# US 7,407,654 B2 · Antibiotics GE 81112 factors A,B,B1, pharmaceutically acceptable salts and compositions, and use thereof

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- Number: US 7,407,654 B2
- Document ID: US7407654B2
- Status: Granted
- Kind: B2
- Application number: 10/496,009
- Filed: 2002-10-04
- Published: 2005-01-27
- Granted: 2008-08-05
- Earliest claimed: 2001-11-28
- Assignees: Vicuron Pharmaceuticals Inc.
- Inventors: Enrico Selva; Flavia Marinelli; Daniele Losi; Linda Cavaletti; Ameriga Lazzarini; Alessandra Marazzi
- Web page: https://patentgrep.com/patent/US7407654B2

## CPC

- C12P 1/04: by using bacteria
- A61P 31/00: Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P 31/04: Antibacterial agents
- Y02A 50/30: Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
- C12P: FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE {(brewing of beer C12C; producing vinegar C12J; producing specific peptides or proteins C07K; producing enzymes C12N9/00; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification C12N15/00; measuring or testing processes involving enzymes or microorganisms C12Q; measuring or testing processes involving nucleic acid amplification reactions C12Q1/6844; fermentation processes to form a food composition, A21 or A23; compounds in general, see the relevant compound class, e.g. C01, C07)}
- A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- Y02A: TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE

## Abstract

The invention relates to an antibiotic substances of microbial origin, arbitrarily denominated antibiotic GE 81112 factor A, factor B1 and factor B, which is produced by fermentation of Streptomyces sp. DSMZ 14386, the pharmaceutically acceptable salts and compositions thereof, and their use as an antibacterial agent having inhibitory activity versus susceptible microbes.

## Claims

### Claim 1

An isolated antibiotic GE 81112 Factor A comprising the following characteristics:
A) Mass spectrum recorded from a 0.01 mg/ml solution in acetonitrile: water 50:50 giving a monoprotonated ion at 644 m/z on a Thermofinnigan® LCQ deca mass spectrum instrument under the following electrospray conditions: Spray Voltage: 4.7 kV; Capillary temperature: 250° C.; Capillary Voltage 9V; Infusion mode 5 μl/min;
B) Infrared Spectrum recorded as a nujol mull with an IFS-48® Fourier Transform infrared spectrophotometer showing the following main bands (cm−1): 3356; 2956; 2855; 2256; 2128; 1663; 1596; 1455; 1378; 1345; 1123; 1050; 1026; 1002; 825; 764;
C) 1H-NMR spectrum recorded at 600 MHz at 25° C. in DMSO-d6, comprising at least one drop of trifluoroacetic acid, showing the following 1H signals:
Chemical Shift (ppm)
Multiplicity
1.52
br d
1.57-1.67
m
1.81
br d
1.88
m
2.87
dd
2.90
m
3.12
dd
3.15
m
3.65
m
3.83
m
3.94
br d
4.40
br s
4.46
m
4.50
dd
4.70
M
5.06
D
6.46
Br
6.94
S
7.29
S
7.99
D
8.17
D
8.30
M
8.57
br d
8.95
D
9.06
br d
14.09
Br
14.27
Br
—
—
D) 13C-NMR spectrum recorded at 150 MHz, at 25° C. in DMSO-d6, comprising at least one drop of trifluoroacetic acid, showing the following 13C signals
Chemical Shift (ppm)
Multiplicity
15.9
t
27.19
t
28.14
t
35.31
t
42.86
t
50.13
d
51.30
d
56.86
d
59.71
d
64.07
d
65.11
d
66.88
d
67.76
T
116.83
d
118.49
d
128.41
s
129.03
s
133.65
d
139.66
s
156.73
s
167.39
s
170.16
s
171.29
s
171.65
s
E) Retention time 14.1 min, determined by using an analytical HPLC pump Shimadzu® LC10-AS equipped with a chromatographic column Altima® RP18, 5 μm, 250×4.6 mm i.d column; isocratic elution performed at 1 ml/min. flow rate, with 40 mM ammonium formate buffer, brought to pH 4.5 by addition of formic acid; UV detection at 230 nm;
F) possesses acid functions and basic functions; and
G) inhibits the growth of Moraxella catarrhalis; inhibits the growth of Escherichia coli and Bacillus subtilis when these bacteria are cultivated in minimal media; shows marginal activity against Streptococcus pneumoniae; does not inhibit growth of Candida albicans, and
wherein the antibiotic GE 81112 Factor A also includes one or more corresponding salts formed with acids and/or bases.

### Claim 2 (depends on 1)

An isolated antibiotic GE 81112 Factor A of claim 1, further comprising the characteristic that the antibiotic GE 81112 Factor A is obtainable by cultivating strain Streptomyces Sp. DSMZ 14386 under aerobic conditions in an aqueous nutrient medium containing assimilable sources of carbon, nitrogen and inorganic salts.

### Claim 3 (depends on 1)

A process for producing the isolated antibiotic GE 81112 Factor A as claimed in claim 1, which comprises:
cultivating Streptomyces sp. DSMZ 14386 or a variant or mutant thereof maintaining the genetic ability to produce antibiotic GE 81112 Factor A under aerobic conditions in an aqueous nutrient fermentation broth medium comprising assimilable sources of carbon, nitrogen and inorganic salts to form an antibiotic mixture;
recovering the antibiotic mixture from the fermentation broth;
purifying the recovered antibiotic mixture and
separating the antibiotic GE 81112 Factor A from the recovered antibiotic mixture.

