overnight. After that it is with water

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and 1Ν sodium bicarbonate solution, dried over sodium sulfate and evaporated to a syrup. The residue is dissolved in methanol and 1N sodium hydroxide solution is added. After 3 hours standing at 25 ⁰ C the solution with dilute sulfuric acid to a pH of 5 is set, and evaporated under reduced pressure to dryness. The residue is extracted twice with hot ethanol. The ethanol extract is mixed with water. After adding 10 $ palladium-on-carbon, the mixture is hydrogenated. After the hydrogenation, the catalyst is filtered off and the filtrate is evaporated under reduced pressure. The syrup obtained is dissolved in water and adjusted to a pH of 3 with dilute sulfuric acid. If ethanol is added slowly, the dipeptide sulfate separates into granular form. is crystals from. The product is filtered off and recrystallized from aqueous ethanol. The homocarnosine sulfate melts at 24-0 ⁰ C with decomposition.

• A column filled with the exchange resin Dowex 50 is filled with IN hydrochloric acid and then washed with water until the eluate reacts neutrally to thymol blue. Then a 10 percent A solution of homocarnosine sulfate is placed on the column and the column is washed with water to separate the sulfate ions, until the eluate reacts neutrally to thymol blue. The homocarnosine is then eluted with 1N ammonia solution.

The eluate is evaporated and the residue is treated with ethanol and left to stand in the cold. This divorces the homocarnosine is crystalline.

Examples of salts of homocarnosine are salts of the carboxyl group and salts of the amino group. There are also salts of both the carboxyl group and the amino group. Examples for salts of the carboxyl group are metal salts, such as the sodium, potassium, calcium, magnesium, zinc and aluminum salts, the ammonium salt and substituted ammonium salts such as trialkylamine salts, e.g., the triethylamine salt. The salts on

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of the amino group are derived from inorganic or organic acids, such as hydrochloric acid, sulfuric acid, phosphoric acid, Acetic acid, propionic acid, lactic acid, tartaric acid, citric acid, Succinic acid, maleic acid, benzenesulfonic acid and Toluenesulfonic acid. The salts can be known per se Way by reacting homocarnosine with the appropriate acid or base in stoichiometric amounts.

The examples illustrate the effectiveness of homocarnosine in the treatment of inflammatory diseases.

Example 1 The increased formation of granulation tissue by homocarnosine was determined using the usual formalin filter paper method (FFP-Mefchode) and the quantitative determination of Hydroxyproline studied. Groups of 5 or 6 week old male Wistar rats with a Body weights of around -170 S each are used. The experimental

animals were shaved on the back and under ether anesthesia a 1 cm incision was made along the dorsal midline. Filter paper was made into circles with a diameter cut from 6.7 mm. The filter paper was in 7 # aqueous Formalin solution immersed. For each rat there were four

Filter paper circles under the skin of the right and left scapular areas as well as under the skin about 4 cm below these areas, namely two filter paper discs each on the right and two on the left. the

Test animals were treated with penicillin in order to reduce the potential for disease

to suppress the possibility of subcutaneous bacterial infection. The cut was closed with clips. On the second day after inserting the soaked with formalin solution The clips were removed from the filter paper disks. On the seventh day the animals were subjected to ether anesthesia opening the carotid killed. The filter paper disks containing the granulation tissue were removed and moist

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weighed. The filter paper disks from the left shoulder blade were used for the quantitative determination of hydroxyproline .

An isotonic solution of homocarnosine was prepared and injected into the animals either subcutaneously or peritoneally once a day for 7 days after wound closure. A 15 # solution was prepared for oral administration, which was administered by means of a gastric tube. The granulation tissue was separated from the filter paper, degreased with a mixture of ethanol and ether and then converted into a gelatinous preparation by autoclaving. Concentrated hydrochloric acid was then added to the preparation and it was saponified ^{at 105 to 110 ° C. overnight.} After evaporation, the residue ⁰ C was dried at 105 to HO. The product was mixed with 0.02N hydrochloric acid and the hydroxyproline content was determined quantitatively with an automatic amino acid analyzer (Hitachi-835) to determine the amount of collagen formed

to estimate.
20th

Table I.

