HU186121B - Process for producing a-ristomycin and/or ristomycin derivative of high purity,and reagent for thrombicyte-aggregation tests containing them - Google Patents

Abstract

The invention relates to a process for the preparation of A-ristomycin and / or ristomycin derivatives for use in platelet aggregation experiments for the diagnosis of Willebrand disease. The aim of the invention is the preparation of A-ristomycin in suitable constitution and high purity. According to the invention, A-ristomycin and / or ristomycin derivatives of the general formula (I) are prepared in which, for example: R is a hydrogen atom, R 1 is an O-alpha-D-Araf (1 -> 2) -O-alpha-D -Manp (1 -> 2) -O-alpha-L-Rhap) - (1 -> 6) -O-beta-D-Glop group, R deep 2 a 2, 3, 6-Tridesoxy-3-amino - (NHY) -alpha-L-ribo-hexapyransyl group, R deep 3 an alpha-D-mannopyranosyl group, R deep 4 an alkyl group containing 1-4 carbon atoms, X the radical of a mineral acid, Y a hydrogen atom.

Description

The present invention relates to a process for the preparation of high purity ristomycin and / or ristomycin derivatives of formula (I) wherein:

R is hydrogen,

R 1 is O-alpha-D-arabinofuranosyl (1- * 2) -O-alpha-D-mannopyranosyl (1- ♦ 2) -O- [alpha-L-rhamnopyranosyl] - (1-6) -O-beta- D-glucopyranosyl group,

R _{2 is} 2,3,6-trideoxy-3-amino- (NH Y) -al-alpha-ribo-hexapyranosyl,

R _{3 is} alpha-D-mannopyranosyl,

R 4 is C 1-4 alkyl,

And X represents a mineral acid residue for hydrogen or C (Z) 3 - (CH ₂ ) "- CO - wherein Z is hydrogen or halogen and n is an integer from 0 to 5,

The invention further relates to platelet aggregation assays, in particular a biology reagent for diagnosing Willenbrand disease.

Platelet aggregation assays are known to provide the most useful data for the assessment of function in screening patients with hypertension. In addition to routinely used aggregating agents (ADP, adrenaline, collagen), a substance called ristocetin, previously used as an antibiotic, has played an important role since 1971. Following HOWARD and FIRKIN, the use of ristocetin allowed rapid and simple recognition of congenital hemorrhage, Willebrand's disease (Thombosis and Diathesis Haemorhagica 26, 362 (1971)).

The essence of Willebrand's disease is coagulation VIII. a molecular defect in the factor complex. The most important physiological role of the Willebrand factor is to promote platelet adhesion to subendothelial structures. Willebrand factor deficiency or quality disorder results in a condition called Willebrand disease, which is the most common form of congenital hemorrhage. The isolation of a Willebrand patient (inheritance, family examinations, therapy) is important, and the detection and screening of such a disease before surgical interventions is particularly important.

In our studies we have found that ristocetin, which is widely used in laboratory practice, is perfectly replaced by another member of the vancomycin antibiotic family, ristomycin. Because of its lower cost of production and the advantageous difference in plasma protein precipitation, we have found ristomycin more suitable for hematology studies. Thus, ristomycin, which is currently used to cure diseases caused by antibacterial infections, could be used in this new field (for diagnostic purposes).

The antibiotic ristomycin is a Soviet researcher of Proactinomyces fructifer var. was isolated from the culture of ristomycin mainly by adsorption on a cation exchange resin of SzDSz-3 or SzDV-3 and elution with acetone in ammonia solution or ammonium acetate buffer (pH 9-10). Later, like ristocetin, ristomycin was separated into two biologically active components, A and B ristomycin. Large-scale production of antibiotics has also been described, and a polarimetric and ultraviolet spectrophotometric method has been developed to determine the active substance content of the culture fluid and in-process products [see U.S. Patent No. 180,753. U.S. Patent No. 6,192, Antibiotiki 8, 392 (1962); Antibiotics 9, 884 (1964); Antibiotics 10 217 (1965); Antibiotics 12, 129 (1967); Antibiotics 16, 786 (1971)].

Determining the exact structure of A-ristomycin [J. Org. Chem. 39, 2971 (1974); J. Antibiot. 39

446 (1979); Tetrahedron Letters 21, 2983 (1980);

After JACS 102, 7093 (1980), our studies focused on the extent to which platelet aggregation is affected by structural alteration of the compound and its degree of purity.