### Claim 4

An isolated antibiotic mixture comprising antibiotic GE 81112 Factor A comprising the following characteristics:
A) Mass spectrum recorded from a 0.01 mg/ml solution in acetonitrile: water 50:50 giving a monoprotonated ion at 644 m/z on a Thermofinnigan® LCQ deca mass spectrum instrument under the following electrospray conditions: Spray Voltage: 4.7 kV; Capillary temperature: 250° C.; Capillary Voltage 9V; Infusion mode 5 μl/min;
B) Infrared Spectrum recorded as a nujol mull with an IFS-48® Fourier Transform infrared spectrophotometer showing the following main bands (cm−1): 3356; 2956; 2855; 2256; 2128; 1663; 1596; 1455; 1378; 1345; 1123; 1050; 1026; 1002; 825; 764;
C) 1H-NMR spectrum recorded at 600 MHz at 25° C. in DMSO-d6 comprising at least one drop of trifluoroacetic acid showing the following 1H signals:
Chemical Shift (ppm)
Multiplicity
1.52
br d
1.57-1.67
m
1.81
br d
1.88
m
2.87
dd
2.90
m
3.12
dd
3.15
m
3.65
m
3.83
m
3.94
br d
4.40
br s
4.46
m
4.50
dd
4.70
m
5.06
d
6.46
br
6.94
s
7.29
s
7.99
d
8.17
d
8.30
m
8.57
br d
8.95
d
9.06
br d
14.09
br
14.27
br
—
—
D) 13C-NMR spectrum recorded at 150 Mhz, at 25° C. in DMSO-d6 and trifluoroacetic acid (drops) showing the following 13C signals
Chemical Shift
(ppm)
Multiplicity
15.9
t
27.19
t
28.14
t
35.31
t
42.86
t
50.13
d
51.30
d
56.86
d
59.71
d
64.07
d
65.11
d
66.88
d
67.76
t
116.83
d
118.49
d
128.41
s
129.03
s
133.65
d
139.66
s
156.73
s
167.39
s
170.16
s
171.29
s
171.65
s
E) Retention time 14.1 min, determined by using an analytical HPLC pump Shimadzu® LC10-AS, equipped with a chromatographic column Altima® RP18, 5 μm, 250×4.6 mm i.d column; isocratic elution performed at 1 ml/min flow rate, with 40 mM ammonium formate buffer, brought to pH 4.5 by addition of formic acid; UV detection at 230 nm;
F) possesses acid functions and basic functions; and
G) inhibits the growth of Moraxella catarrhalis; inhibits the growth of Eseherichia coli and Bacillus subtilis when these bacteria are cultivated in minimal media; shows marginal activity against Streptococcus pneumoniae; does not inhibit growth of Candida albicans, or
mixtures thereof; and
pharmaceutically acceptable salts thereof.

### Claim 5 (depends on 4)

An isolated antibiotic mixture according to claim 4 wherein said antibiotic GE 81112 Factor A, is produced by isolating the antibiotic GE 81112 Factor A, B, B1 or mixtures thereof from the fermentation broth of Streptomyces sp. DSMZ 14386, or the antibiotic GE 81112 Factor A, B, B1 or mixtures thereof producing variant or mutant thereof maintaining the generic ability to produce said antibiotic GE 81112 Factor A, B and/or B1.

### Claim 6 (depends on 5)

A process for producing the isolated antibiotic mixture according to claim 5, which comprises:
cultivating Streptomyces sp. DSMZ 14386 or a variant or mutant thereof maintaining the genetic ability to produce said antibiotic GE 81112 Factor A,
recovering the mixture of antibiotic GE 81112 Factor A, from the culture broth; and
purifying said mixture of antibiotic GE 81112 Factor A.

### Claim 7 (depends on 3)

A process according to claim 3, wherein the Streptomyces sp. DSMZ 14386, or a variant or mutant thereof maintaining the genetic ability to produce the antibiotic GE 81112 Factor A, is pre-cultured.

### Claim 8 (depends on 3)

A process according to claim 3, wherein the step of recovering the antibiotic mixture from the fermentation broth is carried out on an ion exchange resin and the step of purifying the recovered antibiotic mixture is carried out on an adsorption matrix.

### Claim 9 (depends on 3)

A process according to claim 3, wherein the step of separating the antibiotic GE 81112 Factor A from the recovered antibiotic mixture is carried out by a chromatographic technique.

### Claim 10 (depends on 9)

A process as in claim 9, wherein the chromatographic technique is a preparative HPLC technique.

### Claim 11 (depends on 1)

A pharmaceutical composition comprising the isolated antibiotic GE 81112 Factor A as claimed in claim 1.

### Claim 12 (depends on 4)

A pharmaceutical composition comprising the isolated antibiotic mixture as claimed in claim 4.

### Claim 13 (depends on 11)

A pharmaceutical composition according to claim 11 further comprising a pharmaceutically acceptable carrier.

### Claim 14 (depends on 12)

A pharmaceutical composition according to claim 12 further comprising a pharmaceutically acceptable carrier.

### Claim 15 (depends on 11)

A method, which comprises:
manufacturing a medicament comprising the pharmaceutical composition according to claim 11 and
administering to a host in need thereof an effective amount of the medicament.

### Claim 16 (depends on 12)

A method, which comprises:
manufacturing a medicament comprising the pharmaceutical composition according top claim 12 and
administering to a host in need thereof an effective amount of the medicament.

### Claim 17 (depends on 13)

A method, which comprises:
manufacturing a medicament comprising the pharmaceutical composition according to claim 13 and
administering to a host in need thereof an effective amount of the medicament.

### Claim 18 (depends on 14)

A method, which comprises:
manufacturing a medicament comprising the pharmaceutical composition according to claim 14 and administering to a host in need thereof an effective amount of the medicament.