Effect of homocarnosine on the formation of granulation tissue (FFP method)

* Λ Hydroxyproline wet weight

Homocarnosine ^^ (# of control)

100 (20) 100 (9)

3 $ 6.1 ± 5-6 (18) 97-8 ± 10.1 (7)

10 121.3 ± 5 * 7 ^a (.20) I27.6 ± 14.5 (9)

30 I26.6 V 7.3 ^ 19J 123.9 ± 8.0 (8)

50 133.7 +, 7-2 (20)

70 13I-7 + 7-8 (24)

~ ".

'. 90 I35-6 + .7.2 £ 20)

Note: homocarnosine, mg / kg i.p. a ρ <0.01 b ρ ^ 0.025 () number of Gramilome

Example 2

Surgical wound healing by homocarnosine was as follows examined. 6-week-old male Wistar rats, each weighing approximately 170 g, were used as Experimental animals used. The rats were covered on the back shaves a larger area. Then, under ether anesthesia, an incision of about 4 cm led. Penicillin was given to suppress the possibility of bacterial infection. The cut was sutured evenly in three places with surgical suture material.

An isotonic solution of homocarnosine was prepared, applied evenly daily in 8 places on both sides of the incision was injected subcutaneously for 7 days. Physiological saline solution was used as a control. The threads were after 4 days away, and the test animals were sacrificed on the seventh day under ether anesthesia. Three pieces of dorsal skin from each 3 cm length and 1 cm width were prepared so that the incision line was in the center of the skin flap. One side of each skin flap was pinned and the other pulled. The force (tensile strength) required to separate the skin at the seam was determined. 30th

¹ Table II

Effect of homocarnosine on surgical wound healing

\ Tensile strength number of # of

Homocarnosine 'T + standard skin flap control error

454.4 4-29-9 17 100 0.06 436.8 + 27.6 ·. 18 96.1

0.6 - 493-3 + 27.5 15 108.6: -. ' ■ ■ ■. -

6.0 ·. . ·· ._. 539.6 + 41.5 18 .. II8.8

Note: 'mg / rat s.c. a ρ <0.01

Discussion of the test results:

In Table I are the results of the granuloma test after

FFP method summarized. Homocarnosine in an amount of 20th

10, 30, 50, 70 and 90 mg / kg (i.p.) are effective in formation of granulation tissue. The quantitative test for hydroxyproline also shows that its amount increases with Weight amount of granulation tissue increases. In Table II are the effects of homocarnosine on surgically placed Cuts summarized. The effectiveness is shown by the increased tensile strength of the formed Scar tissue on the seventh day after subcutaneous administration of 0.6 or 6.0 mg homocarnosine / rat.

The inflammation of internal organs is named after the afflicted Organ and its pathological condition. The disease is essentially based on inflammation of the connective tissue in these organs. The beneficial effects of homocar- nosin is unspecific for a particular organ. If homocarnosine in sufficient concentration the diseased area

the inflammation begins to heal. That's why Homocarnosine can be effective for the following diseases, for example: corneal injuries, conjunctivitis, wounds of various types (surgery), wounds after tooth extraction, Aphthous ulcers, angulus infectiosus, allergic rhinitis, tonsillitis, inflammation of the respiratory tract, burns, Herpes zoster, decubital ulcers, eczema, allergic dermatitis, ulcers of the stomach, duodenum, des Small intestine, large intestine and rectum, archosyrinx, hemorrhoids and ani ulcer, cervical erosions, ulcerations of the Vagina and external genitalia as well as herpes and ulcerating atheromas.

