CA2024053A1 - Anf-antagonist - Google Patents

Abstract

Abstract The invention is concerned with an antagonist of mammalian atrial natriuretic factor, isolatable from microorganisms of the genus Streptomyces having the DSM Nos. 4777 and 4778, and physiologically compatible salts thereof as well as a process for the isolation of such an antagonist and the production of its salts.

Description

2~2~0~3 RAN 4105/124 Objects of the present invention are an antagonist of mammalian atrial natriuretic factor, its physiologically ;-, compatible salts, microorganisms which contain said antagonist, a process for the production of the antagonist and of its salts, pharmaceutical preparations which contain ~uch an antagonist or its salts and their use for the treatment of illnesses, especially those in which the 10 regulation of blood pressure plays a role. ~, ; ;

Atrial natriuretic factor (ANF) itself is synthesized in the mammalian atrium as a pre-pro-hormone and is stored in granules as a pre-hormone of 126 amino acids. As a 15 eesult of certain stimuli, such as e.g. dilat~on of the :
auricle, the protein is processed to the biologically active peptide consisting of 28 amino acids (99-126) and i8 secreted into the blood circulation.

The preferred target organ of ANF is the kidney where it increases the glomerular filtration rate, renal blood flow and sodium excretion and enlarges the urine volume.
Furthermore, ANF also has vasodilatory activity which leads to a lowering of the blood pressure. Generally, ANF
25 appears to influence the blood pressure regulation mechanisms [Needleman, P. and Greenwald, J.E., New Engl. --J. Med. 314, B28 (1986)].
. -On the other hand, ANF antagonizes the physiologjical 30 effect8 of the renin-angiotensin system at various levels:
by reducing renin secret,ion, by relaxing the blood vessels which are pre-contracted by angiotensin, by blocking angiotensin-induced aldosterone synthesis and by the natriuretic and diuretic effects which counteract 35 aldosterone-induced sodium retention.

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~: 2~ ~ 2 ~ 3 - -An ANF antagonist which can be used in the acute control of severe hypotensions, for example in shock conditions, and of dehydrations has now been found.
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An ANF antagonist in accordance with the invention can be produced by isolating it from microorganisms of the genus Streptomyces, preferably from Streptomycetes, which have been deposited accordinq to the Budapest Treaty on the 30.8.1988 at the Deutschen Sammlung f~r Mikro-organismen in Braunschweig with the DSM Nos. 4777 and 4778 as well as from theie ANF antagonist-containing sub-cultures, mutants and variants.

The aforementioned microorganisms can be cultivated according to known fermentation methods (e.g. Rehm, H.J.
"Indu6trielle Mikrobiologie", Springer-Verlag, Berlin, Heidelberg, New York, 1980) in liquid media, for example those which contain starch, dextrin, glucose, D-mannitol, ribose or glycerol as the carbon source and soya meal, 20 yeast extract or peptone as the nitrogen ~ource. As salts ~or the aforementioned media there preferably come into con~ideration ammonium, magnesium or calcium salts or mixtures thereoe. As further fermentation conditions there are to be considered a tem~erature range of approximately 26 20-37C, an incubation period of 1-6 days under aerobic ~-conditions and the selection of a suitable known antifoam agent such as, for example, one based on polypropylene.

, An ANF antagonist can be isolated from the aforer ~ ~;
30 mentioned microorganisms cultivated under the given conditions either from the crude extract, i.e. an extract of cells and culture medium, from the cell mass or from the culture filtrate by a combination of peptide purifica-tion methods which are known to a person skilled in the 36 art. Por the detection of ~uch an ANF antagonist in mammals such as humans, bovines or rats there can be used . :: . ~, . . ~: - ~ .
, -_ 3 _ ~ 3 a combination of the methods described in detail in Example6 4 and 5 or the determination of a reduced -synthesis rate of cyclic guanosine monophosphate or other detection methods for antagoni6ts which are familiar to a person skilled in the art.
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The following come into consideration as preferred methods for the purification of an ANF antagonist from the aforementioned crude extract: ultrafiltration, liquid- --liquid extraction, e.g. with a mixture of water and 2-butanol, anion exchange chromatography, preferably on DEAE-Sepharose; silica gel chromatography (incl. on reversed phases), affinity chromatography, for example on a chelate affinity resin, preferably on a copper chelate eesin, particularly on a resin having an iminodiacetic acid ligand, and gel permeation chromatography on modified dextran, for example Sephadex LH-20. ~ ;~

An ANF antagonist can be purified from the cell mas6 --using known peptide purification methods, preferably by extraction with low-chain alcohols, for example methanol.