The object of the present invention is therefore to determine the structural structure that can be used in the biological test and to provide a solution for the preparation of the active ingredient in very high purity.

In our experiments, it has been found that, in acidic or alkaline media, partial or complete removal of R 1, R ₂ , R ₃ and R 4 groups and their blocking of hydroxyl functions and R (in ester, ether form) causes an adverse biological effect of the antibiotic, and sometimes an unfavorable change in solubility. so e.g. carboxy-A-ristomycin obtained by substitution of the R4 group with a hydrogen atom did not aggregate human platelet plasma and did not inhibit ristomycin-induced platelet aggregation. The tetra-O-methyl-A-ristomycin (R = R 1 = CH ₃ ) blocked on phenolic hydroxyl groups was also found to be unfavorable. Paracetylated A-ristomycin was insoluble under the conditions required for the assay, so the resulting aglyco-A-ristomycin (R = R 1 -R ₂ = R ₃ = Y = H; RtCH 3) induced agglutination but due to pH and solubility conditions the derivative also did not seem optimal. To the present knowledge, B-ristomycin, which differs only in the absence of the 2-OD-arabinofuranosyl alpha-D-mannopyranosyl moiety in the R) heterotetrasaccharide chain of formula (I), is also unsuitable for the desired diagnostic purpose.

Surprisingly, in our studies, the active ingredient used in the blood coagulation test is optimal if and only if it has a very high degree of purity and is also represented by Y = H in the formula (I) and by Y = CF ₃ -CO-. the pure material is of very good laboratory diagnostic value.

It was concluded on the basis of these initial results suggest that normal human platelet exposition of the general formula (I), the four free phenolic hydroxyl groups (R = H), the C-terminus of a methoxycarbonyl group (R4 = _CH3 ) and intact rostotetrozil (= Ri) side chain is a basic condition.

Thus, it has been found that high purity A-ristomycin and its derivatives of Formula I are well suited for A-ristocetin-induced platelet aggregation assays (Figure 1).

In our experiments, using six different citrate PRPs (Platelet rich plasmaj) and EDTA (ethylenediaminetetraacetic acid) PRPs, the mean values of the curves in Figure 1 were obtained. The curves run the same, with three well-eluted 2I sections. Agglutination was not observed at concentrations lower than 0.8 mg / ml. A linear relationship was observed in the range 0.8-2.0 mg / ml. At concentrations higher than 2.0 mg / ml, the dose curves move towards the peak, increasing the dose further to become an increasingly confusing factor in plasma protein precipitation. The same results can be observed with either citrate or EDTA PRP as substrate. Figure 2 shows that rostomycin is useful for the quantitative determination of the activity of cofactor VIII R: Ag. To measure the plasma factor deficiency in Willebrand's disease, the so-called plasma factor deficiency. ristocetin cofactor assay assays [J. Clin. Invest. 52, 2708 (1973); Thromb. Diath. Haemorrh. 34, 306 (1975)]. Based on our results, ristomycin is well suited for quantitative assays, and instead of the ristocetin-cofactor and ristocetin-cofactor assay, the ristomycin-cofactor and ristomycin-cofactor assay is well-established. The determination of ristomycin cofactor can be performed on both washed (Figure 2 upper curve) and formalin-treated platelets (Figure 2 lower curve). At high concentrations, ristocetin and ristomycin result in plasma protein precipitation. The data obtained for values between 1.5 and 3.5 mg / ml are shown in Table 1.

Table 1

Concentration Light absorption (measurement at 600 nm) (Mg / ml) ristocetin Ristomycin 1.5 0.01 0.01 2.7 0.07 0.02 3.0 0.15 0.55 3.5 0.66 2.02

As can be seen, we have always seen a higher absorption value with ristomycin than we did with ristocetin.

Surprisingly, however, it has been found that the essential condition for the reliability of our assay is that the reagent used for the assay should contain high purity and constant A-ristomycin or derivative. This is probably explained by the fact that although the antibacterial activity of ristocetin and ristomycin is similar, the microorganisms that produce them are significantly different. Thus, depending on the composition of the medium and the metabolic pathways of the biosynthesis, even with the same processing, the contaminants may be different and different. It is further known that both Nocardia lurida NRRL 2430 and Proactinomyees fructiferi var. in the culture fluid of ristomycin, two biologically active components are formed. When used as an antibacterial drug, the presence of component B in A-ristomycin is not a problem since both agents have been shown to be effective against pathogenic microorganisms. Moreover, the bactericidal activity of component B was found to be significantly more active than that of component A.