Homocarnosine works equally well with both oral and parenteral administration. Therefore it can be used, for example, as an injection preparation, Powder, granules, tablet, capsule or tablets or capsules with an enteric coating, ointment, Paste, suppository, enema, inhalation preparation or lozenge can be given. Homocarnosine can either be used alone or given with other drugs. the Dose depends on the route of administration, the preparation and the type and severity of the disease.

Below are typical recipes for homocarnosine supplements given.

Preparation dosage and administration

Injection preparation 0.5 percent homocaroosin I <ö sung is given in a dose of

0.1 to 1.0 ml are given in each of the diseased areas.

Suppositoryen suppositories contain 20 mg

Homocarnosine. The suppositories are in the diseased area introduced.

Ointment An ointment with a homocarnosine

Salary of 1 # is given to the sick Area applied.

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Homocarnosine is practically non-toxic. It is easily soluble in water. Therefore, aqueous isotonic solutions can easily be found with a content of $ 0.3, $ 0.5 or $ 1.0 under aseptic conditions. The solutions can be found under be filled into ampoules with a protective gas. Freeze-dried homocarnosine can be supplied in ampoules under aseptic conditions bottled and isotonic to a 0.3 »0.5 or 1, oprosentigen shortly before the injection. Solution to be prepared. Homocarnosine is produced according to conventional pharmaceutical methods either in the form of a powder, granules, a tablet or a capsule processed for oral administration. Examples of excipients are Binders such as syrup, gum arabic, gelatin, sorbitol, tragacanth or polyvinylpyrrolidone, excipients such as lactose, corn starch, calcium phosphate, sorbitol or glycine, lubricants, such as magnesium stearate, talc, polyethylene glycol, hydroxypropylmethyl cellulose or silica, disintegrants, such as potato starch, and wetting agents such as sodium lauryl sulfate. Tablets can be coated in the usual way. Ointments are usually made by mixing fine

powdered homocarnosine in the desired concentration made with an ointment base. Examples of ointment bases are bleached beeswax, Waalwax, anhydrous lanolin, white petrolatum, higher alcohols, macrogols or plastibase (a hydrocarbon gel ointment base). Furthermore, hydrophilic ointments, water-absorbing ointments or mixtures thereof can be produced. If necessary the medicinal products may contain an oil, such as sesame oil, peanut oil or olive oil, a resinous material, glycerin, propylene glycol, a wetting agent, a germicidal agent, a fungicide or an antioxidant can be added. For the production of homocarnosine ointments, the mixture is kneaded evenly. Homocarnosine suppositories are in nigh made in the same way as the ointments. For example, suppositories are made by adding antiseptics and homo-

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carnosine to the melted suppository base, thorough mixing, pouring into molds and removal of the suppository

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sitoria obtained from the mold after solidification.

Several recipes for processing homocarnosine are given below.

Preparation 1 (injection solution)

Under aseptic conditions, homocarnosine is dissolved in 0.3 percent, 0.5 percent or 1.0 percent aqueous isotonic solution dissolved. The solutions are in ampoules from 10 filled.

Preparation 2 (granules)

A granulate is made from the following components:

Homocarnosine 0.2 ε Lactose 0.34 S. Cornstarch 0.45 G Hydroxypropylmethyl cellulose 0.01 Pt granules 1.00

Preparation J (ointment)

A 1 percent ointment is made from the following ingredients

manufactured:

Homocarnosine 1.0 g

Hydrocarbon gel ointment base 99.0 r ointment 100.0 g

Preparation 4 (suppositories)

Suppositories are made from the following ingredients: 30 homocarnosine 0.02 g

ethyl p-hydroxybenzoate 0.00085 g Hosco S-55 suppository base 1.5 g

For the production of suppositories, the homocarnosine 35 and the p-hydroxybenzoic acid ethyl ester are finely sieved and in small portions into the melt of the suppository base heated to 50 ° S registered. It becomes a uniform

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Mixture obtained. The mixture is poured into a mold ^{at 38 ° C.} After cooling to room temperature, the form is frozen in a refrigerator. The suppository is then removed from the mold and wrapped in paraffin paper.

