NO763361L - - Google Patents

Abstract

Derivatives of the pregnancy-specific 3 - β-glycoprotein, process for their preparation and agents containing them.

Description

The invention relates to derivatives of the pregnancy-specific f^-glycoprotein, methods for their production and immunizing substances, which as essential constituents

part contains a derivative of the pregnancy-specific 3^-glycoprotein, which can be used as an inoculum to prevent a pregnancy. • In DOS 2,157,610, a pregnancy-specific B-^-glycoprotein and a method for its production were discussed. The starting materials for the isolation are human organs or body fluids. When immunizing vertebrates with the pregnancy-specific 3-^-glycoprotein of human origin, an antibody directed against the pregnancy-specific B-^-glycoprotein is found in the plasma of the immunized animals,

which with the starting antigens manages to enter into a specific reaction.

The pregnancy-specific B^-glycoprotein (SP 1)

is characterized by the following parameters:

a) Electrophoretic mobility in agar gel in 3-j_-globulinoma-

Council of Human Plasma Proteins,

b) a sedimentation constant in ultracentrifuge of 4.6 0.5S,

c) a molecular weight determined by gel filtration of 100,000 15,000,

d) an extinction coefficient of E, = 11.6 ± 0.5

0* 1 cm '>J

(determined at 278 mu in 1/15 molar phosphate buffer of pH 7.0),

e) a carbohydrate content of 28.05 1.55-

, On the basis of suitable experiments, according to which it

with antibodies against the pregnancy-specific 3^-glycoprotein, it is possible to prevent conception or a provision of abortion in primates, it was proposed in German patent application no. P 23 45 953.2 to use the pregnancy-specific B^glycoprotein for active immunization of primates or to cause an obstacle to conception resp. provision of abortion by passively applied antibodies. While the passive transfer of antibodies leads

for satisfactory results, the active immunization certainly leaves more to be desired, as when the homologous pregnancy-specific Bj-glycoprotein is used, only small amounts of antibodies are often formed which are not sufficient for a safe prevention of pregnancy. The effect of the proposed immunizing substance is therefore not entirely satisfactory, when the homologous, i.e. from the same species, pregnancy-specific B-^-glycoprotein is used for immunization.

It is known that the glycoprotein corresponding to the pregnancy-specific 3^-glycoprotein can also be isolated from

^opens. After its application in humans, antibodies reacting with the human pregnancy-specific B-^-glycoprotein could be expected in human blood in sufficient quantity. However, the starting material for the production of pregnancy-specific B-^-glycoprotein from monkeys is only available in limited quantities. On the other hand, as stated in the first-mentioned DOS, the pregnancy-specific g-^-glycoprotein can be produced in satisfactory quantity from human deep-frozen placentas or from the serum of pregnant women.

It was therefore set the task of modifying

the pregnancy-specific Bj-glycoprotein, preferably that which was isolated from humans, so that it is considered foreign by the human body and is thus suitable for the induction of antibodies directed against the glycoprotein.

The task is solved according to the invention by producing new derivatives of the pregnancy-specific '3-^-glycoprotein, which can be used as a component of immunizing agents for preventing conception and for providing abortion.

It was surprisingly found that a number of methods lead to a chemical change of the pregnancy-specific Bj-glycoprotein and also to a change in the immunological relationship of the thereby formed derivatives, so that they manage to induce antibodies in the homologous system.

The object of the invention is primarily the derivatives of the pregnancy-specific B-glycoprotein which can be produced by reaction with a protein-modifying compound.

In the case of such transactions, it is based on two basic principles. They both lead to a change in the native structure and thus to a denaturation of the protein: 1. Methods for chemically changing the pregnancy-specific B-^-glycoprotein using reagents which

leads to a change of chemical groups present.

2. Procedures which lead to a chemical change of the pregnancy-specific g-^-glycoprotein by introducing new groups into the molecule or which leads to connection of the molecule with low or high molecular weight compounds.

To the methods mentioned, it is to count them

which in the protein manages to change one or more of the following groups: Amino, guanidyl, imidazole, indole, aliphatic hydroxyl, amide, thioether, disulfide, sulfhydryl, phenol and carboxyl. Here-

to, a number of reactions are known from the literature, for which it is mainly to use the reagents mentioned below under the conditions mentioned for example.

