DE1963326C3 - Process for the production of macromolecular bismuth complexes, as well as such macromolecular bismuth complexes and their use - Google Patents

Description

There are already orally administrable pharmaceutical preparations and processes for their production known, in which proteins are converted into various compounds with a bismuth salt, from some of which are also complex in nature. In general, the protein component is drastically reduced into fragments of much lower molecular weight reduced.

According to DE-PS 5 85 163, the protein is first drastically hydrolyzed with strong alkali and then through Hydrolysis of the protein formed organic acids are then mixed with heavy metals e.g. B. bismuth in salts implemented. The bismuth salts formed in this way are in turn further enhanced by reaction with tannin esters stabilized.

DE-PS 6 66 467 describes the production of metal compounds, ζ. Β. Bismuth compounds, through Coupling reaction between the diazotized metal compounds and certain proteins. The products are also not complexes.

According to DE-PS 5 88 710 and 6 55 684, salts of metals such as bismuth are also formed with sulphydric acid. The latter is not a protein, but an acid formed by the hydrolysis of certain proteins. Also from the German patent specification 1 01 683 the production of bismuth salts with acidic breakdown products of proteins is known.

DE-PS 2 02 955 relates to the production of bismuth salts of paranucleic acid. Paranucleic acids are not proteins but hydrolysis products of much lower molecular weight than the proteins from which they are derived.

The F ^- R-PS 5 77 407 relates to the production of bismul salts of nucleic acids. Nucleic acids are not proteins but hydrolysis products of much lower molecular weight, and the compounds formed are salts and not complexes.

The manufactured according to the above procedures

th preparations are recommended for bacteriostatic purposes, among other things. As far as is known, they have no effect whatsoever in the treatment of inflammation and ulcers, especially ulcers of the Gastrointestinal tract. None of these preparations contain water-soluble non-dialyzable bismuth protein complexes that are reversibly alcohol-precipitable and in which the bismuth is stably bound against dialytic leaching

ίο From the literature (e.g. British Pharmaceutical Codex, Born in 1929) a class of bismuth preparations is known that was previously regarded as pure mixtures Has. These are solutions of bismuth ammonium citrate or tartrate, some of which also contain pepsin and then commercially available as "Bismutammonium citrate cum pepsin" or "Mistura Dspsini et bismutni compositum sine tinetura nucis vomecae et acid hydrocyanicum «are known. Most of these preparations contain a dye like cochineal dye (carmine Preserväermiite! «glycerine resp. Sugar to improve the taste or to stabilize the solution.

Studies have shown that certain bismuth complexes des in these mixtures Pepsins can be present, but in these complex compounds the bismuth is only loosely bound and is immediately washed out by dialysis. Try to heal stomach ulcers with such preparations were inconclusive.

There are stomach or intestinal preparations that contain bismuth, among other things, z. B. Steigerwald stomach tablets (list of specialties). These exist from a mixture of magnesium carbonate, bismuth subnitrate, diethyl papaverine and papain. The bismuth Subnitrate only acts as an antiseptic, astringent and tension reliever (Merck Index 7. Edition, p. 157). Diethyl papaverine has an antispasmotic and narcotic effect. Papain works in the short term digestive. Only the proportionally

♦ ο small amount of magnesium carbonate can also be used

Ulcerations as acid binders some relief

create. In addition, the preparation is not a cure for

Stomach ulcers. In Thom's Handbook of Pharmacy Volume Vl, first

■ »5 half, p. 318 describes the preparation» bismuthose «. Further details of manufacture are the DE-PS 1 17 269 can be found. A completely water-insoluble bismuth protein bond develops. she will for the treatment of various gastric and Intestinal discomfort used, among other things, in Ulcerations. The acid binding capacity of this agent is used.

So far, the healing of gastric ulcers has only been surgically or through careful treatment with a lot Rest and strict diet possible. Basically, the stomach acid content was suppressed here.

As far as preparations containing bismuth were used, they were gastric juice-binding Means that at most contribute to relief

so can. A specific healing effect going beyond that, especially one specific to bismuth traceable ulcer healing effect, was never ascertainable.

Finally, bismuth is also known as a precipitant for proteins. In contrast, it acts

^h "> is water in the present invention to the production of bismuth protein complexes.

