Drug – bio-affecting and body treating compositions – Designated organic active ingredient containing – Carbohydrate doai
Reexamination Certificate
2000-01-11
2002-06-04
Achutamurthy, Ponnathapu (Department: 1652)
Drug, bio-affecting and body treating compositions
Designated organic active ingredient containing
Carbohydrate doai
C536S016800, C536S017200, C536S006400, C435S253500, C435S252350, C435S078000
Reexamination Certificate
active
06399583
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to novel anthracyclines generated by cloning of fused genes in a mutated
Streptomyces galilaeus
strain. The aglycone moieties of the compounds obtained are modified aklavinones, the modifications being caused by the genes introduced into the mutant, and the sugar moieties are those derived from the host strain.
BACKGROUND OF THE INVENTION
Anthracyclines, which are mainly produced by Streptomyces species as secondary metabolites, share a great value as antitumor agents. Generally, the anthracycline molecule comprises an aglycone backbone and one or more sugar moieties attached therein. Aclacinomycins are a group of anthracyclines sharing aklavinone as the aglycone moiety and a sugar residue, as rhodosamine (Aclacinomycin T), wherein 2-deoxyfucose (Aclacinomycin S) and a third sugar, originally rhodinose, can be sequentially attached. Modifications of the third sugar residue are common. The first aclacinomycin complex consisting of Aclacinomycin A (AcmA), B (AcmB), and Y (AcmY) was first isolated from
S. galilaeus
by Oki et al., (1975). Later, aclacinomycin A, Aclarubicin, was passed through clinical trials for use in cancer chemotherapy. Research to obtain aclacinomycins with minor modifications has been carried out to find new candidates for drug development. During these studies compounds produced by the mutated
S. galilaeus
strains possessing modifications in the sugar moiety have been found (Matsuzawa et al., 1981).
The general formula of aclacinomycins (Acm) comprising aklavinone and an aminosugar, rhodosamine (Rhn) is as follows
wherein R is H or a sugar residue being a mono- or a disugar-moiety.
Anthracycline
R
AcmA
dF-CinA
AcmB
dF-CinB
AcmY
dF-Acu
AcmN
dF-Rho
AcmM
dF-Ami
AcmS
dF
AcmT (Aklavin)
H
dF is 2-deoxyfucose, Cin is cinerulose, Acu is aculose, Rho is rhodinose, and Ami is amicetose.
In addition, aklavinone glycosides which have no aminosugar moieties have been produced by
S. galilaeus
strains obtained by mutagenesis treatment (Matsuzawa et al., 1981 and Ylihonko et al., 1994).
Aclacinomycin A, called Aclarubicin, is a unique member of aclacinomycin group used in cancer chemotheraphy. It is mainly used for leukemia. Doxorubicin, an anthracycline representing the daunomycin group, is the most widely used cytostatic antibiotic, showing activity against quite a variety of tumors. Unfortunately, the harmful side-effects such as cumulative cardiotoxicity cause the treatment by doxorubicin to be aborted despite of its effectiveness. Instead, cardiotoxicity is not a common side-effect in aclarubicin treatment. Nevertheless, the lack of activity on solid tumors limits its use mainly to leukemia and lymphomas. For this reason an optimal anthracycline for cancer treatment would possess clinical applicability similar to that of doxorubicin, whereas side-effects should rather be as those of aclarubicin, in accordance with its mode of action. Current status of cancer chemotheraphy perpetuates the need of novel molecules in order to develop new cytostatic drugs.
Matsuzawa et al. (1981) describe production of aklavinone-rhodosamine-2-deoxy-fucose-2-deoxyfucose. This aclacinomycin has been isolated from
S. galilaeus
mutant strains 7U-491 and 7N-1881 derived from the strain
S. galilaeus
ATCC 31133. As well, the strains 7U-491 and 7N-1881 produced aklavinone-Rhn-dF and, in addition, aklavinone-dF-dF (MA144 U9) has been obtained from the culture of 7N-1881.
SUMMARY OF THE INVENTION
We mutated a
Streptomyces galilaeus
wild type strain (ATCC 31615) in order to make the mutant to produce a modified anthracycline. The
S. galilaeus
mutant obtained was named as H075, and deposited under the rules of the Budapest Treaty on Jun. 30, 1997 at the Deutsche Sammlung von Mikroorganismen und Zellkulturen, Mascheroder Weg 1b, Braunschweig, Germany with the accession number DSM 11638.