1.1. Oxidation with iodosobenzoate, porphyrindin, ferrocya--

nitrite or iodine, as it is usual to use a concentration of the oxidizing agent of 0.001 - 0.01 molar, a pH value of 7, a temperature of 0-25°C and a reaction time of 5-30 minutes. The oxidation with iodine is carried out in the presence of iodide in high concentration at a pH value of 1-7. The oxidation can also be carried out with hydrogen peroxide. The preferred conditions are then: The concentration of the oxidizing agent approx. 0.005 molar, .pH value approx. 6,6,

temperature approx. 25°C and reaction duration 0.5-40 hours.

1.2. Reduction with cysteine, thioglycolic acid, thioglycol, cyanide or sulfide, preferably under the following conditions:

Concentration of the reducing agent 0.001-0.1 molar, pH-

value 7-8, temperature approx. 25°C and reaction duration 0.5-

4 hours.

The reaction conditions can be derived in detail from the literature. They are, for example, referred to from H.S. Olcott and H. Fraenkel-Conrat, Chem. Fox. 4l, page 151 et seq

(1947). In addition, part of the reagent and the method conditions can be taken from H.E. Schultze and J.F.■Heremans, Mole-

cular Biology of Human Proteins (1966), pages 40-41 and those therein on page 58 and the following cited references.

With the introduction of new groups in the pregnancy-specific 3-^-glycoprotein, derivatives are produced, which from an immunological point of view are usually referred to as Hapten compounds. According to a recognized definition, a "Hapten" is a protein-free substance, which can react with a specific antibody directed against its configuration, which, however, is not capable of the formation of detectable amounts of antibody. A hapten causes an immune response only in connection with a carrier protein.

Most haptens are low-molecular compounds with a molecular weight below 1000. However, macromolecular compounds such as the pneumococcal polysaccharides are also considered haptens. A hapten-protein connection usually leads to the induction of two types of antibodies, with specificities arising against the main group gene and against the protein carrier. In the case of the pregnancy-specific B-^-glycoprotein, it is true that its hapten compounds also induce antibodies against the carrier protein in the homologous system.

As haptens within the scope of the invention come

it refers to the chemical groups and compounds referred to as such in the literature. In particular, this includes aromatic ring systems, steroids, peptides, purines, pyrimidines, penicillin and its derivatives, but also single molecules such as, for example, iodine and, according to an extended definition made here, the substances that must bind to the carrier of the pregnancy-specific 3-]_- glycoprotein also high-molecular bodies with peptide- or protein and carbohydrate character, which in turn has antigenic properties.

The method for producing derivatives of the pregnancy-specific 3^-glycoprotein according to the aforementioned basic principle (2.) using haptens is known as generally applicable reactions for the introduction of protein-modifying chemical groups and of haptens into egg white bodies during the formation of covalent bonds between chemical substances and proteins. In particular, this relates to reactions that are discussed for the modification of certain groups in the protein molecules. These reactions are also discussed in the works of Olcott and Fraenkel-Conrat and of Schultze-Heremans. Examples of such methods are:

2.1. Alkylation with

iodoacetate, iodoacetamide, (0.05-0.1 molar, pH 7~8, 0-25°C, 0.5-2 hours) or

dinitrofluorobenzene: (0.17 molar, pH 7-8, 25°C, 2 hours).

2.2. Acylation with

ketene (pH 5-8, 0.25°C, 5-30 minutes), acetic anhydride (pH 7-8, 0°C, 30 minutes), carbon suboxide (pH 5-8, 0-25°C, 5"30 minutes), azide, benzoyl-, carbo-

benzoxy or benzene

sulfonyl chloride (pH 7~9, 0-2£°C,' 0.5-2 hours), nitric acid (1 molar, pH 4, 30 minutes), iodine (pH 5-11, -5°-25°C, 0.5-3 hours:

in contrast to the above-mentioned oxidation with a smaller amount of iodine and lower iodine concentration), formaldehyde (1-2 molar, 25°C, at pH 7-8, 1 hour, at pH 11, 10 minutes), epoxides (1-2 molar , pH 5-6, 1-4 days), mustard gas (pH 5-6, 25°C, 0.5-4 hours),

acid/alcohol (mineral acid in absolute alcohol) (0.01-0.1 molar, 0-25°C, 1-2 days) methyl diazoacetate, diazoacetamide XpH 5 (ester), pH 6 (amide), 0°C) p -chloromercuribenzoate (10 _ c -10 _p molar, pH 7, 25 oC,

5-30 minutes),

diazonium compounds (pH 7-9, 2-5°C, 30 minutes) or o-methylisourea (0.5 molar, pH 10.5, 0°C, 3 days).

