NZ198570A - Alpha-amylase inactivator - Google Patents

Description

New Zealand Paient Spedficaiion for Paient Number 1 98570 198570 Prirt"'-'--- -' W 9 >fO -SO* alb £-81 I i. JV J l> 1 <_■. '•j'-'jJ I • • I I I lyi I I 0 ■ I | g 0 f : * Complzlz C--:sci?;cation Filed: 1,1°, .V. r i'vibsiCatlOil C'iit'S: • P.O.'Jcimi-;,' Ho: Mkk wg% ^ f^§ Mfai 111 ijiiMil N-Z. No.

NEW ZEALAND Patents Act 1953 COMPLETE SPECIFICATION .pfrTEWTorncs a* OCT 19*11 " ^-AMYLASE INACTIVATOR, A PROCESS FOR ITS PREPARATION, AN AGENT BASED ON THIS INACTIVATOR AND ITS USE." We, HOECHST AKTIENGESELLSCHAFT, a'corporation organized under the laws of the Federal Republic of Germany,o£ D-6230 Frankfurt/Main 80, Federal Republic of Germany, do hereby declare the invention, for which we pray that a Patent may be granted to us, and the :method by which it is to be performed,to be particularly described in and by the following statement:- ! ; 8 An a-amylase inhibitor, which is obtained by the fermentation of Streptomyces tendae 4158 (ATCC 51210) and its variants and mutants, is described in British Patent Specification 1,599,37 2.. It is a peptide with respect to its chemical structure and has the ability irreversibly to inactivate a-amylase. Owing to these properties, it can be used for regulating the blood sugar level.

It has now been found that by using an improved process for obtaining and purifying the a-amylase inactivator, also designated in the following text as HOE 467, the inactivator can be prepared in relatively high purity by relatively simple rules, and that the highly pure substance comprises two components, designated in the following text as HOE 467-A and HOE 467-B.

If not otherwise stated, a-amylase inactivator is understood, in the embodiments mentioned above and in the following text, as meaning the mixture as well as the individual components.

The invention therefore relates to an a-amylase inactivator comprising the components HOE 467-A and HOE 467-B, or comprising the component HOE 467-A or HOE 467-B, and a process for its preparation and separation into the two components.

The description of the substances given in British Patent Specification 1,599,372 applies for the a-amylase inactivator HOE 467 according to the invention, and for the two' individual components HOE 467-A and HOE 467-B, with the exception of the following essential differences: 198570 9 - 3 - ■ It has an increased enzymatic activity and comprises the 2 components HOE 467-A and HOE 467-B, which are characterised by the following data: HOE 467-A is characterised by the following aminoacid 5 composition: Asp 6 Glu 7 Ala 7 Tyr 6 Lys 1 Thr 8 Pro 3 Val 8 lie 2 Arg 3 Ser 5 Gly 7 Cys 4 Leu 4 His 2 Trp 1 HOE 467-B has the following aminoacid composition: Asp 5 Glu 6-7 Ala 7 Tyr 6 Lys 1 Thr 6-8 Pro 3 Val 7-8 He 2 Arg 3 Ser 4-5 Gly 7 Cys 4 Leu 4 His 2 Trp 1 The determination of Trp was effected by absorption measurement in UV light. The remaining amino-15 acids were determined by hydrolytic cleavage.

