Note: Descriptions are shown in the official language in which they were submitted.
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.
AB/26.7.90
, .
~: 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.
:
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
. :: . ~,
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, -
_ 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.
: ' '"
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
- 4 - ;'~J~
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
- 6 -
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
.,.; ,~.
2~0~ `
- 7 -
'.: '.; . "
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. ~
~- .. -, .
~Q2~
g : . :
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):
OH / CH2-C02H
~ 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).
',' ~, '". '. '
. . - .
''~ :'`"~.-,
2 t~
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):
16
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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- 13 -
Table ICso value8
Ligand IC50 (
Rat ANF (99-126) 0.110
ANP antagonist 860
. .;
.
ExamPle 5
16
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.
. . .
' :~'
3 ::
- 14 -
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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