United States Patent [ l 9J
`Sehgal et al.
`
`3,929,992
`[ 11]
`[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
`
`[22] Filed:
`
`Apr. 12, 1974
`
`[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. CI. ............................... 424/122; 195/80 R
`[51]
`Int. CI.2 .......................................... A61K 35/00
`
`[58] Field of Search ........................ 424/122; 195/80
`
`[56]
`
`References Cited
`OTHER PUBLICATIONS
`Miller, The Pfizer Handbook of Microbial Metabolites
`McGraw-Hill Book Co. I.nc., N.Y., N.Y., 1961, p.
`580.
`
`Primary Examiner-Jerome D. Goldberg
`
`ABSTRACT
`[57]
`Antibiotic rapamycin is producible by culturing Strep(cid:173)
`tomyces hygroscopicus NRRL 5491 in an aqueous nu(cid:173)
`trient medium. Rapamycin has antifungal properties.
`Methods for its preparation and use are disclosed.
`
`4 Claims, 2 Drawing Figures
`
`West-Ward Exhibit 1048
`Sehgal USP '992
`Page 001
`
`

`

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`WAVELENGTH (MICRONS)
`
`8
`
`7
`
`6
`
`4
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`3
`
`0.0
`
`2.5
`
`West-Ward Exhibit 1048
`Sehgal USP '992
`Page 002
`
`

`

`U.S. Patent Dec. 30, 1975
`
`Sheet 2 of 2
`
`3,929,992
`
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`
`West-Ward Exhibit 1048
`Sehgal USP '992
`Page 003
`
`

`

`1
`
`3,929,992
`
`RAPAMYCIN AND PROCESS OF PREPARATION
`
`This application is a continuation-in-part of our ear(cid:173)
`lier application Ser. No. 293,699, filed Sept. 29, 1972 5
`now abandoned.
`·
`
`BACKGROUND OF THE INVENTION
`a. Field of Invention
`This invention relates to an antibiotic, a new compo(cid:173)
`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(cid:173)
`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(cid:173)
`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. Aszalosetal.,J. Anti(cid:173)
`biotics, 2 I , 611 ( 1968) is a triene antibiotic with anti tu(cid:173)
`mor activity which also shows marked activity against
`gram positive organisms and only marginal activity 25
`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(cid:173)
`ics, 20, 329 ( 1967) are probably identical. Both have
`low antifungal activity (MIC against C. albicans: 5
`µ.g/ml) and high toxicity (LD50 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)(cid:173)
`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(cid:173)
`ous nutrient medium. The compound has the property
`of adversely affecting the growth of fungi, for example,
`Candida albicans and Microsporum gypseum. Accord(cid:173)
`ingly, rapamycin may be used to prevent the growth of 45
`or reduce the number of certain fungi in various envi(cid:173)
`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(cid:173)
`cultural Research Service, U.S. Department of Agricul(cid:173)
`ture, Peoria, Ill., 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'(cid:173)
`nitro-N-nitroso-guanidine, manganese chloride, cam(cid:173)
`phor, nitrogen mustards, and the like, as well as poly(cid:173)
`ploids of the various mutants.
`Streptomyces hygroscopicus NRRL 5491 develops
`abundantly in culture media usually employed for culti(cid:173)
`vation of other organisms of the same genus. It is capa(cid:173)
`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 Dennet's agar. On potato slices there is
`no aerial mycelium, but substrate growth is well devel(cid:173)
`oped and buff in color. On all media, the aerial growth
`is at first white then grayish with black spots. Sporo(cid:173)
`phores are often compact, forming a spiral of more
`than ten spores. Substrate growth is light yellow to
`IO almost colorless and in some media pale brown. Occa(cid:173)
`sionally a yellowish pigment is produced. The organism
`is H2S- and melanine-negative.
`Carbohydrate utilization by Streptomyces hygro(cid:173)
`scopicus NRRL 5491 was studied in carbon utilization
`15 agar (ISP Medium 9) according to the procedure stan(cid:173)
`dardized by the International Streptomyces Project
`(ISP).
`The best utilized carbohydrates were D-glucose, ino(cid:173)
`sitol, D-fructose and D-mannitol; less well utilized car-
`20 bohydrates were rhamnose, raffinose, xylose, starch
`and arabinose. Carbohydrates not utilized were sucrose
`and cellulose.
`The environment and nutritional requirements for
`the fermentation of Streptomyces hygroscopicus NRRL
`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
`placed on shaking machines. For industrial production,
`30 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-
`35 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
`40 being included sugars (for example, glucose, D-fruc(cid:173)
`tose, D-mannitol, maltose, arabinose, rhamnose, raffi(cid:173)
`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(cid:173)
`tially or totally defatted), meat flours or animal viscera,
`50 various peptones, casein hydrolysates, soybean hydro(cid:173)
`lysates, yeast hydrolysates, lactalbumin, wheat glutins,
`distillers solubles, corn steeps, inolasses, urea and
`amino acids.
`Mineral salts, such as the chlorides, nitrates, sulfates,
`55 carbonates and phosphates of sodium, potassium, am(cid:173)
`monium and calcium, should be included in appropri(cid:173)
`ate concentrations. The nutritive medium should con(cid:173)
`tain a number of trace elements such as magnesium,
`iron, manganese, and zinc.
`60 The inoculum of the above medium for the fermenta(cid:173)
`tion is provided with a fresh slant of Streptomyces hy(cid:173)
`groscopicus NRRL 5491.
`Under the described conditions and with the temper(cid:173)
`ature of cultivation at about 20°-35°C, preferably at
`65 about 25°C, maximum production of rapamycin in
`tanks is obtained in from about 2 to about 8 days. Al(cid:173)
`ternatively, the pH may be controlled during fermenta(cid:173)
`tion in tanks and maintained at about pH 6.0, and glu-
`
`West-Ward Exhibit 1048
`Sehgal USP '992
`Page 004
`
`

