VOL. XXVIII NO. 10
`
`THE JOURNAL OF ANTIBIOTICS
`
`727
`
`RAPAMYCIN (AY-22,989), A NEW ANTIFUNGAL ANTIBIOTIC
`
`II. FERMENTATION, ISOLATION AND CHARACTERIZATION
`
`S. N. SEHGAL, H. BAKER and Claude VEZINA
`
`Department of Microbiology, Ayerst Research Laboratories
`Montreal, Quebec., Canada
`
`(Received for publication June 17, 1975)
`
`Rapamycin is a new antifungal antibiotic produced by Streptomyces hygroscopicus
`It was isolated from the mycelium by solvent extraction, purified by silica
`NRRL 5491.
`gel column chromatography and crystallized as a colorless solid which melts at 183 --
`1850C and has the empirica1 formula CssHs9NOu. From its characteristic ultraviolet
`absorption spectrum rapamycin can be classified as a triene. It is highly active against
`Its activity is compared with that
`various Candida species, especially Candida albicalls.
`of amphotericin B, candicidin and nystatin.
`
`In a previous publication, 1 > a strain of Strepromyces hygroscopicus, newly isolated from an
`
`Easter Island soil sample, was reported to inhibit Candida albicans, Microsporum gypseum and
`Trichophyton granulosum. The active principle was isolated and found to be a new antibiotic
`of unknown structure; it was named rapamycin.
`In the present paper, we are describing the
`fermentation of rapamycin in agitated-aerated vessels, an improved process for its isolation and
`purification as well as its physical-chemical characteristics. Comparison of its activity with
`that of other antifungal antibiotics is also reported.
`
`Production of Rapamycin
`
`The producing strain, Streptomyces hygroscopicus NRRL 5491, was grown and maintained
`on tomato paste-oatmeal agar, as previously described. 11 Good growth and sporulation were
`obtained in 7,_,15 days of incubation at 25°C. Spores from one Roux bottle were suspended
`in 50 ml of sterile distilled water to constitute the spore inoculum.
`Un baffled, 500-ml Erlenmeyer flasks were filled with 100 ml of an inoculum medium con(cid:173)
`sisting of (g/liter): soybean meal ("Special X,,, Archer Daniels Midland Co., Minneapolis,
`Minn.), 40; "Cerelose" (a pharmaceutical grade of glucose), 20; (NH4) 2S04, 3; CaCO:h 1.5; and
`tap water to 1 liter (pH 7.0). The flasks were sterilized at 121°C for 30 minutes, cooled to
`2S°C and inoculated with 4 ml of the spore inoculum. The inoculated flasks were incubated
`for 24 hours at 25°C on a gyrotory shaker at 240 rev/min, 2" -throw, to constitute the first-stage
`inoculum.
`Unbaffied, 24-liter round bottom flasks were filled with 3.4 liters of the same medium and
`autoclaved at 121°C for 30 minutes. The flasks were agitated to resuspend the solids and
`autoclaved for an additional period of 1 hour at 121°C, cooled to 25°C and inoculated with
`78 ml (2 %) of the first-stage inoculum. The inoculated flasks were incubated for 18 hours at
`25°C on a reciprocating shaker at 65 strokes per minute and 4"-throw. These flasks were used
`to inoculate the production stage.
`
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`Table I. Production of rapamycin in
`aerated-agitated fermenters
`
`Packed cell I Potency
`
`volume (1'6)
`
`Fermentation
`time (hours)
`
`48
`72
`96
`
`pH
`
`6.0
`6.0
`6.3
`
`(.ng/ml)
`
`23
`60
`50
`
`20
`63
`87
`
`Fermenters (model F-250, New Brunswick
`Scientific Co.), 250-liter capacity, equipped with
`automatic antifoam addition system and pH
`recorder-controller, were filled with 160 liters
`of the production medium consisting of (g/liter):
`soybean meal ("Special X"), 30; "Cerelose,"
`20; (NH4 ) 2SO.a, 5; KH2P04 , 5; Mazer DF-143PX
`(antifoam), 0.5 ml; and tap water to 1 liter.
`The fermenters were sterilized at 121°C for 30 minutes under an agitation of 150 rev/min, cooled
`to 25°C and pH of medium adjusted to 6.1 by addition of 10 N NH40H solution. The fermenters
`were inoculated with 3.2 liters (2 96) of the second-stage inoculum. The fermentation was run at
`25;,C under an agitation of 200 rev/min and an aeration of 0.25 v/v/min. Sterile Mazer DF-143PX
`antifoam was added on demand. After 30.-35 hours of incubation the pH started to drop but
`was controlled at 6.0 by addition of 10 N NH 40H solution on demand. After 48 hours of
`incubation, a 40 96 sterile solution of ''Cerelose" was added continuously at the rate of 3.85 96
`per day. The antibiotic titres were determined every 24 hours starting at 48 hours. The maxi(cid:173)
`mum titers were usually obtained in 96 hours. The results of a typical fermentation are shown
`in Table 1.