The invention therefore relates to a method for the production of macromolecular bismuth complexes,

15th

which can be reversibly precipitated from aqueous solution with alcohol, which is characterized in that at pH 9-10 and, if necessary, slightly warming a soluble Wistnut salt with a protein in water-soluble form and a hydroxycarboxylic acid in counter- war of ammonia or ammonium ions

The invention further relates to macromolecular bismuth complexes, which are readily soluble in water, from aqueous Solution can be precipitated reversibly with alcohol and are dialysis-resistant as well as by reaction at pH 9-10 of

a) a soluble bismuth salt,

b) a protein in water-soluble form,

c) ammonia, optionally in ionized form,

d) Malic acid, citric acid, lactic acid or tartaric acid

have been manufactured.

The solutions of these compounds can be stabilized with a glycol compound. Finally concerns the invention also relates to the use of the compounds for combating ulcers on digestive organs.

Bismuth citrate in particular comes into consideration as the soluble bismuth salt.

The protein components that can be used are those which are water-soluble and which, in particular, contain bismuth Form water-soluble complexes. Protein-like substances such as enzymes, hormones, tissue proteins and can also be used as the protein component according to the invention The like in concern. All proteins are included (regardless of whether they are partially or completely denatured, chemically modified or in some other way degraded), which are suitable for the production of bismuth complexes at a pH value of 9 to 10 in water. The water solubility also relates to colloidal solution. As water-soluble proteins come in particular consider pepsin, trypsin and chymotrypsin.

In certain embodiments, the proteinaceous substance is from the class of the spherical Proteins selected that are at least partially chemically modified or denatured or chemically degraded were to release nucleiphilic sites in the protein molecule, which then at least partially with bismuth were occupied. Among the spherical proteins that can be used in this way are also various tissue proteins, enzymes, oxygen-carrying proteins, protein hormones, proteins, globulins, histones come into consideration.

Soluble complexes of the type mentioned are also included if they are made up of protamines (salmines, see above) Klupeinen, Sturinein) were derived. There are also paired proteins, especially nucleoproteins, mucoids and mucoproteins can be used. There are also bismuth complexes with fiber proteins, such as collagen, Elastin and keratin into consideration.

Collagens can be made soluble prior to complex formation by diluting them in water Acids or bases boil. Elastins and keratins can be partially hydrolyzed to make them water-soluble. bO

The proteinaceous substance can be selected with a specific affinity for the tissue present will. For example, it can be < > ■> can be selected. So you can choose them as a bismuth component for the stomach area, with a iso-electrical point below 5, preferably below 2, better still below 1 is preferred. For this offers Pepsin is a good example.

The proteinaceous substance can also be used as an iso-electric point in the alkaline range Bismuth carriers can be selected for the alkaline part of the gastrointestinal tract. Examples of this are trypsin and chymotrypsin.

Ammonium compounds can also be used as the ammonia component.

Particularly suitable for the hydroxy acids are <x-, β-, γ- and δ-hydroxy fatty acids and 2- and 3-basic acids. Tartaric acid and citric acid are particularly preferred.

It is particularly important that the macromolecular Bismuth complex compound precipitates reversibly with the help of alcohol and that is out of the complex Bismuth cannot be removed by dialysis. After adding alcohol, practically all of it solidifies Solution.

The presence of certain dyes, such as carmine or azure dye as an additional ligand for the complex compound is not essential. These dyes can affect the stability or durability influence the complex compound in aqueous solution favorably, but are not decisive in the Medicinal effect involved.

On the other hand, it must be emphasized again that the use of water-soluble proteins or Proteins in macromolecular chemically digested form as nucleophilic ligand therapeutic is particularly effective.

The solutions of the complex compounds according to the invention can with a glycol compound such as Glycerin can be stabilized.

The bismuth compound is preferably based on a bismuth ammonium citrate compound.

The solutions of the complexes according to the invention preferably contain at least 3 mg / nil bismuth.

example 1

6000 g of bismuth citrate are dissolved using a mixture of 400 ml of concentrated (25%) ammonium hydroxide solution and 20 liters of distilled water if necessary, regulates the pH value to 9.6.

2000 g of pepsin are dissolved in a little distilled water and the solution is added to the bismuth solution

80 g of carmine dye are dissolved in the smallest amount of chloroform water, then together with 700 g of potassium citrate dissolved in 22 liters of syrup simplex and added to the previous one. The volume will made up to 80 liters, a further 900 g of potassium citrate are added, and the pH value again with concentrated ammonia adjusted to 9.6.