`

`3,929,992
`
`3
`cose may be added continuously from about 2 days
`after beginning to the end of fermentation, thus obtain(cid:173)
`ing maximum yields in about 4 to 5 days.
`Thereafter, a variety of procedures may be employed
`in the isolation and purification of raparriycin, for ex- 5
`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(cid:173)
`mercial recovery inasmuch as they are less time con- IO
`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- 15
`sfantially water-immiscible solvent, preferably a lower
`alkanol, for example n-butanol, n-pentanol or the com(cid:173)
`mercial mixture of pentanols known as "Pentasol" or
`n-hexanol, or a substantially water-immiscible lower
`alkyl lower alkanoate, for example, ethyl acetate, butyl 20
`acetate, amyl acetate or the commercially available
`mixture of amyl acetates, or a substantially water(cid:173)
`immiscible halogenated aliphatic hydrocarbon, for ex(cid:173)
`ample, chloroform, methylene dichloride or dichloro(cid:173)
`ethane. The extracts are dried and concentrated under 25
`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 30
`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(cid:173)
`taining the mycelium is extracted as described below 35
`under ( c ). The filtrate, i.e. the mycelium-free fermen(cid:173)
`tation mixture, is extracted several times with a sub(cid:173)
`stantially water-immiscible solvent, for example, a
`lower alkanol, lower alkyl lower alkanoate or haloge(cid:173)
`nated aliphatic hydrocarbon as exemplified above in 40
`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 45
`last-named extracts are treated in the same manner as
`described above under (a) to yield an oily residue con(cid:173)
`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(cid:173)
`nol or ethanol. The extract is concentrated by evapora(cid:173)
`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(cid:173)
`drocarbon or a substantially water-immiscible lower
`alkanol as described above or an aromatic hydrocar(cid:173)
`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(cid:173)
`cesses described in sections (a), (b) or (c) is then puri(cid:173)
`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(cid:173)
`ond solvent, more polar than said first solvent.
`
`4
`. DETAILS OF THE INVENTION
`Rapamycin is useful as an antifungal agent against a
`number of pathogenic fungi; for example, Candida albi(cid:173)
`cans, and other Candida species, Microsporum gyp(cid:173)
`seum, Trichophyton mentagrophytes, Aspergillus sp.,
`and Sporotrichum sp ..
`The illhibitory activity of rapamycin is especially
`pronounced against Candida albicans and said last or(cid:173)
`ganism may be used advantageously for assay purposes.
`The antifungal activity of this compound is demon(cid:173)
`strable in standard tests used for this purpose, for ex(cid:173)
`ample, in the tests described in "Antiseptics, Disinfec(cid:173)
`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. Inc., 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(cid:173)
`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(cid:173)
`gally effective amount of the antibiotic may be adminis(cid:173)
`tered orally in solid form containing such excipients as
`starch, sugar, certain types of clay and so forth. Simi(cid:173)
`larly, such an amount may also be administered orally
`in the fqrm of solutions or suspensions, or the antibiotic
`may be injected parenterally. For parenteral adminis(cid:173)
`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 ofsorbitol 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(cid:173)
`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(cid:173)
`ful or deleterious side effects and preferably at a level
`50 that is in a range of from about ! .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 I 0 mg to about 100 mg per kilo per day
`is most desirably employed in order to achieve effective
`55 results.
`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(cid:173)
`ceptable vehicles containing 0.1-5 per cent, preferably
`60 2 per cent of the agent, and may be administered topi(cid:173)
`cally to the infected area of the skin.
`Rapamycin may also be used for cleaning and disin(cid:173)
`fecting laboratory equipment, surgical instruments,
`locker rooms, or shower rooms of sensitive fungus
`65 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
`wate~ containing 0.001-0.1 % of a non-ionic surface-ac-
`
`West-Ward Exhibit 1048
`Sehgal USP '992
`Page 005
`
`