⁵ celt.

Below are the results of clinical studies with homocarnosine preparations in the treatment of tooth extraction wounds, Aphthous stomatitis, angulus infectiosus, herpes zoster, cervical erosion, decubitus, hemorrhoids,

Inflammation of the respiratory tract and various types of dermatitis summarized.

Clinical case # 1

A 0.5 percent homocarnosine injection solution was in

given the tooth extraction wound.

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Fail area

DoalS Table III
Post-operative pain

Initial state.

Additional

Woman, [U ■ Years .

Man, · έΠ years.

Man, JT years

k. Mrs,
. · Ό years

₅ . 'Fr.au, fj

3fl years

diagnosis

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2't h

Edema heating. £ xon

Horizontal ünpaotion

Aiveoiaires

Osteitis more traumatic

iüiocnenbruch.

Tooth root - 0.2 cyst

Aiveoiaires

Osteitis none none 'none

none none

Evaluation criteria: Pain: - no subjective pain

- It is indeed subjective pain
available, but no analgesic was needed

+ Subjective pain; it became a

Given analgesic

Wound healing: ++ Very effective compared to the usual therapy + Effective

- Ineffective

10

15th

25 30 35

Clinical case no.2

Patients with aphthous stomatitis received 0.5 percent homocarnosine solution injected.

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Table IV

At the same time

Fail range diagnostic dose ml pain effect cation term Medicare

1. Man Gingivo-buccale isolated 0.1 - ++ no 18 years connection of Γ3 aphthe

2. Ms. tip of the tongue "0.1 - ++ under 36 years

3. Woman lower surface '0.1 - ++ no 38 years of the tongue

Evaluation criterion: good (++), if the patient immediately after the injection

the homocarnosine solution were painless, also on the next ^ ι Day ceased to complain of pain, and the ulcerated area had become smaller.

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1 Clinical Pall No. 3

A 0.5 percent homocarnosine paste was used in one total dose of 0.8 g applied three times a day to angulus infectiosus (corner of the mouth).

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Case

area

Table V Simultaneous

Diagnosis dose, g pain effect ^ fion

1st wife

29 years

2nd wife

26 years

3rd man

39 years

4th wife

43 years

5th wife

33 years

6th man

36 years

to the right

right and left

Left

right and left

to the right

to the right

Angulus infektiosus 0.8x 3

"0.8 χ 6

"" 0.8 χ 3

"0.8 χ 6

"0.8 χ 3

"" 0.8 χ 3

no no no no no no

Evaluation criteria: To evaluate the effect of the treatment, those cases in who closed the wounds on the day after the homocarnosine administration, labeled ++. The cases where three more applications were required before favorable results were indicated by +.

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Clinical case no.4

5 percent homocarnosine paste was applied to herpes zoster.

Patient: Prau, 63 years old.

Area: Herpes zoster extended from the lower part of the right breast to the side of the body.

Medical history: the patient searched more subjectively after 3 days Pain advice. 1 g of 5 percent homocarnosine paste was applied to the diseased area. The pain completely disappeared within about 1 hour. The same amount was given five times a day (1 g each) applied. The blisters turned discolored and dried up and after 7 days it could the herpes disease can be considered cured.

Clinical case no.5

1.7 g suppositories each containing 20 mg homocarnosine were given to patients suffering from cervical erosion.

Table VI

case

Main complaints

Diagnosis dose effect

1. 40 years Discharge since
2 years Cervical erosion
C II 2 χ
Every day ge
bj applies 2. 38 years Contact bleeding
and discharge
for 3 years Cervical erosion
om _a 1 χ
Every day
while
7 days ge
heals
++ 3. 48 years Contact bleeding
and discharge
for 2 years Cervical erosion
c in _b 2 χ
Every day
while
7 days ge
heals
++ 4th 51 years Contact bleeding
and discharge Cervical erosion
C IH ₀ 2 χ
Every day
while
14 days ge
heals
++

^Γ . ι ₉ . 34276 1

1 Notes:

1. The cytological findings were made according to the following classification:

Class I: absence of atypical or abnormal cells Class II: Atypical cytology, but no evidence of

malignancy

Class III: cytology spectrum, but no clear indication on malignancy (Class III is further divided into Class III, Class III, and Class UI-. Class III_ is rather benign,

while in class III a malignancy

thought obvious. Class IH ^ lies in between.