In a preferred embodiment an ANF antagonist is isolated from the culture filtrate. For this there can likewise be used known protein or peptide purification methods such as, for example, adsorption on a macroporous adsorber resin, preferably based on polystyrene, e.g.
Amberlite, ion exchange chromatography, preferably based on agarose, e.g. DEAE-Sepharose, chromatography on silica gel, including on reversed phases, and affinity chromato-graphy, preferably on a metal chelate matrix, as well as extraction procedures, of which extraction with 2-butanol is especially preferred.

3S In an especially preferred embodiment an ANF
antagonist is isolated in the following manner. The ANF

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antagonist i8 sepa~ated from the culture filtrate using a macroporou6 adsorber resin ba6ed on polystyrene and i8 eluted from this resin using aqueous i6epropanol. The biologically active eluate from this column iB then, after centrifugation, dilution with phosphate buffer and filtration, added to a DEAE-ion exchange column equilibrated at pH 7Ø Elution of the ANF antagonist is effected with a suitable concentration of NaCl. The thus-obtained eluate is then pumped on to a reversed phase 10 silica gel column. Elution of the ANF antagonist therefrom --is effected using a stepwise gradient of water/aceto-nitrile which contains a suitable amount of trifluoro-acetic acid. This eluate, diluted with water, is then added to a preparative C-18 silica gel column. Elution of 16 the ANF antagonist from this column is effected using a linear water/acetonitrile gradient. The eluate is evaporated in the presence of isopropanol. The residue is taken up in isopropanol, separated on a copper chelate gel column eguilibrated with phosphate buffer/NaCl and the ANF
antagonist is eluted with equilibration buffer and a sultable concentration of imidazole. This eluate i~ added to a preparative C-18 HPLC silica gel column. The ANF
antagoni~t can be eluted there~rom in pure form us~ng a linear acetonitrile gradient in water in the presence of a suitable trifluoroacetic acid concentration.
. . . :..
The characterization of an ANF antagonist in accordance with the invention can be carried out usi~g physical-chemical methods which are usual in peptide chemistry. Figures 1-3 show specific data for an ANF
antagonict isolated in accordance with Examples l-S.

Flauro 1: "Fast atomic bombardment" mass spectrum of the ANF antagonist in thioglycerol: m/z , 1870.9 is 3S the (M+H ) peak value with the composition goHlllN2124+H The calCulat molecular weight amounted to 1870.8.

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Fiaure 2: Infrared spectrum of the ANF antagonist in KBr.

Fiaure 3: Ultraviolet spectrum [measured in methanol:
~max = 280 nm (~ = 6923)]-Furthermore, an optical rotation of [a~D ~ +12 (c . 0.9%, in methanol) was determined for this ANF
antagonist. Decomposition of this ANF antagonist occurred from about 200C. Characterization using thin-layer 10 chromatography, analytical high pressure liquid chromato- ~
graphy and amino acid analysis can be carried out in a ~-known manner and preferably as described in detail in Example 3. The 13pecific data from these analyses are also to be found in Example 3.
On the bas i8 of the data determined, the ANF
antagonist in accordance with the invention is a cyclic peptide.
~ ' Cyclic peptides can be synthesized chemically using methods which ace known in peptide chemistry, such as, for example, by classical methods of ~eptide synthesis in solution, by the condensation of fragments, by partial or complete solid phase synthesis, for example as described 26 by Merrifield in J. Am. Chem. Soc. 85, 2149 (1963), and subesquent cyclization. Moreover, the linear analogues of such cyclic peptides can also be produced using -recombinant DNA technology methods which are known to a person skilled in the art, as described, for examplel, in 30 Maniatis et al. (1982) I~Molecular Cloning~, Cold Spring Harbor, and subsequently can be cyclized.
. :
A further ob3ect of the present invention are analogue~ o~ t~e ANF antagonist obtained from 3S Streptomycetes having ANF-antagonizing activity. Under analogues there are to be understood those compounds in which either one or more amino acids of the cyclic peptide is/are chemically modified on the side-groups in a known manner or those in which one or more amino acids i~/are replaced or absent without thereby being detrimental to the ANF-antagonizing activity. Such analogues can be produced according to the methods of peptide chemistry or recombinant DNA technology already described, such as, for example, planned mutagenesis.