`

`3 929 992
`'
`'
`6
`5
`5.8-6~2.The fermenters are inoculated with the second
`tive agent, for example, polysorbate 80 U.S.P., immedi(cid:173)
`stage inoculum described above and incubated at about
`ately before applying it to the objects to be cleaned and
`25°C with· ·agitation and aeration while controlling and
`disinfected.
`maintaining the mixture at approximately pH 6.0 by
`PREPARATION
`5 addition .of 'a base, .for example, sodium hydroxide,
`potassium hydroxide or preferably ammonium hydrox-
`Jn one embodiment of this invention rapamycin is
`ide, as required from tjme to time. Addition of a source
`prepared in the following .manner:
`-of assimilable carbon, preferably glucose, is started
`A suitable fermenter is charged with production me-
`when the concentration of the latter in the broth has
`dium 8KM (see Example I). After sterilization and
`cooling, the medium is inoculated with a first stage IO dropped to about 0.5% ~t/vol, normally about 48 hrs
`after the start of fermentation, and is maintained until
`inoculum preparation of· Streptomyces hygroscopicus
`the end of.the particular run. In this manner a fermen-
`NRRL 5491.
`A maximum titre of 20 to 100 µg/ml of the antibiotic
`tation broth containing about 60 µg/ml of rapamycin as
`is reached in the fermentation mixture after 2-8 days,
`determined by the· assay method described above is
`usually after about 5 days, as determined by the cup 15 .obtafoed in 4-5 days, when fermentation is stopped.
`Filtration of the mycelium, mixing the latter with a
`plate method and Candida albicans as the test organism.
`The mycelium is harvested by filtration with diatoma-
`watet-miscible lower alkanol, preferably methanol,
`ceous earth. Rapamycin is then extracted from the
`followed by extraction with a halogenated aliphatic
`mycelium with a water-miscible solvent, for example a
`hydrocarbon, preferably trichloroethane, and evapora-
`lower alkanol, preferably methanol or ethanol. The 20 tion of the solvents yields a first oily residue. This first
`oily residue is dissolved in a lower aliphatic ketone,
`latter extract is then concentrated, preferably under
`reduced pressure, and the resulting aqueous phase is
`preferably acetone, filtered from insoluble impur!ties,
`extracted with a water-immiscible solvent. A preferred
`the filtrate evaporated to yield a second oily residue
`water-immiscible solvent for this purpose is methylene
`which is extracted with a water-miscible lower alkanol,
`dichloride although chloroform, carbon tetrachloride, 25 preferably methanol, and the latter extract is evapo-
`rated to yield crude rapamycin as a third oily residue.
`benzene, n-butanol and the like may also be used. The
`latter extract is concentrated, preferably under ~e-
`This third oily residue is dissolved in a mixture of a
`duced pressure, to afford the crude product as an 011.
`lower aliphatic ketone and a lower aliphatic hydrocar-
`The product may be purified further by a v~rietY. of
`bon, preferably acetone-hexane, an absorbent such as
`methods. Among the preferred methods of purification 30 charcoal or preferably silica gel is added to adsorb the
`rapamycin, the latter is eluted from the adsorbate with
`is to dis~olve the crude product in a substantially non-
