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`48
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`Inhibition of the immune response by
`rapamycin, a new antifungal antibiotic
`
`R. R. MARTEL, J. KLICIUS, AND S. GALET
`Department of Pharmacology, Ayers! Research. Laboratories,
`Montreal, P.Q., Canada H4R 116
`
`Received June 30, 1976
`
`MARTEL, R. R., Kucrus, J ., and GALET, S. 1977. Inhibition of the immune response by
`rapamycin, a new antifungal antibiotic. Can. J. Physiol. Pharmacol. 55, 48——51.
`Rapamycin, a new antifungal antibiotic, was found to inhibit the immune response in
`rats. It totally prevented the development of two experimental immunopathies (experi-
`mental allergic encephalomyelitis (EAE) and adjuvant arthritis (AA)) and the formation
`of humoral
`(IgE—like) antibody. It was about half as potent as cyclophosphamide in
`inhibiting EAE. In AA and on antibody formation. rapamycin and cyclophosphamide
`were about equipotcnt, whereas mcthotrcxatc was more potent. The immunosuppressant
`activity of rapamycin appears to be related to inhibition of the lymphatic system.
`
`MARTEL, R. R., KLICIUS, J. et GALET, S. 1977. Inhibition of the immune response by
`rapamycin, a new antifungal antibiotic. Can. J’. Physiol. Pharmacol. 55, 48-51.
`La rapamycine, un nouvel antibiotique antifungique,
`inhibe la réponse immunitaire
`chez le rat.
`I1 empéche totalement
`le développement de deux immunopathies experi-
`mentales (encéphalomyélite expérimentale allergique (EEA) et arthrite adjuvantc (AA))
`et la formation d"anticorps humoraux semblables aux lgE. Sa puissance est la moitié de
`celle du cyclophosphamide dans Finhibition dc 1’EEA. ljetfet sur 1’AA et sur la formation
`d’anticorps est approximativement le meme pour la rapamycine et le cyclophosphamide
`alors que le méthotrexate est plus puissant. I.’activité immunosuppressive de la rapamycirle
`pztrait liée 51 une inhibition du systéme lymphatiquc.
`
`[Traduit par le journal]
`
`Introduction
`
`Rapamycin is a recently described (Vézina
`et al. 1975; Sehgal et al. 1975) antifungal
`antibiotic
`extracted from a
`streptomycete
`(Streptomyces hygroscopicus) isolated from an
`Easter Island soil sample. It is particularly ef-
`fective against Candida albicans both in vitro
`and in vivo (Sidorowicz et al. 1975).
`The inhibitory effects of this new antibiotic
`on two experimental immunopathies (EAE and
`AA) and on the formation of IgE-like antibody
`are described in this report.
`
`Methods
`
`EAE
`inbred Wister—Lewis
`EAE was induced in female,
`rats (120440 g). The rats were injected in the left
`hind foot pad with 0.05 ml of an emulsion consisting
`of guinea pig spinal cord (4.2 g) homogenized in a
`mixture of 5.8 ml of 0.5% aqueous phenol and an
`equal volume of complete Freund’s adjuvant contain-
`ing 4.:-1mg/ml of heat-killed, dried Mycobacterium
`
`ABBREVIATIONS: EAE, experimental allergic enceph-
`alitis; AA, adjuvant arthritis; po, per os; EA, egg
`albumin; PCA, passive cutaneous anaphylaxis.
`
`butyricum (Difco). The sensitized rats were treated
`orally with rapamycin or cyclophosphamide according
`to different schedules
`(see Table 1). The animals
`were observed for signs of hindleg paralysis from day
`10 to day 16 (day of sensitization is day 0). The rats
`that did not show paralysis of the hindlegs during the
`observation period were considered protected.
`
`AA
`AA was induced in male inbred Wister—Lewis rats
`(180—200 g). The rats were injected intradermally in
`the foot pad of the left hindpaw (day 0) with 0.05 ml
`of a fine suspension of killed and dried M. butyrfcum
`(Difco) at a concentration of 5 mg/ml in liquid paraflin
`(Freund’s adjuvant). For
`the prophylactic study,
`compounds were administered per 05 daily starting on
`the day of adjuvant injection (day 0) and until day 1.6.
