`(11)
`(191
`United States Patent
` Sehgalet al. [45] Dec. 30, 1975
`
`
`[75]
`
`[54] RAPAMYCIN AND PROCESS OF
`PREPARATION |
`Inventors: Surendra N. Sehgal, Dollard des
`’ Ormeaux; Teodora M. Blazekovic,
`Mount Royal; Claude Vezina,
`Deux-Montagnes,all of Canada
`[73] Assignee: Ayerst McKenna and Harrison Ltd.,
`Montreal, Canada
`Apr. 12, 1974
`[22] Filed:
`(21] Appl. No.: 460,665
`Related U.S. Application Data
`[63] Continuation-in-part of Ser. No. 293,699, Sept. 29,
`1972, abandoned.
`
`‘
`
`.
`[52] U.S. Ch ecereeees 424/122; 195/80 R
`(51] Unt. Che cecceceeseeeseeeees AG61K 35/00
`
`{58] Field of Search.............0....0. 424/122; 195/80
`
`[56]
`
`References Cited
`OTHER PUBLICATIONS
`Miller, The Pfizer Handbook of Microbial Metabolites
`McGraw-Hill Book Co.
`Inc., N.Y., N.Y., 1961, p.
`$80.
`
`Primary Examiner—Jerome D. Goldberg
`[57]
`ABSTRACT
`Antibiotic rapamycin is producible by culturing Strep-
`tomyces hygroscopicus NRRL 5491 in an aqueous nu-
`trient medium. Rapamycin has antifungal properties.
`Methodsfor its preparation and use are disclosed.
`4 Claims, 2 Drawing Figures
`
`West-Ward Pharm.
`Exhibit 1040
`Page 001
`
`West-Ward Pharm.
`Exhibit 1040
`Page 001
`
`
`
`
`
`
`
`(WD)YJGWNNJAVM
`
`3,929,992
`
`IN
`
`West-Ward Pharm.
`Exhibit 1040
`Page 002
`
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`
`
`
`
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`ZL9Sv£4
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`
`U.S. Patent
`
`Dec. 30,1975
`
`Sheetl of 2
`
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`
`
`
`(SNOUDIW)HLONATIJAVM
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`
`West-Ward Pharm.
`Exhibit 1040
`Page 002
`
`
`
`U.S. Patent
`
`Dec. 30,1975
`
` Sheet20f2
`
`3,929,992 |
`
`
`
`
`
`PPM(1)
`
`West-Ward Pharm.
`Exhibit 1040
`Page 003
`
`West-Ward Pharm.
`Exhibit 1040
`Page 003
`
`
`
`1
`
`3,929,992
`
`RAPAMYCIN AND PROCESS OF PREPARATION
`
`This application is a continuation-in-part of our ear-
`lier application Ser. No. 293,699, filed Sept. 29, 1972
`now abandoned.
`‘
`
`BACKGROUNDOF THE INVENTION
`a. Field of Invention
`This invention relates to an antibiotic, a new compo-
`sition of matter calling rapamycin, and to a process for
`its preparation.
`b. Descriptionof Prior Art
`The antibiotic of this invention is readily distin-
`guished from prior art compoundsofits class by its
`profound antifungal activity andits relatively low order
`of toxicity.
`Moreexplicitly, the ultra violet spectrum of rapamy-
`cin, noted herein, indicates that this compoundbelongs
`to the class of antibiotics knownastriene antibiotics. In
`this particular class there are only five compounds
`reported previously. Trienine, A. Aszaloset al., J. Anti-
`biotics, 21,611 (1968) is a triene antibiotic with antitu-
`moractivity which also shows marked activity against
`gram positive organisms and only marginal activity
`against Candidastrains. The antifungal triene reported
`by J. J. Armstrong, et al., Nature, 206, 399 (1965) and
`Mycotrienin reported by C. Coronelli et al., J. Antibiot-
`ics, 20, 329 (1967) are probably identical. Both have
`low antifungal activity (MIC against C. albicans: 5
`pg/ml) and high toxicity (LDs. in mice: 15 mg/kg). The
`remaining two antibiotics - Resistaphylin, S. Aezaiva et
`al., J. Antibiotics, 24, 393 (1971) and Proticin, G.
`Nesemannet al., Naturwissenschaften, 59, 81 (1972)-
`are readily distinguished from the compound of the
`present
`invention in that
`they exhibit antibacterial
`without any antifungal activity.
`BRIEF SUMMARYOF THE INVENTION
`
`Rapamycin is a chemical compound producible by
`culturing a rapamycin-producing organism in an aque-
`ous nutrient medium. The compoundhasthe property
`of adversely affecting the growth of fungi, for example,
`Candida albicans and Microsporum gypseum. Accord-
`ingly, rapamycin maybe used to prevent the growth of
`or reduce the numberofcertain fungi in various envi-
`ronments.
