NO138852B - PROCEDURES FOR THE PRODUCTION OF A NEW HORMONE, LITORALON - Google Patents

Description

The present invention relates to a method for the production of a hitherto unknown hormone produced by the parathyroid gland, called litoralone.

One of the parathyroid hormones, the so-called

parathormone (molecular weight 8,500) with an elevating effect on the blood concentration of calcium, is frequently mentioned in the literature of recent years.

(The Journ. of Biol. Chem. 236 : 759, 196l. Rasmussen et al.:. Isolation and Cha raet erization of Bovine Parathyroid Hormone; Biochemistry 5 : 344, 1966. Hawker et al.: Further Studies on

the Isolation and Cha raeterization of Parathyroid Polypeptides;

Calc. Pee. Res. 4 : 67, 1970. O'Riordan et al.: The Properties

of Porcine Parathyroid Hormone; Annals of Int. Med. 70 : 1243,

1969. Aurbach et al.: Polypeptide Hormones and Calcium Meta-

bolism; Hoppe Seyler's Zrscht. for Phys. Chem. 351 : 1581, 1970.

Nial et al.: The amino acid sequence of Bovine Parathyroid

Hormone; Pro. Nat. Acad. Pollock. (USA) 67 : 1862, 1970. Brewer et

al.: Bovine Parathyroid Hormone: Amino Acid Sequence; Pro. Nat.

Acad. Pollock. (USA) 68 : 63, 1971. Potts et al.: Synthesis of the Biologically Active N-terminal Part of Parathyroid Hormone).

This hormone has a polypeptide-like structure. forward

position takes place by extraction of defatted, finely divided parathyroid glands with hot hydrochloric acid or phenol, or with water at a pH value between 6.5 and 7.5 (BDR explanatory note 1 193 64l).

An impure product is obtained from the extract which at a pH of

4.8 is precipitated by numerous fractionations with a solution

agent and by salting out and precipitation with trichloroacetic acid." The chemically pure calcium-active parathyroid hormone, or its active isohormone, can be produced by complicated gel filtration technology.

The basis of the present invention is the

ruling of the parathyroid gland in addition to those already

known hormones contain one or more substances with a hormonal effect, which are significantly different from the previously known substances. In this description, the new hormone is called "litoralon".

The chemical structure of litoralone has not been clarified. The

is not unequivocally clear whether this concerns a single compound or a group of compounds, or a more physical or physico-chemical modification of a single chemical compound.

The new substance with a hormonal effect is calcium-inactive, i.e. unlike the previously known hormones, it does not affect the blood calcium concentration in either a positive or negative direction, but it increases the blood's silicon content.

Biologically, the highly purified fractions already in a rather weak concentration (below 1 ug per kg body weight) exhibit a broad pharmacological and therapeutic effect which will be described in more detail later.

The new hormone, litoralone, which is produced according to the invention, is an oligopeptide. It is water soluble and insoluble

in apolar solvents, gives no feigning with trichloroacetic acid, it contains a peptide bond, the ninhydrin reaction is positive. Litoralon is hydrolysable with acids and bases. Characteristic groups are the carboxyl and amino group and a group with an acidic character, which has not yet been identified. The substance has an acidic character, at 1500 V voltage and pH 1.8 it migrates during the electrophoresis in the direction towards the anode. It certainly contains serine and aminocarboxylic acids bound peptide-like. Molecular weight: 500-2000. Litoralon also exerts its effect by oral administration. Elemental composition C, N, O, H and S and/or P.

The molecule consists of at least 5 and a maximum of 15 fragments.

Phosphorus is therefore not excluded, as the unknown group with an acidic character can also be a phosphate group.

The most characteristic pharmacological effects: Weight increase of Harder's glands in rats (increase in function), increase in the concentration of the trace elements Si, Cu, Zn and Mn in the blood.

Blood sugar-lowering effect. The blood calcium level is not affected.

AMP and phosphate-depleting effect in the urine.

