DE2335102A1 - PROTEIDS OF BIOLOGICAL ORIGIN, METHOD OF PREPARATION AND PHARMACEUTICAL COMPOSITIONS CONTAINING THESE COMPOUNDS - Google Patents

Description

Dr. F. Zumsteln sen. Dr. E. Assmann Dr..R. Koenlgsberger - Dlpl.-Phys. R. Holzbauer - Dr. F. Zumsteln Jun.

PATENT LAWYERS

TELEPHONE: COLLECTING NO. 225341 8 MUNICH 2, TELEX 529979 BRÄUHAUSSTRASSE 4 / III

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Cas 14O9 / i / d -

ROUSSEL-UCLAF, Paris / France

Proteins of biological origin, processes for their preparation and compounds containing these pharmaceutical compositions

[Addition to patent (patent application P 21 40 353.2)]

The present invention is an addition to that

DT-PS , (patent application P 21 40 353.2) represents the one

Glycoprotein from animal or human serum or from the placenta, which has an isoelectric point of 4.0-0.2 as determined by electrofocalization.

This glycoprotein has a complexing effect on circulating amines and especially a histaminopexisehe Activity.

This glycoprotein is also isolated by its action on the Guinea pig ileum labeled. A unit of effect is expressed as the minimum amount of pure or mixed glycoprotein that, dissolved in 1 ecm of physiological solution according to Tyrode, the contracting effect of histamine on the isolated

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Guinea pig ileum decreased maximally.

Contain the purest fractions containing this glycoprotein 500,000 units piomg.

9 One histaminopexic unit corresponds to 2 nanograms (2 χ 10 g) this glycoprotein.

As in the main patent. (Patent application

P 21 4O 353.2), there was great uncertainty with regard to the type, identity and uniformity of the serum factor capable of binding histamine This main patent described invention is based on the • The fact that new isolation and purification procedures allowed to obtain a fraction responsible for the phenomenon and in a very pure form.

It was also shown in the main patent specification that this Extraction and this purification can be applied to serum proteins of animal origin as well as to the placenta, so that the spectrum of compounds to be used as starting material has been considerably expanded.

The invention was further based on the fact that the obtained, histaminopexically acting fraction other than globulins, immunoglobulins and other proteins chemically defined class could be identified.

The analyzes and determinations carried out have shown that the serum factor responsible for the binding of histamine is a glycoprotein.

The invention was also based on the fact that the fraction obtained is free from any antigenic factors that may arise due to the presence of other proteins or proteins of heterogeneous origin.

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In this cleaning condition could zusammerifassen the physico-chemical properties of this glycoprotein which, to distinguish it ge _r endowed this material previously found:

The material is soluble in O, ln-phytic acid solutions and possesses thus a sour character;

the optimal range for precipitation by ammonium sulfate extends with a p "value of 7.0 from 2.5 m to 2.8 m. This extremely narrow range indicates that the compound obtained is not an immunoglobulin as it is at much lower levels molar concentrations of ammonium sulfate precipitate;

the molecular weight determined by filtration through Sephadex G 200 is slightly larger than that of an immunoglobulin;

the electrophoretic mobility on agarose, cellulose acetate or a sucrose gradient is similar to that of one Prealbumin;

Finally, electrofocalization allows the isoelectric point to be fixed at very close to 4.0.

The present invention relates to histaminopexic glycopro- Teins from human or animal serum or from the placenta, which determines an isoelectric point of 4 i 0.2 by electrofocalization, and which are characterized in that they contain at least 10 histaminopexic units per mg included.

The invention also relates to histminopexic glycoproteins of human origin, which are additionally characterized in that they contain about 56 % proteins, 17% hexoses, 1.3% fucose, 14% osamines and 15% terminal sialic acid _{and have} a molecular weight of about 110,000 .

The present invention also relates to a histaminopexic Glycoprotein of animal origin, which in addition

characterized in that it contains about 47% proteins and 16% hexoses and has a molecular weight of about 120,000.

The protein content is determined by the biuret reaction according to the method determined by Gornall and by Lowry's colorimetric method.

The elemental analysis, on the other hand, allows the glycoprotein content on carbon, hydrogen and nitrogen.

The hexose content can be determined using the method with 5-MethylresDrcin compared with a calibration standard, which can be determined with the aid of a galactose-mannose mixture (2 parts by weight / l part by weight ) was determined.

The content of a specific sugar of fucose and of osamines was also determined according to the method of Elson-Morgan and determined at the terminal sialic acid.