`The rapamycin - producing organism used for this
`invention, Streptomyces hygroscopicus NRRL 5491,
`was obtained from Easter Island soils and samples
`thereof have been deposited without restrictions with
`the Northern Utilization and Research Division, Agri-
`cultural Research Service, U.S. Departmentof Agricul-
`ture, Peoria, [ll., U.S.A.
`It is to be understood that the inventionis not limited
`to the use of the particular organism herein described,
`but includes variations and mutants obtained by natural
`selection or by treatment of the microorganism with,
`for instance, ultraviolet rays, X-rays, N-methyl-N’-
`nitro-N-nitroso-guanidine, manganese chloride, cam-
`phor, nitrogen mustards, and the like, as well as poly-
`ploids of the various mutants.
`Streptomyces hygroscopicus NRRL 5491 develops
`abundantly in culture media usually employed for culti-
`vation of other organisms of the same genus.It is capa-
`ble of growing at temperatures ranging from 20° to
`35°C., preferably at about 28°C, on Czapek’s agar,
`glucose asparagine agar, glycerol asparagine agar,
`
`10
`
`30
`
`35
`
`starch agar and peptonebeefagar. Also, the organism
`growsvery well on yeast extract agar, malt extract agar,
`starch-inorganic salts agar, oatmeal agar, oatmeal-
`tomato agar and Bennet’s agar. On potato slices thereis
`no aerial mycelium, but substrate growth is well devel-
`oped and buff in color. On all media, the aerial growth
`is at first white then grayish with black spots. Sporo-
`phores areoften compact, forming a spiral of more
`than ten spores. Substrate growth is light yellow to
`almost colorless and in some media pale brown. Occa-
`sionally a yellowish pigment is produced. The organism
`is H,S- and melanine-negative.
`Carbohydrate utilization by Streptomyces hygro-
`scopicus NRRL 5491 wasstudied in carbonutilization
`agar (ISP Medium 9) according to the procedure stan-
`dardized by the International Streptomyces Project
`(ISP).
`Thebest utilized carbohydrates were D-glucose, ino-
`sitol, D-fructose and D-mannitol; less wellutilized car-
`bohydrates were rhamnose, raffinose, xylose, starch
`and arabinose. Carbohydratesnotutilized were sucrose
`and cellulose.
`The environment and nutritional requirements for
`, the fermentation of Streptomyces hygroscopicus NRRL
`25
`5491 are similar to those necessary for the production
`of antibiotics by other aerobic microorganisms. Thus,
`aerobiosis can be sustainedin a liquid nutrient medium
`inoculated with a sterile culture incubated in flasks
`placed on shaking machines.For industrial production,
`metal tanks with internal aeration and agitation by
`meansof paddles can be substituted. Rapamycin is also
`produced by surface cultivation. The microorganism
`requires as nutrient elements assimilable carbon and
`organic nitrogenous substances. The presence of min-
`eral salts is desirable. Cultivation is best effected when
`the initial pH of the culture medium is between 6.5 and
`7.5, the optimum pH being around 6.8-7.3.
`The utilizable sources of assimilable carbon for the
`production of the antibiotic are very diverse, there
`being included sugars (for example, glucose, D-fruc-
`tose, D-mannitol, maltose, arabinose, rhamnose, raffi-
`nose,xylose, andthe like), dextrin, starchesofdifferent
`types of origin, glycerol (and other polyalcohols), ino-
`sitol and animal and vegetable fats, as well as esters
`thereof. The sources of organic assimilable nitrogen
`which actively stimulate growth and favor production
`of rapamycin are substances such as soybean meal,
`cotton meal and other vegetable meals (whole or par-
`tially or totally defatted), meat flours or animalviscera,
`various peptones, casein hydrolysates, soybean hydro-
`lysates, yeast hydrolysates, lactalbumin, wheat glutins,
`distillers solubles, corn steeps, molasses, urea and
`aminoacids.
`Mineralsalts, such as the chlorides, nitrates, sulfates,
`carbonates and phosphates of sodium, potassium, am-
`monium and calcium, should be included in appropri-
`ate concentrations. The nutritive medium should con-
`tain a numberof trace elements such as magnesium,
`iron, manganese, and zinc.
`The inoculum of the above mediumfor the fermenta-
`tion is provided with a fresh slant of Streptomyces hy-
`groscopicus NRRL 5491.