The solution is then subjected to the following "ripening" process to give the desired complexes to manufacture:

The ripening process is preferably carried out with brief UV irradiation (10 sec.) With the aid of a normal Laboratory U.V. lamp is initiated, followed by infrared radiation (or other gentle type of energy supply) to slowly raise the temperature of the solution to a To increase the final temperature of 60 - 65 ° C over a period of 5 hours or more, and this Temperature is maintained until a sufficient level of activity is achieved according to the following empirical test:

(i) To a small sample of the solution, add 3 volumes of pure alcohol. As soon

When a noticeable "activity" has arisen, a characteristic red gelatinous precipitate forms immediately, which sinks immediately and which can be dissolved in water as well. If the test is very positive, the next test is carried out:
(H) Leave 12 g of Sephadex G 100 (a crosslinked polydextran suitable for the separation of peptides and spherical proteins in the molecular weight range of about 4,000-150,000) in an appropriate amount of 0.05 M Tris buffer solution pH 9 for a period of 72 Swell for hours and fill a column (the dimensions should be about 35 χ 2 '/ 2 cm after filling) with swollen gel. 2 ml of the "active" solution are added and the column is developed in the usual way with 0.05 M Tris (flow rate £ 0 ml / h) in order to saturate the column. This is repeated with a further 2 ml of "active" solution until the column is saturated and the pink band can be leached and collected. Now add exactly 1.0 ml of "active" solution, develop it with Tris buffer solution, pH 9, (flow rate 25-30 ml / h) and capture the first colored band quantitatively. This fraction is diluted to an appropriate volume with distilled water, an appropriate sample of which is used for the quantitative determination of bismuth.

The preparation is suitable for the treatment of ulcers and inflammation e.g. B. the stomach wall, if a total of 3.0 mg bismuth (or more) per ml of the preparation placed on the column in the entire pink Ties are in place. In practice, values of 5.0 mg bismuth per ml or more are achieved.

The product obtained in this way represents a solution of the compound (obtained by the previously mentioned pink band, which appeared in the activity test) with some bismuth ammonium citrate and unconverted proteinaceous substance. It can be administered directly as a solution (daily Dose 4 times 5 ml half an hour before main meals and at night).

Example 2

A solution is prepared essentially as in Example 1, but instead of pepsin one is used of the following proteins with an isoelectric point in the alkaline range:

Trypsin, chymotrypsin.

A concentrate of proteinaceous bismuth complexes is made by adding four times the volume Alcohol to the solution, a precipitate of the complexes forming.

The precipitate is encapsulated, each capsule containing 50 mg of bismuth and of the type that it dissolves immediately after passing through the stomach.

The capsules are administered three times a day, with a single dose for the treatment of Ulcers on the duodenum, jejunum and small intestine, between 1 and 3 capsules.

Example 3
Comparative experiments with different proteins

Under the experimental conditions as in Example I, bismuth-protein complexes of the following proteins manufactured: trypsin, pepsin, ovalbumin, bovine serum A! bumin, gelatin, casein and papain.

The following quantities of raw materials were used:

Ammonia (25%) 17 ml, bismuth citrate 35 g, potassium citrate 15 g, protein 7.5 g

M ethyl parahydroxybenzoic acid 6.0 g

ίο N-propyl parahydroxy

benzoic acid 0.6 g

as a preservative

Amaranth sol. (Bp.) 6.2 ml, carmine dye 1.25 ml, 60% sugar solution 117 ml

Final volume 560 mL

In all cases the final pH was adjusted to 9-9.5. After the reaction, the solutions looked like this:
Clear solutions were obtained with pepsin, trypsin, serum albumin and ovalbumin *. Papain gave a slight cloudiness. Severe turbidity was noted with casein. Severe precipitate formed with gelatin. (However, complex compounds formed according to the invention also remained in solution _b -.)

10 ml each of the filtered solutions were mixed with 3 ml Ethyl alcohol (96%) added

A heavy fluffy precipitate formed immediately with pepsin and quickly settled. With trypsin and Papain initially resulted in a heavy, whitish-reddish cloudiness, which then settled as a precipitate, (but slower than in the case of pepsin). With serum albumin and ovalbumin there was also one fine whitish-reddish precipitate which settled even more slowly than in the case of trypsin and papain.