`Conventional paper disc-agar diffusion assays were used to determine the antibiotic titre.
`A 10-ml sample of fermentation broth was centrifuged at 2,500 rev/min for 15 minutes. The supernate
`was discarded and the mycelial pellet suspended in 250 ml of methanol and shaken vigorously.
`The extract was filtered. Filter paper discs, 13 mm in diameter, were dipped in the extract and
`placed on filter paper to dry. Similar discs were dipped in standard solutions containing 10,
`5, 2.5 and 1.25 µg rapamycin/ml. All the discs were deposited on agar plates seeded with the
`test strain of Candida albicans AY F-598. The inhibition zone diameters obtained for the
`standard solutions after overnight incubation were plotted against log concentration on semi(cid:173)
`logarithmic paper and titre of fermentation broths read from the standard curve and corrected
`for dilution.
`
`IsoJation of Rapamycin
`
`The fermentation broth was ad justed to pH 4.0 with a 30 96 sulfuric acid solution and
`filtered on a vacuum rotary filter coated with Celite. The mycelium, containing the antibiotic,
`was extracted twice by stirring for 1 hour with ll volume of trichloroethane. The tri(cid:173)
`chloroethane extracts were pooled and evaporated to a small volume under reduced pressure,
`dehydrated with anhydrous sodium sulfate and further concentrated to an oily residue. A
`typical 160-liter fermentation run yielded about 500 g of oily residue. The residue was extracted
`twice with one volume of methanol. The methanolic extracts were pooled and evaporated to
`dryness to yield about 50 g of oily residue containing rapamycin. The residue was dissolved
`in 10 v/w of a solvent mixture consisting of 15 96 acetone in hexane. To this solution, 2 weights
`of silica gel G (Merck) per weight of oil were added and the mixture stirred gently for 50 minutes.
`The mixture was filtered and silica gel with adsorbed rapamycin washed onto a column with
`several volumes of 15 96 acetone in hexane. The antibiotic was eluted with 25 96 acetone in
`
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`THE JOURNAL OF ANTIBIOTICS
`
`729
`
`hexane and the eluant evaporated to dryness. The residue was dissolved in ether from which
`pure rapamycin crystallized out. The recoveries were about 40 96 based on broth assay.
`
`Physical and Chemical Properties of
`Rapamycin
`
`Fig. 1. Ultraviolet absorption spectrum of rapa(cid:173)
`mycin (A Y-22,989).
`
`Rapamycin is a white crystalline solid
`melting at 183 ...... 185°C.
`It is freely soluble
`in methanol, ethanol, acetone, chloroform,
`methylene dichloride, trichloroethane, dimethyl
`formamide, dimethyl sulphoxide; sparingly solu(cid:173)
`ble in ether, and practically insoluble in water.
`Rapamycin analysed for C56H 89N014 (E.W.
`999). Calcd: C, 67.2; H, 8.9; N, 1.4; Found:
`C, 67.24; H, 8.93; N, 1.39.
`
`225
`
`325
`
`35onm
`
`Fig. 2.
`
`Infrared spectrum of rapamycin (AY-22,989).
`
`4
`
`5
`
`6
`
`7
`
`8
`
`9
`
`10
`
`15
`
`30
`
`50)J
`
`2.5
`o.o
`
`0.1
`
`•
`
`~ 0.2
`0
`.Q
`
`!
`
`~0.4
`
`0.6
`o.e
`r.o
`Ui
`~..._ _ _ _,_ _ _ _._ _ _ __._ _ _ _ _ L.-. _ _ . . _ _ _ _._ _ _ _,_ _ _ - - - ' _ _ _ _ . _ _ _ _._ _ _ _,_ _ _ __. _ _ _ _ ,
`
`4000
`
`3500
`
`3000
`
`2500
`
`2000
`
`1800
`
`1600
`
`1400
`
`1200
`
`1000
`
`800
`
`600
`
`400
`
`200 cm·•
`
`Fig. 3. NMR (200 MHZ) spectrum of rapamycin (A Y-22,989).
`4.