The isoelectric points of the two components differ by a trivial amount and are dependent on the ionic strength and the methods of determination (Biochemisches Taschenbuch (Biochemical Handbook), edited by H.M. Rauen, 20 Springer Verlag, 1964). The values obtained by isoelectric focusing (R.C. Allen, H.R. Maurer: Electrophoresis and Isoelectric Focusing in Polyacrylamide Gel, V. de Gruyter, Berlin, 1974) are: HOE 467-A: 4.35+0.15 25 _ HOE 467-B: 4.53+0.15 The highly pure a-amylase inactivator HOE 467-A according to the invention also has aspartic acid as its end group, comprises 74 aminoacids with the following sequence 198570 Asp-Thr-Thr-Val-Ser-Glu-Pro-Ala-Pro-Ser-Cys-Val-Thr-Leu-Tyr-Gln-Ser-Trp-Arg-Tyr-Ser-Gln-Ala-Asp-Asn-Gly-Cys-Ala-Glu-Thr-Val-Thr-Val-Lys-Val-Val-Tyr-Glu-Asp-Asp-Thr-Glu-Gly-Leu-Cys-Tyr-Ala-Val-Ala-Pro-Gly-Gln-Ile-Thr-Thr-Val-5 Gly-Asp-Gly-Tyr-Ile-Gly-Ser-His-Gly-His-Ala-Arg-Tyr-Leu- i Ala-Arg-Cys-Leu, and has disulfide bridges between Cys 11 and Cys 27 and between Cys 45 and Cys 73. Its mole cular weight is calculated on the basis of the composition to be 7958.

For determining the aminoacid composition, HOE 467-A was hydrolytically cleaved, and the corresponding acids Glu and Asp were formed from glutamine (Gin) and asparagine (Asn).

The inactivator HOE 467-B is a degradation pro-15 duct of HOE 467-A, shortened at the N-terminal end of the chain. Thus, for example, serine can be the terminal group in one of the possible degradation products.

For the two components HOE 467-A and HOE 467-B, 20 the specific inhibitory action against the a-amylase from pig's pancreas is 1.7 . 10 AlU/g.

The inhibitory action was determined in the amylase test described below: ' Amylase test An amylase inhibitor unit (AIU) is defined as the quantity of inhibitor which is capable, under the . test conditions, of a 50% inhibition of two amylase units (AU). According to international agreement, an amylase unit is the enzyme quantity which cleaves, in 1 98570 one minute, 1 ji equivalent of glucosidic bonds in starch. The \i equivalents of cleaved glucosidic bonds are determined photometrically, using dinitro-salicylic acid, as |i equivalents of reducing sugar.

The data are calculated as p. moles of maltose, which are determined by means of a maltose calibration line. The tests are carried out as follows: a-Amylase from pig's pancreas and the solution to be tested are pre-incubated together in 1.0 ml of 20 10 mM phosphate buffer, pH 6.9, and 10 mM NaCl for 10 - 20 minutes at 37°C. The enzymatic reaction is started by the addition of 1.0 ml of soluble starch, according to Zulkowski (0.25% in the buffer indicated). After exactly 10 minutes, the reaction is stopped with 2.0 ml 15 of dinitrosalicylic acid color reagent (according to Boehringer Mannheim: Biochemica Information II) and the mixture is heated for 5 minutes in a boiling water bath to develop the color. After the mixture has cooled, the extinction is measured at 546 nm against the blank 20 reagent. By using various quantities of inhibitor, the 50% inhibition in comparison to the uninhibited enzyme reaction is determined graphically by plotting the probability.

In the preparation of the a-amylase inactivator 25 according to the invention, the strain Streptomyces tendae 4158 (ATCC 31210) or the derived strain Streptomyces tendae HAG 1266 (DSM 1912) is used according to the invention. The inactivator is obtained from the culture solution. The strain Streptomyces tendae 198570 HAG 1266 is preferably used.

Furthermore, the invention also relates to the strain Streptomyces tendae HAG 1266.

The new strain differs in important properties 5 from the strain Streptomyces tendae ATCC 31210. The different morphological characteristics of the two strains and properties, which are also new and are important for the fermentative preparation and the isolation of the a-amylase inactivator according to the invention, such as 10 the more rapid growth and the a-amylase inactivator production capacity which is increased by about 40%, are striking. The melanine formation, which manifests itself in the case of Streptomyces tendae ATCC 31210 by a brown-black coloration of the culture solution and of 15 the culture filtrate and presents certain purification problems for the working-up, was reduced to about 1/lOth in the case of the new strain HAG 1266 with the aid of genetic interventions and also changed qualitatively by a shift in the color. The melanine pigments now exhibit a reddish-yellow color spectrum. The elimi nation of this type of melanine in the working-up is unproblematic.

