Inhibition of the immune response by
`aapamycin, a new an~fungal antibiotic
`
`R. R. MARTEE, J. KLICIUS, AND S. GALET
`Department o f Plzarn~aco[ogy, Ayerst Research Laboratories,
`~Vorzrreak, P.Q., Ccavzadu HI4R 166
`Received June 30, 1976
`
`MARTEL, R. R., KLICIUS, J., and GAI-ET, S. 1977. Inhibition of the immune response by
`rapamycin, a new antifungal antibiotic. Can. J. Physiol. Pharmacol. 55, 48-5 1.
`Mapamycin, a new antifungal antibiotic, was found to inhibit the immune responsc in
`rats. It totally prcvented the development of two experimental immunc~pathies (experi-
`mental allergic encephalomyelitis (EAE) and adjuvant arthritis (AA) ) and the formatic~n
`s f humoral (IgE-like) antibody. It was about half as potent as cyclophosphamide in
`inhibiting EAE. In AA and on antibody formation, rapamycin and cyclophcsspharnide
`were about equipotent. whereas mcthotrexate was more potent. The im~raunosuppressant
`activity of rapamycin appears to be related to inhibition of the Hyrnphatic system.
`
`MARTEL, R. R., KLICIUS, J. et GALET, S. 1977. Inhibition of the immune response by
`rapamycin, a new antifbangal antibiotic. Can. J. Pkysiol. Pharmacol. 55, 48-51.
`La rapamycine, un nouvel antibiotique antifeingique, inhibe Ba
`rkponse immunitaire
`chez le rat. I1 empkche totalemerat le d6veloppement de deux i~nmunopathies expkri-
`mentales (encOphalomydlite expirimentale allergique (EEA) et arthrite adjuvantc (AA) )
`et la formation d'anticorps humoraux semblables aux IgE. Sa puissance est Ba moitik de
`celle du cyclophosphamide dans l'inhibition de 1'EEA. k'effet sur I'AA et sur la formation
`d'anticorps est approximativement le m2me pour la rapamycine et le cyclophosphamide
`alors que He mithotrexate est plus puissant. L'activitk immunosiappressive de la rapanaycine
`parait like h une inhibition du systeme lyrnphatique.
`
`[Traduit par le journal]
`
`Introduction
`Rapamycin is a recently described (Vkzina
`et al. 1975; Sehgal et ul. 1975) antifungal
`f~-om a streptsrnycete
`antibiotic extracted
`(Streptosazyces hygroscopicus) isolated from an
`Easter Island soil sample. It is particularly ef-
`fective against Candida albicans both in vitro
`and in vivo (Sidorowicz et ul. 1975).
`The inhibitory effects of this new antibiotic
`on two experimental irnmunopathies (EAE and
`AA) and on the formation of IgE-like antibody
`are described in this report.
`
`Methods
`
`EAE
`EAE was induced in female, inbred Wister-Lewis
`rats (12B-140 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 rnl of 0.5% aqueous phenol and an
`equal volume of complete Freund's adjuvant contain-
`ing 4.4 mg/mI of heat-killed, dried Mycobacterium
`
`ABBREVIATIONS: EAE. experimental allergic enceph-
`alitis; AA, adjuvant arthritis; po, per 0s; EA, egg
`albumin; PCA, passive cutaneous anaphylaxis.
`
`brltyricurn (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.
`
`A A
`AA was induced in male inbred Wister-Lewis rats
`( 180-200 g). The rats were injected intradernlally in
`the foot pad of the left hindpaw (day 0 ) with 0.05 mB
`
`of a fine suspension of killed and dried ,%I. butyricum
`(Difco) at a con cent ratio^^ of 5 mg/ml in liquid paraffin
`(Freund's adjuvant). For
`the prophylactic study,
`compounds were administered per os daily starting on
`the day of adjuvant i~ljection (day 0 ) and until day 16.
`For the therape~atic study, treatment was started on
`day 14 and continued until day 22. Hindleg volurne
`was determined by mercury displacement 2 h after
`the last treatment. Hindlegs were dipped in mercury
`up to the hair linc. The mercury displaced represents
`the volume of the hindlcgs ( 1 3.6 g of mercury = I ml).