It is also possible after hydrolysis with hydrochloric acid the. Carry out analysis of the amino acids present in the polypeptide chain of the glycoproteins and their distribution determine.

The molecular weight is determined using a concentration gradient over methylene bis (polyacrylamide) gel compared to a comparative albumin of known molecular weight.

The glycoproteins of human or animal origin that are able to block histamine differ chemically insignificant, although they show certain parallels in terms of their constituent elements.

The antigen / antibody reaction is very slight and suggests an identity with regard to the immunological effect of the

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close both glycoproteins.

The glycoproteins of the present invention have the same pharmacological properties Activity for histamine-induced contraction of the isolated guinea pig ileum

like the one in the main patent specification. »(Patent application

P 21 40 353.2) described connections. The difference is that the effect is considerably stronger.

The invention also relates to the use of the glycoproteins mentioned, that are able to block histamine as drugs. They find use in therapy, in particular in that they produce counter-effects against pathological phenomena caused by histamine, such as nettle rash, hay fever and various allergies.

They can also be used to combat disorders that are a consequence of the excess of circulating biological Amines, such as the catecholamines, are. Because of their strong effect and their great purity, they make the medicine of choice.

The invention also relates to pharmaceutical compositions which contain the defined glycoprotein as an active ingredient a pharmaceutically inert binder suitable for parenteral, rectal, topical or permucous administration contain.

To do this, they are in the form of injectable solutions or suspensions, in the form of suppositories, sublingual tablets, the Skin-penetrating ointments or aerosols.

The dosage ranges depending on the therapeutic Indication and route of administration.

The invention also relates to a method for producing the glycoproteins according to the invention which are capable of histamine

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- b

to block, which is essentially characterized in that serum proteins of human or animal origin or placental origin are subjected to diafiltration over a membrane based on non-ionic polymers with selective porosity, the protein fraction retained in the diafiltration cell is separated off and chromatography over carboxymethyl cellulose gel at a _pH value in the range of 4.2 cleans and separating the non-retained fraction from the column.

The method of the invention is further through the following preferred embodiments:

1) As a membrane based on non-ionic polymers

with selective porosity is used under the designation

AMICON XM 100 A or AMICON XM 300 from AMICON Corporation, Lexington, Massachusetts / USA.

2) The carboxymethyl cellulose gel is previously in particular with Using an acetate buffer (aceto-acetic acid buffer) with a p "value set from 4.2 to a p" value that is slightly higher than the isoelectric point of the histaminopexic Glycoproteins.

3) The starting material can be a human or animal serum Origin or a serum placental origin or can also consist of serum proteins that were previously carried out by Precipitation with ammonium sulfate and redissolution obtained and are present in the supernatant after phytic acid precipitation.

4) These cleanings can also be carried out using other cleaning methods can be combined, such as purification by electrofocalization and by chromatography over Sephadex gel (chemical cross-linked dextran).

The following examples are intended to explain the invention further without, however, restricting it.

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example 1

Production of histaminopexic glycoproteins from Pig serum

A. Salting out with ammonium sulfate

. One dilutes 12.5 l of serum with an equal volume of one
0.4% sodium chloride solution before salting out with
Ammonium sulphate.

There are three in terms of molarity of ammonium sulphate different salting out areas:

0 to 1.9 Ri p _H = 7
1.9 to 2.4 mp _H = 7
2.4-3.3 mp _H = 7

Only the third fraction is dissolved in 0.8% sodium chloride solution dissolved, dialyzed well against demineralized water and concentrated.

B. Precipitation with phytic acid

The sample obtained has a volume of 1.5 liters and a pirotein concentration of 8.0%. It is acidified with 3 volumes of demineralized water verdünni ^ Roit little 2N hydrochloric acid to a p ~ value of 2.10 _H, and finally with stirring with 6 volumes of 0, In-phytic acid solution _(pH-value = 2.10)
offset.

Before the mixture is left to ^{stand at +4 0} C for 24 hours,
one checks whether the supernatant liquid with phytic acid
another precipitate results "

After filtration or centrifugation, the supernatant is adjusted phytic acid solution with about 2N sodium hydroxide to a _pH value of about. 6

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C. Dialysis using the membrane with selective porosity (AMICON XM 100 A)

The obtained 15 l of the supernatant phytic acid solution are under a pressure of 0.7 bar with about 10 volumes demineralized water diafiltered.

A concentrated to 200 ml is obtained by ultrafiltration Sample which, in the freeze-dried state, yields 5 g of a powder.