`polar, first solvent, for exami:ile petr~leum. ether ?r
`a similar but more polar solvent mixture, for exan:iple a
`hexane, and to treat the resultmg solution with a smt-
`mixture as above but containing a higher proportion of
`able absorb.e~t, _for example charcoal or silica gel, so
`the aliphatic ketone, the eluates are evaporated and the
`that the anttb10~1c becomes absorbed on the absorbant. 35 residue is crystallized from diethyl ether, to yield pure
`crystalline rapamycin. In this manner a total of 45-5~%
`T?e absorbant 1s then separated and washed or eluted
`with a second solvent more polar than t~e firs~ solvent,
`of the rapamycin initially present in the fermentation
`for example ethyl acetate, methylene d1chlonde, or a
`mixture is recovered as pure crystalline rapamycin.
`mixture of methylene dichloride and ether (preferred).
`CHARACTERIZATION
`Thereafter, concentration of the wash solution or elu- 40
`a. Purified rapamycin is a colourless crystalline com-
`ate affords substantially pure rapamycin. Further puri-
`pound, m.p. 183°-135°c after recrystallization from
`fication· is obtained by partial precipitation with a non-
`polar solvent, for example, petroleum ether, hexane,
`ether;
`b. rapamycin is soluble in ether, chloroform, acetone,
`pentane and the like, from a solution of the rapamycin
`in a more polar solvent, for example, ether, ethyl ace- 45 methanol and dimethylformamide; very sparingly solu(cid:173)
`tate, benzene and the like. Still-further purification is
`ble in 'hexane and petroleum ether and .substantially
`obtained by column chromatography, preferably em- ·
`insoluble in water; ·
`ploying silica gel, and by crystallization of the rapamy-
`c. rapamycin shows a uniform spot on thin layer
`plates of silica gel G (E'. Merck A. G., Darmstadt)
`cin from ether.
`In another preferred embodiment of this invention a
`developed with a variety of thin layer chromatography
`first stage inoculum of Streptomyces hygroscopicus
`solvent systems; for example; ether.-hexane 40:60 (Rf=
`NRRL 5491 is prepared in small batches in a medium
`0.42 ),·isopropyl alcohol"benzene 15:85 (Rf= 0.5) and
`containing soybean flour, glucose, ammonium sulfate,
`ethanol-benzene 20:80 (Rf= 0.43);
`and calcium carbonate incubated at about 25°C at pH
`d .. rapamycin obtained' from four successive fermen-
`7 .1-7 .3 for 24 hrs. with agitation, preferably on a gyro- 55 .tation batches. gave the following values on repeated
`tary shaker. The growth thus obtained is used to inocu-
`elemental analyses:
`late a number of somewhat larger batches of the s~me
`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- 60
`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(cid:173)
`termined level and for continuous metered addition of 65
`nutrient. They are charged with a medium containing
`soybean flour, glucose, ammonium sulfate, and potas(cid:173)
`sium phosphate, sterilized, and the pH is adjusted to pH
`
`rapamycin exhibits the following characteristic
`~-
`absorption maxima in its ultraviolet absorption spec·
`trum (95% ethanol):
`
`50
`
`67.24,
`
`66.14,
`
`67.26,
`
`66.72.
`
`%
`%
`
`%.
`
`8.93,
`
`1.39,
`
`8.72,
`
`1.37,
`
`8.92,
`
`1.28,
`
`8.9.
`
`1.28.
`
`AVER(cid:173)
`AGE
`66.84
`
`8,84
`
`1.37
`
`West-Ward Exhibit 1048
`Sehgal USP '992
`Page 006
`
`

