Drug – bio-affecting and body treating compositions – Antigen – epitope – or other immunospecific immunoeffector
Reexamination Certificate
2002-02-27
2004-01-20
Peselev, Elli (Department: 1623)
Drug, bio-affecting and body treating compositions
Antigen, epitope, or other immunospecific immunoeffector
C424S168100, C435S030000, C435S072000, C435S124000, C435S253100, C514S023000, C514S028000, C514S450000, C536S001110, C536S007100, C536S018700, C549S266000
Reexamination Certificate
active
06680055
ABSTRACT:
TECHNICAL FIELD OF THE INVENTION
The present invention relates to pharmacoactive polyketide macrolides obtained from
M. ulcerans
, semi-synthetic derivatives thereof, aseptic mixtures of polyketide macrolides obtained from
M. ulcerans.
and methods of using the same.
BACKGROUND OF THE INVENTION
Mycobacteria ulcerans
is the causative agent of buruli ulcers. Buruli ulcers are progressive necrotic skin lesions that can persist for decades without treatment and which are not uncommonly suffered by persons living in tropical climates, including parts of Africa. Buruli ulcers are painless, not accompanied by symptoms of systemic disease, and are generally not accompanied by an initial acute inflammatory response. Several articles have shown evidence that
M. ulcerans
produces a toxin, although the identity of this toxin or toxins has heretofore remained unknown. Read et al.,
Infect. Immun.,
9, 1114 (1974), reported that the sterile filtrate of an
M. ulcerans
liquid culture has cytopathic activity on cultured murine fibroblasts. Pimsler et al.,
J. Infect. Dis.,
157, 577 (1988), reported that the sterile filtrate of an
M. ulcerans
has an immunosuppressive property. George et al.,
Infect. Immun.,
66, 587-593 (1998), reported that a lipid toxin that is soluble in acetone could be isolated from
M. ulcerans
culture supernatant by organic extraction and causes a cytopathic effect by arresting L929 murine fibroblasts in the G
I
phase of the cell cycle.
SUMMARY OF THE INVENTION
The present invention provides a polyketide macrolide that can be isolated from virulent cell cultures of
Mycobacterium ulcerans
. The inventive polyketide macrolide has the formula of formula 1:
Also provided are pharmacologically acceptable derivatives and prodrugs of the polyketide macrolide of Formula 1.
Suitable pharmacologically acceptable esters, ethers and prodrugs of the polyketide macrolide include those of Formula 2:
wherein R
1
-R
5
are the same or different and each is independently selected from the group consisting of hydrogen, R
6
, a C(O)R
7
, a C(S)R
7
, a C(O)NHR
7
, and a C(S)NHR
7
, each occurrence of R
6
is independently selected from the group consisting of a C
1
-C
6
alkyl, a C
5
-C
12
aryl, and a sugar, each occurrence of R
7
is independently selected from the group consisting of hydrogen, a C
1
-C
6
alkyl, and a C
5
-C
12
aryl, and wherein R
1
and R
2
, R
2
and R
3
, and/or R
4
and R
5
can be taken together to form a ketal ring.
The present invention also provides an aseptic mixture of macrolides comprising (i) a polyketide macrolide isolated from
Mycobacteria ulcerans
wherein the fraction corresponding to the distance the polyketide macrolide travels from the origin divided by the distance the solvent front travels past the origin (Rf) is greater than 0.15 and less than 0.60 when chromatographically separated by silica-gel thin-layer-chromatography employing a solvent system of 90:10:1 of chloroform:methanol:water and (ii) a pharmaceutically acceptable carrier.
The present invention further provides methods of using a polyketide macrolide to inhibit cancer in a mammal and to suppress an inflammatory response in a mammal. These methods comprise administering an effective amount of an isolated polyketide macrolide or an aseptic mixture of macrolides to the mammal so as to inhibit cancer or to suppress an inflammatory response, respectively.
Further provided is a method of inducing an immune response to
Mycobacteria ulcerans
without inducing a buruli ulcer. The method comprises inoculating a mammal with an immune response-inducing amount of
M. ulcerans
cells that produce less than about 5% of polyketide macrolides per cell in comparison to a fresh culture of a virulent isolate of
M. ulcerans
1615, wherein said polyketide macrolides have an Rf of greater than 0.15 and less than 0.60 when chromatographically separated by SG-TLC employing a solvent system of 90:10:1 of chlorform:methanol:water. The mammal has an immune response to
M. ulcerans
and does not develop a buruli ulcer.