Litoralon prevents or reduces the symptoms of experimental lathyrismus and fluorosis.

The most characteristic clinical effects;

Litoralon reduces the symptoms of Rhinolaryngo-pharyngitis, Sjøgren syndrome, Periodontitis, Mucovisci dose, Vitiligos and radiation damage.

The invention relates to a method for the production of a concentrate containing the new hormone litoralone which is produced by the parathyroid gland, in which parathyroid glands isolated from mammals are dried, minced and possibly defatted, and the product thus obtained is extracted with an aqueous solution, preferably with water, and the method is characterized by the fact that egg whites and polypeptides with a molecular weight above 1000 are first removed from the extract, preferably by fining with trichloroacetic acid, and then the excess fining agent is removed and the crude concentrate obtained is optionally purified. Cell cultures derived from mammalian parathyroid glands are also suitable as starting materials.

In known extractions of biologically active substances from parathyroid glands, they are not extracted with water alone, and it will already be understood from this that the product obtained from the method according to the invention is different from the known ones. Regarding the relationship between litoralone and the substances extracted by the known methods, it should be briefly added that a method for the production of liquids containing hormones from parathyroid glands is known from Danish patent document no. 48044; the active principle is extracted with aqueous alcohol acidified to pH 4.6-5; it will be seen from the above that in the present method extraction is carried out in a neutral medium. US Patent No. 1 680 660 relates to a method for the production of a concentrate of the parathormone which raises the calcium content of the blood and prevents tetany symptoms. The extraction takes place with an aqueous solution of metaphosphoric acid followed by neutralization, filtration and removal of inorganic salts. Litoralon is calcium inactive.

US Patent No. 1,890,851 describes recovery of the parathormone by hot, acidic extraction. US Patent No. 3,256,151 relates to the production of the hormone calcitonin from glandular tissue from the parathyroid gland. The hormone lowers the blood's calcium content and thus partially has the opposite effect of the parathormone. It has later been shown that calcitonin, which has a polypeptide character, is formed by the C cells in the thyroid gland, not in the parathyroid gland. Calcitonin can be easily distinguished from litoralone on chemical criteria, i.a. it can be combined with trichloroacetic acid and other protein precipitants, and it has a higher molecular weight than litoralone. Litoralon, on the other hand, can be recovered by water extraction at a neutral pH value.

The concentrates produced by the present method (aqueous solutions or lyophilized powders) are well suited for the production of pharmaceutical preparations, which exhibit the pharmaceutical or biological effects described below.

More active concentrates can also be produced, with the aim being a product that contains the active compound in a more concentrated or purer form, and/or that the accompanying calcium-active hormone is deactivated. The cleaner products are better suited to the manufacture of pharmaceutical preparations both from an aesthetic point of view and from a dosage point of view (the cleaner products give a more unambiguous dosage).

As a precipitating agent for egg whites, e.g. trichloroacetic acid or sulfosalicylic acid is used. Removal of the egg white substances can also be done with the help of solvents and by salting out. Precipitation with trichloroacetic acid has been found to be most convenient and successful. A disadvantage, however, is that it is relatively difficult to remove excess precipitating agent. This can conveniently be done by serial extractions with ether, or also by heating the aqueous solution freed from egg whites (experience has shown that litoralon is not particularly sensitive to heat). In this way, 90% of the trichloroacetic acid has been split. (When heated, chloroform and C02 are formed).

It is also known that separation of egg whites, polypeptides and other fractions is carried out with good results by means of gel filtration.

In this case, the desired substance can easily be obtained in a relatively high degree of purity in the corresponding fraction. Purification by gel filtration of the aqueous solution after removal of egg white is particularly suitable, where a littoralone fraction of relatively high purity can be recovered from a narrow fraction on a column with high fractionation capability, possibly by repeated gel filtration.

The concentrate of active substance can also be purified in other ways, for example by chromatography, dialysis, extraction, precipitation, adsorption or by combinations of these methods.

For the destruction of the calcium-active parathormone, the most appropriate method is to treat with compounds that easily react with egg whites, respectively polypeptides.