In contrast, for both antibiotics, only component A is considered valuable for haematological tests. This was reported by JENKINS et al. (Thromb. Res. 7, 531 (1975)) who found that B-ristocetin did not aggregate normal human platelets.

It is known that under the alkaline conditions of fermentation and ion exchange purification, both ristomycin molecules can undergo degradation. To clarify this, carboxy-A-ristornycin was prepared as a model compound for our experiments and was found to no longer aggregate nor inhibit A-ristomycin-induced aggregation. Thus, the hydrolysis of the C-terminal methoxycarbonyl group of the highly sensitive glycopeptide molecule by alkali (and the subsequent retro-aldol cleavage on both beta-hydroxyphenylserine units) can render the A-ristomycin prepackage completely worthless for hematological purposes. In our experience, we have not been able to get rid of these unwanted auxiliaries by the recrystallization of water-alcohol used earlier.

In addition, the ristomycin base also has extremely hygroscopic properties. For example, it can absorb more than 15% moisture on its own weight when standing in the air for a few minutes. This is why accurate weighing of the preparation is almost impossible. However, we were surprised to find that the aggregate activity of A-ristomycin samples during irrigation of phosphorus pentoxide in everyday laboratory practice is irreversibly impaired.

It is beyond doubt that biological or ultraviolet spectrophotometric determination of drug content and specific optical rotation value measurement alone can be used to detect any impurities accompanying the preparation, specifically the B component and various alkaline degradation products, during the recovery of the antibiotic. Namely, said auxiliaries also have ultraviolet light absorption at 280 mm and a similar optical optical capacity.

According to our invention, the highly pure Formula I Risomycin and / or ristomycin derivative is prepared by preparing 30-50% aqueous solution of either the crude ristomycin base and the pH of this solution. with a mineral acid, preferably dilute sulfuric acid, between 6.7 and 7.0, preferably 6.8; or the pH of the aqueous solution of the contaminated A-ristomycin monosulfate with an alkaline solution, preferably ammonium hydroxide. After adjusting between 6.7 and 7, 20-50% (v / v) of water-miscible low-carbon carbonitrile, preferably acetonitrile, is added to the solution at room temperature. optionally, the resulting crystals are dissolved in an organic solvent, preferably a C 1 -C 5 alkanol, and then acylated with an acid anhydride, preferably trifluoroacetic anhydride, at a temperature of 0 to 20 ° C. The resulting product (s) are then isolated.

The A-ristomycin or its derivative obtained in i'y way is dissolved in aqueous buffer solutions, preferably in an aqueous solution of tris-hydroxyaminomethane, for platelet aggregation assay, in particular in Willebrand3.

A reagent for diagnosing disease -3186121 is prepared.

In a preferred embodiment of the process according to the invention, a crude solution of 30-50% is prepared by dissolving the crude ristomycin base obtained in known fermentations in distilled water at room temperature. The pH of the solution 6) is then adjusted to 7-7.0 with mineral acids, preferably dilute sulfuric acid.

Alternatively, starting from crude A-rostomycin sulfate, after reconstitution, the desired pH is adjusted with a dilute alkaline solution, preferably ammonium hydroxide solution. Optimal pH is achieved by instrumental measurement, or preferably by a blue-to-blue conversion of the bromothymol blue indicator. The solution thus prepared is then treated with 20-50% (v / v) water-miscible alkyl nitrile, preferably acetonitrile, at which time crystallization begins.

However, it is also possible to crystallize the higher purity crude product by first adding 15-50% (v / v) acetonitrile to the aqueous solution of A-ristomycin sulfate prepared as described above, followed by the desired pH of 6.7-7 The solution was heated to dissolution and allowed to cool slowly to room temperature.

The desired crystals were washed on the filter with acetone and dried in a vacuum desiccator over calcined calcium chloride and paraffin chips by constant suction to constant weight. The process produces non-hygroscopic, high purity and crystalline A-ristomycin or sulfate, with excellent aggregation ability, in 60-80% yield, depending on the starting material content.

In a further preferred embodiment of the invention, the crude A-ristomycin is dissolved or suspended in an organic solvent, preferably an alkanol, followed by cooling with an acylating agent, e.g. trifluoroacetic anhydride was added dropwise. The crude product is purified, if desired, by recrystallization.