For some of the above-mentioned groups, the procedure is detailed below: a) Iodination (Literature: Kabat and Mayer's Experimental Immunochemistry, sec. edition 1961, 816-818).

Iodine is reacted as iodinate with tyrosine residues of proteins, as one or tp. jpdatoms are introduced into the tyrosine molecule and the iodine protein is formed. In the same way, the iodine compound of the pregnancy-specific 3-^-glycoprotein

is produced.

b) Diazotization and coupling (Literature: Kabat and Mayer's

■ Experimental Immunochemistry,

Sec. edition 1961, 798-799).' To this end, a compound with an aromatic amino group, for example arsanilic acid or sulfanilic acid, is diazotized, and the diazonium compounds are then reacted with the protein. Thereby, tyrosine residues in particular, but also histidine and lysine residues of the protein form a covalent bond with the aromatic component. According to this method, the diazo-arsanilic acid or diazobenzene-sulfonic acid compound of the pregnancy-specific 3-[_-glycoprotein can be prepared.

c) Reaction with vinyl sulfone compounds (in analogy to the reaction of reactive dyes, which as reactive components

contains the vinyl sulfone group, with cellulose or wool). Aliphatic or aromatic vinyl sulfone derivatives and sulfuric acid half-esters of γ-hydroxyethyl sulfones can be brought into reaction with the amino groups and/or hydroxyl groups of the protein. In this way, for example, the hapten connection of the pregnancy-specific 6^-glycoprotein can be obtained

with 1-aminobenzene-43-hydroxyethyl sulfonic acid esters.

d) Reaction with the isocyanate and isothiocyanate compounds (Literature: Kabat and Mayer's Experimental Immunochemistry,

Sec. edition 1961, 809-811). This reaction, which takes place over the protein's free amino groups, enables, in analogy to the known methods from protein chemistry, the conversion of haptens with the corresponding chemical groupings also with

the pregnancy-specific 3^-glycoprotein.

e) Pinitrophenylation (Literature: Carsten, M.E.; Eisen, H.N., J. Am. Chem. Soc. 77, 1273 (1955). The reaction usually carried out with dinitrobenzenesulfonate or dinitrofluorobenzene leads to conversion of the free amino groups of protein during formation of dinitrophenyl derivatives. In this way, dinitrophenylated pregnancy-specific 3-L_glycoprotein is obtained. f) Reaction with mixed anhydrides (Litte>rature: Kabat and Mayer's, Experimental Immunochemistry, sec. edition 1961,

813-815). This method is suitable for connecting

a number of different haptens, which primarily need to be used in acylation. is transferred in a mixed anhydride with amino groups of the protein. Moreover, by direct reaction of proteins with anhydrides, it is possible to prepare acid amides with the general formula -R-CO-NH-CI^-protein in a known manner, where R can be a desirable hapten. By means of this reaction, it is possible, for example, to produce the pregnancy-specific 3^-glycoprotein hapten compound

with R = benzyl as the hapten.

g) Reaction with carbodiimides (Literature: Makino, T. et al., Contraception 8 (2), 133 (1973). The carbodiimides with the

general formula R-N=C=N-R (R = an arbitrary organic residue) is suitable for connecting carboxyl group-containing haptens with amino groups of proteins. This also results in reacting

tion product the compound R-CO-NH-protein, where R can be any desired hapten having a carboxyl group. By means of this reaction, for example, hapten preparation of the pregnancy-specific 3-β-glycoprotein with cyclohexanecarboxylic acid as the hapten is obtained.

According to the invention, the immunological relationship of the pregnancy-specific g-glycoprotein can also be changed by linking the glycoprotein with a peptide or protein. The linking can either be carried out covalently or by complex formation. As reaction participants, peptides can be used here, for example also those with their own biological function, e.g. decapeptides such as LRP (luteinizing hormone releasing factor). LRP is, for example, to be linked in the same way with the pregnancy-specific g-i^-glycoprotein as this is discussed for its linking with albumin by T. Makino and others in Contraception 8, 2 (1973)» page 133 et seq.

The underlying reaction goes back to the carbodiimide activation of the carboxyl groups with the carbodiimide compounds.

A further possibility for changing the immunological relationship of the pregnancy-specific 6-^-glycoprotein consists in linking the protein with another•protein substance, thus as this is known for example for linking enzymes with proteins, since the pregnancy-specific 3-j_-glycoprotein in the reaction can replace one of the two participants.. For the connection of the two proteins with each other, a number of methods are common. They use certain groups of activating substances such as carbodiimides, cyanuric chloride, p.,p-difluoro-m,m-dinitrophenylsulfone or glutardialdehyde.

