VOL. XXXI NO. 6
`
`THE JOURNAL OF ANTIBIOTICS
`
`539
`
`RAPAMYCIN (A Y-22,989), A NEW ANTIFUNGAL ANTIBIOTIC
`
`III.
`
`IN VITRO AND IN VIVO EVALUATION
`
`H. BAKER~ A. S100Row1cz, S. N. SEHGAL and CLAUDE VEZINA
`Department of Microbiology, Ayerst Research Laboratories,
`Montreal, Quebec, Canada
`
`(Received for publication March 11, 1978)
`
`The activity of rapamycin, a new anti-Candida antibiotic, was not affected by pH values
`between 6 and 8; at pH 4, however, activity was abolished. The MIC of rapamycin did not
`vary drastically with the size of inoculum: a ten-fold dilution of the inoculum reduced the MIC
`only two-fold. Serum binding was extensive. Serum levels obtained in mice were higher on
`subcutaneous injection than with oral administration. Dogs absorbed rapamycin after oral
`administration.
`Rapamycin cured systemic candidosis in mice: PDso s. c. was 9.5 mg/kg; PD50 p. o. \\'as
`In the same experimental infections amphotericin B and nystatin exhibited PDso
`11 mg/kg.
`values of < 0.25 mg and > 4,000 units/kg respectively. Rapamycin and amphotericin B,
`administered at 1, 4 and 24 hours after infection, gave approximately the same percent survival
`after 30 days of observation. When the above treatment was extended by an additiona1 daily
`rrcatment for 6 days, rapamycin by the subcutaneous route yielded a higher percentage of
`survival than either rapamycin or amphotericin B, administered orally, after a 30-day obsen•a(cid:173)
`tion period. Vaginal candidosis in female rats was treated efficiently (91 % cure) by rapamycin
`administered orally. No increase of resistance of C. albicans was observed during treatment.
`
`Rapamycin is an antifungal antibiotic produced by Streptomyces hygroscopicus NRRL 5491 10 ).
`It was isolated in pure, crystalline form and found to be mainly active against Candida species; C.
`It has no activity against bacteria, Trichomonas vagina/is, T.
`albicans is the most sensitive species6 '.
`foetus, and Protheca segbwema (pathogenic alga); it is moderately active against filamentous fungi,
`including some Dermatophytes, and weakly active against dimorphic fungi. Acute toxicity is low7> =
`LDso in mice is 597 (i. p.) and > 2,500 mg/kg (p. o.); LDso in rats is > 1,600 (i. p.), 40 (i. v.), and > 1,600
`mg/kg (p. o.).
`The present study deals with the influence of pH, serum and inoculum size on rapamycin activity,
`its bioavailability in mice and dogs, and the protection it affords to mice and rats against both systemic
`Its in vivo activity is compared to that of amphotericin B3•8> and nystatin~).
`and vaginal candidoses.
`A method is described for studying the effect of antifungal agents on vaginal candidosis in rats.
`
`Materials and Methods
`
`Test antibiotics
`Nystatin and amphotericin B were generously provided by E. R. Squibb and Sons, Ltd., Montreal,
`Que. Pure rapamycin was prepared as previously reported&'.
`Candida albicans strains
`C. albica11s strain AY F-598 is the test organism for rapamycin assay. C. a/bicans ATCC 11,651
`(A Y F-634) was used to produce systemic infections in mice and vagina] infections in rats.
`Culture media and inocu1a
`Candidal strains were maintained as lyophilized cultures. Cultures were grown on BBL-SABOURAUD
`
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`JL:"E 1978
`
`Dextrose Agar (Baltimore Biological Laboratory, Inc., Baltimore, Maryland) at 37'JC for 18 hours,
`then transferred to BBL-SABOURAUD Liquid Broth (modified) and incubated under the same conditions.
`These Jiquid cultures were diluted ten-fold, and the resulting diluted suspensions served as inocula for
`SABOURAUD Liquid Broth used for MIC (minimum inhibitory concentration) determinations, and BBL(cid:173)
`Nystatin Assay Agar (Antibiotic Medium No. 12) for serum assay of rapamycin 1
`., •
`
`MIC determination
`The minimum inhibitory concentration was determined by the conventional two-fold serial broth
`dilution method. Culture tubes containing 10 ml of the medium were inoculated with 0.1 ml of the
`inoculum described in the preceding section, and incubated at 37°C for 7 days. Tubes were examined
`for visible turbidity at 2 and 7 days. To determine the influence of pH on MIC, HCI or NaOH was
`added to SABOURAUD Liquid Broth before sterilization to give pH values of 4, 6, 7, and 8 at the time of
`inocu1ation. MI C's were also determined in the presence of 5 % sterile horse serum added to SABOURAUD
`Liquid Broth before inoculation. The effect of inoculum size on MIC was studied using an inoculum
`prepared as above, and adjusted by plate counts to contain 52 x 106 celJs/ml; MI C's were read at 48 hours,
`then each tube was subcultured on SABOURAUD Dextrose Agar plates to determine the minimum fun(cid:173)
`gicidal concentration (MFC).