The ANF antagonist in accordance with the invention and its analogues as well as their physiologically compatible salts can be used for the production of pharmaceutical preparations, primarily of those for the treatment of illnesses in which the regulation of blood pressure plays a role. In addition, an ANF antagoni~t in accordance with the invention - where desired or required in combination with other pharmaceutically active substances - can be processed with conventionally used solid or liquid carrier materials in a known manner. The ao dosage of such preparations can be effected taking into consideration the usual criteria in analogy to already known ~roparaeions of similar activity and structuce.

Since the invention has been described hereinbefore in 25 ~eneral terms, the following Examples are intended to illustrate the invention in more detail, although they are not intended to limit its scope in any manner.

ExamP l e 1 ~ ~ ., ~ ' ,. ', ' ., Fermentation 10 ~haking flasks each with 100 ml of Medium 644 (2S
full-fat soya meal, 2t D-mannitol: the pH was adjusted to 35 7~4 with NaOH prior to sterilization of the medium at 121C for 20 minutes: polypropylene glycol in a .,.; ,~.

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concentration of 1 ml/l was also added to the medium prior to the sterilization) were inoculated with spores or mycelium of the Streptomycetes having DSM No. 4778 and incubated aerobically at 30C for 48 hours. These culture~
6 were then trans-inoculated into a fermenter which contained 10 1 of Medium 644 and which was operated at 30C for 24 hours, with an aeration of 0.4 vvm and while stirring at 360 rpm. This fermenter was then used to inoculate a 200 1 production fermenter containing 200 1 of Medium 644. This was oeerated at 30C for 96 hours, with an aeration of 0.3 vvm and while stirring at 600 rpm.

5 fermentations each of 200 1, which were carried out in this manner, together gave a yield of 6.1 g of ANF
antagonist tdetermined using the binding test from ~ -Example 4) in the culture filtrate. -`

ExamDle 2 I801ation of the ANF antaaonist Step 1:
The nutrient brot~ from a 200 1 fermenter in accordance with Example 1 was filtered over a suction 25 filter, which gave 130 1 of culture filtrate.
~ . , Step 2:
The filtrate from Step 1 was pumped at 20 l/h through a,chromatography column (bed dimension: 15 x 50 cm) filled 30 with Servachrom XAD-2 (~lerva, Heidelberg, Germany, particle si2e 0.3-0.9 mm) and subseguently washed with 20 1 of water and 20 1 of 10% aq. isopropanol. The ANF
antagonist was eluted from the re~in with 25 1 of 50%
isopropanol. The eluate was concentrated at 20C in a 35 vacuum and gave 1 1 of biologically active concentrate.
The adsorber resin was regenerated by 6ubsequent washing with 10 1 of isopropanol, 10 1 of 50% isopropanol and 30 1 ~-of watee.