The reagent of the present invention comprises a standardized, high purity rhizomycin or ristomycin derivative prepared by the above process, which is a weight-stable, stable compound. The reagent of the invention (tradename: AGGRISTIN-kit) preferably contains 15 mg dosage units. However, the free phenolic hydroxyl groups of the reagent may, under prolonged storage, in the presence of oxygen in the air, under sunlight, exhibit an unfavorable quinidial structure for platelet aggregation. This is evidenced by our long-term (40 hours) ultraviolet light irradiation of the solid preparation. Therefore, the reagent of the present invention should be packaged in a hermetically sealed vial of brown glass in the Hematology Diagnostic Kit.

The main advantages of the invention are summarized below:

1. The process of the present invention provides high purity compounds of formula (I) which are excellent for use in blood coagulation assays.

2. The reagent of the present invention is excellent for use in quantitative blood plasma tests and provides a more favorable range of measurement than prior art methods.

3. The reagent of the invention is relatively stable, stable in weight, sterilized at 100 ° C, and can be stored.

Stability studies have undoubtedly clarified that distilled aqueous solution of crystalline A-ristomycin monosulphate can be sterilized for 0.5 to 1.0 hours at 100 ° C without significant damage. It was also found that a solution of 15 mg / ml in TRIS buffer (pH 7.4) in the AGGRISTIN kit can be stored for 3 months at + 4 ° C without decomposition.

The invention is illustrated by the following non-limiting examples.

Example 1

Preparation of A-ristomycin sulfate (R, R 1, R 1, R ₃ are as defined in claim 1, R 4 - CH 3, X = HSO ₄ , YH)

2 g of a crushomycin base crude product prepared in a known manner (see U.S. Patent No. 180,753) at room temperature are 5.0 ml distillate. water was carefully adjusted to pH 6.8 with 1N sulfuric acid. The yellowish, slightly opalescent solution was clarified with a small amount of charcoal and filtered. Preferably, 15 to 20% (v / v) acetonitrile was added dropwise to the crude filtered solution dropwise until cloudy, and the mixture was allowed to stand at room temperature until crystallization occurred. If necessary, this operation was repeated several times with additional small volumes of acetonitrile for two to three days, with the crystallization finally terminated by cooling in a refrigerator at + 4 ° C for 24 hours.

The resultant square whitish crystals were collected on a filter, washed first with 2x1.0 mL of an ice-cold mixture of acetonitrile-water (1: 1) and cold with 2 mL of acetone, and then in a desiccator containing calcined calcium chloride and paraffin at room temperature under suction. , dried to constant weight. Yield: 71%.

Example 2

Preparation of A-ristomycin sulfate

1.5 ml to 2.0 g of crude A-ristomycin sulfate used as starting material at room temperature with constant shaking 4.0 ml. Dist. water, and to the highly effervescent solution, 1.0 mL of acetonitrile was added, clarified with a small amount of activated carbon, and filtered. Subsequently, the pH of the medium was neutralized with 2N ammonium hydroxide in the presence of bromothymol blue indicator (yellow to sky blue) to a tap (checked by sampling). Additional acetonitrile was added to the solution until turbid, and after crystallization began, the precipitated crystals were re-dissolved by heating in a water bath and allowed to cool slowly to room temperature.

In the following, the procedure of Example 1 was followed. Yield: 65%.

The A-ristomycin monosulphate prepared by both methods had very good platelet aggregation properties and met high quality requirements:

(a) Mettler FP-5 (photocell recorder) melting point, product starts to decompose at 165-170 ° C.

b) High performance liquid chromatography (Hewlett-Packard 1082, HPLC) gave a high quality chromatogram of the material.

(c) The material is homogeneous under layer chromatography conditions and has a loge value in the aqueous solution at 280 nm of 4,0 to 4,5 and a sulphate ash content not exceeding 0,25%. With a water jet pump (about 15 mmHg), with continuous aspiration at the boiling point of acetone for 4 hours, the weight loss of A-ristomycin monosulfate does not exceed

1-2.5%.

Thin layer chromatography of the crude product and of A-ristomycin monosulphate prepared in Examples 1 and 2 was carried out as follows.

I. Freshly prepared aqueous solution of 1-2% A-ristomycin

II. 1. DC-Alurolle cellulose (Merck. 0.1 mm) thin film

2. Fixion 50 x 8 Disc (REANAL)

III. Mixtures of solvents to run

(a) n-butanol-pyridine-acetic acid-water (15: 10: 3: 12) or

b) The upper organic phase of n-butanol-butane-n-propanol-acetic acid-water (20: 10: 5: 3: 32) is prepared as described in Table II. For thin film 1.