`Underthe described conditions and with the temper-
`ature of cultivation at about 20°-35°C, preferably at
`about 25°C, maximum production of rapamycin in
`tanks is obtained in from about 2 to about 8 days. Al-
`ternatively, the pH may be controlled during fermenta-
`tion in tanks and maintained at about pH 6.0, and glu-
`
`55
`
`60
`
`65
`
`West-Ward Pharm.
`Exhibit 1040
`Page 004
`
`West-Ward Pharm.
`Exhibit 1040
`Page 004
`
`
`
`3
`cose may be added continuously from about 2 days
`after beginning to the end of fermentation, thus obtain-
`ing maximum yields in about 4 to 5 days.
`Thereafter, a variety of procedures may be employed
`in the isolation and purification of rapamycin, for ex-
`ample, solvent extraction, partition. chromatography,
`silica gel chromatography, liquid-liquid distribution in
`a Craig apparatus, and crystallization from solvents.
`Solvent extraction procedures are preferred for com-
`mercial recovery inasmuchas they are less time con-
`suming and less expensive.
`Generally speaking, rapamycin may be harvested by
`one of the following methods.
`a. The fermentation mixture is extracted with.a sub-
`stantially water-immiscible solvent, preferably a lower
`alkanol, for example n-butanol, n-pentanol or the com-
`mercial mixture of pentanols known as ‘“‘Pentasol” or
`n-hexanol, or a substantially water-immiscible lower
`alkyl lower alkanoate, for example, ethyl acetate, butyl
`acetate, amyl acetate or the commercially available
`mixture of amy! acetates, or a substantially water-
`immiscible halogenated aliphatic hydrocarbon,for ex-
`ample, chloroform, methylene dichloride or dichloro-
`ethane. The extracts are dried and concentrated under
`reduced pressure to yield an oily residue which is in
`turn extracted with a water-miscible solvent, preferably
`a lower alkanol, for example methanolor ethanol. Said
`last-named extracts are filtered through diatomaceous
`earth (‘‘Celite”), and the filtrate concentrated under
`reduced pressure to yield an oily residue containing
`crude rapamycin.
`.
`b. The fermentation mixtureis filtered through a pad
`of diatomaceousearth (Celite) and the filter cake con-
`taining the mycelium is extracted as described below
`under (c). Thefiltrate, i.e. the mycelium-free fermen-
`tation mixture, is extracted several.times with a sub-
`stantially water-immiscible solvent,
`for example, a
`lower alkanol, lower alkyl lower alkanoate or haloge-
`nated aliphatic hydrocarbon as exemplified above in
`section (a). The extracts are dried and concentrated
`under reducedpressure to yield an oily residue which is
`extracted with a water-miscible solvent, preferably a
`lower alkanol, for example methanol or ethanol. Said
`last-named extracts are treated in the same manner as
`described above under (a) to yield an oily residue con-
`taining crude rapamycin.
`.
`c. The mycelium is separated from the fermentation
`mixture and extracted with a suitable water-miscible
`solvent, preferably a lower alkanol, for example metha-
`nol or ethanol. The extract is concentrated by evapora-
`tion to the aqueous phase, which in turn is extracted
`with a substantially water-immiscible solvent, such as a
`loweralkyl lower alkanoate, halogenatedaliphatic hy-
`drocarbon or a substantially water-immiscible lower
`alkanol as described above or an aromatic hydrocar-
`bon, for example benzeneor toluene. The latter extract
`is evaporated under reduced pressure to yield an oily
`residue containing crude rapamycin.
`4
`The crude rapamycin obtained by any of the pro-
`cesses described in sections (a), (b) or (c) is then puri-
`fied by a variety of methods, for example, see above.
`Preferred methods include absorption of. the crude
`rapamycin on an absorbent, for instance charcoal or
`silica gel from a solution in a substantially non-polar,
`first solvent, followed by elution therefrom with a sec-
`ond solvent, more polar than said first solvent.
`
`20
`
`25
`
`30
`
`40
`
`45
`
`50
`
`55
`
`_DETAILS OF THE INVENTION
`Rapamycin is useful as an antifungal agent against a
`numberof pathogenic fungi; for example, Candida albi-
`cans, and other Candida species, Microsporum gyp-
`seum, Trichophyton. mentagrophytes, Aspergillus sp.,
`and Sporotrichum sp..
`The inhibitory activity of rapamycin is especially
`pronounced against Candida albicans and said last or-
`ganism maybe used advantageously for assay purposes.
`The antifungal activity of this compound is demon-
`strable in standard tests used for this purpose, for ex-
`ample, in the tests described in “Antiseptics, Disinfec-
`tants, Fungicides andSterilization”, G. F. Reddish, Ed.,
`2nd ed., Lea and Febiger, Philadelphia, 1957 or by D.