For this purpose, nitric acid, ethylene oxide,

formalin etc.

As previously stated, the various details concerning litoralon have not been clarified. It has been established that different active "litoralone fractions" can be separated by different chemical and physiochemical treatments. thus different fractions can be obtained by precipitation of solvents, different chromatographic methods, gel filtration, dialysis, extraction, fractional salting out, treatment with ion exchangers. For finer separation, combinations of these methods can be used.

Litoralone or litoralone fractions produced according to the present invention can be used as an active ingredient in therapeutic and/or preventive preparations. The amount of active ingredient can be varied within wide limits and depends on the stated conditions. The amount of active ingredient can be 0.05-10 mg per dose. These preparations are produced according to known methods from litoralone-containing solid or liquid concentrates, respectively. mixtures, in the form of tablets, suppositories, solutions, ointments, powder mixtures or sprays.

With preparations containing litoralone, extensive, clinical-therapeutic investigations were subsequently carried out. The preparation was generally administered orally in doses of 3 times 0.1 -

0.5 mg litoralone concentrate. Harmful side effects with a patient material of 3,000 were practically not observed, »in some cases, however, an urticaria-like rash on the skin surface was noticeable, resp. nausea and vomiting. This occurred in a maximum of 5% of the treated patients.

Characteristic of the new connection, resp. compounds with hormonal action, the following physiological effects are:

a) Pharmacological effect

Blood serum concentration of calcium is practically not

affected. Increase in blood silicon concentration and changes in the concentration of other important trace elements (e.g. copper, fluoride, etc.).

The new substance causes phosphaturia and an increase in organic monophosphate and cyclic monophosphate concentration (cyclic AMP) in urine.

Lowering blood sugar concentration.

Effect on blood coagulation factors, increased lypolysis.

Impact on the hypothalamic-pituitary-adrenal system.

(Production of an adoption syndrome resistance state: Increase in serum cortisone concentrations, sensitization of the adrenal cortex to ACTH, protection against shock from overproduction of steroids by the adrenal cortex, inhibition of endogenous ACTH release by the pituitary gland, protection against adrenal cortical hypertrophy from sustained ACTH dosing).

With chronic (annual) dosing, the zonaglomerulosa hypertrophies slightly, the zonafasciculata, on the other hand, shows a slight hypofunction. This has been proven by special histological examinations. The myeloid and erythroid substance ratio in the bone marrow is increased.

Repetition of mesenchyme-activating effect: stimulation of growth and proliferation of fibroblasts, build-up of connective tissue substance and fibrils of the phase system, delayed wound healing which is prevented by cortisone treatment.

Increased concentration of glycoprotein and seromucoid content in serum.

Impact on the healing of bones and bone substance

and in smaller concentrations activation of osteoblasts and chondro-scythes. It is necessary for the physiological function of bone matrix cartilage as well as tendons in bands and synovial membranes. In the case of an intracellular effect, it increases the electron density in the Golgi apparatus and the endoplastic reticulum in cells that build up bones and cartilage. It prevents experimental lathyrism. Lowering the serum citrate concentration and lowering the alkaline phosphatase concentration.

It protects the organism against harmful effects from electro-magnetic radiation. It causes faster regression of skin erythema, and significantly increases the lifespan of rats irradiated with lethal doses of X-rays. Reduction of the mitosis-inhibiting effect of X-rays on bone marrow and myelocytes. It not only has a prophylactic, but also a therapeutic effect on radiation damage.

Considerable increase in weight of the rat's Harder glands,

which glands contain the extra retinal photoreceptors in the rat.

Influence on the biological-energetic effect of light (solar) best rawling. Increased concentration of C, , labeled glucos-amines in deep-lying (metular) kidney tubules, and pronounced increase in mucopolysaccharide concentration in kidney tissue. Increased diuresis.

Substantial reduction of histamines' lowering effect on rectal temperature and regulatory effect on arteriovenous anastomoses.