`IgE-like A~zriRsdy Formation
`A modification of the method described by Mota
`(1964) was based to produce IgE-like antibody in the
`rat. The eEwt of raparnycin on this respcmse was
`studied. Male Charles River rats ( 180-200 g ) were
`injected intrapcritoneally with 1 ml of killed Bordetella
`pertussis cells ( 2 x 10'" cells/mH) and intramuscularly
`ARGENTUM
`Exhibit 1021
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`TABLE 1. Effect of rapamycin on EAE
`
`Treatment,
`daily
`
`Duration,
`d
`
`Dose,
`mg /kg, po
`
`Rats paralyzeda
`Rats sensitized % protected
`
`Control
`Rapamycin
`
`0-1 3
`0-1 3
`8-1 3
`0- 6
`7-1 3
`
`"Rats which showed hindleg paralysis between day 10 and 16.
`
`with 0.3 ml of EA 10 rng/ml). Oral administration
`of rapamycin and other compo~ands was started at the
`same tirne (day 0) and conai~~ucd daily until day 1 I .
`On day 12, the animals were anesthetized with ether
`and bled from the abdominai aorta. The presence of
`EA antibody in the serums of control and treated
`groups (eight animals in each group) was detsrrnined
`by the PCA rraethod. The pooled undiluted serum (0.1
`m l ) of each group was injected intradermalay in six
`rats (three sites per rat). Forty-cight hours later the
`anin~als were challenged intravenously with 11.25 mg
`of EA per kilogram in 1% Evans hiue dye in saline
`(0.9 ml per BOO g ) . After 30 nain the rats were killed
`by COa inhalation and the diameter sf the wheals
`formed on the underside of thc skin was measured.
`The cyclophosphnmide used in these studies was
`purchased from ICN Pharmaceutical, Hnc., and the
`mcthotrexate was generously supplied by Lederle
`Laboratories.
`
`Efject 012 EAE
`EAE is characterized by paralysis of the hind
`quarter. Rapamycin ( 10 mg/kg. PO), admin-
`istered daily for 84 d starting on the day of
`sensitization (day 0 ) completely prevented the
`development of hindleg paralysis (Table 1 ) .
`None of these rats (10 ing/kg) showed Bate
`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 I ) .
`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-
`thaIe et nk. 1969), appeared to be about twice
`as potent as rapamycin in these experiments.
`
`Efleet on A A
`AA is characterized by a severe inflammatory
`
`reaction of the hindlegs. RFhen 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 (RFalz et al. 1971; Rosenthale
`1974).
`Rapamycin ( 5 mg/ kg), cyclophosp hamide
`(5 mg/kg), and methotrexate (0.25 mg/kg),
`when administered orally, completely blocked
`the secondary immune response (day 16) in
`both laindlegs. 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 sf control and
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`50
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`CAN. J. PKYSIOL. PHARMACOL. VOL. 55, I977
`
`TABLE 2. Effect of rapamycin on AA
`
`Treatmenta
`
`Normal control
`Arthritic control
`Raparnycin, 5 mg /kg
`Cyclophospharnide,5mg/kg
`Methotrexate, 0.25 mg /kg
`
`Day 3
`
`2.1 f 0.02
`3.7 f 0.08
`3.7 f 0.10
`3 . 4 f 0 . 1 0
`3.9 + 0.20
`
`=Orally from day 0 to day 16 (8-10 rats per group).
`
`Hindleg volume, ml f SE
`
`Injected
`
`Day 16
`
`2.3 f 0.06
`4.9 _+ 0.24
`3.1 f 0.02
`3 . 6 f 0 . 2 6
`3.3 _+ 0.15
`
`Day 22
`
`2.4 f 8.06
`5.7 + 0.22
`2.9 f 0.24
`3 . 3 f 0 . 2 1
`3.2 f 0.12
`
`Noninjected
`Day 22
`
`Day 16
`
`2.3 f 0.06
`3.3 f 8.24
`2.2 f 0.12
`2.1 + 0.02
`2.2 f 0.04
`
`2.4 f 8.06
`4.2 + 0.23
`2.3 f 0.19
`2.2 + 0.22
`2.2 f 0.04
`
`TABLE 3. Effect of rapamycin on IgE-like
`antibody formation
`
`Treatment,
`daily
`
`Control
`Rapamycin
`
`Cyclophospharnide
`
`Dose,
`mg /kg, PO
`
`10
`3
`10
`3
`0.3
`25
`
`Discussion
`Rapamycin, a new antifungal antibiotic, has
`been found to inhibit the immune response.