`Bioavailability study
`Swiss albino Wistar male mice (25- 30 g) were administered micronized rapamycin as a suspension
`in 5~,~ acacia; the suspension was given by gavage (p. o.) or by subcutaneous injection. Mice, in groups
`of three. were bled by cardiac puncture at 0, 1, 2, 3, and 4 hours; therefore, the sera of three animals were
`combined to constitute one bleeding.
`A cross-over design was used in the dog (10 kg each) study. On day 0, one dog received 250 mg and
`the other dog 500 mg of rapamycin contained in a gelatin capsule. After one week rest, the same doses
`were reversed in the same dogs. Blood was sampled from the jugular vein in the neck at 0, l, 2, 4 and 6
`hours after rapamycin administration, allowed to clot, and the serum collected for assay.
`Sera were assayed on the day of bleeding by a modification of the agar diffusion method of BENNETT
`> with C. albicans A Y F-598 as the test organism and Nystatin Assay Agar as the assay medium.
`et a/. 1
`The liquefied assay medium (200 ml) was inoculated with 0.25 ml of a ten-fold dilution of a 18-hour
`broth culture of the test organism. Standard solutions of rapamycin were prepared in appropriate
`serum from a stock solution containing 100 /tg ofrapamycin per ml (in methanol) to give final concentra(cid:173)
`tions of 5, 2.5, 1.25, 0.625 and 0.3125 pg/ml of serum. These standard sera were pipetted in quadrupli(cid:173)
`cate into agar wells according to a randomized arrangement. Serum samples, undiluted, were similarly
`distributed. All plates were then incubated at 37°C for 18 hours. Zones of inhibition were measured,
`averaged and the average of each standard plotted against rapamycin concentration on semi-Jog graph
`paper to draw the standard curve. Concentration of the unknowns was obtained by interpolation from
`the standard curve.
`~~~tc~i_on ilga_i!l~. systemic c~ndidosis
`Preliminary experiments had shown that the intra venous injection of 3 - 7 x I 06 cells of C. a/hicans
`ATCC 11,651 killed untreated Swiss albino Wistar male mice (25- 30 g) within 48 hours.
`ln subse(cid:173)
`quent studies, the infecting dose ranged from 5 to 7 x 106 cells.
`The treatment consisted of three doses, administered either orally or subcutaneously at l, 4 and
`:24 hours after infection.
`In one study, after the initial three treatments, a single daily treatment was
`continued for G days. Deaths were recorded, and survivors kept under observation for 7, 18 and 30
`days, depending on the study.
`Infected, non-trcatedt as well as non-infected, treated mice served as
`controls for mortality and drug toxicity. Each treatment group comprised at least 10 mice. PD:o was
`calculated according to REED and MuENc11 5>.
`Rapamycin was administered as a suspension in 5 ~ .. ~acacia. Mycostatin (nystatin) a.nd Fungizone
`(amphotericin B) were administered as the commercial products according to the recommendations of
`the manufacturer.
`Pr~tection againsl vaginal canc:!i_dos~s
`Sprague-Dawley female rats (150 g) \\'ere infected intravaginally by means of specially prepared
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`VOL. XXXI :"iO. 6
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`THE JOURNAL OF ANTIBIOTICS
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`541
`
`airfoam sponge plugs previously contaminated with C. albicans ATCC 11,651. Sterile cylindrical air(cid:173)
`foam sponge plugs (7I16" in length x 5/ 16 11 in diameter) were placed in 125-ml Erlenmeyer flasks (25
`plugs/flask) containing 75 ml of SABOURAUD Liquid Broth inoculated with C. albicans ATCC J 1,651.
`Flasks were incubated without agitation at 37°C for 18 hours. After incubation, the plugs were removed
`to sterile Petri plates and the broth culture centrifuged to recover the candidal cells which were then
`resuspended in 7.5 ml of sterile 10% glucose solution. Each plug was saturated with 0.2 ml of this
`suspension. One infected plug was inserted and remained in the rat vagina for the duration of the experi(cid:173)
`ment. Presence of candidal cells was ascertained by microscopic smear examination and cultures.