It affects the endothelium of the vein wall, respectively mucopolysaccharide synthesis in the intima.

b) Clinical effect

The results of investigations at 7 different Hungarian university institutes can be summarized as follows: 1) Litoralon had a good therapeutic effect in 63% of the cases of diseases of the locomotor organs that respond poorly to treatment with the commonly used drugs. 2) Litoralon is suitable for the symptomatic treatment of sicca syndrome of any origin in the upper respiratory tract. In the non-constitutional forms of the disorder, i.e. sicca symptoms accompanying infections, tracheostomy, the effect begins after a relatively short time. In constitutional sicca syndrome beginning at a young age, chronic treatment is required to maintain a symptom-free state. In the case of ozaena, treatment with litoralone, even in the most severe forms, leads to relief of suffering and improvement of the objective condition. 3) Litoralon is effective in subacute and chronic diseases of the respiratory organs. Oral treatment with 5 mg is well tolerated. 4) The localized form of scleroderma responded favorably to litoralon, while the systemic form showed a poor response. In more than half of the vitiligo patients, repigmentation occurred to a degree that has not been observed with other drugs. 5) In mild forms of xerophthalmia, litoralon had a beneficial influence. In true sicca cases, litoralone can be used as substitution therapy, but after the end of the treatment, the symptoms return after 1 week to 1 month. Changes in the general symptoms, e.g.

Sjøgren's syndrome does not always run parallel to the ocular condition. Iatrogenic xerophthalmia caused by steroid eye drops and ointments improves more quickly if, after the end of the treatment, litoralone is used in parallel with causal therapy than if, after the end of the steroid treatment, causal therapy is used alone. There appears to be a causal link between litoralone treatment and xerophthalmia. Gastric side effect occurs. 6) The radioprotective effect of litoralon could be demonstrated by measuring local dermal reactions that occurred at reduced intensity and were restored in a shorter time. 7) In the case of periodontal atrophy of the 1st and 2nd degree, an optimal effect was achieved after 1 month of treatment consisting of a 50% reduction in the looseness of the tooth attachment.

The pharmacological and chemical investigations have so far been carried out at 8 university teaching sites, state institutes and pharmacological factories, while the clinical investigations have been carried out systematically in 20 university clinics, state institutes and Budapest hospitals for 2 years. The therapeutic investigations of various disease patterns have so far taken place with registered medical records of ^ 000 people.

In certain disease cases, the therapeutic effect can take several weeks or months to manifest (eg Bechterew's disease), but it can also produce a relatively quick effect (eg ozena).

In certain cases the remission is persistent, in other cases strong hormone dependence occurs and if the treatment is interrupted, the patient quickly relapses Cf.eg. rhinopharyngitis sicca).

The purer fraction of hormones exhibits its therapeutic effect already in fairly small concentrations. (At a concentration of 0.001 ppm or less which corresponds to an oral dose of 1 microgram per kg of body weight).

Surprising is the wide spectrum and the apparently unrelated diseases on which litoralone has had a beneficial therapeutic effect. In this context, one can refer to cortisone and its derivatives, which have shown a therapeutic effect on hundreds of diseases. The therapeutic effect of litoralone can only be compared in its scope and importance to cortisone derivatives, and opens a new chapter in therapeutic pharmacology and biochemistry. They not only differ from cortisone derivatives in the significantly different indications (indication for dosage of the drug), these are in several cases the opposite of hydrocortisone, but also in their significantly lower toxicity and mode of action. In its generality, litoralone causes a gradually developing effect in contrast to the often rapid action of cortisones and unfolds in its field in chronic, degenerative and sclerotizing progressions and not with any beneficial influence on worsening, flaring stage of diseases. In the course of clinical use, no significantly harmful side effects were observed with continued treatment up to 1 year.

The fact that litoralone significantly affects the silicon content in the blood, and in this regulation plays a decisive role (blood concentration of silicon increased during litoralone treatment in rats and hares by more than 100$ within 30 days), draws attention emphatically to the particularly silicon has an important physiological role in the synthesis of glyco-mucoproteins and ensuring their physiological function.