`

`3,929,992
`
`1% 541) and 288
`
`5
`
`7
`267 nm (E 1cm 1% 417), 277 nm (E 1c111
`nm (E 1cm 1'f0 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- 1;
`Further infrared absorption bands are characterized
`by the following data given in reciprocal centimeters
`with (s) denoting a strong, (m) denoting a medium, and 10
`( w) denoting a weak intensity band. This classification
`is arbitrarily selected in such a manner that a band is
`denoted as strong (s) if its peak absorption is more than
`two-thirds of the background in the same region; me(cid:173)
`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.
`
`8
`EXAMPLE I
`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 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(cid:173)
`lum.
`The first-stage inoculum medium consisted of Emer(cid:173)
`son broth [R. L. Emerson et al., J. Bacteriol, 52, 357
`( 1946)] 0.4%; peptone, 0.4%; sodium chloride, 0.25%;
`yeast extract, 0 I.%; and glucose, I%; pH 7 .O; flasks
`containing the above medium were inoculated with I%
`20 of the spore suspension described above. The inocu-
`- - - - - - - - - - - - - - - - - - - - - - - lated flasks were incubated for 30 hrs. at 28°C on a
`2990 cm-• (m)
`1158 cm-• (m)
`reciprocating shaker set at 65 r.p.m. ( 4 inch stroke).
`2955 cm-• (s)
`1 l29 cm-• (s)
`2919 cm- 1 (s)
`1080 cm-• (s)
`2858 cm-1 (s)
`1060 cm-• (s)
`2815 cm-1 (m)
`1040 cm-• (m)
`1440 cm-1 (s)
`1020 cm-1 (m)
`1365 cm-• (m)
`978 cm- 1 (s)
`1316 cm-• (m)
`905 cm-1 (m)
`1272 cm-• (m)
`888 cm-• (w)
`1178 cm-• (s)
`866 cm-• (w)
`
`Production stage
`25 The production stage was run in 250-liter New
`Brunswick fermenters Model F-250, equipped with
`automatic antifoam addition system and pH recorder(cid:173)
`controller. The fermenters were charged with 160 liters
`of an aqueous production medium(8 KM) consisting of
`30 the following constituents:
`
`35
`
`40
`
`soluble starch
`(NH 4),S04
`K2HOP4
`glucose ( Cerelose)
`MgSO,
`ZnS04
`MnS04
`FeS04.7H20
`CaC03
`"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%
`
`g. the nuclear magnetic resonance spectrum of rapa(cid:173)
`mycin in deuterochloroform is reproduced in FIG. 2;
`h. the minimum inhibitory concentration of rapamy(cid:173)
`cin against various microorganism is listed below:
`
`Organisms
`
`Candida albicans ( 5 strains)
`C. caten,ulata
`C. lipoly ti ca
`C. stellatoidea
`C. tropicalis
`C. pseudtropicalis
`C. parapsilosis
`C. morrera
`C. intermedia
`M. gypseum
`T. mentagrophytes
`
`Rapamycin:
`(JLg/ml)
`
`0.02 to 0.1
`<0.1
`2.5
`<0.1
`0.1
`>5.0
`<0.1
`<O.l
`<0.1
`12.5
`>1000
`
`i. rapamycin exhibits a LD50 (i.p., mice) of 597.3 ±
`28.1 mg/kg and a LD50 (p.o., mice) of >2,500 mg/kg.
`In protection studies, mice were infected by intrave(cid:173)
`nous injection of C. albicans ATCC 11651. At 1,4 and
`24 hours after infection, mice were administered I 0
`mg/kg (s.c.) of rapamycin. At this dose 50% of the 55
`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(cid:173)
`vived, and at 25 mg/kg complete protection was ob- 60
`served.
`A I% 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 65
`applied to a rabbit's eye caused no irritation.
`The following Examples illustrate further this inven-
`tion.
`
`45
`
`The fermenters were sterilized at 121°C for 45 min(cid:173)
`utes, cooled and inoculated with one flask (2% inocu(cid:173)
`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 µ,g/ml, determined by microbiologi-
`50 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(cid:173)
`formed by one of the following methods:
`
`Extraction
`a. The fermentation mixture was extracted twice with
`I v/v of n-butanol. The combined butanol extracts were
`washed with 1 v/v of water, dried with anhydrous so(cid:173)
`dium sulfate and evaporated to dryness under reduced
`pressure to yield a residue. The oily residue was ex(cid:173)
`tracted 3 times with 2 liters of methanol. The combined
`methanol extracts were passed through diatomaceous
`earth (Ce lite) and evaporated to dryness to yield an
`oily residue containing crude rapamycin.
`b. The fermentation mixture was filtered over diato(cid:173)
`maceous earth (Celite). The filtrate was extracted
`twice with I v/v of ethyl acetate. The ethyl acetate
`extra.cts were washed with 1 volume of water, dried
`
`West-Ward Exhibit 1048
`Sehgal USP '992
`Page 007
`
`