`The treatment consisted of a single oral dose of rapamycin (50 mg/kg) or Mycostatin (40,000 units/
`kg). administered daily for 6 days. The course of infection was monitored by vaginal swab cultures
`taken on days J, 4 and 6 of treatment, and on days 2 and 3 after termination of therapy. The amount
`of candidal growth was estimated semi-quantitatively on a scale from 0 (no growth) to 4 + (heavy
`growth). The response to treatment was evaluated by the decrease of number of animals having vaginal
`cultures wirh moderate (3 +) and heavy (4 +) growth as compared to untreated controls.
`
`Results
`
`Effect of pH. Scrum and Ino;:;ulum Size
`At pH 6 and 7, the MIC of rapamycin against C. alhicans was < 0.02 pg/ml as read after 2 and 7
`days of incubation in SAROURAUD liquid broth. At pH 4, the MIC increased to > 10 pg/ml; at pH 8.
`it was found to be < 0.02 pg/ml after 2 days of incubation, but 0.16 pg/ml after 7 days of incubation.
`In the presence of 5 ~,~horse scrum, the MIC of rapamycin was > 1011g/ml as compared to < 0.02
`;1g/ml in the absence of scrum; observation was at 2 and 7 days of incubation in SABOURAUD liquid broth.
`The MIC value of rapamycin against C. albicans was not affected by inoculum sizes between IO and
`50 x 10· cells/ml. For an inoculum of 5.2 x 10~ cells/ml or less, a two-fold decrease in MIC was noted.
`The M FC remained constant at 0.05 /tg/ml with all inoculum sizes tested.
`
`Bioavailability in Mice, Rats and Dogs
`Rapamycin was administered to male mice at a dose or 15 mg/kg. Results are illustrated in Fig.
`1. Serum levels were higher for the subcutaneous than for the oral route of administration. Distinct
`
`Fig. I. A \"eragc scrum concentration of rapamycin
`in mice after oral or subcutaneous administration
`or 15 mg;kg.
`
`Fig. 2. A vcrage scrum concentration of rapamycin
`in dogs after oral administration of 250 and 500 mg/
`dog: two-dog cross-over experimen l.
`
`/'\500mg
`I
`\
`I
`I
`I
`,.,
`../
`,,...
`/
`.........
`/
`',...,,..,;...
`
`\.
`
`',
`'
`\,
`
`\
`
`\
`
`1.0
`
`o.e
`~
`g: 0.6
`c:: ·u
`>.
`E 0.4
`D
`a.
`0
`0::
`
`0.2
`
`I
`I
`I
`
`'
`
`I
`I
`I
`I
`I
`I
`I
`I
`I
`I
`I
`
`0'-----..1..-----L----.....__ __ ~
`0
`I
`2
`3
`4
`Time after adminis1ration(hoursJ
`
`2
`4
`3
`5
`Time ofter odminisfrarion {hoursJ
`
`6
`
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`542
`
`THE JOURNAL OF ANTIBIOTICS
`
`JUNE 1978
`
`peaks were observed in both curves. Rapamycin was not eliminated very rapidly from blood; 4 hours
`after subcutaneous or oral administration, the concentration of rapamycin stiJl present in blood was
`4.3 and 1.85 Jlg/ml respectively; these values are welJ above the MIC and MFC of rapamycin against
`C. albicans ATCC 11,651.
`Rapamycin was administered orally to two dogs in a cross-over study at two doses, 250 and 500 mg/
`dog. The results are illustrated in Fig. 2. For the Jower dose a single peak of 0.55 µg/ml was obtained
`at 2 hours; the concentration then decreased to reach a minimum at about 4 hours, after which it increas(cid:173)
`ed slightly to 0.21 p.g/ml at 6 hours, probably toward a second peak. For the higher dose two peaks
`were observed as in mice, one peak of0.75 µg/ml at 2 hours, and another peak of0.98 µg/ml at 4.5 hours.
`At 6 hours the concentration was 0.61 µg/ml.
`In summary, subcutaneous or oral administration of rapamycin to mice and dogs led to rapid ab(cid:173)
`sorption and yielded blood serum concentrations well above the MIC and MFC values of the antibiotic
`against C. a/bicans ATCC 11,651.