In the case of casein and gelatin, there was one

white milky cloudiness, which occurs in the case of casein settled as a heavy white precipitate after 24 hours. In the case of gelatine, the Precipitation not settled even after 24 hours. All precipitates were soluble in water.

Electrophoresis

The Beckmann microzone electrophoresis system with cellulose acetate membranes was used for this comparative experiment In all cases, protein fractions containing bismuth (i.e. complex compounds) were used found whose mobilities and isoelectric points were clearly different from those of the Starting proteins. With the exception of the protein complexes migrated under the experimental conditions dof trypsin complex connection to the anode. The trypsin complex wandered to the cathode.

Animal testing

Eighty young adult rats were matched in 8

ω groups distributed, one for each of the seven preparations and one as a control group. After folding for one day, gastric ulceration became apparent in all of the test animals with phenylbutazone (twice on the same day

administered under light ether anesthesia) induced

The test preparations were all diluted in the same way with water, and three times every six hours administered (1 ml of the undiluted preparation per animal). The first application took place two hours after

the last phenylbutazone treatment. The control animals received the corresponding one instead of the preparation Amount of saline solution.

All animals were sacrificed 24 hours after the last administration of the preparation. The stomachs were removed, rinsed thoroughly with saline and with 15 ml IO% formalin under slight pressure filled and bloated. The organs that had been pretreated were examined for ulceration under strong light examined and the degree of ulceration recorded according to the following scheme:

Ulcer index

I to 3
4 to 6
7 to 9
10

Injuries

no injuries
bloody spots
small ulcers
large ulcers
Perforation

According to the above scheme, the following ulcer indices were determined for the individual test groups:

Protein used Ulcer index pepsin 2.4 Ovalbumin 5.1 Bovine serum albumin 4.1 gelatin 7.0 casein 5.9 Trypsin 2.9 Papain 3.1 control 62

Therapeutic effectiveness

Long-term clinical and laboratory tests had shown that the phenylbutazone induced in rats -to Gastric ulcers do not allow absolute conclusions to be drawn about the therapeutic effectiveness in humans allow the values obtained in this way to provide very useful comparative values. Based on these Experience can clearly be derived as follows:

a) All preparations are therapeutically effective against ulcer formation in the stomach.

b) There is a clear connection between

(i) Water solubility of A;: gang protein,
(ii) water solubility of the compound compound,
(iii) therapeutic efficacy.

c) The proteins were deliberately chosen so that that different protein classes as good as possible

r, were represented. Are pepsin, papain and trypsin

Enzymes and representatives of the group of spherical proteins. They are particularly water-soluble and result in particularly effective preparations. Also the Albumins (ovalbumin and serum albumin) are spherical proteins and are easily soluble in water. These too have a fairly high therapeutic effectiveness. Casein represents both that Group of globulins as well as that of conjugated proteins. Its water solubility is essential lower and, accordingly, the therapeutic effectiveness is also lower. Gelatin is a Ki.-ilagen and falls into the class of fibrous Proteins. These are almost insoluble in water. If one first determines the water solubility by means of

jo table pretreatment is also here with a correspondingly improved effectiveness

In all cases (with the exception of the control experiments) it was found that the stomachs of the test animals had taken on a darker color (bismuth precipitate). This bismuth precipitate was much stronger in the peripheral areas of the ulcer formation. The observation corresponds completely to the clinical observations on human patients.

Claims (4)

Patent claims: 1. Process for the preparation of macromolecular bismuth complexes, which from aqueous solution with Alcohol can be precipitated reversibly, characterized in that at pH 9-10 and possibly slight heating a soluble bismuth salt with a protein in water-soluble Reacts form and a hydroxycarboxylic acid in the presence of ammonia or ammonium ions 2. Macromolecular bismuth complexes, which are easily soluble in water, from aqueous solution with alcohol are reversibly precipitable and dialysis-resistant, as well as by reaction at pH 9-10 of a) a soluble bismuth salt, b) a protein in water-soluble form, c) ammonia, optionally in ionized form, d) Malic acid, citric acid, lactic acid or tartaric acid have been produced. 3. Solutions of the compounds according to claim 2, characterized in that they are stabilized with a glycol compound. 4. Use of the compounds according to Claim 2 for combating ulcers on digestive organs.