`For the therapeutic study,
`treatment was started on
`day 14 and continued until day 2.2. Hindleg volume
`was determined by mercury displacement 2h after
`the last treatment. Hindlegs were dipped in mercury
`up to the hair line. The mercury displaced represents
`the volume of the hindlcgs (13.6 g of mercury : 1 ml).
`
`IgE-like Antibody Formation
`A modification of the method described by Mota
`(1964) was used to produce IgE-like antibody in the
`rat. The effect of rapamycin on this response was
`studied. Male Charles River rats (l80—200 g) were
`injected intrapcritoneally with 1 ml of killed Bordeteila
`pertussis cells (2 x 10” cells/ml) and intramuscularly
`
`West-Ward Pharm.
`Exhibit 1021
`
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`Exhibit 1021
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`MARTEL ET AL.
`
`49
`
`TABLE 1. Effect of rapamycin on EAE
`
`Treatment,
`daily
`
`Duration,
`(1
`
`Dose,
`mg /kg, po
`
`Rats paralyzed“
`
`Rats sensitized % protected
`
`Control
`Rapamycin
`
`Cyclophosphamide
`
`O—13
`(P13
`0-13
`(% 6
`7-13
`
`0-13
`0-13
`0-13
`
`10
`5
`2.5
`10
`10
`
`5
`2 .5
`1
`
`33 /41
`0/ 12
`9/18
`11/17
`2/12
`7/12
`
`1/19
`9/19
`4/6
`
`20
`100
`50
`35
`83
`42
`
`95
`53
`33
`
`“Rats which showed hindleg paralysis between day 10 and 16.
`
`with 0.3 ml of EA (10 mg/ml). Oral administration
`of rapamycin and other compounds was started at the
`same time (day 0) and continued daily until day 11.
`On day 12, the animals were anesthetized with ether
`and bled from the abdominal aorta. The presence of
`EA antibody in the serums of control and treated
`groups (eight animals in each group) was determined
`by the PCA method. The pooled undiluted serum (0.1
`ml) of each group was injected intradermally in six
`rats (three sites per rat). Forty-eight hours later the
`animals were challenged intravenously with 11.25 mg
`of EA per kilogram in 1% Evans blue dye in saline
`(0.9 ml per 100 g). After 30 min the rats were killed
`by C02 inhalation and the diameter of the wheals
`formed on the underside of the skin was measured.
`The cyclophosphamide used in these studies was
`purchased from ICN Pharmaceutical,
`Inc., and the
`methotrcxate was generously supplied by Lederle
`Laboratories.
`
`Results
`
`Eflect on EAE
`EAE is characterized by paralysis of the hind
`quarter. Rapamycin (10mg/kg, po), admin-
`istered daily for 14 (1 starting on the day of
`sensitization (day 0) completely prevented the
`development of hindleg paralysis (Table 1).
`None of these rats (l0mg/kg) showed late
`paralysis when
`observed
`until
`day
`21.
`Rapamycin was more active when administered
`early (day 0 to 6) in the course of EAE than
`when administered from day 7 to 13 (Table 1).
`These data suggest that rapamycin exerts most
`of its effect during the induction of EAE.
`The
`immunosuppressive drug cyclophos—
`phamide, a potent inhibitor of EAE (Rosen-
`thale et al. 1969), appeared to be about twice
`as potent as rapamycin in these experiments.
`
`Efleet on AA.
`AA is characterized by a severe inflammatory
`
`reaction of the hindlegs. When the adjuvant
`(mycobacteria in oil) is injected into a hindpaw
`(day 0) and treatment started on that day
`(prophylactic treatment), drugs can be evalu-
`ated for their effect on two distinct inflammatory
`phases of the disease: (a) and early phase in the
`injected paw which peaks around day 3 and is
`mainly dependent on an acute inflammatory
`reaction to the adjuvant, and (b) a late phase
`(starting around day 10)
`in the injected and
`the noninjected hindlegs, resulting from a de-
`layed or cellular-type hypersensitivity reaction
`to
`some
`constituent of
`the mycobacteria
`(Rosenthale 1974). The immunosuppressive
`agents inhibit only the late or immune phase,
`whereas the anti-inflammatory drugs inhibit
`both phases (Walz et al. 1971; Rosenthale
`1974).