`C. Grove and W. A. Randall in “Assay Methods of
`Antibiotics”, Med. Encycl. inc., New York 1955.
`Whenthe antibiotic of this invention is employed as
`an antifungal agent in warm-blooded animals,e.g.rats,
`it may beused alone or in combination with pharma-
`ceutically acceptable carriers, the proportion of which
`is determined by the solubility and chemical nature of
`the compound, chosen route of administration and
`standard biological practice. For example, an antifun-
`gally effective amount ofthe antibiotic may be adminis-
`tered orally in solid form containing such excipients as
`starch, sugar, certain types of clay and so forth. Simi-
`larly, such an amount may also be administeredorally
`in the form ofsolutions or suspensions,or the antibiotic
`-may be injected parenterally. For parenteral adminis-
`tration the antibiotic may be used in the form of a
`sterile solution or suspension containing other solutes
`or suspending agents, for example, enough saline or
`glucose to makethesolution isotonic, bile salts, acacia,
`gelatin, sorbitan monoleate, polysorbate 80 (oleate
`esters of sorbitol and its anhydrides copolymerized with
`ethylene oxide) and thelike.
`The dosageofthe present antibiotic will vary with the
`form of administration and the particular compound
`chosen. Furthermore, it will vary withthe particular
`host under treatment. Generally, treatmentis initiated
`with small dosages substantially less than the optimum
`dose of the compound. Thereafter, the dosage is in-
`creased by small increments until the optimum effect
`under the circumstances is reached.
`In general,
`the
`compoundofthis invention is most desirably adminis-
`tered at a concentration level that will generally afford
`antifungally effective results without causing any harm-
`ful or deleterious side effects and preferably at a level
`that is in a range of from about !.0 mg to about 250 mg
`perkilo per day, although as aforementioned variations
`will occur. However, a dosagelevel thatis in the range
`of from about 10 mg to about 100 mg per kilo per day
`is most desirably employed in orderto achieve effective
`results.
`_
`In addition, the agent may be employed topically. For
`topical application it may be formulatedin the form of
`solutions, creams, or lotions in pharmaceutically ac-
`ceptable vehicles containing 0.1-5 per cent, preferably
`2 per cent of the agent, and may be administered topi-
`cally to the infected area of the skin.
`Rapamycin mayalso be used for cleaning anddisin-
`fecting laboratory equipment, surgical
`instruments,
`locker rooms, or shower rooms of sensitive fungus
`organisms. For such purposes it is preferred to use
`0.1-10% solutions of rapamycin in a lower alkanol,
`preferably methanol, diluted with 10-100 volumes of
`water containing 0.001-0.1% of a non-ionic surface-ac-
`
`3,929,992
`
`4
`
`60
`
`65
`
`West-Ward Pharm.
`Exhibit 1040
`Page 005
`
`West-Ward Pharm.
`Exhibit 1040
`Page 005
`
`
`
`3;929,992
`
`:
`5
`tive agent, for example, polysorbate 80 U.S.P., immedi-
`ately before applyingit to the objects to be cleaned and
`disinfected.
`
`PREPARATION
`
`—_ 0
`
`20
`
`6
`5.8~6:2.'The fermenters are inoculated with the second
`stage inoculum described above and incubated at about
`25°C with: agitation:and aeration while controlling and
`“maintaining the mixture at approximately pH 6.0 by
`addition ‘of ‘a base, for example, sodium hydroxide,
`potassium hydroxide or preferably. ammonium hydrox-
`ide, as required from timeto time. Addition of a source
`-of -assimilable carbon, preferably glucose,
`is started
`when the concentration of the latter in the broth has
`dropped. to about 0.5% wt/vol, normally about 48 hrs
`after.the start of fermentation, andis maintained until
`the end of the particular run. In this manner a fermen-
`tation broth containing about 60 yg/ml of rapamycin as
`determined by thé assay method described above is
`obtained in 4-5 days, when fermentation is stopped.
`Filtration of the mycelium, mixing the latter with a
`water-miscible lower’ alkanol, preferably methanol,
`followed by extraction with a halogenated aliphatic
`hydrocarbon, preferably trichloroethane, and evapora-
`tion of the solvents yields a first oily residue. This first
`oily residue is dissolved in a lower aliphatic ketone,
`preferably acetone,filtered from insoluble impurities,
`the filtrate evaporated to yield a second oily residue
`- which is extracted’with a water-miscible lower alkanol,
`preferably methanol, and the latter extract is evapo-
`rated to yield crude rapamycinas a third oily residue.