The two strains which produce a-amylase inactivator are compared in the following table: 1 70 Table • ATCC 31210 HAG 1266 Color of the substrate yellow-brown reddish mycelium Color of the sporulated gray-brown white-yellow aerial mycelium Morphology of the spore Retinaculum Flexibilis chains apertum (RA) (F) Spore morphology spherical, spherical, slightly sharp-edged verrucous to tubercles , smooth surface, size 0 1.3 fim size 0 1 yum Melanine formation on positive negative * peptone medium Nitrate reduction positive positive Substrate Glucose ++ +++ evalua Fructose + ++ Saccharose + +++ tion Maltose + + spectrum Lactose + - Galactose + - Rhamnose + - - Sorbose - - Xylose + - Arabinose + ++ « Inositol ++ - Mannitol ++ - Raffinose + - Cellulose _.

The strain Streptomyces tendae HAG 1266 has been deposited in the Deutschen Samrnl ung von Mikro-organismen (DSM) (German Collection of Microorganisms) under the registration No. DSM 1912.

The fermentation is advantageously carried out in a manner analogous to that described in British Patent Specification No. 1,599,372.

The prepared fermentation solutions of Streptomyces tendae ATCC 31210 and HAG 1266 contain enzymes which can reduce the active ingredient concentration considerably during the working-up. In addition, substances such as, for example, melanine can be present, which can only be separated off with difficulty by the known processes.

It has now been found that the necessary separation of the inactivator from the culture solution can readily be carried out using so-called adsorption resins (U. S Patent Specifications 3,531,463 and 3,531,963).

The process, according to the invention, for the preparation of the c-amylase inactivator comprises cultivating Streptomyces tendae ATCC 31210 or HAG 1266 and separating off the inactivator from the culture with the aid of adsorption resins or reverse-phase chromatography, and subsequently purifying the inactivator.

Commercial resins, such as, for example, polystyrene resins, are suitable. The resin is used by bringing it into contact with the culture filtrate. The pH value of the culture liquids is adjusted to 2 .198570 to 8, preferably 4 to 6. The solid resin selectively adsorbs the inactivator KOE 467, whereas the undesired enzymes and the major part of the impurities can be removed by filtration since they remain unbound in solu-5 tion. The recovery of the HOE 467 from the resin is advantageously effected either by washing with suitable aqueous buffer solutions, such as, for example, phosphate buffer, or with aqueous solutions of organic solvents, such as, for example, lower aliphatic alcohols, acetone, 10 acetonitrile or others. The further purification of the now concentrated inactivator HOE 467 is effected according to known processes.

The separation with adsorption resins which have been described above i s b as ed m the principle of the parti 15 tioning of different polar compounds. Still further methods of partitioning can be used for the required rapid separation. Thus, partitionings in the con text of reversed phase chromatography are also suitable (G. Schwendt, Chromatographische Trennmethoden (Chroma N tographic Separation Methods), Georg Thieme Verlag, Stuttgart, 1979), either commercial carriers or self- \ " prepared, for example silanized, carriers being used. Finally, liquid/liquid separation processes may also be pointed out, for example using aqueous polyethylene 25 glycol, aqueous phosphate buffer systems, as described in principle, for example, in U.S. Patent Mo. 4,144,130. rGS I 511 3'7'^ It has been reported in fa aI'mani 0,701j0C)0 that ion exchangers, such as, for example, DEAE 1 985 70 cellulose, are suitable for the purification of the a-amylase inactivator HOE 467. In this process, as is customary in ion exchange chromatography, pH values were selected which provide the required degree of ionization.

It has now be en found t surprisingly^ that if purifications are carried out - contrary to the rule - in the proximity of the isoelectric point, that is to say at a low degree of ionization, HOE 467 is separated into two components. The pH range from 4.4 to 6, preferably from 4.8 to 5.3, 10 is suitable for the separation. In this process, one component of the active ingredient is retained by the ion exchanger and can be detached from this ion exchanger only by changing the pH value or by increasing the salt concentration. This substance is called HOE 467-A.

Under the conditions mentioned, the other component remains more or less unbound by the ion exchanger and can thus readily be washed from the carrier. This component is called HOE 467-B.

