Drug – bio-affecting and body treating compositions – Designated organic active ingredient containing – Having -c- – wherein x is chalcogen – bonded directly to...
Reexamination Certificate
1994-09-23
2002-03-19
Kifle, Bruck (Department: 1624)
Drug, bio-affecting and body treating compositions
Designated organic active ingredient containing
Having -c-, wherein x is chalcogen, bonded directly to...
C540S456000
Reexamination Certificate
active
06358969
ABSTRACT:
The present invention relates to a novel compound and derivatives thereof, to processes for their production, to pharmaceutical formulations containing them, to their use in medical therapy, particularly in the treatment of bacterial and fungal infections, and also to their use as immunosuppressants.
Rapamycin is a known compound and was first isolated as an extract of the fungus
Streptomvces hygroscopicus
and reported to have antifungal activity (British Patent 1436447). Subsequently rapamycin has been implicated as an immunosuppressant (Martel R. R. et al Can. J. Physiol. Pharmacol. 55, 48-51, 1977).
A large number of microorganisms have been found to produce a variety of metabolites which have subsequently been isolated and have been shown to possess useful therapeutic properties. One such compound is 29-desmethylrapamycin. This is believed to be a novel compound and has been found to have useful antifungal activity and also immunosuppressant properties.
Accordingly the present invention provides 29-desmethylrapamycin and derivatives thereof.
The invention in a second aspect, further provides a process for the production of 29-desmethylrapamycin which comprises cultivating a producing microorganism and subsequently isolating 29-desmethylrapamycin or derivatives thereof.
29-desmethylrapamycin is believed to have the following structure:
It has the following characteristics:
i) it has an apparent molecular weight of 899 by fast atom bombardment (FAB) mass spectroscopy,
ii) it may be obtained by the cultivation of a microorganism from the genus Streptomyces,
iii)
13
CNMR spectroscopy reveals 50 carbons in the molecule,
iv) it shows antifungal activity against
Candida albicans.
v) it shows immunosuppressant properties.
29-desmethylrapamycin may be obtained by the cultivation of a producing organism and the recovery of it or a derivative thereof from the culture.
The term ‘cultivation’ (and derivatives of that term) as used herein means the deliberate aerobic growth of an organism in the presence of assimilable sources of carbon, nitrogen, sulphur and mineral salts. Such aerobic growth may take place in a solid or semi-solid nutritive medium, or in a liquid medium in which the nutrients are dissolved or suspended. The cultivation may take place on an aerobic surface or by submerged culture. The nutritive medium may be composed of complex nutrients or may be chemically defined.
It has been found that suitable microorganisms for use in the cultivation process according to the invention include bacterial strains belonging to the genus Streptomyces which are capable of elaborating 29-desmethylrapamycin. It has further been found been isolated from nature and also mutants thereof.
The term ‘mutant’ as used herein includes any mutant strain which arises spontaneously or through the effect of an external agent whether that agent is applied deliberately or otherwise. Suitable methods of producing mutant strains including those outlined by H. I. Adler in ‘Techniques for the Development of Microorganisms’ in ‘Radiation and Radioisotopes for Industrial Microorganisms’, Proceedings of a Symposium, Vienna, 1973, page 241, International Atomic Energy Authority, and these include:
(i) Ionizing radiation (e.g. X-rays and &ggr;-rays), u.v. light, u.v. light plus a photosensitizing agents (e.g. 8-methoxypsoralen), nitrous acid, hydroxylamine, pyrimidine base analogues (e.g. 5-bromouracil), acridines, alkylating agents (e.g. mustard gas, ethyl-methane sulphonate), hydrogen peroxide, phenols, formaldehyde, heat, and
(ii) Genetic techniques, including, for example, recombination, transformation, transduction, lysogenisation, lysogenic conversion, protoplast fusion and selective techniques for spontaneous mutants.