`This third oily residue is dissolved’ in a mixture of a
`loweraliphatic ketone. and a loweraliphatic hydrocar-
`bon, preferably acetone-hexane, an absorbent such as
`charcoal or preferably silica gel is added to adsorb the
`rapamycin,thelatter is eluted ftom theadsorbate with
`a similar but morepolarsolvent mixture, for example a
`‘mixture as above but containing a higher proportion of
`the aliphatic ketone, the eluates are evaporated and the
`residueis crystallized from diethyl ether, to yield pure
`crystalline rapamycin. In this mannera total of 45-58%
`of the rapamycin initially present in the fermentation
`mixture is recoveredas pure crystalline rapamycin.
`CHARACTERIZATION
`a. Purified rapamycin is a colourless crystalline com-
`pound, m.p. 183°-185°C after recrystallization from
`ether;
`—
`:
`:
`b. rapamycinis soluble in ether, chloroform, acetone,
`methanol and dimethylformamide; very sparingly solu-
`ble in ‘hexane and petroleum: ether and substantially
`insoluble in water;
`c. rapamycin shows a. uniform spot on thin layer
`, plates of silica gel G (E. Merck. A. G., Darmstadt)
`50
`developed with a variety of thin layer chromatography
`-solvent systems; for example, ether-hexane 40:60 (Rf=
`' 0.42), isopropyl alcohol-benzene 15:85 (Rf= 0.5) and
`ethanol-benzene 20:80 (Rf = 0.43);
`d. rapamycin obtainedfrom four successive fermen-
`55
`_ tation. batches. gave. the following values on repeated
`elemental analyses:
`
`In one embodiment of this invention rapamycin is
`prepared in the following manner:
`“
`A suitable fermenter is charged with production me-
`dium 8KM (see Example 1). After sterilization and
`cooling, the medium is inoculated with a first stage
`inoculum preparation of: Streptomyces ‘hygroscopicus
`NRRL 5491.
`eo
`=
`A maximum titre of 20 to 100 ug/mlof the antibiotic
`is reached in the fermentation mixture after 2-8. days,.
`usually after about 5 days, as determined by the. cup
`plate method and Candida albicans as the test organism.
`The mycelium is harvested by filtration with diatoma-
`ceous earth. Rapamycin is then extracted-from the
`mycelium with a water-miscible solvent, for example a
`lower alkanol, preferably methanol or ethanol. The
`latter extract is then concentrated, preferably under
`reduced pressure, and the resulting aqueous phase is
`extracted with a water-immiscible solvent. A preferred
`water-immiscible solvent for this purpose is methylene
`dichloride although chloroform, carbon tetrachloride,
`benzene, n-butanol and the like may also be used. The
`latter extract
`is concentrated, preferably: under re-
`duced pressure, to afford the crude product ds anoil.
`The product may be purified further by a variety of.
`methods. Amongthe preferred methodsofpurification
`is to dissolve the crude productin a substantially non-
`polar, first solvent, for example petroleum ether or
`hexane, and to treat the resulting solution with a suit-
`able absorbent, for example charcoalorsilica gel, so
`that the antibiotic becomes absorbed on the absorbant.
`The absorbantis then separated and washed or eluted
`with a second solvent more polar than thefirst solvent,
`for example ethyl acetate, methylene dichloride, or a
`mixture of methylene dichloride and ether (preferred).
`Thereafter, concentration of the wash solution or elu-
`ate affords substantially pure rapamycin. Further puri-
`fication is obtained by partial precipitation with a non-
`polar solvent, for example, petroleum ether, hexane,
`pentane andthelike, from a solution of the rapamycin
`in a more polar solvent, for example, ether, ethyl ace-
`tate, benzene andthe like. Still-further purification is
`obtained by column chromatography, preferably em- °
`ploying silica gel, and by crystallization of. the rapamy-
`cin from ether.
`;
`In another preferred embodimentofthis invention a
`first stage inoculum of Streptomyces hygroscopicus
`NRRL5491 is prepared in small batches in a.medium
`containing soybean flour, glucose, ammonium sulfate,
`and calcium carbonate incubated at about 25°C at pH
`7.1-7.3 for 24 hrs. with agitation, preferably on a gyro-
`tary shaker. The growth thus obtained is used to inocu-
`late a number of somewhatlarger batches of the same
`medium as described above which are incubated at
`about 25°C and pH 7.1-7.3 for 18 hrs. with agitation,
`preferably on a reciprocating shaker, to obtain a sec-
`‘ond stage inoculum whichis used to inoculate the pro-
`duction stage fermenters.
`The production stage fermenters are equipped with .
`devices for controlling and maintaining pH at a prede-
`termined level and for continuous metered addition of 65
`nutrient. They are charged with a medium. containing
`soybean flour, glucose, ammonium sulfate, and potas-
`sium phosphate, sterilized, and the pH is adjusted to pH
`
`35
`
`40
`
`45
`
`60
`
`%
`:
`%
`
`67.24,
`.