The pregnancy-specific 3-L~SlykoProte:i-n can e.g. by means of glutardialdehyde in a reaction analogous to that described by S. Avrameas: Immunochemistry 6, 1969, 43~52, for the coupling of gamma globulin with peroxidase is carried out with a number of proteins. Preferably, within the framework of the invention, the pregnancy-specific glycoprotein prescribed as an immunizing substance is combined with a protein substance, which is itself effective as a grafting substance component, such as e.g. tetanus toxoid.

With the above list of methods, it should only be shown, for example, that all reactions such as those mentioned in the literature are suitable for derivatization. of proteins can be used for the production of the products according to the invention.

The object of the invention is generally the methods for producing derivatives of the pregnancy-specific Bj-glycoprotein, as these are characterized by subjecting the pregnancy-specific B^-glycoprotein to a process for oxidation, reduction, alkylation or acylation of proteins, to produce a covalent bond between protein and haptens and between proteins with each other.

The reaction products formed are transferred according to known techniques in preparations suitable for parenteral injection and, as usual with inoculants, are optionally equipped with stabilizing additives and inorganic or organic immune adjuvants, in order to be able to use them as immunizing agents for birth control. These immunizing substances are likewise the subject of the present invention. Stabilizing additives include substances that improve the durability of the preparations, in particular low molecular weight carbohydrates or egg whites or their derivatives which break down and re-cross-link collagen, for example the gelatin product available under the trade name "Haemaccel", possibly with further additives such as amino acid salts, e.g. sodium glutamate. The amount of such additives suitably amounts to 0.5 to 5$, sometimes up to 10%. The immunizing substance can then be made available liquid or in dry, suitably freeze-dried form. The latter is to dissolve before application in water or a physiologically tolerable medium.

The following experiments carried out with the products which

can be obtained according to examples 1 or 2 shows the result of the active immunization of monkeys with the derivatives according to the invention of the pregnancy-specific B-j^-glycoprotein.

Test description.

Sexually mature female monkeys (Cynomolger) were immunized over a period of 6 weeks by intravenous and/or subcutaneous administration of a total of 3 mg of a derivative of the pregnancy-specific ftj-glycoprotein in isotonic saline solution with finely divided aluminum hydroxide as adjuvant, alternately intravenously and subcutaneously. All animals developed antibodies against the pregnancy-specific B-[_-glycoprotein, which in a gel diffusion test using pregnancy-specific 8-^-glycoprotein was detected qualitatively and determined quantitatively with the radial immunodiffusion

■sion. On average, a titer of 0.09 mg antibody/ml was achieved. After the immunization, the female animals were brought together with a fertile male animal for mating each time at the time of ovulation for 3 days, and the result of the copulation was then examined. Half of the animals received monthly between copulation or during pregnancy Booster injection (0.4 mg protein). Compared to untreated control animals, the female monkeys immunized against pregnancy-specific 6^-glycoprotein were severely inhibited in their ability to reproduce. Table 1 shows the results of the immunized animals. In some monkeys, even after several copulations, no pregnancies occurred, others only became pregnant after the third or fourth mating. The monkeys that became pregnant at the first or at one of the following copulations aborted almost all in the first or second third of the pregnancy. Only one of the monkeys that had become pregnant at the first copulation showed a normal pregnancy with a normal delivery. Another had, after the third copulation, carried out a normal pregnancy.

Control for comparison:

Of 39 monkeys, the third copulation was at the latest

31, i.e. Q0% pregnant. Of these pregnancies, only one ended in a spontaneous abortion, the others all progressed normally.

The invention shall be explained in more detail with help

of some examples.

Example 1.

Modification of pregnancy-specific B-^-glycoprotein with diazotized sulfanilic acid.

a) Preparation of the diazonium salt.

1 g of sulfanilic acid dissolves at room temperature

in 100 ml of 0.1 N HC1, then placed in ice water and cooled with stirring to Ooc. The sulphanilic acid is thereby precipitated as a salt. 50 ml of a cold 1% solution of sodium nitrite in water is then allowed to drip into the suspension slowly (over the course of an hour) with stirring; the end of the reaction is indicated by blue staining of potassium iodide-starch paper.

b) Preparation of hapten compound.