Still further provided is a composition comprising
M. ulcerans
cells that produce less than about 5% of polyketide macrolides per cell in comparison to a fresh culture of a virulent isolate of
Mycobacteria ulcerans,
wherein the polyketide macrolides have an Rf of greater than 0.15 and less than 0.60 when chromatographically separated by SG-TLC employing a solvent system of 90:10:1 of chloroform:methanol :water.
DETAILED DESCRIPTION OF THE INVENTION
The present invention provides an isolated polyketide macrolide that can be isolated from virulent isolates of
Mycobacterium ulcerans.
The polyketide macrolide has the formula of Formula 1:
Also provided are pharmacologically acceptable derivatives and prodrugs of the polyketide macrolide of Formula 1.
Suitable pharmacologically acceptable esters, ethers and prodrugs of the polyketide macrolide include those of Formula 2:
wherein R
1
-R
5
are the same or different and each is independently selected from the group consisting of hydrogen, R
6
, a C(O)R
7
, a C(S)R
7
, a C(O)NHR
7
, and a C(S)NHR
7
, each occurrence of R
6
is independently selected from the group consisting of a C
1
-C
6
alkyl, a C
5
-C
12
aryl, and a sugar, each occurrence of R
7
is independently selected from the group consisting of hydrogen, a C
1
-C
6
alkyl, and a C
5
-C
12
aryl, and wherein R
1
and R
2
, R
2
and R
3
, and/or R
4
and R
5
can be taken together to form a ketal ring. For example, R
1
and R
2
can be taken together as a C
1
-C
6
alkyl. Suitable sugars in the context of the present invention include, but are not limited tetroses, pentoses, hexoses, heptoses, and disaccharides and polysaccharides comprising tetroses, pentoses, hexoses, and heptoses. Suitable aryl substituents of the present invention include heteroaryl substituents comprising a nitrogen, oxygen, or sulfur heteroatom.
The structure of mycolactone (Formula 1) has been unambiguously identified by multiple analytical methods including, but not limited to, proton NMR (including GMQCOSY, TOCSY, HSQC, and ROESY), and mass spectrometry. Both the observed and calculated m/z ratio by mass spectrometry (HRMS) was 765.4912 (&Dgr;0.1 ppm). The long-range correlations between the proton signals at 2.02 and 2.41 ppm (2-CH
2
) and the carbon signal at 173.3 ppm confirmed the position of one of the ester carbonyls at C1. The long-range coupling of olefinic methyl protons to the olefinic carbons 3 bonds away allowed the joining of spin systems separated by methyl-bearing quaternary carbons. The long-range correlations between the methyl proton signal at 1.71 ppm (22-CH
2
) and the carbon signals at 46.4 (C7) and 123.8 ppm (C9), and the methyl proton signal at 1.64 ppm (24-CH
3
) and the carbon signals at 44.3 (C12) and 133.9 ppm (C14), allowed the establishment of the dodecanoic chain. The long-range correlations between the olefinic protons at 5.94 (2′-CH) and 7.92 (3′-H) and the carbonyl signal at 166.0 ppm confirmed the unsaturated ester carbonyl at 1′. The olefinic proton signal at 6.35 ppm (5′-H), which is directly attached to the carbon resonating at 141.8 ppm (C5′), showed long-range correlations to carbon signals at 143.1 (C3′) and 134.8 ppm (C7′), while the methyl proton signal at 1.91 ppm (19′-CH
3
) showed long-range correlations to carbon signals at 139.9 (C9′) and 134.6 ppm (C11′), respectively, completing the hexadecanoic chain. The proton signal at 4.71 ppm (5-CH) showed long-range coupling to the carbon signal at 166.9 ppm (C1′) indicating that the hydroxyl function at C5 is acylated with the hexadecyl moiety. The protons on the remaining 6 hydroxyl-bearing methine functions resonated at 4.9 (C11), 4.28 (C12), 3.99 (C15′), 3.96 (C19), 3.68 (C13′), and 3.5 (C17) ppm. Based on chemical shifts, it is evident that the hydroxyl function at C11 is involved in the formation of the lactone. This was further confirmed by the presence of NOE correlations between 11-H (4.9 ppm) and 2-CH
2
(2.02, and 2.41 ppm)
George Kathleen M.
Small Pamela L. C.
Peselev Elli
The United States of America as represented by the Department of
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