United States Patent
`
`[191
`
`[111
`
`3,929,992’
`
`
`
` Sehgal et al. [45] Dec. 30, 1975
`
`[75]
`
`[54] RAPAMYCIN AND PROCESS OF
`PREPARATION ,
`lnventors: Surendra N. Sehgal, Dollard des
`' Ormeaux; Teodora M. Blazekovic,
`Mount Royal; Claude Vezina,
`DeuX_MomagneS, an of Canada
`[73] Assignee: Ayerst McKenna and Harrison Ltd.,
`Montreal, Canada
`
`[58] Field of Search ...................... .. 424/122; 195/80
`
`[56]
`
`Ref°l‘°“¢95 Cited
`OTHER PUBLIC AT]QNs
`
`Miller, The Pfizer Handbook of Microbial Metabolites
`McGraw~Hill Book Co.
`lnc., N.Y., N.Y., 1961, p.
`580.
`
`[22] Filed;
`
`Apt 12’ 1974
`
`‘
`
`Primary Examiner——Jerome D. Goldberg
`
`[21] Appl. No.: 460,665
`
`Related U'S‘ Apphcanon Data
`[63l C0“‘l““a‘i°“'l“‘Pa" Of 55‘? N0- 293.699. Sept 29.
`1972i abandomd‘
`‘
`
`.
`[52] U.S. Cl. ............................. .. 424/122; 195/80 R
`[51]
`Int. Cl.”........................................ .. A6lK 35/00
`
`[57]
`ABSTRACT
`Antibiotic rapamycin is producible by culturing Strep-
`tomyces hygroscopicus NRRL 5491 in an aqueous nu-
`trient medium. Rapamycin has antifungal properties.
`Methods for 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
`
`

`

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`West-Ward Pharm.
`Exhibit 1040
`Page 002
`
`West-Ward Pharm.
`Exhibit 1040
`Page 002
`
`

`

`U.S. Patent Dec.30, 1975
`
`Sheet2of2
`
`3,929,992
`
`4'0
`
`9.0
`
`8.0
`
`7.0
`
`PPM(1')5.060
`
`
`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.
`'
`
`BACKGROUND OF 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. Description‘ of Prior Art
`The antibiotic of this invention is readily distin-
`guished from prior art compounds of its class by its
`profound antifungal activity and its relatively low order
`of toxicity.
`More explicitly, the ultra violet spectrum of rapamy-
`cin, noted herein, indicates that this compound belongs
`to the class of antibiotics known as triene antibiotics. In
`
`this particular class there are only five compounds
`reported previously. Trienine, A. Aszalos et al., J . Anti-
`biotics, 21, 61 1 (1968) is a triene antibiotic with antitu-
`mor activity which also shows marked activity against
`gram positive organisms and only marginal activity
`against Candida strains. 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
`ug/ml) and high toxicity (LD_.-,0 in mice: 15 mg/kg). The
`remaining two antibiotics - Resistaphylin, S. Aezaiva et
`al., J. Antibiotics, 24, 393 (1971) and Proticin, G.
`Nesemann et 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 SUMMARY OF THE INVENTION
`
`Rapamycin is a chemical compound producible by
`culturing a rapamycin-producing organism in an aque-
`ous nutrient medium. The compound has the property
`of adversely affecting the growth of fungi, for example,
`Candida albicans and Microsporum gypseum. Accord-
`ingly, rapamycin may be used to prevent the growth of
`or reduce the number of certain 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, US. Department of Agricul-
`ture, Peoria, lll., U.S.A.
`It is to be understood that the invention is 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,
`
`2
`starch agar and peptone beef agar. Also, the organism
`grows very well on yeast extract agar, malt extract agar,
`starch-inorganic salts agar, oatmeal agar, oatmeal-
`tomato agar and Bennet’s agar. On potato slices there is
`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 are often 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 H2S- and melanine-negative.
`Carbohydrate utilization by Streptomyces hygro-
`scopicus NRRL 5491 was studied in carbon utilization
`agar (ISP Medium 9) according to the procedure stan-
`dardized by the International Streptomyces Project
`(ISP).
`The best utilized carbohydrates were D-glucose, ino-
`sitol, D-fructose and D-mannitol; less well utilized car-
`bohydrates were rhamnose, raffinose, xylose, starch
`and arabinose. Carbohydrates not utilized were sucrose
`and cellulose.
`
`10
`
`15
`
`20
`
`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 sustained in a liquid nutrient medium
`inoculated with a sterile culture incubated in flasks
`
`30
`
`35
`
`40
`
`45
`
`50
`
`55
`
`60
`
`65
`
`placed on shaking machines. For industrial production,
`metal tanks with internal aeration and agitation by
`means of 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, and the like), dextrin, starches of different
`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 animal viscera,
`various peptones, casein hydrolysates, soybean hydro-
`lysates, yeast hydrolysates, lactalbumin, wheat glutins,
`distillers solubles, corn steeps, molasses, urea and
`amino acids.
`Mineral salts, 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 number of trace elements such as magnesium,
`iron, manganese, and zinc.
`The inoculum of the above medium for the fermenta-
`tion is provided with a fresh slant of Streptomyces hy-
`groscopicus NRRL 5491.
`-
`Under the 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-
`
`
`
`West-Ward Pharm.
`Exhibit 1040
`Page 004
`
`
`West-Ward Pharm.
`Exhibit 1040
`Page 004
`
`