D. Chromatography over carboxymethyl cellxilose

It is known that carboxymethyl cellulose (sodium salt) is a weak cation exchanger.

The desired glycoprotein has an isoelectric point of 4.0 ± 0.1, determined with the aid of the electrofocalization process. The predominant impurity that needs to be removed, on the other hand, is albumin with an isoelectric point of 4.9.

If the protein is placed in a buffer with a ρ value of 4.2, the protein, based on its isoelectric point, is in the basic range and is therefore negatively charged and not bound to the exchanger. Of the serum proteins only Orosomucoid has leads to a negative charge an isoelectric point (2.7), the ~ at a p _H value of 4.2, is not bound thereby, this material of the ion exchanger. The other serum proteins, which are located with respect to their isoelectric point in an acidic medium, v / v, however earth bonded and / ground from the column using three buffers with increasing _pH value is washed at constant ionic medium.

Procedure:

The four eluents used were as follows

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1. Acetate buffer 0, Im p _H = 4.2

2. Acetate buffer 0.1 m, p _H = 4, S

3. Acetate buffer 0.1m p _H = 5.5

4. Sodium hydroxide solution O, O25n.

The one available under the name CMC 52 (Whatmann), as Carboxymethyl cellulose (sodium salt), which acts as an ion exchanger, is in a pre-swollen state. One leads to caution run a course of treatment before first use. Then equilibrate the material with buffer # 1.

The chromatography is carried out in a descending phase in a column with a diameter of 35 mm and a length of 60 cm, which at both ends with a regulating piston and a porous Disc is equipped.

The applied protein sample is dissolved in buffer no. 1 at a concentration of 2.5%.

Two consecutive chromatographies are carried out with two samples performed, with:

A. in the diafiltration of pig serum across the membrane XM 100 A obtained residue and

B. residue obtained in the diafiltration of pig serum through the XM 300 membrane.

The throughput is controlled by positive pumping with the help of a Perpex LKB pump (which makes it possible to avoid the recirculation of the eluent), with a throughput of 15 ml / hour / cm is maintained.

For each chromatography, the elution buffer is used for each appropriate fractions collected after collection, dialyzed, concentrated and freeze-dried.

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The column is then washed sequentially with the three buffers (buffer p "= 4.2, buffer _TJ p = 4.8, and O, O25n-Natriurr; hydroxide solution) using a discontinuous gradient eluted. The beast of the buffer took place when the eluate no longer absorbs roughly at 240.

The slowly emerging first fraction contains the histamine-blocking glycoprotein and yields after freeze-drying 300 mg of the product.

The following table shows that in pig serum there is an enrichment by a factor of 130 OCO and an increase in health activity by a factor of about 19.

Starting
serum Final fraction after the
Chromatography over
Carboxymethyl cellulose Pig Serum Glyco proteins Weight or volume 12.5 1 300 mg Weight of protein-
substance 103 7 g 140 mg χ (ng)
l / x (U / mg)
'P / x 13.7 χ 10 ³
73
7.5 χ 10 ⁷ 0.1
10 ⁷
140 χ 10 ⁷

In this table, χ means the weight of one in the fraction contained histaminopexic unit, the expression "l / x" stands for the number of units per mg of the protein, while the term "p / x" stands for the total number of histarninopexic Units per sample.

Example 2

Production of histaminopexic glycoproteins from human serum after the proteins have been precipitated

The starting material used is 5CO g of freeze-dried

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human serum.

A. Salting out with the aid of ammonium sulfate

The serum is dissolved in 20 liters of 0.45% sodium chloride solution. One precipitates using ammonium sulfate solution with three different concentrations. from;

O - 1.9 m Ph ⁼ 1.9 m - - 2.4 m Ph ⁼ ^ 2.4 m - - 3.3 m Pr, = ⁷

The last precipitate obtained is isolated, the, in 0.8 percent Sodium chloride solution dissolved, for a long time ν, id with frequently renewed demineralized water dialys ;! kind and finally careful of ammonium sulfate is released, which would be particularly harmful in the following stage,

B. Precipitation mi t Phyt ynoic

The sample volume obtained during the salting out is 11, while the protein concentration is 6.5%. The reaction medium is diluted with 3 liters of demineralized water, acidified with an approximately 2N hydrochloric acid solution with a p value of approximately 2.10 and then slowly with stirring with 6 l In-phytic acid (p _H value = 2.10 ) offset.

After making sure that an aliquot If the supernatant liquid does not produce any precipitate on further addition of phytic acid, then medium 24 becomes Left at +4 C for hours.