Conjugation of pregnancy-specific Bj-glycoprotein with the diazotized sulphanilic acid takes place at slightly alkaline pH. One dissolves 100 mg of pregnancy special

fixed 8i~glycoprotein in 10 ml of a 0.2 molar sodium phosphate^buffer of pH 8.2 and cools the solution to 4°C. 0.57 ml of the diazonium salt solution is then added (the molar ratio between pregnancy-specific 8-β-protein and diazotized sulphanilic acid in the reaction mixture is in this case 1:20) and stirred for 4 hours at 4°C. The pH value, which should not fall below 8.0, is thereby corrected by the addition of 0.2 N

NaOH. The solution is then allowed to stand for 15 hours at 4°C.

The modified protein is then dialyzed for several days

thoroughly against water and lyophilized.

The reaction of pregnancy-specific B-^~glycoprotein with the diazotised sulphanilic acid results in an orange coloring of the protein. Furthermore, the introduction of acidic groups changes the electrophoretic mobility. During electrophoresis in agar or in polyacrylamide (PAA-)) gel, the modified protein travels faster to the anode than the negative pregnancy-specific B-[_-glycoprotein.

A dose of the immunizing agent has the following composition: 0.2 mg of the derivative of the pregnancy-specific

B-^-glycoprotein dissolved in 3 ml isotonic saline solution containing 0.05% A1(OH)^, 5% "Haemaccel" and as a preservative 0.15 mg sodium timerfonate.

Example 2.

Preparation of the hapten compound with 1-aminobenzene-4B-hydroxyethylsulfonic acid ester.

The starting material is the l-aminobenzene-48-hydroxyethyl sulfonic acid ester, which in the alkaline pH range (pH 9-10) splits off sulfuric acid and thereby gives a reactive aromatic vinyl sulfonic compound, the so-called "Parabase", which then reacts with the amino or hydroxyl group in the protein.

100 mg of the pregnancy-specific g-^-glycoprotein is dissolved in 10 ml of a 2.5% solution of ^2^0^.2 HoO (pH 9.25). For this, 3.1 ml of a 15 µg solution of the "Parabase" ester in water is added while stirring at room temperature (molar ratio: Pregnancy-specific B^-glycoprotein to "Parabase" = 1:100). The mixture is stirred for 4 hours at room temperature, thereby keeping the pH constant at 9.2 by adding 0.1 N NaOH. It is then allowed to stand for 15 hours at 4°C, then thoroughly dialyzed against H2O and lyophilized the modified protein.

A dose of the immunizing agent has the following composition: 1 mg of pregnancy-specific g-^-glycoprotein-hapten compound dissolved in 5 ml of isotonic saline solution. Example 3.

Coupling of the pregnancy-specific B^-glycoprotein with tetanus toxoid.

19 mg pregnancy-specific B^-glycoprotein

and 54 mg of tetanus toxoid (formalin-treated tetanus toxin) are dissolved in 5.4 ml of water, adjusted to pH 5.0 by the addition of 1/10 N hydrochloric acid and then mixed with stirring with 6.3 mg of N-ethyl-N' (3-Dimethylaminopropyl)-carbodiimide-HCl. The mixture is stirred for 3 hours at approx. 2o8c and then 15 hours at 4°C, then neutralized and dialyzed against isotonic sodium chloride solution.

When monkeys (cynomolgus) are immunized with this coupling product within the area of the experimental description, the reproductive capacity of these animals is inhibited.

A dose of the immunizing agent has the following composition: 0.5 mg protein in 3 ml isotonic saline solution and 1.5 mg γ-Al(OH)

Claims (3)

1. The derivative of the pregnancy-specific 8-j^-glycoprotein which can be produced by reaction with a protein-modifying compound and which is to be characterized by the parameters a) electrophoretic mobility in agar gel in the B-^ -globulin region of human plasma proteins, b) a sedimentation constant in ultracentrifuge of 4.6 ± 0.5S,. c) a molecular weight determined by gel filtration of 100,000 ± ^ qQq d) an extinction coefficient of E, = 11.6 ± 0.5 (determined at 278 mu in 1/15 molar phosphate buffer of pH 7.0). e) a carbohydrate content of 28.05 ± 1.55.. 2. Method for producing compounds according to claim 1, characterized in that the pregnancy-specific 6^-glycoprotein is subjected to a method for oxidation, reduction, alkylation or acylation of proteins, to produce a covalent bond between proteins and haptens or between proteins with each other . 3. Immunizing agent for the parenteral application containing a compound according to claim 1 in admixture with a pharmaceutical usual carrier for the inoculant.