`

`3,929,992
`
`4
`
`,DETAILS OF THE INVENTION
`
`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 inasmuch as 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 amyl 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 methanol or 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 mixture is filtered through a pad
`of diatomaceous earth (Celite) and the filter cake con-
`taining the mycelium is extracted as described below
`under (c). The filtrate, 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 reduced pressure 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
`lower alkyl lower alkanoate, halogenated aliphatic hy-
`drocarbon or a substantially water-immiscible lower
`alkanol as described above or an aromatic hydrocar-
`bon, for example benzene or toluene. The latter extract
`is evaporated under reduced pressure to yield an oily
`residue containing crude rapamycin.
`‘
`,
`.
`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.
`
`Rapamycin is useful as an antifungal agent against a
`number of 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 may be 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 and Sterilization”, 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. lnc., New York 1955.
`When the antibiotic of this invention is employed as
`an antifungal agent in warm-blooded animals, e.g. rats,
`it may be used 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 of the 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 administered orally
`in the form of solutions 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 make the solution isotonic, bile salts, acacia,
`gelatin, sorbitan monoleate, polysorbate 80 (oleate
`esters of sorbitol and its anhydrides copolymerized with
`ethylene oxide) and the like.
`The dosage of the present antibiotic will vary with the
`form of administration and the particular compound
`chosen. Furthermore, it will vary with the particular
`host under treatment. Generally, treatment is 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
`compound of this 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 1.0 mg to about 250 mg
`per kilo per day, although as aforementioned variations
`will occur. However, a dosage level that is in the range
`of from about 10 mg to about 100 mg per kilo per day
`is most desirably employed in order to achieve effective
`results.
`
`10
`
`15
`
`.20
`
`25
`
`30
`
`35
`
`40
`
`45
`
`50
`
`55
`
`60
`
`65
`
`In addition, the agent may be employed topically. For
`.
`topical application it may-be formulated in 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 may also be used for cleaning and disin-
`fecting laboratory equipment, surgical
`instruments,
`locker rooms,‘ or shower rooms of sensitive fungus
`organisms. For such purposes it
`is preferred to use
`0.1—l0% solutions of rapamycin in a lower alkanol,
`preferably methanol, diluted with /10-100 volumes of
`water containing 0.00l—0. 1 % ofa non-ionic surface-ac-
`
`West-Ward Pharm.
`Exhibit 1040
`Page 005
`
`
`West-Ward Pharm.
`Exhibit 1040
`Page 005
`
`