As a result of litoralone research, silicic acid and its complexes are probably being raised to a high rank in our hitherto limited physiological system. (Building up of bradytrophies: Tissues with reduced metabolism, elastic connective tissues, regulation of bone formation, etc.).

The difference between the two hormones from the parathyroid gland: between parathormone and litoralon is most compactly and clearly explained by the fact that parathormone affects vitamin D characteristics, while litoralon affects vitamin A.

It seems likely that hormones with a litoralone effect are produced in the so-called eosinophilic cells of the parathyroid gland.

Example 1

10 g of lyophilized and ground gland powder was degreased in the following known manner: The powder was mixed with 25 ml of absolute acetone at room temperature and then filtered on a glass filter G^. Then the well-aspirated gland powder was again mixed with 25 ml of chloroform at room temperature, and filtered on a glass filter G^. The operation was repeated. The aspirated substance was then finally suspended in 25 ml of absolute acetone at room temperature and filtered through a glass filter G^. The gland powder obtained was dried in vacuum at room temperature. The weight of the dry substance varied between 7 and 9 g depending on the fat content of the starting material.

10 g of the defatted, anhydrous gland powder was then extracted 3 times, each time with 100 ml of distilled water, at room temperature and over the course of 1 hour. Between the extraction steps, the gland powder was centrifuged on a laboratory centrifuge at high rotation speed. The extract was stored in a nitrogen atmosphere at 3 - 5°C for further processing. The aqueous extract was ninhydrin, biuret and trichloroacetic acid positive.

The aqueous extracts were then combined and a 50 volume, aqueous trichloroacetic acid solution was then added dropwise with good stirring, until the final concentration of trichloroacetic acid was 15%. After 1 hour's rest at 3 - 5°C, the trichloroacetic acid precipitate was centrifuged off.

The upper layer was a clear, pale yellow aqueous solution that was biuret and trichloroacetic acid negative, and ninhydrin positive.

The excess of trichloroacetic acid was removed by several extractions at room temperature with an aqueous ether solution (1 -

1.2 parts by volume ether : 1 part by volume water). Usually 17 - 18 extractions are necessary to remove the trichloroacetic acid. In practice, 20 - 25 extractions are carried out.

After the last extraction, the aqueous phase must have a pH between U-.5 and 5.

By means of a water jet pump and at a temperature which

did not exceed 30°C, the ether was separated from the aqueous phase.

The removal of ether took approx. 6 hours and resulted in a yol umform reduction. (Part of the aqueous phase was also distilled off).

The removal of ether was checked by sample smelling.

The aqueous solution was then lyophilized and stored in a tightly closed vessel. The solution is hygroscopic. Yield calculated on the total gland powder (for degreasing) was 10 - \%

(depending on the fat content).

Example 2

As indicated in example 1, an extract was prepared from

an aqueous extract from defatted bisque cartilage, which extract was free of egg whites and precipitating agent. 5 g of a trichloroacetic acid inactive lyophilic batch was gel filtered on a 'Bephadex G-15<IL> containing soil. The mass of the flush was 4.3 cm by 144 cm. Water was used as eluent. The flow rate was 60 ml per hour. The fraction size was 15 ml.

Weight of the total and lyophilized sample:

The main fraction, row number 46 - 83 with, a content of 913 mg was relatively rich in litoralone and had a distinct physiological, respectively therapeutic effect at a dosage of 5 - 10 micrograms per kg body weight.

This is the substance contained in both of the first peak points during the gel filtration.

Separation of the fractions can conveniently be carried out by the following method: a) Spectrophotometric absorption measurement at 280 millimicrons. b) Spectrophotometric absorption measurement at 234 millimicrons. c) Spectrophotometric absorption measurement at 36° millimicron.

d) Measurement of electrical conductivity.

e) Qualitative examination, on chloride ion with solvnitrate reagent. f) pH measurement of the combined fractions belonging to the individual peak point.

g) Examination with ninhydrin reagent.

h) Examination with the bare bye with regard to translucency and color (yellowish).