S. galilaeus
H075 itself produces aklavinone-rhodosamine-2-deoxyfucose-2-deoxyfucose (H075-1a) as the major product, and minor amounts of aklavinone-dF-dF-dF (H075-1d), an anthracycline which has no aminoglycosides. H075 thereby differs from the producer strains of Matsuzawa et al. (1981), since a novel compound, aklavinone-deoxyfucose-deoxyfucose-deoxyfucose is produced as well. This mutant strain was used as the host strain to express the known genes for nogalamycin (sno) and rhodomycin (rdm) biosynthesis to produce novel hybrid anthracyclines. The sno1-3 genes derived from
S. nogalater
cause a replacement of an ethyl group at C-9 to a methyl group of aklavinone. The rdmB and E genes derived from
S. purpurascens
are responsible for hydroxylation of the positions 10 and 11, respectively, and rdmC gene product decarboxylates the position 10 in the aglycone moiety. This results in novel compounds useful in cancer chemotherapy.
A specific object of this invention is thus a process for producing new hybrid anthracyclines by transforming the genes for nogalamycin (sno) and/or rhodomycin (rdm) biosynthesis to a
S. galilaeus
strain, preferably to the H075 mutant.
The compounds produced by expressing said genes in
S. galilaeus
H075 to change the substituents at positions 9, 10 and 11 in aklavinone are included in this invention.
DETAILED DESCRIPTION OF THE INVENTION
The
S. galilaeus
mutant H075 (DSM 11638), which is used as the host for production of novel anthracyclines according to this invention is an overproducer of aklavinone glycosides. The general formula II for the said compounds is
wherein
R
1
=CH
2
CH
3
or CH
3
,
R
2
=COOCH
3
, H or OH and
R
3
=OH or H.
TABLE 1
Novel compounds of formula II
Aglycone
Substituents
Glycoside
H075-2
R
1
= CH
2
CH
3
R
2
= OH
R
3
= H
a,b,c,d,e
H075-3
R
1
= CH
3
R
2
= COOCH
3
R
3
= H
a,d,e
H075-4
R
1
= CH
2
CH
3
R
2
= COOCH
3
R
3
= OH
a,b,c,d,e
H075-5
R
1
= CH
3
R
2
= COOCH
3
R
3
= OH
a,b,c,d,e
H075-6
R
1
= CH
3
R
2
= OH
R
3
= H
a,b,c,d,e
H075-7
R
1
= CH
3
R
2
= H
R
3
= H
a,b,c,d,e
H075-8
R
1
= CH
2
CH
3
R
2
= H
R
3
= H
a,b,c,d,e
H075-9
R
1
= CH
2
CH
3
R
2
= H
R
3
= OH
a,b,c,d,e
H075-10
R
1
= CH
2
CH
3
R
2
= OH
R
3
= OH
a,d,e
H075-11
R
1
= CH
3
R
2
= OH
R
3
= OH
a,b,c,d,e
H075-12
R
1
= CH
3
R
2
= H
R
3
= OH
a,b,c,d,e
In the following description the compounds are designated according to formula II above in the manner that a number is given to each aglycone based on the R-groups listed above, whereas small letters refer to sugar moieties according to formula II, a=Rhn-dF-dF; b=Rhn-dF; c=Rhn; d=dF-dF-dF; e=dF-dF. (Rhn=rhodosamine, dF=deoxyfucose).
The transformed strains obtained using
S. galilaeus
H075 as a host produce altogether 5 (sugar moiety)×11 (aglycone modifications) different anthracyclines.
The invention concerns specifically the new compounds H075-2a,b,c,d,e, H075-3a,d,e, H075-4a,b,c,d,e, H075-5a,b,c,d,e, H075-6a,b,c,d,e, H075-7a,b,c,d,e, H075-8a,b,c,d,e, H075-9a,b,c,d,e, H075-10a,d,e, H075-11a,b,c,d,e, H075-12a,b,c,d,e as given above in Table 1, and in specific the novel compounds H075-2a, -2b and -2d; H075-3d; H075-4a; H075-5e; H075-6b and -6c; H075-8a; H075-10a; and H075-11b and -11e, as defined in claim 1.
The prior known compounds related to the compounds of the present invention—as named according to the formula II—are:
H075-1a (MA144 U1, Matsuzawa et al., 1981),
H075-1b (AcmS, Oki et al., 1977),
H075-1c (Aklavin, AcmT, Streliz et al., 1956),
H075-1e (MA144 U9, Matsuzawa et al., 1981),
H075-3b (Auramycin C, Hoshino et al., 1982),
H075-3c (Auramycin D, Hoshino et al., 1982),
H075-10b (Betaclamycin S, Yoshimoto et al., 1984) and
H075-10c (Betaclamycin T, Yoshimoto et al., 1984).
The present invention enables the drug development of compounds of the formula II. According to the invention the compounds of the present study are active against the MES-SA cell
Hakala Juha
Kunnari Tero
Ylihonko Kristiina
Achutamurthy Ponnathapu
Crowell & Moring LLP
Galilaeus Oy
Kerr Kathla
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