`

`‘3;9'29,992,
`
`:6
`5.8-6:2.-The fermenters are inoculated with the second
`
`5
`tive agent, for example, polysorbate 80 U.S.P., immedi-
`ately before applying it to the objects to be cleaned and
`disinfected.
`
`PREPARATION
`
`ln one embodiment of this invention rapamycin is
`prepared in the foll0wing.manner:
`V
`-
`‘
`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.
`~
`
`A maximum titre of 20 to 100 p.g/ml of 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 as an oil.
`The product may be purified further by a variety of .
`methods. Among the preferred methods of purification
`is to dissolve the crude product in 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 charcoal or silica gel, so
`that the antibiotic becomes absorbed on the absorbant.
`
`stage inoculum described above and incubated at about
`= 25°C :w°ith'-'agitation‘and aeration while controlling and
`'mai‘nt»ai~'ning‘? the mixture at approximately pH 6.0 by
`addition loffa base, for example, sodium hydroxide,
`potassium hydroxide or preferably ammonium hydrox-
`ide, as required from time to 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 __fe_rr_nentation, and is maintained until
`the end of the particular run. In this manner a fermen-
`tation broth containing about 60 pg/ml of rapamycin as
`determined by the ‘assay method described above is
`‘obtained in 4-5 days, when fermentation is stopped.
`' Filtration of themycelium, 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 extractedjwith a water-miscible lower alkanol,
`25
`preferably methanol, and the latter extract is evapo-
`rated to yield crude rapamycin as a third oily residue.
`This third oily residue is dissolved‘ in a mixture of a
`lower aliphatic ketone and a lower aliphatic hydrocar-
`bon, preferably acetone-hexane, an absorbent such as
`charcoal or preferably silica gel is added to adsorb the
`rapamycin, the latter is eluted from the’ adsorbate with
`a similar but more polar solvent mixture, for example a
`’ mixture as above but containing a higher proportion of
`the aliphatic ketone, the eluates are evaporated and the
`residue is crystallized from diethyl ether, to yield pure
`crystalline rapamycin. In this manner a total of 45-5 8%
`of the rapamycin initially present in the fermentation
`mixture is recovered’ as pure crystalline rapamycin.
`
`l0
`
`l5
`
`20
`
`30
`
`35
`
`The absorbant is then separated and washed or eluted
`with a second solvent more polar than the first 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 and the like, from a solution of the rapamycin
`in a more polar solvent, for example, ether, ethyl ace-
`tate, benzene and the like. Still-further purification is
`obtained by column chromatography, preferably em- '
`ploying silica gel, and by crystallization of the rapamy-
`cin from ether.
`.
`
`40
`
`CHARACTER‘lZATION
`
`45
`
`a. Purified rapamycin is a colourless crystalline com-
`pound, m.p.
`l83°—l85°C after recrystallization from
`ether;
`’
`»
`*
`b. rapamycin is soluble in ether, chloroform, "acetone,
`methanol and dimethylformamide; very sparingly solu-
`ble in ‘hexane and petroleum. ether and.substantially
`insoluble in water; i
`'
`’
`c. rapamycin shows a uniform spot on thin layer
`50
`‘ plates of silica gel G (E. Merck A. G., Darmstadt)
`developed with a variety of thin layer chromatography
`i 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 f'—- 0.43);
`55
`d., rapamycin obtained‘: from four successive fermen-
`_ _tation batchesgave the following values on repeated
`elemental analyses:
`
`£7
`'0
`%
`
`67.24,
`
`66.l4,
`
`8.93,
`1.39.
`
`.
`
`8.72,
`H
`1.37,
`
`67.26.,
`
`,
`
`3.92,
`1.23,
`
`60
`
`65
`
`AVER-
`. AGE
`.. 66.84
`
`'
`
`« 8,84
`1.37
`
`66.72.
`
`.
`
`8.9.
`.
`1.23.
`
`e. _raparnycin exhibits, the following characteristic
`absorption maxima in its ultraviolet absorption spec-
`trum (95% ethanol):
`‘
`
`West-Ward Pharm.
`Exhibit 1040
`Page 006
`
`
`ln another preferred embodiment of this invention a
`first stage inoculum of S treptomyces hygroscopicus
`NRRL 5491 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 somewhat larger 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 reciprocatingshaker, to obtain a sec-
`"ond stage inoculum which is 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
`
`nutrient. They are charged with a medium containing
`soybean flour, glucose, ammonium sulfate, and potas-
`sium phosphate, sterilized, and the pH is adjustedto pH
`
`
`
`West-Ward Pharm.
`Exhibit 1040
`Page 006
`
`