II. 2. Thin layer equilibration and run was performed with pH 6 phosphate buffer in deionized water:

7.0 g of citric acid 4.0 g of sodium hydroxide

81.9 g of sodium chloride and

100 ml of methylcellulose was dissolved in a 1000 ml volumetric flask and distilled. water up to the mark.

ARC. Developer: Pauly's reagent 0.05 g of diazotized sulfanylic acid was dissolved in 10 ml of sodium carbonate and used freshly.

The pure crude product appears on the cellulose layer in solvent mixture a) at R _f = 0.59 and in b) at R _f = 0.52. Fixion 50x8 plates were assayed for Rf = 0.61 in citrate buffer pH 6. The reagent is highly sensitive, indicating virtually all degradation products and has a lower limit of detection of 0.5-1.0 / rg.

High performance liquid chromatography was performed on a Hewlett-Packard 1082 isocratic HPLC instrument. A Cg-Lichrosorb (10 µm) loading (4.6 x 20 mm) analytical column was used for our assays. Active substance content of A-ristomycin samples was indicated by a UV detector operating at 254 nm. 1 M solutions were prepared from the samples and 20-30 μl aliquots were injected into the column. The most suitable eluent composition (I and II), the flow rate and the recording paper speed for the preparation of chromatograms are given below:

Eluent I: Sörensen pH 6.3 citrate buffer containing 12% methylcellulose. Flow rate: 2.0 ml / min. Recording paper speed: 0.1 cm / min.

II. eluent: pH 7.30 ammonium formate solution containing 10% acetonitrile (formic acid free)

Flow rate: 30 ml / min Recording paper speed: 0.3 cm / min

The stability of crystalline A-ristomycin monosulfate prepared according to Examples 1 and 2, UV light irradiation of solid samples, heat sterilization of aqueous solutions of various concentrations, and standing in TRIS buffer at + 4 ° C.

(a) 50-100 mg of A-ristomycin was irradiated with ultraviolet light in a stoppered bottle for 40 hours so that the sample container contained approximately 50 ml of the sample. 20 cm from the lamp. After 40 hours of irradiation, the solution was slightly deepened and human platelets were only slowly aggregated at 15 mg / ml.

b) In water distilled from A-ristomycin, two similar dilutions (0.39 mg / ml and 0.35 mg / ml) and one more concentrated solution (6.8 mg / ml) were prepared in a water bath at 100 ° C for 3 hours. held. From time to time after evaporation of the evaporating water, samples were taken and after cooling to room temperature, EJS values were determined from the absorbance at 280 nm. It can be seen that the absorbance of the solutions at 100 ° C increases non-linearly. The substances recovered by lyophilization in the 0-1 hour range have not yet indicated decomposition by thin layer and high performance liquid chromatography. The substances are properly aggregated at normal concentrations in normal human platelets.

c) A 15 mg / m 'solution of ristomycin was prepared in TRIS buffer (pH 7.4) and stored in a + 4' 'Con refrigerator for 12 weeks. The stock solution was subjected to the usual platelet aggregation assay every two weeks, or appropriate dilution buffer (0.2 ml to 10 ml), and the Ek * value was determined. The aggregation activity and Ekm value of A-ristomycin solution remained unchanged throughout the study.

Example 3

Preparation of Bis-Ν, Ν'-trifluoroacetyl-A -ristomycin

206 mg of A-ristomycin in 10 ml of abs. slurried in methanol and 0.28 mL of trifluoroacetic anhydride was added dropwise under ice-cooling. The mixture was allowed to stand at room temperature and then evaporated to dryness in vacuo. Abs. redissolved in methanol and evaporated again. This operation was then repeated three more times and dried in a vacuum desiccator over calcined calcium chloride and paraffin chips to constant weight. Yield: 90%.

Hist-Ν, Ν-trifluoroacetyl-A-ristomycin did not have a characteristic melting point. Standardization: on the cellulose layer according to III. a) Chromatography in solvent mixture (see Example 2) Rf = 0.45.

Analysis CwHicsO-jsNgFe (2258) to calculated F, 5.05; N, 4.96. found: F% = 4.91; % N, 4.86; 4.88 4.74

-5186121

The compound prepared above aggregated normal platelets in the same way as A-ristomycin.