`8.93,
`1.39,
`
`66.14,
`
`8.72,
`-
`1.37,
`
`67.26,
`.
`8.92,
`
`1.28,
`
`AVER-
`. AGE
`66.72... 66.84
`
`8.9.
`
`1.28.
`
`:
`
`- 8,84
`
`1.37
`
`e., rapamycin exhibitsthe following characteristic
`absorption maximain its ultraviolet absorption spec-
`trum (95% ethanol):
`:
`
`West-Ward Pharm.
`Exhibit 1040
`Page 006
`
`West-Ward Pharm.
`Exhibit 1040
`Page 006
`
`
`
`7
`267 nm (Ejem!® 417), 277 nm (Eyem'” 541) and 288
`nm (Eyem!” 416);
`f. the infrared absorption spectrum of rapamycin in
`chloroform is reproduced in FIG. 1 and shows charac-
`teristic absorption bands at 3560, 3430, 1730, 1705
`and 1630-1610 cm™!;
`Further infrared absorption bands are characterized
`by the following data given in reciprocal centimeters
`with (s) denoting a strong,(7) denoting a medium, and
`(w) denoting a weak intensity band. This classification
`is arbitrarily selected in such a mannerthat a bandis
`denotedas strong(s) if its peak absorption is more than
`two-thirds of the background in the same region; me-
`dium (m) if its peak is between one-third and two- 15
`thirds of the background in the same region; and weak
`(w) if its peak is less than one-third of the background
`in the same region.
`
`10
`
`1158 em™(m)
`2990 cm™ (m)
`1129 em™! (s)
`2955 cm™ (s)
`1080 cm“ (s)
`2919 cm™(s)
`1060 cm™! (s)
`2858 cm™(s)
`1040 cm™ (m)
`2815 cm7 (m)
`1020 cm=! (m)
`1440 cm(s)
`978 cm™(s)
`1365 cm™ (m)
`905 cm(m)
`1316 cm™ (m)
`888 cm™(w)
`1272 cm™(m)
`
`1178 cm™ (s) 866 cm“! (w)
`
`g. the nuclear magnetic resonance spectrum of rapa-
`mycin.in deuterochloroform is reproduced in FIG. 2;
`h. the minimum inhibitory concentration of rapamy-
`cin against various microorganismis listed below:
`
`Rapamycin:
`
`Organisms
`(ug/ml)
`Candida albicans (5 strains)
`0.02 to 0.1
`C. catenulata
`.
`<0.1
`C.lipalytica
`2.5
`C, stellatoidea
`<0.1
`C, tropicalis
`0.1
`'_C. pseudtropicalis
`>5.0
`C. parapsilosis
`<0.1
`C. morrera
`<0.1
`C. intermedia
`<0.1
`M. gypseum
`12.5
`T. mentagrophytes
`> 1000
`
`:
`
`20
`
`25
`
`30
`
`35
`
`40
`
`45
`
`3,929,992
`
`8
`EXAMPLE1
`
`Microorganism
`Streptomyces hygroscopicus NRRL 5491 was grown
`and maintained on oatmeal-tomato paste agar slants
`(T. G. Pridham, et al., Antibiotic Annual 1956-1957,
`Medical Encyclopedia Inc., New York, p. 947) and in
`Roux bottles containing the same medium. Good
`growth wasobtained after 7 days of incubationat 28°C.
`Spores from one Roux bottle were washed off and
`suspended into 50 mlofsterile distilled water. This
`suspension was used to inoculate the first stage inocu-
`lum.
`The first-stage inoculum medium consisted of Emer-
`son broth [R. L. Emerson etal., J. Bacteriol, 52, 357
`(1946)] 0.4%; peptone, 0.4%; sodium chloride, 0.25%;
`yeast extract, 01.%; and glucose, 1%; pH 7.0; flasks
`containing the above medium were inoculated with 1%
`of the spore suspension described above. The inocu-
`lated flasks were incubated for 30 hrs. at 28°C on a
`reciprocating shaker set at 65 r.p.m. (4 inchstroke).