`
`Protection against Systemic Candidosis
`Rapamycin was compared to amphotericin B and nystatin in protecting mice against a systemic
`infection with C. albicans A TCC 11,651. Antibiotics were administered orally 1, 4 and 24 hours after
`an intravenous injection of 6.5 x 106 candidal cells. The results are tabu1ated in Table 1. The PDso's
`for rapamycin, amphotericin B and nystatin were respectively 11 mg/kg, < 0.25 mg/kg and > 4,000
`units/kg. When the antibiotics were administered subcutaneously (Table 2) the results were essentially
`the same; PDso of rapamycin was slightly lower (9.5 mg/kg) for subcutaneous than for oral (11 mg/kg)
`administration, which corroborates the results of bioavailabiJity studies (Fig. l). STEINBERG et al. 8)
`
`Table 1. Protective effect of rapamycin, amphoteri(cid:173)
`cin B and nystatin administered orally to mice
`systemically
`infected with C. a/bicans A TCC
`11,651
`
`Table 2. Protective effect of rapamycin, amphoteri(cid:173)
`cin B and nystatin administered subcutaneously
`to mice systemically
`infected with C. a/bicans
`ATCC 11,651
`
`Antibiotic
`
`Dose
`(mg/kg)a
`
`I Survival I PDso
`(mg/kg)c
`( %)b
`
`Antibiotic
`
`Dose
`(mg/kg)"'
`
`I Survival
`( %)b
`
`PD so
`(mg/kg)c
`
`1 l.O
`
`Rapamycin
`
`0
`7.5
`10.0
`12.5
`15.0
`20.0
`
`0
`22.7
`52.9
`90.0
`96.5
`97.3
`
`9.5
`
`Rapamycin
`
`A mphoterici n
`B
`
`Nystatin
`(unit/kg)
`
`I
`I
`I
`I
`
`-~---~-·-
`
`0
`7.5
`10.0
`12.5
`15.0
`I 20.0
`! 0
`0.25
`0.50
`1.00
`
`0
`2,000
`3,000
`4,000
`
`0
`12.5
`30.0
`63 .1
`86.3
`100.0
`
`0
`72.7
`94.7
`96.4
`
`0
`7 .1
`14. 2
`30.7
`
`<0.25
`
`>4,000
`(units/kg)
`
`Amphotericin
`B
`
`Nystatin
`(units/kg)
`
`0
`0
`81.8
`0. 25
`95.0
`0.50
`96.2
`1.00
`- - - - - - - - ' - - - - - - - - - - - -
`0
`0
`2,000
`0
`3,000
`0
`4,000
`0
`
`<0.25
`
`>4,000
`(units/kg)
`
`a Treatment: 1, 4 and 24 hours after infection.
`b Observation at 7 days.
`Infectious dose: 6.5 >: 106 cells.
`c
`
`a. Treatment: 1, 4 and 24 hours after infection.
`b Observation at 7 days.
`Infectious dose: 7 x t 0 6 cells.
`c
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`VOL. XXXI NO. 6
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`THE JOURNAL OF ANTIBIOTICS
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`
`Table 3. Protective effect of rapamycin (20 mg/kg) and amphotericin B ( 1 mg/kg) administered orally or
`subcutaneously to mice systemically infected with C. a/bicans ATCC 11,651
`
`Antibiotic and route
`of
`administration
`
`Treatment A
`I
`
`18 days
`
`30 days11
`
`Percent survival
`I
`I
`
`18 days
`
`Treatment B
`I
`
`---
`30 daysb
`
`--
`
`--
`
`26.6
`66.6
`33.6
`
`93.3
`93.3
`100.0
`
`I
`I
`I
`I
`
`Infected
`
`Non-infected
`control
`
`Rapamycin, oral
`Rapamycin, s. c.
`Amphotericin B, oral
`
`46.6
`93.3
`93.3
`
`33.3
`46.6
`40.0
`
`I
`
`I
`
`--
`
`.
`
`Rapamycin, oral
`Rapamycin, s. c.
`Amphotericin B, oral
`
`100
`100
`ND
`
`100
`100
`ND
`
`93.3
`86.6
`53.3
`
`93.3
`93.3
`100.0
`
`I,
`Infectious dose: 3.15~<106 cells.
`n Treatment A: 1, 4 and 24 hours after infection.
`b Treatment B: I, 4 and 24 hours, and 2, 3, 4, 5, 6 and 7 days after infection.
`Infectious dose: 5.0 x 106 cells.
`ND: not determined.