`

`3,929;992
`
`S
`
`10
`. a,gyrqtary sha~er (2 in.ch stroke) at 240 ·rpm for 24 hrs .
`' :;it .25°.C,
`. .. .
`.
`' .
`..
`.
`.
`Secon.cl Stag~ lnoculum.· Twenty-four .liter flat -bot(cid:173)
`tom flasks containing 3.2 I of the inoculum medium
`described abov¢ at'cpH 7.1..,7.3 are sterilized'.'by a~to­
`claving at 121° for 3:5'riiinutes, shaken to rei;u~1wnd the
`insoluble material and:resterilized. for another 45 min(cid:173)
`utes. The flasks are: cooled to 25° and inoculated with
`64 ml of first stage inoculum, placed ~n a reciprocating
`shaker ( 4 inch stroke). set at 65 rpm and incubated for
`18 hrs at 25°C.
`·
`Production Stage. The produ<.;tion stage is run in 250
`liter New Brunswic~fermenters Model F-250 equipped
`with. automatic; antifoam addition. system and pH re(cid:173)
`corder-controller. The fermenters are charged with
`160 liters of an aqueous production medium consisting
`of th.e follo-..ving constituents:
`'
`
`Soybean flour ( Arc'her-Dani.els Co.,
`Midland, Mich., "Special X")
`Glucose ( Cerelose)
`Ammonium sulfate
`Potassium phosphate (monobasic)
`Antifoaming Agent ("DF-1.43 - PX"
`Mazer Chemicals, Inc., Gurnee, Ill.,)
`
`= 3%
`11 wt/vol
`wt/vol
`. = 2%
`· = 0.1% wt/vol
`= 0.5% wt/vol
`
`= 0.05% wt/vol
`
`.
`. 9
`\:Vith anhydrous so.ciium .sulfat~ aO:d evaporated ·under
`reduced pressure to dryness. The residue'ias extracted
`twice with I liter of methanol.The methanol extracts
`were evaporated under reduced pressure to yield-,an
`oily residue containing crude rapamycin:
`c. The mycelium obtained as described under section
`(b) was washed with I to 2 volumes of water. The
`washed mycelium was extracted 3 times with 5 volumes
`of methanol per weight of' wet mycelium' each time;
`The methanolic extracts were pooled and cbricentrated IO
`under reduced pressure to a small 'volume of an aque(cid:173)
`ous phase containing approximately I 0% v /v of metha(cid:173)
`nol. This aqueous phase was extracted 3 times with I
`vol. of methyh:ne chloricie; the methylene chloride IS
`extracts were combined, dried with anhydrous sodium
`sulfate and evaporated to yield an oily residue.
`The oily residue .was diluted with I volume of pet~o­
`leum ether, and 30% w/v of charcoal (Darco G60) was
`added. The mixture was stirred for half an hour and 20
`filtered. The charcoal, which retained'substantialiy all
`of the product, was washed twice wiih one v9lume of
`petroleum ether. The charcoal was eluted three times
`with 5 vol. (based on the weight of the charcoal) .of a .
`mixture of methylene chloride and ether (50:5.0). The 2S
`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
`from the ether solution with cold petroleum ether.
`Alternatively, the oily residue obtained by any of the
`extraction procedures described above was diluted
`with I 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(cid:173)
`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(cid:173)
`orated to a small volume and crude product obtained
`by precipitation from the ether solution with cold pe(cid:173)
`troleum ether.
`
`The Jermenters are sterilized at 121°C for 30 min(cid:173)
`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-
`30 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
`35 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 da