Although the components are produced in suffi-20 ciently pure form for pharmaceutical use, an additional purification can still be carried out.

Thus, the inactivator HOE 467-A is purified, for example, by chromatography in the presence or absence of a dissociating buffer, preferably urea (6-8 M aqueous 25 solution), over DEAE or cation exchanger columns, such as, for example, carboxymethylcellulose columns, and is • isolated by customary methods. This additional puri fication is carried out, for example, before the determination of the aminoacid sequence. 198570 The a-amylase inactivator according to the invention is resistant to enzymatic degradation. Its properties are of interest with regard to its use as a medicament, particularly as a therapeutic for diabetes 5 and prediabetes, as well as adiposity, and with regard to its use as a dietary support. When used, the individual components as well as a mixture of HOE 467-A and HOE 467-B can be employed.

The invention therefore also relates to a 10 pharmaceutical agent containing the amylase inactivator according to the invention and to its use.

Starch containing foodstuffs and luxury consumables lead to an increase in the blood sugar in animals and man, and thereby also to an increased insulin secre-15 tion of the pancreas. Hyperglycemia arises from the cleavage of the starch in the digestive tract, under the action of amylase and maltase, to give glucose.

In diabetics, this hyperglycemia is particularly pronounced and prolonged.

In adipose subjects, the increased insulin secre tion acts on the lipogenesis and reduces the lipolysis.

The alimentary hyperglycemia and the hyper-insulinemia after the uptake of starch can be reduced by the amylase inactivator claimed. The action is depen-25 dent on the dose. The amylase inactivator according to the invention can therefore be employed as a therapeutic for diabetes, prediabetes and adiposity, and also as a dietary support. For this purpose, an admini stration at mealtimes is particularly recommended. 198570 The dosage, which should be in accordance with the weight of the patient and the individual requirement, is about 10,000 to 300,000 AIU, but can also be above or below these values in cases requiring this.

The amylase inactivator according to the inven tion is particularly suitable for oral administration. It can be used as the pure substance and also in the form of a pharmaceutical preparation, using the customary auxiliaries and excipients, such as, for example, talc, 10 magnesium stearate, lactose, starch or polyethylene glycol. Tablets, push-fit capsules or even granules are suitable forms for administration.

A combined use with other medicaments, such as substances which lower the blood sugar level or which 15 lower the lipid level, can also be of advantage.

Since higher-molecular peptides are not resorbed as such, or are not significantly resorbed as such, from the digestive tract, no toxicologically unacceptable side effects are to be expected from the substance 20 according to the invention. Owing to the not uncommon aminoacid composition, any proteolytic cleavage products can also be regarded as physiologically acceptable. Accordingly, no striking symptoms could be recognized in the oral administration of even higher 25 doses of the amylase inactivator to experimental animals. Also in the case of intravenous application to mice (l g/kg), the inactivator according to the invention was tolerated, in a 24 hour observation period, without a recognizable toxic effect. 198570 To test the pharmacological action of the amylase inactivator, male Wistar rats which had fasted and which had a weight of between 200 and 250 g received, in an oral administration, the inactivator according to the 5 invention simultaneously with 2 g of starch per kg of body weight, after a blood sample for determining the starting blood sugar value had been taken immediately before. Further samples of blood were taken from the caudal vein after 15 and 30 minutes and after 1, 2, 3 10 and 5 hours. The blood sugar determinations were carried out in an autoanalyzer, according to the method of Hoffman (J. Biol. Chem. 120, 51 (1937)).

NZ0 mice have a disturbed glucose tolerance.

They are therefore particularly well suited for investi-15 gations in which the blood glucose level is affected. The experimental arrangement corresponds to that for rats. The blood samples are taken from the orbital venus plexus. The blood sugar trend is monitored over a period of 3 hours.

The active ingredient is proved on NMRI mice in an analogous manner. The blood samples are also taken from the orbital venus plexus and the blood sugar trend is monitored over a period of 3 hours.