`
`reported the PDso of amphotericin B in experimental C. albicans infection of mice to be < 0.55 mg/kg,
`per os, and < 0.32 mg/kg, subcutaneously.
`In another study, rapamycin per os and subcutaneously, or amphotericin B, per os, were given at
`the doses that were shown, in the previous experiments, to give almost complete protection to mice
`systemically infected with C. albicans, i.e. 20 mg rapamycin/kg and l mg amphotericin B/kg, given at
`1, 4 and 24 hours after infection. Animals were observed for 18 and 30 days (Treatment A). The
`results are presented in the first and second columns of Table 3. The highest percentage of deaths oc(cid:173)
`curred between day 7 (Tables 1 and 2) and day 18 (Table 3).
`It would appear that rapamycin controlled
`the infection for a period of 7 days with a subsequent mortality of 53 % between days 7 and 18. Rapa(cid:173)
`mycin, given subcutaneously, and amphotericin B, per os, protected 93.3 % of the mice for 18 days (Table
`3). The survival rate at 30 days were essentially the same for rapamycin, given oraJly or subcutaneously,
`and amphotericin B, given orally.
`In a paralJel study, after the initial three treatments, a single daily dose was administered for 6 days
`and the animals were observed for 18 and 30 days (Treatment B). The results are reported in the third
`and fourth columns of Table 3. The additional daily treatment (for 6 days) with rapamycin given orally
`extended the percent survival rate from 7 days to 18 days. The daily treatment with rapamycin admin(cid:173)
`istered subcutaneously did not significantly affect the percentage of survivors over the initial treatment
`after 18 days of observation. After 30 days of observation, the percent survival was higher with subcu(cid:173)
`taneous treatment than with oral treatment, but not much higher than the initial subcutaneous treatment
`(Treatment A). The percentage of survivors with amphotericin B after daily treatment and 18 days of
`observation was significantly lower than that of Treatment A; this may be due to the combination of drug
`toxicity and infection. After 30 days of observation, the percentage of survivors with amphotericin B
`In conclusion, we would suggest that in the in vfro
`(Treatment A) was not significantly different.
`evaluation of an anti-Candida agent, irregardless of treatment, the test animals should be kept for 30
`days after infection, and the survivors observed after 7, 18 and 30 days.
`In the course of these experiments C. a/bica11s was regularly reisolated from animals treated with
`rapamycin; the cultures showed no increase in MIC, an indication that the resistance of candidal cells
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`
`did not develop during treatment.
`
`Fig. 3. Protective effect of rapamycin and nystatin
`against vaginal candidosis in rats.
`
`~ Rapamycin !SOmg/ko fllrafs1
`t=:J Nysfatin 40,000units/kg 112 rats}
`~ No treatment rinfected control)
`Ill rats)
`
`12
`
`Protection against Vaginal Candidosis
`A number of female
`rats were infected
`intravaginally by means of lhe sponge plug
`technique described in Materials and Methods
`and 34 pro,·ed to carry the inrection (Fig. 3, day-
`1>. They were grouped as follows: group A (11
`rats) received no treatment (control); group B (J 2
`rats) was treated with Mycostatin; group C ( 11
`rats) \\'as treated with rapamyi.::in. Oral treat(cid:173)
`ments started on clay 0 and lasted for 8 days.
`A II animals \Vere examined on days 1, 4, 6, 8 and
`11 ( 3 days after cessation of therapy) after the
`beginning of therapy. The histogram in Fig. 3
`reports the number of animals having vagina)
`cultures with moderate (3 +) and heavy (4-r-)
`growth of C. nlbicaus. At the end of therapy (day 8) the cure rates were: 6 of 12 (50~~) for Mycostalin,
`8 of 11 (73 ° ~) for rapamycin, and 3 of 11 (27 ~~) for spontaneous cure. Three days after cessation of
`treatment (day 11) no change was recorded for Mycostatin. but 10 of the 11 rats <91 ~-~) treated with
`rapamycin \Vere cured.
`Histological examination of infected vaginas and normal vag!nas in which sterile plugs had been
`inserted (non-infected, non-treated control) showed some degree of irritation cu used by these plugs, but
`the puruknt exudate was present only in the vaginas containing infected plugs.
`
`-I
`
`8
`II
`4
`j End of therapy
`in1erapy initiated
`Days of
`therapy
`
`6
`
`Discussion
`
`It \Vas previously reported'·: that rapamycin is more active than candicidin and nystatin against
`clinical isolates of C. albicaus; the MIC"s obtained with candicid!n and nystatin were within the range
`reported by 0ROUHET2>.