`,......:;;.2.~o,..._,.., .......... ~3.~0~...--.4~.o ................ ........,s.o ................. ~6~.o .......................... ~o...-rr ....... ......,ao ......... ...,.,.........,9D......,~~O~p~pm(JI
`
`7.0
`
`6.0
`
`5.0
`
`4.0
`
`2.0
`
`J.0
`
`o ppmf8)
`
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`
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`
`The ultraviolet spectrum (Fig. I) shows i.mn:i: at 288, 277 and 267 nm with EU~ 416. 514
`and 417 respectively.
`The infrared spectrum (Fig. 2) shows OH at 3500, a band at 1730 (possibly lactone carbonyl)
`and at 1700 (carbonyl), and a band between 1610 and 1630 cm- 1 (C=C).
`NMR spectrum (200 MHZ) of rapamycin is shown in Fig. 3.
`It shows vinylic protons
`between i3 s ...... 6.5, methoxyl between o 3. J ...... 3.6 and viny1ic at o 1.8.
`Optical rotation is [a]i-f-58.2 in methanol. Rapamycin forms a yellow chromophore when
`dissolved in 0.1 N methanolic NaOH and heated at 60°C; this property is the basis of a
`colorimetric assay.
`
`Table 2. Comparison of rapamycin with other trienes
`
`I
`I
`
`Antibiotics Appearance
`
`m.p.
`
`iMolecular \ Molecular or
`or
`. 1
`equivalent; equiva ent
`formula
`: weight
`j
`
`RaJJamycin Colorless,
`(A Y-22, 989) crystalline
`
`lt83-J85::.C I
`I
`
`I
`
`999
`
`CssHs9NOu
`
`I
`
`U.V. I.max
`(nm)
`
`Trienine
`
`Mycotrienin 'Yellow
`powder
`Off-white
`powder
`-
`
`Proticin
`
`MM8
`
`Yellow
`powder
`
`149-lSO'C
`
`683
`
`CasHsoN20s
`
`15
`
`163-165:iC
`
`1,400
`
`-
`
`j257' 267 '278 Anti tumor
`
`-
`
`-
`
`-
`
`:
`i
`
`582
`
`-
`
`Ca1H.u01PNa 264,272,284 Antibacterial > 150
`(i. v.)
`-
`
`-
`
`2
`
`3
`
`4
`
`5
`
`1:i-oxic~tyl
`Anti-
`microbial 1.p. mice: Ref
`;(LDso
`\
`·
`spectrum I mg;kg)l
`600
`267,277,288 Antifungal:
`strongly_
`candiciaal
`262,272,282 Antifungal
`
`262,270,282 Anti fungal
`against
`filamentous
`fungi
`230, 267, 275, Antibacterial
`285
`
`Resistaphylin Colorless,
`crystalline
`
`191-
`
`92°C
`
`I
`
`462.S C2-iHa.iN201
`I
`I
`On the basis of its ultraviolet spectrum, rapamycin can be classified as a triene, but com(cid:173)
`parison with other known trienes (Table 2) shows it to be novel compound.
`
`-
`
`6
`
`Antimicrobial Activity of Rapamycin
`
`Rapamycin inhibits yeasts and filamentous fungi. Results reported in Table 3 using the
`agar-diffusion assay indicate that rapamycin would be as active against the dermatophytes as it
`is against Candida albicans. However, activity expressed as minimum inhibitory concentration
`in broth has already been shown11 to be much higher against yeast than against the dermatophytes;
`
`Table 3. Antifungal activity of rapamycin
`
`Zones of inhibition (mm)
`
`Candida albicans
`
`Microsporum
`gypseum
`
`I
`
`I
`
`Trichophyton
`mentagrophytes
`
`i
`
`Asper gm us
`fumigatus
`
`0
`21
`23
`27
`28
`
`0
`18
`22
`26
`32
`
`0
`20
`25
`30
`32
`
`0
`>40
`-
`-
`-
`
`Concentration
`(µg/ml)
`
`0 (control)
`o.s
`1.0
`2.5
`5.0
`
`not determined.
`
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`

`VOL. XXVIII NO. 10
`
`THE JOURNAL OF ANTIBIOTICS
`
`731
`
`Table 4. Activity of rapamycin and ampbotericin
`B against clinical isolates of various Candida
`species
`
`Table 5. Activity of rapamycin compared to that
`of nystatin and candicidin against various strains
`Candida albicans
`
`Minimum inhibitory:
`concentration (µg/ml)
`
`Rapamycin Amphoteri(cid:173)
`cin B
`
`Candida
`albicans
`Strain
`Nos.
`
`Incuba-
`ti on
`time
`(hour
`or day)
`
`Minimum inhibitory
`concentration (MIC) (µg/ml)
`
`Rap~my- !Nystatin
`Ctn
`
`Can-
`dicidin
`
`Candida species
`(AY Nos.)