Yield of the main fraction 46 - 83 with regard to dry, non-defatted parathyroid gland powder was calculated to be approx. 2 - 2.5$.

Example

Example 2 was repeated with the change that both of the first main quantities were taken out in separate main fractions. Thus, 302 mg in main fraction No. 1 and 555 mg in main fraction II were obtained from 5 g of egg white substance f-free aqueous extract.

Main fraction II was then subjected to gel filtration. 456 mg of substance was removed from the flask, and water was used as eluent. The flushing mass was 4.3 times 144 cm, the flow rate 50 ml per hour, and the fraction size 10 ml. Filling: "Sephadex G-15".

In this way, main faction II was divisible into 3 peaks. Factions belonging to the middle peak were united. Weight of these

after lyophilization was 237.5 mg.

Yield calculated on the basis of non-defatted parathyroid powder was 0.6 - 0.8$.

Characteristic properties: Yellow hygroscopic substance with a content of C H N 0 Sog/or P. Characteristic groups: -SH, -C00H, -0H. In a mass spectrographic examination, the following fragments were characteristic: C^H^N^S; C^H^N^O^ and C^NO.

It is assumed that a molecule has a side chain with sulfhydryl content and that it is a purine derivative.

The faction contained additional structures.

This fraction showed a physiological or therapeutic effect already at a dose of 1 microgram per kg body weight.

The product could be further purified by chromatographic or other methods.

Using a cellulase or ion exchange column, the contaminating amino acids (lysine, glutamic acid) can be removed. Thus, with a yield of 0.3 - 0.<L>>$ still contaminated litoralon can be obtained. which, however, is effective in a concentration below 1 microgram per kg body weight. Yield is calculated on tort parathyroid gland powder.

Example 4

The substance described in Example 1 was gel filtered on "Sephadex G-15" ("Pharmacia" Swedish product, particle size 90-120 microns) with descending technology.

The elution diagram was measured at 280 nm extinction. When determining the volume of fluid flow through as a function of extinction, the second peak counted from the front contains the litoralone.

The characteristic parameters of the gel filtration are the following:

Starting amount: 5.1 g of substance (Example 1).

Column diameter: 4.85 cm.

Column height: 149 cm.

Flow rate: 0.78 ml/min.

Time: 10 minutes.

Size of the fractions: 7.8 ml/rbr.

Top B contains the active ingredient.

The elution volume of 104 tubes amounts to 8l4 ml.

The total volume of top B is 188 ml.

Between peaks D and E, the chlorine reaction is positive.

The dry weight of the lyophilized top B varies between 0.43 - 0.53 g when starting from 5 g of substance.

Identification by gel filtration

After the gel filtration on "Sephadex G-15", the substance appears in the eluate at (0.67-0.02) x V , when the determination takes place in aqueous solution by absorption at 280 nm. VQ means the volume at which the salt appears in the eluate (positive chlorine reaction).

Peak B is fractionated on a "Dowex' 50x2" column at pH 1.8.

301 mg of substance in a 30$-ig aqueous solution is applied to a

132 x 1 cm "Dowex 50x2" column pre-equilibrated with a formic acid-acetic acid mixture at pH 1.8. Flow rate: 13 ml/hour. Fraction size: h ml. The fractions are determined at 280 nm. 9 elution peaks were obtained:

The majority of the active substance was in fraction E-^, but some of the active substance may also be present in fractions D^, D2 and E2.

The above fractions are applied to Whatman 3 MM paper in a width of 22 cm and subjected to electrophoresis at pH 1.8 with 1500 V

for 90 minutes.

Material application 0.5 mg/2 cm. Composition of the puffer:

h0 ml of 98% formic acid and 160 ml of glacial acetic acid diluted with distilled water to 2 litres.