`It was also reported that rapamycin was more active than amphotericin B
`against eight species of Candida; only C. pseudotropicalis was more sensitive to amphotericin B.
`In the present study, rapamycin was found not to be affected by pH between 6 and 8, but at pH 4
`there was complete loss of activity. The MIC and MFC of rapamycin against C. a/bicans were not in(cid:173)
`In the presence of 5 % horse serum, the antibiotic lost its in vitro
`fluenced by the size of inoculum.
`activity possibly due to the binding of rapamycin to serum components. This binding must be rever(cid:173)
`sible, since bioavailabiUty data in mice and dogs demonstrated that rapamycin is present in the blood at
`a level well exceeding its MIC against C. a/bicans (Figs. 1 and 2), and was sufficient to eradicate severe
`systemic candidosis in mice (Tables 1 and 2).
`In the ill l'ivo studies rapamycin, given orally or subcutaneously, was compared to amphotericin B,
`an antibiotic effective in the treatment of systemic candidosis. The dose selected was based on an initial
`study in which both antibiotics yielded 96.2,..., 100% survival after 7 days of observation.
`Rapamycin at 20 mg/kg, per os and subcutaneously, and amphotericin B at 1 mg/kg, oralJy, adminis(cid:173)
`tered at 1, 4 and 24 hours after infection gave approximately the same percent survival after a 30-day
`observation period (Treatment A). When the above treatment was extended by an additional daily
`treatment for 6 days (Treatment B), the percent survivals for rapamycin and amphotericin B given orally
`were similar, but subcutaneous administration of rapamycin gave higher survival after 30 days of
`observation. Rapamycin was also found very active orally in the treatment of an experimental vaginal
`candidosis in rats. The high in i•fro activity of rapamycin, combined with its low acute toxicity (lower
`
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`THE JOURNAL OF ANTIBIOTICS
`
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`
`than that of amphotericin 8 9>) and good oraJ absorption, makes it a possible candidate for the treatment
`of vagina) candidosis, as well as acute and chronic systemic candidosis.
`
`References
`
`1) BENNETT, J. V.; J. L. BRODIE, E. J. BENNER & W. M. M. KIRBY: SimpJified, accurate method for antibiotic
`assay of clinical specimens. Appl. Microbiol. 14: 170-- 177, 1966
`2) DROUHET, E.: Some biological activities of antifungal antibiotics and their mode of action. "Ciba
`Foundation Symposium on Systemic Mycoses''. G. E.W. WOLSTENHOLME & R. PORTER Eds., J. & A.
`Churchill, London, pp. 206-240, 1968
`3) GoLD, W.~ H. A. STOUT, J. F. PAGANO & R. DoNov1cK: Amphotericins A and B. antifungal antibiotics
`produced by a streptomycete. I.
`In w"tro studies. Antibiotics Ann. 1955/1956: 579.-586, 1956
`4) HAZE~. E. L. & R. BRow:-.i: Fungicidin, an antibiotic produced by a soil actinomycete. Proc. Soc. Exp.
`Biol. 76: 93-97, 1951
`5) REED, L. J. & H. MUENCH: A simple method of estimating 50 percent end points. Amer. J. Hyg. 27:
`493-497. 1938
`6) SEHGAL, S. N.; H. BAKER & C. VF.ZINA: Rapamycin (A Y-22,989), a new antifungal antibiotic. II. Fermen(cid:173)
`tation, isolation and characterization. J. Antibiotics 28: 727-732, 1975
`7) SmoRowrcz, A.; H. BAKER & C. VF:ztNA: Rapamycin (AY-22, 989), a new antifungal antibiotic: in l1itro
`(Sept. 24- 26, 1975,
`and in i•fro studies. Abst. 26, 15th Intcrsci. Conf. Antimicrob. Agents & Chemother.
`Washington, D. C.).
`8) STEINBERG, B. A.; W. P. JAMBOR & L. 0. SUYDAM: Amphotericins A and B: two new antifungal antibiotics
`possessing high activity against deep-seated and superficial mycoses. Antibiotics Ann. 1955/1956: 574
`-578, 1956
`9) UTZ, J.P.; J.E. BENNEIT, M. W. BRANDISS, W. T. BUTLER & G. J. H1LL: Amphotericin B toxicity. Ann.
`Intern. Med. 61 : 334- 354, 1964
`10} VEZINA, c.; A. KUDELSKI & 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-
`726, 1975
`
`Breckenridge Exhibit 1047
`Baker
`Page 007
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