Step 3:
6 For the further purification, a chromatography column (bed dimension: 14 x 26 cm) was filled with 4 1 of DEAE--Sepharose FF ~Pharmacia, Uppsala, Sweden) and washed with 12 1 of 100 mM sodium phosphate buffer (pH 7.0), followed by 8 1 of 10 mM sodium phosphate buffer (pH 7.0). The 10 column was operated at a flow rate of 4-5 l/hr. 1 1 of the ~ -concentrate from Step 2 was diluted with 7 1 of 10 mM -sodium phosphate buffer (pH 7.0), centrifuged (30 min., 10,000 rpm), filtered (fluted filter) and pumped through the column. The column was then washed with 16 1 of io mM
16 ~odium phosphate buffer (pH 7.0) and the ANF antagonist was eluted with 16 1 of 10 mM sodium phosphate buffer (pH 7.0), 100 mM sodium chloride. The column was washed with ~ 1 of 10 mM sodium phosphate buffer (pH 7.0), -lM ~odium chloride, followed by 8 1 of O.lM sodium ;; 20 hydroxide solution in order to regenerate the ion exchanger.

Five purification runs over Steps 1 to 3 gave 62 1 of eluate containing ANF antagonist.
2g Ste~ 4 ` 62 1 of eluate from Step 3 were pumped through a ;~
chromatography column which had been filled with Silica "~ RP-18 from ICN Biomedicals (Eschwege, Germany) (particle 30 size: 32-63 ~m bed dimensions: 3.7 x 58 cm: flow rate:
;~ 3~6 l/hr.). Subsequently, the column was washed in ~
sequence with 1 1 of water, 8.5 1 of O.lM ~odium phosphate ~ -buf~er (pH 3.0) and 4 1 of water. The column was then Washed with a stepwise gradient of water/acetonitrile, 35 5 mM teifluoroacetic acid, whereby the ANF antagonist was eluted with 40-60~ acetonitrile. 5.7 1 of eluate were -` ;
collacted. ~
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Step s:
The eluate from Step 4 was diluted to 10 1 with water and pumped through a steel column which had been filled with Prep C-18 (Waters, Milford, USA) (particle size:
50-105 ~m: bed dimengion: 5 x 30 cm; flow rate:
80 ml/min.). Then, a mixture of water/acetonitrile containing 5 mM trifluoroacetic acid was pumped through the column. The amount of acetonitrile was ZO% for Z5 minutes, 20-40~ (linear gradient) for 30 minutes and finally 40% for 75 minuteg, wheceby the ANF antagonist wag eluted. 1.21 1 of eluate wece collected and were evaporated at 40C in a vacuum, whereby isoproeanol was added portionwise in order to keep the product in golution. There were obtained 3.99 g of evaporation 16 cesidue which corresponded to 2.44 g of ANF antagonigt (determined by amino acid analygis).

Step 6:
A chelate gel of the following structure was prepared 20 from Se~harose CL 6~ FF according to Hochuli, E. [Chimica 40, qO8 (1986):

~ 26 Sepharose (6B)-O-CH2-CH-CH2-N
;~ 2 2 .. .. . .
0.8 1 thereof was filled into a chromatography column 30 (bed dimengions: 7 x 21 cm). The gel was prepared by sequential washing with the following solutions at a flow eato o~ 5 l/h: 2 1 o~ 50 mM co~er sulphate solution: 2 1 of water: 2 1 of 50 mM sodium acetate solution (pH 3.5), :
lM sodium chloride; 2 1 of water and 2 1 o~ 50 mM sodium 3S phosphate buffer (pH 7.5), 0.5M godium chloride (equilibration buffer). 2 g of the material obtained in 2 ~ 3 - 1 0 - "

Step 5 were dissolved in 75 ml of isopropanol, diluted with 1 1 of equilibration buffer and pumped through the chelate gel column at a flow rate of 2 l/h. Subsequently, the column was washed with 4 1 of equilibration buffer.
The ANF antagonist was then eluted with 4.4 1 of equilibration buffer containing 3 mM imidazole, followed by 2.6 1 of equilibration buffer containing 6 mM imidazole at a flow rate of 2 l/h. From the entire 3.99 g of the material from Step 5 there were obtained in two runs on the chelate column 3.4 1 of eluate which contained only ANF antagonist according to HPLC analysis (see Example 3).
Por regeneration, the chelate gel was washed with the following soluttons at a flow rate of 5 l/hr.: 2 1 of 50 mM EDTA solution (pH 8.0), 2 1 of water; 2 1 of 0~.2M
1S 80dium hydroxide solution 5 1 of water. ;~