The active substance is ninhydrin-positive and migrates evenly from the starting line towards the positive pole (5 cm/90 rnin/l^OO V).

The product isolated by electrophoresis is eluted with water and tested for its biological activity. Yield of the isolated product calculated on dry glandular powder was 0.01-0.02$.

The litoralon obtained in this way can be considered sorn

chemically pure, and its biological activity was proven by the following experiment:

The weight of the Harder glands in the rats was significantly increased

a dosage of 3/120 g subcutaneously. At the same dosage, the blood sugar content was significantly reduced.

At a dosage of oral tablets of 7 micrograms three times a day, the symptoms of Rhinolarryngopharyngitis sicca in humans were cured and the AMP-raen.gd.en excreted in the urine increased significantly.

Example 5

The final product obtained and lyophilized according to Example 1 was subjected to dialysis. 700 mg of suostans was dissolved in 33 ml of distilled water and dialyzed in a dialysis membrane hh. It was dialyzed against 2 times 100 and then 13 times 1^0 ml of water in such a way that the outer aqueous phase was changed every 3.5 hours. In each of the 3 intervening nights, the replacement time was 16 hours. The dialysis was carried out at 5°C.

Within the membrane remained hl ml of the Ibsningen (non-dialysing part).

Dry weight after lyophilization was 35 mg.

The outer phase was evaporated in vacuum at 3U°C and after lyophilization gave 662 mg of tdr substance.

Example 6

The starting point was 20 g of glandular powder (lyophilized, non-defatted) and following the procedure according to example 1, the aqueous solution separated from egg white and ether was evaporated to 1/3 volume, and then decanted 3 times with chloroform (1:1) after which the chloroform was removed in vacuo. The chloroform-free aqueous solution was further evaporated in vacuo to 20 ml, and at room temperature 5 parts by volume of acetone were added. The mixture was allowed to stand overnight on ice.

From the practically goodbye acetone phase separated out a

yellow oily part. The acetone phase was decanted off, and the yellow oily substance was evaporated in vacuo to constant weight. 1.1 g of a yellow, resinous concentrate A was obtained.

The acetone solution was evaporated in vacuo to dryness. 1 g of a resinous concentrate B was obtained.

Example 7

The starting point was 50 g of lyophilized gland powder which was degreased as described in example 1. 38 g of the fat-free gland powder was mixed with 400 ml of a 10% urea solution. After standing for 1 hour, the mixture was mixed with 400 ml of glacial acetic acid and 400 ml of acetone. The mixture was allowed to stand. for 1 hour, after which it was poured over with 1200 ml of acetone and 11.2 ml of N sodium hydroxide solution. the mixture was filtered through a layer of gas, to the urea- and acetone-containing extract 2 1 of ether was added. the mixture was allowed to stand overnight and then decanted. The suspension was centrifuged and washed 2 times with 50 ml of acetone-ether (1:1). The precipitate obtained was mixed with 200 ml of a 10% acetic acid solution, whereby the precipitate was dissolved. A precipitate was precipitated from the solution by mixing in 10 g of sodium chloride. The precipitate was taken up in 50 ml of water.

One dialyzed with 6 times 2 1 of water, emptied the dialysis water and lyophilized the remaining product. 732 g of a solid concentrate were obtained. After precipitation with NaCl and centrifugation in solution, the remaining substance (197 ml) was treated with 40 ml of 45% trichloroacetic acid and then centrifuged. The dry phase was dialyzed 4 times, each time with 3 1 of water, evaporated in vacuo and lyophilized. 86 g of a concentrate (substance D) were obtained.

Claims (1)

Process for the production of a concentrate containing the new hormone litoralone produced by the parathyroid gland, in which parathyroid glands isolated from mammals are dried, minced and possibly defatted, and the product thus obtained is extracted with an aqueous solution, preferably with water, characterized in that then egg whites and polypeptides with a molecular weight above 1000 are first removed from the extract, preferably by precipitation with trichloroacetic acid, and then excess precipitation agent is removed and the crude concentrate obtained is optionally purified.