Then the precipitate is filtered off or centrifuged off.

The supernatant liquid is adjusted with about 2n-Natriumhydroxvdlösung on eir.ci-n _H p ~ value of about. 6 The excess phytic acid contained in the solution is v / ird. eliminated by diafiltration.

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Diafiltration through a membrane with selective porosity (AMICON XM 100 A)

The cell used has a volume of 2 1 (AMICON- ■ ' Cell No. 2000). It is connected to a reservoir of 10 1. The 10 1 of the supernatant phytic acid solution will be diafiltered under a pressure of 0.7 bar with about 100 l of demineralized water. Then the protein solution concentrated by ultrafiltration in the same cell to a volume of 200 ml. By freeze drying the concentrate 8.20 g of a powder are obtained.

D. Chromatography over carboxymethyl cellulose

The carboxymethyl cellulose ion exchanger (CM 52) is used in Delivered in a swollen condition. After getting the material equilibrium with an 0.1 m buffer with a p n value of 4.2 has brought and removed the fine particles, the column used for the chromatography is filled (diameter 35 mm, length 60 cm), which is closed on both with a piston and a porous disk.

The protein sample is dissolved in the 0 lra buffer having a _pH value of 4.2 g in a concentration of 4 per 100 ml, dialyzed for 48 hours against the same buffer, then centrifuged and the supernatant obtained in the centrifugation is introduced into the column by positive pumping at a rate of 15 ml / hour / cm, ie in the present case in an amount of 120 ml / hour.

The elution of the column is followed by determining the absorption at 280 nm using a photometer (Uvicord II LKB), which is connected to a recorder (LKB).

The following eluents are used:

an 0.1m buffer with a p _R value of 4 ^ 1, an 0.1m buffer with a ρ value of 4.8 and a 0.025N sodium hydroxide solution.

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The eluate is collected in a fraction collector, whereupon the fractions belonging together are dialyzed against demineralized water, concentrated and freeze-dried.

The first fraction containing the desired protein corresponds to 720 mg of a powder.

Chromatography on carboxymethyl cellulose gel was used also for comparison purposes when using two diafiltration samples carried out, of which one by the diafiltration through the membrane XM 100 A and the other by the diafiltration via the XM 300 membrane.

Results; Pharmacological effect

It shows only an effect of not retained on the exchanger fraction (the fraction which was eluted with a 0, lm buffer having a _pH value of 4.2).

The following table shows the effects of various treated Samples before "and after transfer over carboxymethyl cellulose specified.

Starting
serum Final fraction after the.
Chromatography over
Car boxym eihylcellulo se Human serum glycoproteins 500 g
350 g
2 χ 1O ³
500
17.5 χ 10 ⁷ 720 mg
403 mg
0.1
10 ⁷
403 χ 10 ⁷ Weight of the powder
Weight of egg white
substances (biuret)
χ (ng)
l / x (U / mg)
Ρ / χ

From the table above it can be seen that in the human Serum an enrichment by the factor (from the comparison

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the values of the unit of effect) and an increase in the overall activity by a factor of 23 (by comparing the number of units per mg protein).

Physicochemical investigation

Two glycoproteins were made, one from human serum called F 947 and the other one based on pig serum, which is subsequently referred to as F 950 (first production) and F 952 (second production) abbreviated to.

I. Purity criteria

a) electrophoresis

The electrophoresis took place at a p "value of 8.6 when using a veronal buffer over cellulose acetate for 3/4 hours at a voltage of 200 volts. Dyeing amido black does not produce any coloring, even if you work in excess.

Attempts have been made to stain with Comassie blue (Bleu de Comassie) at a concentration of 1 % , the sensitivity of which, five times greater than that of amido black, allows to stain a zone of albumin mobility.

Periodic oxidation and re-staining of the Schiff's reagent can show that this Protein with albumin motility is a glycoprotein.

b) immunoelectrophoresis

Immunoelectrophoresis were carried out on agarose, the migration in a veronal buffer with a p "value of 8.2 was reached over 1 hour and 15 minutes at a voltage of 280 volts.

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1) The preparation F 947 (of human origin) shows with a total human antiserum immune serum difficult recognizable precipitation zone in the albumin area.

With a human anti-albumin antiserum one results extremely diffuse zone which is difficult to decolorize without, however, suggesting a precipitate.

With an anti-IgG immune serum, there is no precipitation arch or range.