Example 4

Preparation of the reagent

15.00 g of crystalline A-ristomycin monosulphate and 100 ml of distilled water (pH 6.8) were suspended in a 1 liter Erlenmeyer flask. An additional 400 to 500 ml of active ingredient is added to the wall of the flask under constant agitation. Dist. water. The resulting homogeneous solution was filtered through a 1000 mL volumetric flask, the flask and the filter were quantitatively washed and finally made up to the mark. The solution prepared above contained 15 mg / ml of A-ristomycin monosulfate. (In the case of sterile formulation, the solution was kept at 100 ° C for 30-60 minutes and then cooled to room temperature.) Then, using a dispensing machine (aseptic conditions), volumes of exactly 1.00 to 1.00 ml were brown. vials and lyophilized after freeze-drying and sealed.

To check the correct dosing and the completeness of lyophilization, the content of A-ristomycin (15 mg) per charge was checked by HPLC and weight measurement.

B. Preparation of the reagent solution

15 mg of lyophilized A-ristomycin monosulfate prepared in A can be dissolved with the following diluents:

1. Creating TRIS Buffer:

2.42 g of TRIS-hydroxymethylaminomethane give 8.64 g of NaCl per 1000 ml of pH 7.4

2. Preparation of Tyrode solution

^ concentration i was (relative to anhydrous salt) NaCl 0.80 KC1 0.02 CaCfe 0.02 MgCl ₂ 0.01 NaHCOj 0.10 NaH ₂ PO ₄ 0,036 glucose 0.10 Concentration of ions in maeg / l Na ⁺ 144 K ⁺ 3 Ca ⁺⁺ 4 Mg ⁺⁺ 2 Cl 146 HCOj 12 Η ₂ ΡΟϊ 3 glucose 6 mM

3. Preparation of Ringer's Solution ("Elevator"): Concentration of salt (relative to anhydrous salt)%

NaCl 0.90 KC1 0,040 CaCl ₂ 0,025 NaH ₂ PO ₄ 0,020 Concentration of ions in maeg / l Na ⁺ 156 K ⁺ 5 Ca ⁺⁺ 5 Cl 164 HCOí 2 -

4. Preparation of the Locke solution:

salt (relative to anhydrous salt) %-in NaCl 0.90 KC1 0.042 CaCl ₂ 0,024 NaHCOj 0,020 glucose 0.10 Concentration of ions in maeg / l Na ⁺ 156 K ⁺ 6 Ca ⁺⁺ 4 Cl 164 HCOj 2 glucose 6 mM

Patent claims

Claims (2)

Patent claims Process for the preparation of high purity ristomycin and / or ristomycin derivatives of formula I - wherein R is hydrogen, R, O-alpha-D-arabinofuranosyl (1- * 2) -O-alpha-D-mannopyranosyl (1- ^> 2) -O- [alpha-L-rhamnopyranosyl] - (1-6) -O-beta -D-glucopyranosyl group, R _{2 is} 2,3,6-trideoxy-3-amino- (NH Y) -alpha-L-ribo-hexapyranosyl, R 1 is alpha-D-mannopyranosyl, R _{4 is C} 1-4 alkyl, X mineral acid residue And Y is hydrogen or C (Z) j - (CH ₂ ) n -CO-, where Z is hydrogen or halogen, and n is an integer from 0 to 5, characterized in that the crude ristomycin 30-50% aqueous solution of the base by treatment with sulfuric acid, preferably diluted with mineral acid, to pH 6.7-7.0, preferably pH 6.8, or a 30-50% aqueous solution of contaminated A-ristomycin monosulfate in an alkaline solution, preferably ammonium hydroxide pH 6.7-7.0 20-50% (v / v) water-miscible lower aliphatic carboxylic acid nitrile, preferably acetonitrile, is added at room temperature and then crystallized at <4 ° C, optionally in an organic solvent, preferably It is dissolved in a C 1 -C 5 alkanol and then acylated with an acid anhydride acylating agent, preferably trifluoroacetic anhydride, at 0 to 20 ° C, and the product (s) is isolated. A reagent suitable for platelet aggregation assay and / or for the diagnosis of thrombolytic processes, preferably Willebrand's disease, DIC (diffuse intravascular coagulation), characterized in that it is prepared according to the method of claim 1, -6186121 standardized high purity ristomycin and / or ristomycin derivative in aqueous solution at a concentration of 8 to 20 mg / ml, preferably 12 to 15 mg / ml, if desired, buffering agent, preferably tris-hydroxymethylaminomethane 0.5 -5.0 mg / ml and inorganic salts, preferably sodium chloride, potassium chloride, calcium chloride or sodium bicarbonate, at a concentration of 0.01-0.01 mg / ml.