`Rapamycin (5 mg/ kg), cyclophosphamide
`(5 mg/kg), and methotrexate (0.25 mg/kg),
`when administered orally, completely blocked
`the secondary immune response (day 16) in
`both hindlegs. However, they did not decrease
`significantly the primary nonimmune phase in
`the injected paw. The protective effect of the
`three compounds was still complete on day 22,
`6 d after treatment was stopped (Table 2). The
`doses reported in the table are about the lowest
`that will inhibit AA completely.
`In established arthritis (six controls and six
`treated), rapamycin (10 mg/ kg) was inactive.
`It did not prevent further swelling of the hind-
`paws. However, the swelling was slightly less
`than in the untreated arthritic rats.
`
`Eflect of IgE-like Antibody Formation
`Twelve days after injection of EA and B.
`pertussis,
`the pooled serums of control and
`West-Ward Pharm.
`Exhibit 1021
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`
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`Exhibit 1021
`Page 002
`
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`50
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`CAN. J. PHYSIOL. PHARMACOL. VOL. 55, [977
`
`TABLE 2. Effect of rapamycin on AA
`
`Hindleg volume, ml i SE
`
`Injected
`
`Day 16
`
`2.3 i 0.06
`4.9 i 0.24
`3.1 i0.02
`3.6 i 0.26
`3.3 i 0.15
`
`Noiiinjected
`
`Day 22
`
`Day 16
`
`Day 22
`
`2.4 i 0.06
`5.7 i 0.22
`2.9i0.24
`3.3 i 0.21
`3.2 1- 0.12
`
`2.3 i 0.06
`3.3 i 0.24
`2.2i0.l2
`2.1i 0.02
`2.2 1 0.04
`
`2.4 i 0.06
`4.2 i 0.23
`2.3:0.19
`2.2 i 0.22
`2.2 1- 0.04
`
`Treatment”
`
`Normal control
`Arthritic control
`Rapamycin,5mg/kg
`Cyclophosphamide, 5 mg/kg
`Methotrexate, 0.25 mg/kg
`
`Day 3
`
`2.1 i 0.02
`3.7 i 0.08
`3.7iO.10
`3.4 i 0.10
`3.9 jg 0.20
`
`“Orally from day 0 to day 16 (8-10 rats per group).
`
`TABLE 3. Effect of rapamycin on IgE-like
`antibody formation
`
`Treatment,
`dafly
`Control
`Rapamycin
`
`cyclophosphamide
`
`D056,
`mg/kg, 90
`
`10
`3
`10
`3
`0_3
`25
`
`Diameter of
`Skin Wheal,
`mm t SE
`23_1 i 0_8
`0
`10_2 i 0.2:;
`O
`12_0 i 0.4:;
`O
`27_() i ()_3
`
`Methotrexate
`phgnflbugazone
`M, < Om
`NOTE: The skin was sensitized with 0.1 ml of the pooled serum of
`eight rats per group.
`
`treated rats were assayed for the presence of
`EA antibodies by the 48-h PCA method. As
`shown in Table 3, the serum of the rats treated
`with 10mg of rapamycin per kilogram and
`cyclophosphamide produced no skin wheals.
`A smaller dose of rapamycin and cyclophospha-
`mide partially inhibited the response, while
`methotrexate was more potent. Rapamycin
`(100 mg/kg, po, administered to sensitized rats
`1 h previous to challenge) or diluted anti-EA
`serum (one part with three parts of serum ob-
`tained from unsensitized rats
`treated with
`
`rapamycin, 50 mg/kg orally for 2 (1) had no
`effect on the PCA response. From these results
`it was concluded that rapamycin, similar to the
`immunosuppressive agents cyclophosphamide
`and methotrexate,
`inhibited antibody forma-
`tion. The nonsteroidal anti-inflammatory drug
`phenylbutazone had no effect. No drug-related
`adverse eflects could be noted, apart from a
`depression of the growth curve with rapamycin
`and the other immunosuppressive agents.
`
`Discussion
`_
`_
`.
`_
`_
`Rapamycin, a new antifungal antibiotic, has
`been found to inhibit the immune response.
`Small, well-tolerated
`doses,
`5-10 mg/kg,
`(Sidorowicz et al. 1975) of this antibiotic totally
`prevented the development of cellular immunity
`(EAE and AA), as well as the formation of
`IgE-like antibody. In EAE, rapamycin appeared
`to be half as_ potent as eyclophosphamide.