`Production stage
`The production stage was run in 250-liter New
`Brunswick fermenters Model F-250, equipped with
`automatic antifoam addition system and pH recorder-
`controller. The fermenters were charged with 160liters
`of an aqueous production medium(8 KM) consisting of
`the following constituents:
`
`soluble starch
`(NH,)2SO,
`K,HOP,
`glucose (Cerelose)
`MgSO,
`ZnSO,
`MnSO,
`FeSO,.7H,O
`CaCO,
`“Blackstrap” molasses
`hydrolyzed casein (NZ-Case,
`Sheffield Chemical, Norwich,
`New York)
`lard oil (Larex No. 1, Swift
`Canadian Co., Toronto)
`pH 7.1 to 7.3
`
`1.0%
`0.5%
`0.5%
`1.5%
`0.025%
`0.005%
`0.001%
`0.002%
`0.2%
`0.5%
`
`0.5%
`
`0.2%
`
`55
`
`ji. rapamycin exhibits a LDso (i.p., mice) of 597.3 + 50
`28.1 mg/kg and a LDgo (p.o., mice) of >2,500 mg/kg.
`In protection studies, mice were infected by intrave-
`nousinjection of C. albicans ATCC 11651. At 1,4 and
`24 hours after infection, mice were administered 10
`mg/kg (s.c.) of rapamycin. At this dose 50% of the
`mice were protected. Treatment with 25 mg/kg (s.c.)
`offered complete protection. When rapamycin was
`administered orally, at 10 mg/kg 4 out of 10 mice sur-
`vived, and at 25 mg/kg complete protection was ob-
`served.
`A 1% suspension (0.2 ml) of rapamycin in water
`containing 1.5% polysorbate 80 (Tween 80), when
`injected intradermally into a rabbit’s ear caused no
`irritation. Similarly, two drops of a 0.5% suspension
`applied to a rabbit’s eye caused noirritation.
`The following Examplesillustrate further this inven-
`tion.
`
`60
`
`65
`
`The fermenters were sterilized at 121°C for 45 min-
`utes, cooled and inoculated with one flask (2% inocu-
`lum) of first stage inoculum. Incubation temperature:
`28°C; aeration: 0.5 vol/vol/min.; agitation: 250 r.p.m.
`A titre of ca. 20 g/ml, determined by microbiologi-
`cal assay on agarplates seeded with Candida albicans,
`was reached in 5 days. The fermentation was stopped.
`Extraction and isolation of the antibiotic was per-
`formed by one of the following methods:
`Extraction
`
`a. The fermentation mixture was extracted twice with
`1 v/v of n-butanol. The combined butanol extracts were
`washed with 1 v/v of water, dried with anhydrous so-
`dium sulfate and evaporated to dryness under reduced
`pressure to yield a residue. The oily residue was ex-
`tracted 3 times with 2 liters of methanol. The combined
`methanol extracts were passed through diatomaceous
`earth (Celite) and evaporated to dryness to yield an
`oily residue containing crude rapamycin.
`b. The fermentation mixture wasfiltered over diato-
`maceous earth (Celite). The filtrate was extracted
`twice with 1 v/v of ethyl acetate. The ethyl acetate
`extracts were washed with 1 volume of water, dried
`
`West-Ward Pharm.
`Exhibit 1040
`Page 007
`
`West-Ward Pharm.
`Exhibit 1040
`Page 007
`
`
`
`‘10
`
`=3% 11 wt/vol
`= 2%
`wt/vol
`=0.1% wt/vol
`=0.5% wt/vol
`~ = 0.05% wt/vol
`
`.
`
`20
`
`te 5
`
`40
`
`3939992
`with anhydrous sodium sulfateand siapieatadunder
`“ a;gyrotaryshakerQ@ inch stroke) at 240°rpm for 24 hrs ::..
`at 25°C,
`.
`reduced pressure todryness. The residue‘was extracted
`“Second Stage Inoculum::Twenty--four Jiter flat bot-
`twice with | liter of methanol.’ The, methanol éxtracts
`tom flasks containing3.2:1 of the inoculum medium
`were evaporated under reduced pressure: to yield-;an '-
`described above at ‘:pH7.1-7.3 are stérilized:‘by auto-
`oily residue containing crude rapamycin: :
`claving at 121° for 35’minutes,shaken to' resuspend the
`c. The mycelium obtained as‘described under séction
`‘insoluble material:and:resterilizedfor another 45-min-
`(b) was washed with.1 to 2 volumes of water. The
`"utes. The flasks aré:Gooled to25° and inoculated with
`washed mycelium was extracted 3 times with 5 volumes
`64 mloffirst stage inoculum, placedon areciprocating
`of methanol per weight of’ wet mycelium ‘each time:
`shaker (4.inch.stroke):set at 65 rpm.and incubated for
`The methanolic extracts werepooled andcoricentrated —
`18 hrs at 25°C.
`,
`under reduced pressure to a small volume of an aque-
`Production Stage..The production stage is run in 250
`ous phase containing approximately 10% v/v of metha-
`liter New Brunswickfermenters Model F-250 equipped
`nol. This aqueous phase was extracted 3 times with1
`with automatic ‘antifoamaddition system and pH re-
`vol. of methylene chloride;
`the methylene chloride.