Using the methods of Becker B. Lechevalier M. P., Gordon R. E., Lechevalier H. A., 1964, Appl. Microbiol. 12, 421-423 and Williams S. T., Goodfellow M, Wellington E. M. H., Vickers J. C., Alderson. G., Sneath P. H. A., Sackin M. J., and Mortimer M. 1983 J. Gen. Microbiol. 129, 1815-1830, Sp. NCIB 40319 has been identified as a previously unreported, atypical, strain of Streptomyces and therefore also forms a part of the present invention, particularly in biologically pure form. It has been deposited at the National Collections of Industrial and Marine Bacteria Ltd. (N.C.I.B), Aberdeen, Scotland under number 40319 on Sep. 14, 1990.
Strain NCIB 40319 has been characterised as follows:
The method of whole-cell amino acid analysis was that described by Becker et al (1964). Identification media used for the characterisation of the culture were as described by Williams et al (1983). In addition, starch casein agar (Waksman S. A., 1961. The Actinomycetes Vol. 2 Williams and Wilkins Co. Baltimore ppl-363) was used for the morphological description of the culture.
The microorganism was characterised by inoculating agar blocks from a well grown plate into Y broth (see Table 1) and incubating for three days at 28° C. on a shaker. It was then centrifuged for 20 minutes at 3660 rpm, washed twice with distilled water, then finally resuspended in phosphate buffered saline (Dulbecco A). This inoculum was plated onto media commonly used for the identification of members of the Actinomycetales as above. Plates were incubated at 28° C. and the results were read at varying times but most were commonly taken at 14 days. The colours are described in common terminology but exact colours were determined by comparison with colour chips from the Methuen Handbook of colour (3rd Edn).
Results:
Cell Wall analysis
The whole-cell hydrolysates contained LL-diaminopimelic acid. The observations of growth and appearance of the organism were as follows:
Yeast extract-Malt extract Aear (ISP 2 Difco)
Growth good, cream 22a), with a white powdery centre. Colonies raised and rather wrinkled, no sporulation.
Inorganic Salts Starch Agar (ISP4 Difco)
Growth good, white with pale grey to grey (11b, 11c) aerial mycelium. Colonies quite flat with slightly raised centre. Reverse cream (22a).
Glycerol Asparagine Agar (S. A. Waksman, 1961, p328). medium No. 2.
Growth moderate to good, white with grey centre (11d).Colonies flat, reverse cream (22a).
Starch Mineral Salts Agar
Growth very poor, opaque small colonies. No aerial mycelium.
Starch Casein Agar
Growth good, white with light grey to grey central area (11c, 11d), occasional small patch of white non-sporulating mycelium in grey sporulating areas. Tiny colourless droplets over the grey areas. Colonies fairly flat and gently rounded. Small black hygroscopic patches may occur after 4 weeks incubation.
Morphological Properties
These were observed after two weeks incubation on starch casein agar: spore mass in grey colour-series; spore chains in section spirales, tightly coiled or slightly open, of small diameter, generally 2-6 coils, occasionally more, may aggregate into hygroscopic masses. There was no fragmentation of vegetative mycelium.
Biochemical Properties
See Table 2 for full details. In summary, melanin not produced; nitrate not reduced to nitrite in organic nitrate broth; H
2
S produced in peptone-yeast extract iron broth; no growth on inhibitors; degradation only of arbutin, antibiosis only against
Bacillus subtilis.
Carbohydrate utilization glucose, cellobiose, fructose, inositol, mannitol, raffinose, rhamnose and xylose. Nitrogen sources used: asparagine, histidine and hydroxyproline, a-amino-butyric acid used only slightly.
Determination of Identification Scores
These were obtained using the Matiden program (Sneath P. H. A., 1979. Computers and Geosciences 5 195-213) which provides the best identification scores for known or unknown strains against the percent probability matrix of Williams et al (1983). Willcox Probability—the nearer the score reaches 1.0, the better is the fit of an unknown with a group in the matrix (scores of >0.85 acceptable) Taxonomic distance—low scores indicate relatedness (scores <0.3 acceptable). The organism had acceptable identification scores with cluster 32 (violaceoniger) which contains
Streptomyces hygroscopicus
species.
Co
Banks Rhona Mary
Shelley Peter Robin
Kifle Bruck
SmithKline Beecham p.l.c.
Stereho Yurly F.
Venetanner Stephen
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