`In AA and antibody formation, the potency of
`rapamycin and cyclophosphamide appeared
`comparable, whereas methotrexate was more
`potent. Similarly, Walz et al. (1971) used 20-
`times less methotrexate than cyclophosphamide
`to block AA, and Rosenthale et al.
`(1969)
`showed methotrexate to be much more potent
`than cyclophosphamide in inhibiting paralysis
`in EAE.
`
`the inhibitory
`All evidence indicates that
`effect of rapamycin in EAE and AA depends
`on suppression of
`the
`immune
`response.
`Rapamycin produced a complete and long-
`lasting inhibition of EAE. Prophylactic treat-
`ment with rapamycin in the AA model
`in-
`hibited only the immune-mediated phase of
`inflammation and the inhibition lasted after
`
`treatment was discontinued. In established AA,
`10mg of
`rapamycin per kilogram,
`a dose
`which completely prevented the development
`of EAE and AA, was inactive. This profile was
`reported for immunosuppressive agents such
`as azathioprine, methotrexate,
`cycloleucine,
`and cyclophosphamide by Rosenthale (1974).
`Nonsteroidal anti-inflammatory drugs do not
`produce full protection in EAE (Komarek and
`Dietrich 1971). In AA, the steroidal and non-
`steroidal anti-inflammatory drugs inhibit both
`the early nonspecific and the late immune
`
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`
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`MARTEL ET AL.
`
`5]
`
`phases of inflammation (Walz et al. 1971; Wax
`\et al. 1974). They are about equipotent when
`assayed by the preventive and the therapeutic
`methods (Perper et al. 1971; Walz et al. 1971;
`Wax er al. 1974; Martel and Klicius 1976).
`Arthritis occurs readily after closing is stopped
`(Perper et al. 1971). Furthermore, rapamycin
`inhibited antibody ‘formation, whereas the non-
`steroidal anti—inflammatory drug pl1enylbuta-
`zone did not.
`
`The mechanism of action of rapamycin on
`the immune system is unknown at the present
`time. However,
`long-term toxicity studies in
`dogs (Hemm, R. D., and Authier, L., personal
`communication)
`have
`demonstrated
`that
`rapamycin caused hypoplasia of lymphatic tis-
`sues (lymph nodes, spleen, thymus). Thus, it
`appears that the activity of this antibiotic on
`the immune response depends on an inhibition
`of the lympathic system.
`
`Acknowledgments
`
`The authors gratefully acknowledge the able
`technical assistance of Mrs. Lise Brisebois and
`Miss Josée Caron.
`
`KOMAREK. A., and DIETRICH, F. M. 1971. Chemical
`prevention of experimental allergic encephal0mye-
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`MARTEL, R. R., and KLICIUS, J. 1976. Anti-inflamma-
`tory and analgesic properties of etodolic acid in rats.
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`and
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`J. R., ALVAREX, B., COLOMBO, C.,
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`ROSENTHALE, M. E. 1974. Evaluation for immuno-
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`ROSENTHALE, M. E., DATKO, L. J., KASSARICH, J., and
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`SEHGAL, X. N., BAKER, H., and VEZINA, C. 1975.
`Rapamycin (AY-22,989), a new antifungal anti-
`biotic. II. Fermentation. isolation and characteriza-
`tion. J. Antibiot. 28, 727-732.
`SIDOROVVICZ, H., BAKER, H., and V15.ziNA, C. 1975.
`Rapamycin (AY-22,989), a new antifungal anti-
`biotic.: in vitro and in vivo studies. 15th Interscience
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`therapy, abstract 26.
`VEZINA, C., KUDELSKI, A., and SEHGAL, S. N. 1975.
`Rapamycin (AY-22,989), a new antifungal anti-
`biotic. I. Taxonomy of the producing streptomycete
`and isolation of the active principle. J. Antibiot. 28,
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`WALZ, D. T., DIMARTINO, M. J., and MISHER, A. 1971.
`Adjuvant—induced arthritis in rats. II. Drug effects
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`biochemical
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`WAX,
`J., WINDER, C. V., TESSMAN, D. K.,
`and
`STEPHENS, M. D. 1974 Comparative activities, toler-
`ances and safety of non—steroidal anti-inflammatory
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`
`West-Ward Pharm.
`Exhibit 1021
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