2) The preparation F 950 (made from pig serum) shows a total pig antiserum immune serum in the albumin range a fuzzy precipitation zone.

c) polyacrylamide

By migration in a polyacrylamide gel compared to a total human serum, in the case of samples F 947 and F 950, it is the same as that given because of the better sensitivity Staining with Comassie blue to determine the presence of a band with albumin motility.

d) Gel concentration gradient

Attempts have been made to determine the molecular weight of the hista- minopexic glycoproteins to be more clearly identified. The migration in a gel concentration gradient shows one Migration from cathode to anode, i.e. migration from larger meshes of the gel structure to smaller meshes of the gel framework.

Comparison with pure human albumin (Behring)

More distinct bands result up to the main band. The graph shows different polymers with decreasing molecular weight until the monomer with

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a molecular weight of 69,000 - appears.

Preparation F 947 (of human origin)

A band with low intensity in the area of the albumin monomer, which suggests an albumin contamination,

a much more intense band in the range of one molecular weight of 110,000, which corresponds to the desired protein,

two or three bands at an even higher molecular weight, which are due to either impurities or polymers of the can be attributed to both of the above fractions.

Preparation F 950 (obtained from pig serum)

A band with low intensity results in the area of the albumin monomer, which here also indicates an albumin contamination lets close, and

a more intense band in the region of a molecular weight from about 120 00 * 0. The porcine protein used as the starting material appears to have a molecular weight that is slightly larger than the protein of human origin.

Preparation F 952 (obtained from pig serum)

No clear band can be seen in the area of the albumin monomer,

while a band in the region of the molecular weight of about 120,000 confirms the value already determined.

e) Quantitative Immunoelectrophoresis

The quantitative immunoelectrophoresis makes it possible for the protein examined in the course of the already mentioned Investigations to prove suspected or detected impurities as well as the type of these impurities to determine and, if necessary, to determine the quantity.

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1) Two-dimensional immunoelectrophoresis method

A sample of 0.6 mg of the preparation F 947 is taken in a volume of 3 μl. In an immune serum-free Gel containing 1% agarose, an initial electrophoretic migration is carried out on one for 1 hour Voltage gradient of 10 volts / cm through.

A band of 2.5 cm of the gel obtained, corresponding to the electrophoretic migration zone, is taken and transferred to the cathode side of a new plate. The remaining surface of the plate is covered with 12 ml of an agarose solution containing 1 % rabbit immune serum (Behring-Werke, Lot T 1585 AZ) and total human antiserum. A migration is made in a direction perpendicular to that which occurred first.

A slight voltage gradient is used for 16 hours of 5 volts / cm. In this way two phenomena arise at the same time, namely the electrophoretic one Migration and antigen / antibody precipitation.

Results:

The presence of a band with albumin motility can be clearly ascertained. It is furthermore to see a precipitate line with β mobility corresponding to very low contamination.

2) Combination with albumin and polyvalent human antiserum immune serum

It is to be determined whether the protein with albumin motility Corresponds to albumin or the protein examined,

To do this, you do a hike in two stages.

In the first stage, the samples of different types migrate, the starting points of which are offset by 1 cm,

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in an agarose gel free of immune serum on the same line. One causes four parallel migrations of the same kind on the same Layer. After the migration is over, cut off four equal bands, each of the parallel ones Walks correspond.

In the second stage, one of the bands already mentioned is repeated on a new layer, starting from the cathode side. An agarose gel containing immune serum is poured onto the free layer. The second hike is then carried out in a direction perpendicular to the first. The presence of a single band with a significant surface area but a blurred edge can be seen which in all likelihood corresponds to the protein being investigated.

3) Combination with albumin and human anti-albumin immune serum

In order to better define the common antigen, the same investigation was repeated, but with the amounts of comparative albumin were reduced and a specific human anti-albumin immune serum was used.

method

You work in the same way as described above.

The albumin sample corresponds to 0.7 pg albumin standard (Behring-Werke) in 4 μl. The sample examined contains 0.8 mg of preparation F 947 in a volume of 4 μl.

The first hike takes place over 1 hour with a voltage gradient of 10 volts / cm.

Four bands are developed on the first layer, corresponding to each line of migration.

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Transfer one of the bands to a second layer
near the cathode. Then 12 ml agarose gel containing 0.8% by weight rabbit immune serura human anti-albumin (Behring-Werke T 2139 A) is poured on.

The second migration, which takes place in the vertical direction, lasts for 16 hours with a voltage gradient of 2.5 volts / cm.

The presence of two distinct confluent bands can be observed, one of which
the comparative albumin and the other of the presence
corresponds to an impurity that is certainly albumin-like.