`
`C. albicans F-598
`C. albicans F-634
`(A TCC 11651)
`C. catenulata F-662
`C. intenzodia F-670
`C. /ipolytica F-669
`C. monosa F-664
`C. parapsilosis F-665
`C. pseudotropicalis
`F-666
`C. stellatoidea F-661
`C. tropicalis F-668
`
`<0.02 to 0.2
`0.08 to 0.32
`
`<0.1
`<0.1
`2.5
`<0.1
`<0.1
`>5.0
`
`<0.1
`<0.1
`
`2.5
`2.5
`
`2.0
`<0.1
`4.0
`0.2
`> 1.0
`3.0
`
`0.7
`2.5
`
`F-612
`
`F-615
`
`F-619
`
`F-620
`
`F-621
`
`F-623
`
`F-624
`
`F-626
`
`F-004
`
`48 hours
`8days
`48 hours
`8days
`48 hours
`Sdays
`48 hours
`8days
`48 hours
`8 days
`48 hours
`8days
`48hours
`8days
`48 hours
`8 days
`48 hours
`8days
`
`0.08
`0.0025
`5.0
`0.02
`1.2
`>10.0
`0.0025
`5.0
`0.16
`0.62
`0.32
`>10.0
`0.08
`5.0
`<0.00063
`0.04
`>10.0
`1.25
`5.0
`<0.00063
`0.08
`0.02
`0.62
`>10.0
`>10.0
`2.5
`>10.0
`>10.0 > 10.0
`>10.0
`0.00125
`10.0
`0.04
`0.02
`>10.0
`0.16
`<0.00063
`5.0
`0.08
`0.00125
`>10.0
`1.25
`<0,00063 1
`5.0
`0.02
`0.01
`I >10.0
`0.04
`2.5
`0.04
`0.02
`>10.0
`JO.O
`0.04
`
`I
`
`these contradictory results were explained by
`the instability of the antibiotic in broth over
`long period of incubation required for the
`growth of dermatopbytes: uninoculated SABOURAUD dextrose broth containing 5 pg of rapamycin
`per ml lost 80 % of its activity after 7 days of incubation.
`Rapamycin was compared to amphotericin B against clinical isolates of various Candida
`species, and the results are shown in Table 4;
`the minimum inhibitory concentration of
`rapamycin is much lower than that of amphotericin B, except for Candida pseudotropicalis.
`When compared with nystatin and candicidin using the same method, rapamycin again appeared
`somewhat more active against clinical isolates of Candida albicans (Table 5). Rapamycin was
`also found active against candidal infections in mice; the results will be reported in a subsequent
`publication. 71
`
`The authors wish to express their thanks to Dr. G. SCHILLING and his group for analytical data.
`Technical assistance of Mr. RENE SAUCIER, Mr. K. PANDEY and Mrs. T. BELANGER is acknowledged.
`
`Acknowledgements
`
`References
`
`1) VE.zrNA, C.; A. KuoELSKI & S. N. SEHGAL: Rapamycin (A Y-22,989), a new antifungal antibiotic.
`I. Taxonomy of the producing streptomycete and isolation of the active principle. J. Antibiotics
`28: 721 ....... 126, 1975
`2) CoRONELLI, C.; R. C. PASQUALUCCI, J.E. THIEMANN & G. TAMONI: Mycotrienin, a new polyene
`antibiotic isolated from streptomyces. J. Antibiotics, Ser. A 20: 329--333, 1967
`3) ASZALOS, A.; R. s. ROBISON, P. LEMANSKI & B. BERK: Trienine, an antitumor triene antibiotic.
`J. Antibiotics 21: 611-615, 1968
`4) PRAVE, P.; D. SuKATSCH & L. VERTENY: Proticin, a new phosphorus-containing antibiotic. J.
`Antibiotics 25: 1-4, 1972
`
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`5) ARMSTONG, J. J.; J. F. GROVE, W. B. TURNER & G. W.Aao: An antifungal triene from Streptomyces
`sp. Nature 206: 399--400, 1965.
`6) AIZAWA, S.; M. SHIBUYA & S. S1-11RATO: Resistaphylin, a new antibiotic. I. Production, isolation
`and properties. J. Antibiotics 24: 393--396, 1971
`7) BAKER, H.; A. SID0Row1cz, S. N. SEHGAL & C. VEZINA: Rapamycin (AY-22,989), a new antifungal
`antibiotic.
`III. Protection studies in mice. Antimicr. Agents & Chemoth. (in press)
`
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