Step 7:
The eluate from Step 6 (3.4 1) was pumped through a steel column filled with Prep C-18 (Waters) (same ex~erimental parameters a~ in Step 5). Subsequently, the column wa~ washed with 5 mM trifluoroacetic acid until the eluate showed an acidlc pH value. The ANF antagoni~t was then eluted from the column in 60 minutes with a linear ,gradient of 0-80t acetonitrile/5 mM trifluoroacetic acid.
25 There were collected 250 ml of eluate which was concentrated at 40C in a vacuum and, after lyophiliza- ~ -~
tion, gave 1.25 g of ANF antagonist as a white powder. ~ -.. ..
ExamDle 3 :
Characterization of the ANF antaaoni~t Thin-layer chromatography: a spot at Rf . 0.66, which decolorized with chlorine/tolidine, was obtained on 3S ~ieselgal 60 F254 (Merck) with n-butanol/acetic acid/ --water as the eluent in the ratio 80/20/20 (v/v/v).
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High pressure liquid chromatography (HPLC): a signal with a retention time of 4.46 min. was obtained on a ~Bondapak C-18 column (3.9 x 300 mm) from Waters with an eluent mixture of acetonitrile/water in the ratio 1:1, 5 mM trifluoroacetic acid, with a flow of 1 ml/min. and detection at 228 nm.

The amino acid analysis was carried out according to Spaekman, D.H. et al. lAnalyt. Chem. 30, 1190 (1958)]. The hydrolyses were carried out with 6N HCl at 110C in 24 hours or with 4N methanesulphonic acid. The deter-mination of the amino acid composition of the hydrolyzate gave the following results with the Liquimat III amino aeid analyzer (Kontron AG):

A~ino acidAmount foundTheoretical amount His 0.96 NH3 1.23 20 Asx 3.00 3 Ser 0.85 aly 4.99 5 Tle 1.92 2 Tyr 1.02 25 Pbe 2.96 3 Trp 1.1 The value for Asx was equated at 3. Special analyses, which were also carried out according to Spackman, D~H. et 30 al. ~Analyt. Chem. 30, 1190 (1958)~, gave for Asx ~ 2 Asp and 1 Asn. The configuration of the amino acids was determined as the ~L)-confiquration by hydrolysis and subsequent derivatization with (+)-l-(9-fluorenyl)ethyl chloroformate.
, .~ ' The primary 6tructure was determined by automatized Edman degradation of eeptides obtained by enzymatic as well as chemical cleavage, by NMR spectroscopy of the entire molecule with the concurrent used of various 2D pulse techniques such as "ROESY", "NOESY" and "RELAYED
COSY" and by "FAB" mass spectroscopy. The structure is set forth in Figure 4 using the abbreviations for amino acids which are conventional in peptide chemistry. It is thus a substituted 1,4,7,10,13,16,19,22-octaazacyclopentacosane.
ExamPle 4 . ~, In vitro detection of the ANF antaaonist ~ ;

The displacement of radioactively-labelled ANF from its receptor by the ANF antagonist was measured in a binding test according ~o B~rgi6ser et al. [Biochem.
Biophys. Res. Comm. 133, 1201 (19~5)]. A membrane preparation of bovine adrenal glands was used as the receptor preparation. To 100 ~1 of this preparation in 50 mM Tri~/HCl, 500 mM MgC12, 1 mM EDTA, 0.5% bovine ~erum albumin and 1 mM o-phenanthroline, pH 7.6, were added I-ANF (20,000 cpm, 18.2 pM) in 145 ~1 of this bu~fer and the ligand in 5 ~1 of dimethyl sulphoxide.
25 This mixture was incubated at 4C for 24 hours and bound ANF was separated from free ANF by rapid filtration over a - ~ -Whatman GF/C glass fibre filter. After washing the filter three times with the above buffer (3 x 4 ml) the filter was shredded and its radioactivity was measured in a 30 gamma-counter. The binding inhibition was equated quantieatively with the IC50, under which there is to be under~tood that concentration of cocresponding ligand at which the binding of I-ANF is inhibited by half.