Under these experimental arrangements, the ani-25 mals treated with the inactivator according to the invention showed a smaller, more protracted blood sugar increase compared to untreated animals.

Example 1 The strain Streptomyces.tendae HAG 1266 is 1985 70 inoculated on slant tubes with a nutrient medium of the following composition: oat flakes 50 g H20 to 1,000 ml pH 7.2 The inoculated tube is incubated for 10 days at 28°C and is thereafter stored at +4°C. The spores which are readily detached from the yellowish aerial mycelium are suspended in 10 ml of sterilized distilled 10 water with 0.2 ml of Tween 80. 10® - 10^ spores, that is to say 1 ml of the suspension, is used for inoculating a 300 ml Erlenmeyer flask which is charged with 35 ml of sterilized nutrient solution with a pH value of 7.2 and the following composition: 15 1% of glucose 1% of soya flour 0.25% of NaCl The sterilization time is 45 minutes at 121°C and 1 bar. The flask is shaken on a shaking machine at 250 rpm and at an amplitude of 3.5 cm for 40 hours at +30°C. 5 ml of this preculture, in each case, are transferred to several Erlenmeyer flasks which are charged with 50 ml of sterilized nutrient solution and have a pH value of 7.4. The composition of this main 25 culture is as follows: soluble starch 4% soya flour 0.4% cornsteep liquor 0.4% skimmed milk powder 0.7% 198570 - 15 -glucose 1.0% (NH4)2HP04 1.2% The sterilization time is 45 minutes at 121°C and 1 bar. The main cultures were shaken on a shaking 5 machine at 250 rpm and at an amplitude of 3.5 cm for 96 -120 hours at 25°C. On the third, fourth and fifth day of culture, the content of a-amylase inactivator is determined according to the test instructions.

Under the test and culture conditions, the strain 10 Streptomyces tendae HAG 1266 yields 1,900 AlU/ml at an end pH of 6.4.

Example 2 The mixture is as in Example 1, but the main fermentation is carried out in a fermenter of 15 1 total 15 volume with a charge of 10 1. The following nutrient solution composition is used: starch 4% soya flour 0.4% cornsteep liquor 0.4% skimmed milk powder 0.7% glucose 1.0% (NH4)2HP04 1.2% After the sterilization, the pH value should be 6.8, and it is adjusted, as required, to this value 25 using sterilized acid (2N H^P04) or alkali solution (2N NaOH). The main stage is inoculated with 1 1, corresponding to 10%, of a preculture as described under Example 1.

The fermentation is carried out for 50 to 70 198570 hours at 30°C. The aeration is 300 l/hour at a rate of stirring of 250 rpm and an elevated pressure of 0,3 "bar.

The course of the fermentation is controlled and monitored with respect to the inhibitor activity, the 5 degradation of carbohydrates, the development of biomass and the physical behavior of the culture solution (surface tension, viscosity, density and osmotic pressure) by taking samples.

The maximum inhibitor activity is reached from 10 the 60th hour of culture, with an average value of 1,800 AlU/ml. The content of the fermenter is then fed to the working-up process.

Example 3 6.5 1 of culture filtrate, obtained by filtering 15 off.under suction the Streptomyces culture obtained according to Example 2, we re adjusted to pH 4.9, whilst stirring, with glacial acetic acid, and the mixture was introduced onto a prepared glass column. The column contained 230 g of polystyrene adsorption resin (for 20 example Diaion^R^ HP 20) suspended in water. The dimensions of the column were 22 x 5 cm, corresponding to a volume of 430 ml. The throughput of liquid was regulated so that 6.5 1 had run through after 2 hours. The adsorption process had then ended and the resin 25 could first be washed with pure water and then eluted with water to which increasing quantities of isopropanol had been added. The liquid flowing through the column was collected in fractions of 1 1 each, and was tested with respect to the enzyme-inhibiting action. The 198570 -inactive fractions were collected and were concentrated to 100 ml by distillation in vacuo. The concentrate contained 110,000 AlU/ml.