2. In all of the above clinical cases the result of the biopsy showed that no malignancies

ity existed.

3- The following criteria were used to evaluate the effect of ⁵

Homocaraosine uses: ²⁰ - no improvement

+ no discharge and no contact bleeding in the eroded area of the cervix. The whole area has returned to normal.
++ The macroscopic examination and the histology

confirm that the patient has healed completely (Class I). No discharge and no contact bleeding. Normal vagina with no further complications.

Clinical case # 6

l # ige Hom applied.

Oily homocarnosine ointment was applied to a decubital ulcer

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- 2Ό -

case

Area (length x width)

Table VII

Period from the onset of the disease

dose

effect

1st man

move

45 years (2 χ 4 cm)

2nd man

30 years (2x3 cm)

6 months

3 months

0.5-1.0 g ++ once a day for 4 days 0.5-1.0 g ++ once a day during days

Evaluation criteria: - no effect

+ effective ++ very effective

Clinical case no.

1.7 g heavy suppositories with a content of each 10 mg homocarnosine was given to patients with hemorrhoids.

case

Table VIII Main Complaints Diagnosis Dose Effect

1st man pain and biurectal 1 χ ++ 29 years of age bleeding daily during bowel movements *

for 1 year during

4 days

2nd man pain and internal bleeding 1 χ ++ 40 years with stool haemorrhagic daily for 3 years rhoids during

14 days

Note: * given together with 0.5 percent homocarnosine

Ointment. Evaluation criteria: - ineffective

+ effective ++ very effective.

- 2T- '

1 clinical case no.

0.1 percent homocarnosine solution in physiological saline solution was given to patients suffering from inflammation of the respiratory tract. The solution was found in the respiratory

5 nebulized area

case

Table IX
Diagnosis Fogged crowd effect

1st wife

10 23 years

2nd wife

41 years

3rd man

32 years

4th wife

15 28 years

5. ViBTlT)

25 years

allergic rhinitis

0.8 ml χ 2 1.0 ml χ 1

inflamed respiratory tract

inflamed 1.0 ml χ 3 respiratory tract

Tonsillitis 1.0 ml χ 3

asthma

1.0 ml twice ++ daily for 10 days

20 evaluation criteria: - ineffective

+ effective ++ very effective

Clinical case Kr. 9

25 0.5 percent homocarnosine ointment was given to various patients Types of dermatitis given

1 Table X

Case diagnosis dose effect

1. Woman allergic 1.0g twice a day ++ 27 years of age dermatitis

2. Man gerxatric 0.5 S ^before sleep. ++ 70 years of itching A partial ulcer

recovery was observed after one application.

3. Man eczema 1.0 g twice a day ++ 4-1 years for 2 days

4 ·. Man burn 1.0 g once a day ++ 25 years of the left loaned for 4 days

Applied from the back of the hand to the back of the left hand.

Evaluation criteria: - ineffective 15 + effective

++ very effective

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Claims (1)

Claims My homocarnosine and its salts for use as therapeutic agents. 2. homocarnosine and its salts according to claim 1 for promoting the healing of wounds, ulcerations and inflammatory processes. 5. Medicinal products containing homocarnosine or its salt and customary carriers and / or auxiliaries and / or diluents. 4. Medicines to promote the healing of wounds, ulcerations and inflammatory processes, containing homocarnosine or its salt and usual carriers and / or auxiliaries and / or diluents. 5. Medicament according to one of claims 3 and 4- in the form an injection preparation, granules, an ointment or a suppository. L J