4) Determination by Laurell's method

Because the albumin as impurities in the preparations
is contained, its amount can only be determined immunochemically. The Laurell method is used for this.

method

The calibration scale is determined with standard albumin (Behring-Werke) with applied amounts of 4 pl.

The sample F 947 is applied parallel to the calibration scale in the same volume.

The gel containing 1% by weight agarose is made with human anti-albumin rabbit serum (Behring works approach T 1585 AZ) added in such an amount that there is a concentration of 0.5% by weight results.

The hike takes place for 2 hours with a tension gradient of 7.5 volts / cm.

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Results and discussion

The amount of albumin in the sample tested is 0.182 µg, which suggests that the concentration of foreign albumin in the preparation is 0.11%. Two conclusions can be drawn from this:

The albumin contamination is so small that it can be neglected and that certain components can be found can determine to this sample for initial studies regarding the effect of the protein on To use histamine or the ileum.

In general, the sensitivity of the method allows von -Laurell to quantify the presence of 0.182 pg albumin.

II. Determination of certain components and determination of the composition

The impurities present are too low to lead to significant errors in the determination of the constituents of protein. The following determinations can therefore be made.

a) carbon, hydrogen, nitrogen

The results obtained are given in Table A below and show that there was only a slight difference exists between the two fractions examined.

b) protein substances

The determination of the proteins with the biuret technique according to Gornall gives a value for the preparation F 94 7 of 56% and for the preparation F 950 a value of 42.8%.

The determination by the Lowry method gives a value of 66.6% for the preparation F 94 7 and for the

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Preparation F 950 has a value of 46.7 %.

At Lowry's trial, presence interfered with the determination of considerable carbohydrates and leads to erroneous results.

c) sugar

The determination of the components of the carbohydrates is carried out colorimetrically (CS-methylresorcin + ailorwassersiTOffäure). The results obtained are summarized in Table B below.

Table A.

Analysis results of the preparation of human origin (F 947) and the preparation obtained from pig serum (F 950).

947 % C % H % N Combustion
Residue F. 43.91 6.39 3, 05 0.9 950 43.72 6.32 8th, 91 0.7 F. 41.83 6.2O 8th, 09 j, ... 41.66 6.04 8th, 06 3.7

Table B.

Percentage of the components of the carbohydrates of the two preparations F 947 and F 950 (scale: Gal-Kan: 2 / l).

% Hexose,%
(Orcinol) ' % Fucose % Osamine % Sialic acid F 947
F 950 16.9
16 1.3 14.3 15th

5-methylresorcinol

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d) amino acids

After hydrolysis with 6N hydrochloric acid during 24 hours at 100 C in vacuo and after chromatography Using an ion exchanger (Technicon auto-analyseur), the values in the following table were used for each preparation C specified amino acid compositions determined. These results allow the following conclusions:

It can be seen that certain amino acid ratios vary considerably (ARG / hiS - or especially THR / seR),

The CYS values are very different from a Pror be to the other, but the values obtained can be compared, since a "change in this molecule in the course of hydrolysis, in particular oxidation of this molecule can occur,

the amount of MET also seems to be incorrect due to oxidation in the course of hydrolysis,

on the other hand, it should be noted that on the recording strip Chromatography contains a significant amount of a true classical amino acid or other molecule, that reacts with ninhydrin. Because of the position of this x material (directly after valine) is to be assumed, that it may be galactosamine or man;:, amine, which is not in contrast to the obamine composition found stands,

since there is no special colorimeter to determine the Pro lin staining caused by ninhydrin is kei. very accurate value of the amount of this acid obtained.

III. Summary of the physicochemical results:

The preparations obtained contain an impurity which, in the case of preparation F 947, could be identified as albumin. However, it appears that the relatively small amount (0.1 %) of this impurity is a first approximation of the

composition permitted.

In the case of the protein of human origin, a molecular weight of 110,000, a protein content of 56% with the amino acid composition shown in Table C and a carbohydrate content as shown in Table B were found It should also be noted that the sum of the protein and carbohydrate cat content (around 3.5%) is above 100%, which is probably a consequence of the errors of the analysis.

In the case of the protein obtained from pig serum, it is found that it has a molecular weight of about 120,000, a protein content of 42.8% and the amino acid composition given in Table C. The carbon - hydrate content has not been precisely determined due to the lack of raw material.

Differentiate between the proteins exhibiting certain parallels in terms of the constituents (especially the amino acids) slightly.

The low antigen / antibody reaction does not allow up to conclude a complete absence of antigen in common between human glycoprotein and porcine glycoprotein,

Table C.