The solution thus formed, which was now stable to 5 salt, was directly introduced onto a DEAE ion exchanger column prepared with 1/30 phosphate buffer of pH 7.5, and was purified according to German Auslegeschrift 2,701,890, Example 5. 4 g of substance with 2,500 AlU/mg resulted.

Example 4 For further purification, a glass column with a diameter of 2 cm and a height of 22 cm, corresponding to a capacity of 70 ml, was filled DEAE cellulose which . had previously been equilibrated with 1/10 M sodium 15 acetate buffer of pH 4.9 and 0.02% of sodium aziae. The substance obtained according to Example 3 was now dissolved in 10 ml of the same buffer and was introduced onto the column. The column content was first washed with 100 ml of acetate buffer, and sodium chloride was 20 then gradually mixed with this eluting agent at such a rate that a continuous gradient was ensured. When the column throughput was collected in fractions, HOE 467-B occurred in the first fractions which were still free of sodium chloride, whilst the HOE 467-A component 25 could be detected in the fractions in which the NaCl concentration was 0.25 to 0.3 molar. The fractions containing the B component and the fractions containing the A component were collected separately, dialyzed against distilled water and freeze-dried. The 198570 colorless substances each had an activity of 1.7 .. 10^ AlU/mg.

The aminoacid analysis of the products, after hydrolysis with hydrochloric acid for 20 hours, gave the 5 following percentage compositions, with the aid of a Beckman Multichro analyzer: HOE 467-A HOE 467-B Aspartic acid 8.51 7.90 Threonine 10.07 8.17 Serine 5.41 5.98 Glutamic acid 11.30 12.39 Proline 3.28 3.39 Glycine 5.61 6.19 Alanine 6.85 7.41 Cysteine 4.68 .^3,33 Valine 8.69 8.54 Methionine Isoleucine 2.93 3.28 Leucine 5.91 6.61 Tyrosine 11.28 12.69 Phenylalanine Histidine 3.49 3.72 Lysine 1.78 2.05 Arginine 6.12 6.52 Example 5 The a-amylase inactivator HOE 467-A and HOE 467-B are obtained from the culture solution of Streptomyces tendae ATCC 31210 in an analogous manner. 198570 Example 6 a) Preparation of the a-amylase inactivator HOE 467-A being uniform at the N-terminal end; HOE 467-A obtained according to Example 4 or 5 5 is purified by chromatography over a DEAE or carboxy-methylcellulose column in the presence of aqueous 8M urea solution as the dissociating buffer, and is isolated according to customary methods. b) Preparation of tryptic peptides: The a-amylase inactivator HOE 467-A which is obtained according to a) and which has a pure end group is either oxidized with performic acid or reacted with ethyleneimine to cleave the disulfide bridges (see C.H.V. Hirs, J. Biol. Chem. 219, 611-621 (1956) and 15 M.A. Raftery and R.D. Cole, J. Biol. Chem. 241, 3457- 3461 (1966)), and tryptic cleavage is carried out according to customary methods. The tryptic hydrolysis products are chromatographically separated and purified. c) Sequence analysis: The sequence analysis was carried out according to the film technique (P. Edmann and G. Begg, Eur. J. Biochem. 1, 80-91 (1967)), using the following program: a) Quadrolprogram: The a-amylase inactivator HOE 467-A reacted according to b) with ethyleneimine, and an 25 activator A which had been subjected to a limited tryptic cleavage were degraded. p) Propyne program: The remaining peptides were degraded in this process (see G. Braunitzer and B. Schrank, Hoppe-Seyler's Z. Physiol. Chem. 351, 88 (197 ) and

Claims (8)

