Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing heterocyclic carbon compound having only o – n – s,...
Reexamination Certificate
2001-08-03
2003-05-13
Lilling, Herbert J. (Department: 1651)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing heterocyclic carbon compound having only o, n, s,...
Reexamination Certificate
active
06562602
ABSTRACT:
FIELD OF THE INVENTION
The present invention provides methods for preparing mycolactones by fermentation and novel mycolactone compounds useful in the treatment of disease conditions. The invention relates to the fields of chemistry, molecular biology, animal and human health sciences, and medicine.
BACKGROUND OF THE INVENTION
Mycobacterium ulcerans
causes a severe skin disease, Buruli ulcer, which is characterized by extensive necrosis in the absence of an acute inflammatory response. The causative agent of the disease was first identified as a diffusible toxic agent (see Read et al., June 1974, Cytotoxic activity of
Mycobacterium ulcerans, Infection
&
Immun.
9(6): 1114-1122, incorporated herein by reference). Further analysis identified that the agent is a polyketide (see George et al., Feb. 5, 1999, Mycolactone: A polyketide toxin from
Mycobacterium ulcerans
required for virulence,
Science
283: 854-857, incorporated herein by reference). Recently, the complete structural determination of two structurally related agents, called mycolactones A and B, has been reported (see Gunawardana et al., 1999, Characterization of novel macrolide toxins, mycolactones A and B, from a human pathogen,
Mycobacterium ulcerans, JACS
121: 6092-6093, incorporated herein by reference).
The cytotoxic nature of the mycolactones, as well as their ability to suppress the immune response, indicates that the compounds have therapeutic potential as anti-cancer agents and immunosuppressants (see the references cited supra and Pimsler et al., March 1988, Immunosuppressive properties of the soluble toxin from
Mycobacterium ulcerans, J. Infect. Dis.
157(3): 577-580, incorporated herein by reference). However, despite extensive efforts to develop fermentation conditions suitable for large-scale growth of
Mycobacterium ulcerans
and related organisms such as
M. bovis
(see e.g. Mve-Obiang et al., 1999, Growth and cytotoxic activity by
Mycobacterium ulcerans
in protein-free media,
FEMS Microbiol. Lett.
181: 153-157; Nyabenda et al., 1988, The production of mycobacterial antigens by homogeneous culture in a fermentor,
J. Biol. Standard.
16: 259-267; Palomino & Portaels, February 1998, Effects of decontamination methods and culture conditions on viability of
Mycobacterium ulcerans
in the BACTEC system,
J. Clin. Microbiol.
36(2): 402-408; and Palomino et al., November 1998, Effect of oxygen on growth of
Mycobacterium ulcerans
in the BACTEC system,
J. Clin. Microbiol
36(11): 3420-3422, each of which is incorporated herein by reference), cultivation of the organism remains difficult, limiting the availability of the compounds for testing and clinical trials.
There remains a need for fermentation processes by which large scale cultures of
Mycobacterium ulcerans
can be obtained and from which useful quantities of mycolactones could be prepared. The present invention meets that need and provides as well, as one important benefit, novel naturally produced mycolactones in purified form that were heretofore undiscovered due to the lack of suitable fermentation processes.
SUMMARY OF THE INVENTION
In a first embodiment, the present invention provides methods for the production of mycolactones by fermentation. The methods are readily scalable and can be used to produce the mycolactones in amounts sufficient for clinical trials and commercialization. The methods enable the cultivation of
Mycobacterium ulcerans
in a dispersed suspension culture (e.g., fermentation or spinner flasks) and the production of the mycolactones at concentrations greater than that observed in T flasks.
In a second embodiment, the present invention provides improved media formulations that allow scale-up of cultures for the production of mycolactones and improve the production of mycolactone from
Mycobacterium ulcerans.
In a third embodiment, the present invention also provides a robust and scalable method for the purification of mycolactones based on extraction and chromatography.
In a fourth embodiment the present invention provides novel mycolactone compounds isolatable from
Mycobacterium ulcerans
in purified form.
These and other embodiments, modes, and aspects of the invention are described in more detail in the following brief description of the figure, detailed description of the invention, the examples, and claims set forth below.
REFERENCES:
Cadapan, L. et al. (2001)FEMS Microbiology Letters205(2):385-389.
George, K. et al. (1998)Infection and Immunity66(2):587-593.
George, K. et al. (1999)Science283(5403):854-857.
Report of the 3rd WHO Advisory Group Meeting on Buruli Ulcer (Mar., 2000), On Line!, XP002201041. Retrieved from the Internet: <URL:http:/www.who.int/gtb-buruli/activities/PDF/2000_meeting_report_(BU).pdf> retrieved on Jun. 3, 2002! pp. 59-60.
Report of the 4th WHO Advisory Group Meeting on Buruli Ulcer (Mar., 2001), On Line!, XP002201042. Retrieved from the Internet: >URL:http:/www.who.int/gtb-buruli/activities/PDF/2001_Final_Report.pdf> retrieved on Jun. 3, 2002! pp. 57-59.
George et al., Science (1999) 283:854-857.
Gunawardana et al., JACS (1999) 121:6092-6093.
Mve-Obiang et al., FEMS Microbiol. Lett. (1999) 181:153-157.
Nyabenda et al., J. Biol. Standard (1988) 16:259-267.
Palomino et al., J. Clin. Microbiol. (1998) 36(11):3420-3422.
Palomino and Portaels, J. Clin. Microbiol. (1998) 36(2):402-408.
Pimsler et al., J. Infect. Dis. (1988) 157(3):577-580.
Read et al., Infection & Immun. (1974) 9(6):1114-1122.
Arslanian Robert
Cadapan Lawrence
Carney John
Licari Peter
Aparicio Jose
Kaster Kevin
Kosan Biosciences, Inc.
Lilling Herbert J.
Morrison & Foerster / LLP
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