`

`3,929,992
`
`8
`EXAMPLE 1
`
`Microorganism
`
`.
`
`7
`267 nm (E,c,,,‘% 417), 277 nm (E,,,,,‘% 541) and 288
`nm (Emn‘”’° 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, (In) denoting a medium, and
`(w) denoting a weak intensity band. This classification
`is arbitrarily selected in such a manner that a band is
`denoted as strong (3) 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-
`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.
`
`
`1158 cm" (m)
`2990 cm“ (m)
`H29 cm“ (s)
`2955 cm“‘ (5)
`1080 cm“ (s)
`2919 cm" (5)
`1060 cm“ (s)
`2858 cm“ (s)
`1040 cm“ (in)
`2815 cm“ (tn)
`1020 cm" (in)
`1440 cm“ (s)
`978 cm“ (s)
`1365 cm“ (m)
`905 cm“ (in)
`1316 cm“ (m)
`888 cm" (w)
`1272 cm" (m)
`
`1178 cm“ (s) 866 cm“ (w)
`
`10
`
`15
`
`20
`
`25
`
`g. the nuclear magnetic resonance spectrum of rapa-
`mycinin deuterochloroform is reproduced in FIG. 2;
`h. the minimum inhibitory concentration of rapamy-
`cin against various microorganism is listed below:
`
`Rapamycin:
`
`Organisms
`(p.g/ml)
`Candida albicans (5 strains
`0.02 to 0.1
`C. caterrulala
`.
`<0.l
`C. Iipolylica
`2.5
`C. stellalaidea
`<0.l
`C. tropicalis
`0.1
`' C. pseudtropicalis
`>5.0
`C. parapsilosis
`<0.l
`C. marrera
`<0.l
`C. inlermedia
`<0.l
`M. gypseum
`12.5
`
`T. mentagrophytes >l000
`
`A
`
`i. rapamycin exhibits a LD5o (i.p., mice) of 597.3 :1:
`28.1 mg/kg and a LD5o (p.o., mice) of >2,50O mg/kg.
`In protection studies, mice were infected by intrave-
`nous injection 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 no irritation.
`The following Examples illustrate further this inven-
`tion.
`’
`
`30
`
`35
`
`40
`
`45
`
`50
`
`55
`
`60
`
`65
`
`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 was obtained after 7 days of incubation at 28°C.
`Spores from one Roux bottle were washed off and
`suspended into 50 ml of sterile 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 et al., J. Bacteriol, 52, 357
`(1946)] 0.4%; peptone, 0.4%; sodium chloride, 0.25%;
`yeast extract, 0l.%; 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 inch stroke).
`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 160 liters
`of an aqueous production medium(8 KM) consisting of
`the following constituents:
`
`soluble starch
`(NH.,)2SO.
`K2HOP.,
`glucose (Cerelose)
`MgSO.,
`ZnSO,
`MnSO.,
`FeSO..7H2O
`CaC0;,
`“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%
`
`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 ug/ml, determined by microbiologi-
`cal assay on agar plates 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 was filtered 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
`
`