Amino acid composition of the preparations F 947 (of human origin) and F 950 (obtained from pig serum) (indicated as µmoles of amino acids per mg sample).

F 947
(Ν-α-amino acids: 6.5%) F 950
(Na amino acids: 7.4%) ASP 0.350 0.236 THR 0.238 0.108 SER 0.114 0.135 GLU 0.427 0.261 GLY 0.114 0.122 ALA
VAL)
unknown) 0.145
0.540 0.153
0.090
0.273 CYS-SH 0.034 0.006 MET 0.008 0.012 ILE 0.109 0.076 LEU "0.230 0.140 TYR 0.121 0.047 PHE 0.121 0.063 LYS 0.184 0.133 HIS 0.049 0.053 ARG 0.011 0.062 PER 0.200 (?) 0.166 (?)

Pharmacological effect

It is known that the isolated guinea pig ileum in the pharmacological examination in the presence of the necessary histamine glycoprotein fixed. It was investigated whether this bond actually requires the presence of histamine.

investigation

Treat the ileum with 1 DMA / ml active protein and remove then this solution no.1.

The ileum is rinsed with 10 ml of physiological fluid Tyrode, which is solution # 2.

Then the reduction in biological activity is determined ■ of the histamine in solution no. 1. There is no pharmacological a detectable reduction can be determined.

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Proceed in the same way with solution no. 2, a reduction of 15 to 20 % being observed.

(The term DMA stands for the minimum active dose of the product. )

The glycoprofcein is thus bound to the ileum in the absence of histamine. The DMA value remains substantially the same (the difference between the corresponding DMA value 20% of the decrease in the biological action and the 15 to 20% of that produced by the solution no. 2 reduce a result of loss of the sample during the Handling is).

It should be noted that there was no change in contact with the ileum of the protein, which would alter its pharmacological properties.

One can also determine the type of binding and the release of glycoprotein and, in particular, in this area the influence of the presence or absence of histamine.

To do this, the ileuai is brought into contact with 1 DMA glycoprotein per ml of the solution and 0 _f 2nM histamine. To the? solution no. 1 is removed. This is followed by a first rinse with 5 ml of physiological fluid according to Tyrode, which contains 0.2nM / ml histamine, and this solution no. 2 is kept. This is followed by a further rinse with 5 ml of physiological fluid according to Tyrode, which does not contain any histamine. This solution no. 3 is also kept. This is followed by a determination of the pharmacological activity of each of the solutions «

Solutions No. 1 and No. 2 do not call for a reduction in biological Activity of histamine. Only solution no. 3 shows a reduction in histamine activity by 20%.

The glycoprotein is therefore bound to the ileum in the presence of histamine and remains bound to the ileum when this is flushed with a physiological fluid according to Tyrode which contains histamine. In contrast, the first Srülunc ¹ , di.e

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was carried out without histamine, the release of the protein brought about, this detachment being quantitative, since it is a maximum Decrease (20%) of the biological activity of histamine adjusts.

investigation

It is known that the antihistamines are only effective when they come into contact with the ileum before treatment with histamine stand. They do not work if one previously incubated with the ileum bath Antihistamine product and histamine added. The question arises, what happens if you go to the ileum bath beforehand Incubated glycoprotein plus histamine added. Therefore, an incubation of 1 DMA / ml protein took place for 10 minutes at 37 ° C and 0.2nM / ml histamine in 5 ml physiological fluid according to Tyrode. This incubation liquid is brought with the isolated guinea pig ileum in contact and determined its Contraction. There is a reduction of 20%, related on histamine as a reference substance.

Results

The previous incubation does not prevent the histaminopexif.che Behavior that remains unchanged, especially in quantitative terms.,

explorati hung

Attempts have also been made to determine the difference between the antihistamines by determining whether the glycoprotein has a protective effect against the contracting effect of histamine on the ileum. To do this, 0.2nM / ml of histamine is introduced into the ileum bath and a contraction of 100 % is established. Then a negligible volume of active protein is added so that the concentration in the 3ad is 1 DMA / ml. The contractions are only 80 %.

Schlußf o lgerung

The protein develops a protective effect against histamine, as whether this occurs in a fifth of the free spaces in the accelerator

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2335-02

would urge. The material is therefore completely different from the antihistamines.

investigation

An attempt was made to determine if there was any interaction with the lining of the ileum that takes place

on the one hand through the mucous membrane itself, since if this binds the histamine, too small an amount in the determination the contracting effect of an ileum / histamine bath itself would result

or by lysis products of the mucous membrane, whereby in this case, if these products were present and these sought-after pharmacological effects were to develop, they

could interfere with conclusions regarding the presence or absence of the protein in the ileum bath,

the effect of which could add to the effect of the protein examined or

the value of the 100% contraotion by histamine is a contraction could be assumed, which is already reduced by a "percentage" due to these lysis products.