198570 # - 20 - G. Braunitzer, A. Stangel and 0. Scheithauer, Hoppe-Seyler's Z. Physiol. Chem. 359, 137-146 (1978)). d) Aminoacid analysis: The analysis was carried out by hydrolysis, 5 using 6 N hydrochloric acid, of the inactivator obtained according to a). e) Determination of the disulfide bridges: For this purpose, the substance obtained according to a) was treated, in dilute formic acid, with 10 pepsine during the course of 18 hours, and the cleavage products were purified according to customary methods. On the basis of the operations carried out, it was found that the substance obtained according to a) comprises 74 aminoacids, the sequence of which is as 15 follows: Asp-Thr-Thr-Val-Ser-Glu-Pro-Ala-Pro-Ser-Cys-Val-Thr-Leu-Tyr-Gln-Ser-Trp-Arg-Tyr-Ser-Gln-Ala-Asp-Asn-Gly-Cys-A1a-Glu-Thr-Val-Thr-Val-Lys-Val-Val-Tyr-Glu-Asp -Asp-Thr-Glu-Gly-Leu-Cys-Tyr-Ala-Val-Ala-Pro-Gly-Gln-Ile-Thr-Thr-Val-Gly-Asp-Gly-Tyr-Ile-Gly-Ser-His-Gly-His-Ala-Arg-Tyr-20 Leu-Ala-Arg-Cys-Leu, and has disulfide bridges at the positions mentioned. WHAT#/WE CLAIM IS: - 21 - >198570

1. An a-amylase inactivator comprising HOE 467-A with the aminoacid composition: Asp 6 Glu 7 Ala 7 Tyr 6 Lys 1 Thr 8 Pro 3 Val 8 lie 2 Arg 3 Ser 5 Gly 7 Cys 4 Leu 4 His 2 Trp 1 rOf pH and the isoelectric point'4.35 £ 0.15, or comprising HOE 467-B with the aminoacid composition: Asp 5 Glu 6-7 Ala 7 Tyr 6 Lys 1 Thr 6-8 Pro 3 Val 7-8 lie 2 Arg 3 Ser 4-5 Gly 7 Cys 4 Leu 4 His 2 Trp 1 C- Of pH and the isoelectric pointr4.53 £ 0.15, or comprising .defined. the two components HOE 467-A and HOE 467-B as hereinbefore]

2. An a-amylase inactivator as claimed in claim 1, wherein HOE 467-A has a molecular weight of 7,958 and the 74 aminoacids have the following sequence Asp-Thr-Thr-Val-Ser-Glu-Pro-Ala-Pro-Ser-Cys-Val-Thr-Leu-Tyr-Gln-Ser-Trp-Arg-Tyr-Ser-Gln-Ala-Asp-Asn-Gly-Cys-Ala- r Glu-Thr-Val-Thr-Val-Lys-Val-Val-Tyr-Glu-Asp-Asp-Thr- 'tfk. Glu-Gly-Leu-Cys-Tyr-Ala-Val-Ala-Pro-Gly-Gln-Ile-Thr- ^ Thr-Val-Gly-Asp-Gly-Tyr-Ile-Gly-Ser-His-Gly-His-Ala-Arg-Tyr-Leu-Ala-Arg-Cys-Leu, and disulfide bridges allocated between Cys 11 and Cys 27 and between Cys 45 and Cys 73.

3. A process for the preparation of the a-amylase inactivator as claimed in claim 1, 'which comprises cultivating Streptomyces tendae ATCC 31210 or HAG 1266 and separating off the inactivator from the culture, with the aid of adsorption resins or reversed phase - 22 - | °l %b~70 chromatography, and then purifying the inactivator.

4. A process as claimed in claim 3, wherein the inactivator which has been separated off is purified with the aid of ion exchangers in the pH range between 4.4 and 6, and is separated during this process into the [defined. two components HOE 467-A and HOE 467-B as hereinbefore1

5. A process as claimed in claim 3, wherein the inactivator which has been separated off is purified with the aid of ion exchangers in the pH range between 4.4 and 6, and is separated during this process into the two components HOE 467-A and HOE 467-B, and HOE 467-A is further purified and isolated by chromatography on DEAE-or carboxymethyl-cellulose columns, in the presence of a dissociating buffer.

6. A pharmaceutical preparation containing the "-amylase inactivator as claimed in claim 1.

7. A process according to claim 3 substantially as hereinbefore described or exemplified.

8. A preparation according to claim 6 substantially as herein described or exemplified. HOECHST AKTIENGESELLSCHAFT By Their Attorneys HENRY HUGHES LIMITED Per :