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Table ICso value8 Ligand IC50 ( Rat ANF (99-126) 0.110 ANP antagonist 860 . .;
.
ExamPle 5 In vivo activit~ of the ANF antaaonist Spontaneously-hy~eetensive rats (SHR) or normotensive -~athogen-free (SPF) rats 20 weeks old were used for the in vivo te8t. 2.5 ~g/kg/min of ANF were infused into consciou~ SHR, which lowered the blood pressure by 4S ~ Hg. A subsequent administration of bolus of 1 mg/kg ANF antagonlst increased the blood ~ressure again to the initial value of 220 mm Hg. Angiotensin II (0.15 ~g/kg/
25 min) was infused into anaestheti2ed SPF rats without central reflexes ("pithed rats~) in order to raise the ;~; blood pressure to 100-110 mm Hg. Then, a bolus of 10 ~g/kg of ANF was given, which lowered the blood pressure by 20 mm Hg. A subsequent intravenou6 administra 30 tion of 1 mg/kg of ANF antagonist increased the blood pressure again to the initial value. This experiment shows that the blood pressure increase under the ANF antagonist is not due to an activation of sympathetic reflexes, but to the action of an ANF antagonist.
. . .

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ExamPle 6 Dimethvl ester of the ANF antaaonist 40 mg of the ANF antagonist isolated in accordance with Example 2 were dissolved in 40 ml of methanol which contained 30 mg of 96% sulphuric acid and left to stand at room temperature for 65 hours. Thereafter, the solution was diluted with 120 ml of water and pumped with a flow of 5 ml/min through a steel column (2 x 25 cm) filled with Nucleosil C-18, 10 ~m (from Macherey-Nagel, D~en, Germany). Sub~equently, the column was washed with 30 acetonitrile in water, 5 mM trifluoroacetic acid. The dimethyl ester was eluted with 50% acetonitrile in water, 5 mM trifluoroacetic acid. The eluate was lyophilized and qave 31 mg of the dimethyl ester as a colourless powder.
The IC50 value of the dimethyl ester determined in --accordance with Example 4 was 2500 nM.

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Claims (12)

1. An antagonist of mammalian atrial natriuretic factor, isolatable from microorganisms of the genus Streptomyces having the DSM Nos. 4777 and 4778.

2. A compound in accordance with claim 1. which is a cyclic peptide having a molecular weight of about 1870 or a physiologically compatible salt of such as peptide.

3. A compound in accordance with claim 2, which has the amino acid sequence set forth in Figure 4.

4. A compound in accordance with any one of claims 1-3 as a therapeutically active agent.

5. A compound in accordance with any one of claims 1-3 for the treatment of illnesses in which the regulation of blood pressure plays a role.

6. A process for the production of a compound in accordance with any one of claims 1-3, which process comprises cultivating microorganisms of the genus Streptomyces of DSM Nos. 4777 and 4778 and then isolating a compound in accordance with at least one of claims 1-3 from the crude culture extract, the culture cell mass or the culture filtrate and, if desired, converting said compound into a physiologically compatible salt.

7. Pharmaceutical preparations which contain a compound in accordance with any one of claims 1-3, if desired in combination with one or more additional therapeutically active substances and/or non-toxic, inert, therapeutically compatible carrier materials.

8. A pharmaceutical preparation for the treatment of illnesses in which the regulation of blood pressure plays a role, said preparation containing a compound in accordance with any one of claims 1-3, if desired in combination with one or more additional therapeutically active substances and/or non-toxic, inert, therapeutically compatible carrier materials.

9. The use of a compound in accordance with any one of claims 1-3 for the treatment of illnesses.

10. The use of a compound in accordance with any one of claims 1-3 for the treatment of illnesses in which the regulation of blood pressure plays a role.

11. A compound in accordance with any one of claims 1-3, whenever produced according to the process as described in claim 6.

12. The invention as hereinbefore described.