A solution Mr. 1 of physiological fluid is therefore made according to Tyrode, which is left in contact with the isolated guinea pig ileum and 0.2 nM / ml histamine for 15 minutes. Then you remove this solution and determine the contracting effect, based on a comparison product that is below normal Conditions was obtained. An activity of 100% is determined.

In the same way, a solution no. 2 is prepared in which one leave the guinea pig ileum for 15 minutes. Then removed solution no. 2 and determine the protective effect against the contracting effect caused by histamine on one new ileum, showing no protective effect.

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Conclusion

The lining of the ileum does not bind histamine, and the contraction that is said to be 100% active is real
a consequence of the entirety of the introduced histamine.

The same mucous membrane does not release any lysis products that develop the desired pharmacological effect. If you take into account that there is a protective effect of 20 % against the
shows a contracting effect of histamine on the isolated guinea pig ileum, there is no further interaction with active products other than with the glycoprotein.

The observed phenomenon is therefore actually the result of an interaction of the following three materials: protein, receptive site - the ileum and histamine, so that the pharmacological phenomenon can only occur in the presence of these three partners. It is probable that other mucous membranes of analogous structure are evidence of a phenomenon of the same nature
to lead.

"Sephadex G" is the trade name for a gel
from in the presence of epichlorohydrin in alkaline
Polymerized dextran environment. The designation "200"
indicates the amount of water that 10 g of Sephadex
can be absorbed to form a gel.

"AMICON XM 100 A" and "AMICON XM 300" are ultrafilter membranes, made from a non-cellulosic synthetic Polymer exist.

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Claims (9)

Claims 1.) Histaminopexic glycoproteins from human or animal or placental serum with an isoelectric Point of 4 i 0.2, determined by electrofocalization, characterized in that it is at least 1 000 000 histaminopexic Units per mg included. 2.) Histaminopexic glycoprotein of human origin according to claim 1, characterized in that it contains about 56 % proteins, 17% hexoses, 1.3% fucose, 14 % osamines and 15% terminal sialic acid residues and has a molecular weight of about 110,000 . 3.) Histaminopexic glycoprotein of animal origin according to claim 1, characterized in that it contains about 47% proteins and 16 % hexoses and has a molecular weight of about 120,000. 4.) Pharmaceutical compositions, characterized in that it consists of at least one _t 'histaminopexischen Gylkoprctein according to any of claims 1 to 3 as an active ingredient and one for the parenteral, rectal, percutaneous or permuköse administration suitable pharmaceutically inert excipient or carrier material. 5.) Process for the preparation of the histaminopexic glycoproteins according to claim 1, characterized in that a serum of human or animal origin or a serum of placental origin is treated with ammonium sulfate and a protein precipitate is obtained, the precipitate is converted into an aqueous solution, this solution with Treated phytic acid, filtered and the filtrate containing the glycoproteins subjected to diafiltration through a membrane based on non-ionic polymers of selective porosity, which separates the protein fraction retained in the diafiltration cell, at a p "value in the range of about 4.2 a purification Chromatography over carboxymethyl 309804/1400 cellulose gel undergoes and those not retained on the column Separate faction. ' 6.) The method according to claim 5, characterized in that the starting material is a human or placental serum Originally used and a histaminopexic glycoprotein according to claim 1 is obtained. 7.) The method according to claim 5, characterized in that an animal serum is used as the starting material and a histaminopexic glycoprotein according to claim 1. 8.) The method according to claim 5, characterized in that one is a serum of human or animal origin or placental Origin of a diafiltration over a membrane based on non-ionic polymers with selective porosity, which separates the protein fraction retained in the diafiltration cell a p "value of about 4.2 by chromatography on carboxyric ethyl cellulose gel purifies and separates the fraction not retained on the column. 9.) Process according to claim 5, characterized in that one serum proteins human or animal or placental Origin of a diafiltration over a membrane based on non-ionic polymers with selective Undergoes porosity, which separates the protein fraction retained in the diafiltration cell, this at one p n value of about 4.2 from purification by chromatography undergoes over carboxymethyl cellulose gel and which is not y \: the fraction retained in the column separates. 303384/1400