`

`3",‘9i’2‘:i9:':',‘-'ic,i'i59i"2
`
`’10
`inch stroke) at 240 ‘rpm for 24 hrs , -
`,, a.:'g.yr;o,tary_.shal<;er
`t
`if
`,
`,
`'
`,
`ii?1_t.,25.°_C..;
`.
`f:Secon,d Stage Inoculu‘r_n.~Twenty-‘four _liter flat=,bot-
`tom flasks containing.’ 3.21 of the inoculum _me‘dium
`described above at ipl-lo 7.'l-.7.3 are sterilizedi-"by auto-
`claving at 121° for 375-"rr‘i,inutes,'shaken to“ resuspend the
`insoluble material “a‘nd::resterilized_ for another 45min-
`’ utes.iThe flasks are‘fc'o_oled to‘ 25° and inoculated with
`64 ml of first stage inoculum, placed on a reciprocating
`shaker‘(4 -i_nch_’str,ol'<e),set at 65 rpm and incubated for
`18 hrs at 25°C.
`'
`I
`Production Stage. The production stage is run in 250
`liter New Bru_r1swicl§f’ferrnentersiModelF-250 equipped
`with, automatic 3antifoa_m':additionsystem and pH re-
`corder-controller.‘ The fermenters are ‘charged’ with
`l60 liters of anaqueous production medium consisting
`J of the followingeonstituents:
`
`‘
`
`10
`
`15
`
`.
`
`with anhydrous sodium (sulfate and ‘evaporatediunder
`reduced pressure to dryness. The residue”"was extracted
`twice with l liter of methanol."The.methanolextracts
`were evaporated under reduced pressure to y'ield-.,an »
`oily residue containing crude. rapamycin; V
`c. The mycelium obtained asdescribed under section
`(b) was washed with.1 to 2 volumes ofwater. The
`washed mycelium was extracted 3 times with 5 volumes
`of methanol per weight of‘wet myceliumiyeavch time;
`The methanolic extracts werepooled and concentrated
`under reduced pressure to a small volume of an aque-
`ous phase containing approximately 10% v/v of metha-
`nol. This aqueous .phase was extracted ".3 times withfl
`vol. of methylene chloride;
`the methylene chloride.
`extracts were combined, dried withranhydrousi sodium
`sulfate and evaporated to yield an oily residue.
`A
`The oily residue ‘was diluted‘ withil volume of petrol-
`leum "ether, and 30% w/v of charcoal (Darco;G60) was
`added. The mixture was stirred for half an hoursand
`filtered. The charcoal, which retainedfsubstantially all
`of the product, was washed twice with one volume of
`petroleum ether. The charcoal was elutedthree-times
`with 5 vol’. (based on the weight of the charcoal) of a .-
`mixture of methylene chloride and ether (50:50). The 25
`methylene chloride-ether extracts were evaporated to
`dryness and the residue dissolved in a small amount of
`ether. The crude product was obtained by precipitation 4
`from the ether solution with cold petroleum ether.
`30
`Alternatively, the oily residue obtained by any of the
`extraction procedures described above was diluted
`with 1 vol. of hexane and passed through a preparative
`column of silica gel G. The product was adsorbed on
`the column. The silica gel G containing adsorbed prod-
`uct was washedlwith 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.
`
`20
`
`‘ Soybean flour (Archer-Daniels Co.,
`,Midland, Mich., “Special X")
`A. Glucose (Cerelose)
`-
`Ammonium sulfate
`Potassium phosphate (monobasic)
`Antifoaming Agent‘(“DF-1,43 - PXf‘
`Mazer Chemicals, Inc., Gurnee, lll.,)
`
`.
`
`-
`
`,.
`
`= 3% ll wt/vol
`, = 2%
`wt/vol
`= 0.1% wt/vol
`= 0.5% wt/vol
`’
`= 0.05% wt/vol
`
`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
`one flask (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
`
`volume per day and continued until the end of fermen-
`tation. A titer of about 60 ug/ml, determined by micro-
`biological assay on agar plates seeded with Candida
`albicans is reached in 4 to 5 days. The fermentation is
`stopped at this point.
`Extraction and isolation of the antibiotic is per-
`formed by the following procedure: .
`The fermentation mixture is filtered over diatoma-
`ceous earth (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 I vol/wt of methanol by agitation and the
`mixture‘ is 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 by filtration. 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
`1 vols of methanol respectively. The combined metha-
`nol extracts are filtered and the remaining oil is dis-
`carded. The methanol extract containing rapamycin is
`e