`corder-controller. The fermenters are charged’ with
`extracts were combined, dried with anhydrous sodium
`160 liters of anaqueousproduction medium consisting
`sulfate and evaporated to yield an oily residue.
`of |the following¢Constituents:
`Theoily residue was dilutedwith.1 volume of petro-
`leum ether, and 30% w/v of charcoal (DarcoG60) was
`, Soybean flour (Archer-Daniels Co.,
`added. The mixture wasstirréd for half an‘ hour and
`Midland, Mich., “Special X°*)
`filtered. The charcoal, which retained’‘substantially all
`:.. Glucose (Cerelose)
`m
`Ammonium sulfate
`of the product, was washed twice with one volume of
`Potassium phosphate (monobasic)
`Antifoaming Agent (“DF-143 - PX”
`petroleum ether. The charcoal was eluted. three times
`Mazer Chemicals, Inc., Gurnee, Il.)
`with 5 vol. (based on the weight ofthe charcoal).of a .
`25
`mixture of methylene chloride and ether (50:50). The
`methylene chloride-ether extracts were evaporated to
`dryness and the residue dissolved in a small amountof
`ether. The crude product was obtained by precipitation —
`from the ether solution with cold petroleum ether.
`Alternatively, the oily residue obtained by any of the
`extraction procedures described above was diluted
`with | vol. of hexane and passed through a preparative
`column ofsilica gel G. The product was adsorbed on
`the column. Thesilica gel G containing adsorbed prod::
`uct was washed with several volumes of hexane and
`50:50 hexane-ether mixtures. The product was eluted
`from the column with ether. The ether eluant was evap-
`orated to a small volume and crude product obtained
`by precipitation from the ether solution with cold pe-
`troleum ether.
`
`The.fermenters are sterilized. at.121°C for 30 min-
`utes, cooled, and the pH is adjusted to 5.8 to 6.2 with
`ammonium hydroxide. They are then inoculated with
`oneflask (2%) of second stage inoculum and fermenta-
`tion is allowed to proceed at 25°C, with aeration at 0.25
`v/v/min and agitation at 200 rpm.
`The pH of the fermentation broth starts to drop at
`30-35 hours and is controlled at 6.0 until the end of
`fermentation by the automatic, on demand, addition of
`ammonium hydroxide. At about 48 hrs. of propagation
`the glucose concentration in the broth drops to about
`0.5%, and continuous addition of 40% glucose solution
`is started at a rate of 3.75% of fermentation mixture
`volumeper day and continued until the end of fermen-
`tation. A titer of about 60 pg/ml, determined by micro-
`biological assay on agar plates seeded with Candida
`albicans is reached in 4 to 5 days. The fermentation is
`stoppedat this point.
`Extraction and isolation of the antibiotic is per-
`formed by the following procedure: .
`The fermentation mixture is filtered over diatoma-
`ceousearth (Celite) to recover the mycelium. A typical
`400 liter batch obtained from three fermenters yields
`about 60 kg of wet mycelium. The wet mycelium is
`mixed with | vol/wt of methanol by agitation and the
`mixtureis extracted twice with 2 vol of trichloroethane
`(methyl chloroform), yielding about 250 liters of tri-
`chloroethane extract containing about 22-24 g of rapa-
`mycin. The trichloroethane extract is evaporated to
`dryness under reduced pressure to yield 1 to 1.4 kg of
`oily residue. This residue is added slowly with agitation
`to 5 vols of acetone and the resulting precipitate is
`separated byfiltration. The acetone solution is evapo-
`rated to dryness under reduced pressure to yield an oily
`residue. This oily residue is extracted twice with 2 and
`Soybean flour (Archer-Daniels Co.,
`=4% wt/vol
`Midland, Mich.“Special X")
`1 vols of methanol respectively. The combined metha-
`:
`=2% wt/vol
`Glucose (Cerelose)
`
`Ammonium sulfate =0.3%—wt/vol
`nol extracts are filtered and the remaining oil is dis-
`Calcium carbonate
`= 0.15% wt/vol
`carded. The methanolextract containing rapamycin is
`Water to volume, pH 7.1 to 7.3
`evaporated to dryness under reduced pressureto yield
`200 to 300 g ofoily residue. This residue is dissolved in
`5 v/wt of 15% acetone in hexane:To this solution of the
`oily residue, silica gel (Merck) is added in an amount
`equal to twice the weight of the oily residue and the
`West-Ward Pharm.
`Exhibit 1040
`Page 008
`
`Purification
`
`The aforeme