`Diameter of
`Small, well-tolerated
`doses, 5-10 mg/kg,
`skin wheal,
`mm f SE
`(Sidorowicz et al. 1975 ) of this antibiotic totally
`28. + oe8 prevented the development of cellular immunity
`(EAE and AA), as well as the formation of
`0
`IgE-like antibody. In EAE, rapamycin appeared
`10.2 f 0 . 2a
`to be half as potent as cyclophosphamide.
`0
`In AA and antibody formation, the potency of
`12.0 + 0.4n
`rapamycin and cyclophosphamide appeared
`0
`comparable, whereas methotrexate was more
`27.0 f 0.8
`potent. Similarly, Walz et al. (1971 ) used 20-
`times less met hotrexate than cyclophosphamide
`to block AA, and Rosenthale et al. (1969)
`showed n~ethotrexate to be much more potent
`than cyclophosphamide in inhibiting paralysis
`in EAE.
`A11 evidence indicates that the inhibitory
`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,
`10 mg 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
`
`Methstrexate
`Phenylbutazone
`a p < 0.01.
`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
`E A antibodies by the 48-h PCA method. As
`shown in Table 3, the serum of the rats treated
`with 10 mg 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 d ) 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 effects could be noted, apart from a
`depression of the growth curve with rapamycin
`and the other immunosuppressive agents.
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`MARTEL ET AL.
`
`51
`
`phases of inflammation ( Walz et al. 197 1 ; Wax
`et al. 1974). They are about equipotent when
`assayed by the preventive and the therapeutic
`methods (Perper et nl. 1971 ; Walz et nl. 1971 ;
`Wax et nl. 1974; Martel and Klicius 1976).
`Arthritis occurs readily after dosing is stopped
`(Perper et al. 197 1 ) . Furthermore, rapamycin
`inhibited antibody formation, whereas the non-
`steroidal anti-inflammatory drug pllenylbuta-
`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 (I-Iemm, R. D., and Authier, L., personal
`communication)
`have demonstrated
`that
`rapamycin caused hypoplasia of lymphatic tis-
`sues (Iymph nodes, spleen, thymus). Thus, it
`appears that the activity of this antibiotic on
`the immune response depends on an inhibition
`of the ly~npathic system.
`
`Acknowledgments
`The authors gratefully acknowledge the able
`technical assistance of Mrs. Lise Brisebois and
`Miss J o k e Caron.
`
`KOMAREK, A., and DIETRICH, F. M. 1971. Chemical
`prevention of experimental allergic encephalomye-
`litis in rats: a quantitative evaluation of steroids
`and various non-steroid drugs. Arch. Int. Pharma-
`codyn. 193,249-257.
`MARTEL, R. R., and KLICIUS, J. 1976. Anti-inflamma-
`tory and analgesic properties of etodolic acid in rats.
`Can. J. Physiol. Pharmacol. 54, 245-248.
`
`MBTA, I. 1964. The mechanism of anaphylaxis. I.
`Production and biological properties of mast cell-
`sensitizing antibody. Immunology. 7, 68 1-699.
`PERPER, J. R.. ALVAREX, B., COLOMBO, C., and
`SCHRODER, H. 1971. The use of a standardized
`adjuvant arthritis assay
`to differentiate between
`anti-inflammatory and immunosuppressive agents.
`Proc. Soc. Exp. Biol. Med. 137, 506-5 12.
`ROSENTHAI-E, M. E. 1974. Evaluation for immuno-
`suppressive and antiallergic activity. In Anti-inflam-
`matory agents, chemistry and pharmacology. Vol.
`11. Edited hy R. A. Scherrer and ha. W. Withehouse.
`Academic Press Inc., New York. pp. 123-192.
`ROSENTHALE, M. E., DATKO, L. J., KASSARICH, J., and
`SCEINEIDER, F. 1969. Chemotherapy of experimental
`(EAE) . Arch.
`Int.
`allergic encephalomyelitis
`Pharmacodyn. 179,251-275.
`SEIPGAL, X. N., BAKER, H., and VBZINA, C. 1975.
`Kapamycin (AY-22,989). a new antifungal anti-
`biotic. 11. Fermentaticn, isolation and characteriza-
`tion. J. Antibiot. 28, 727-732.
`SIDOROWICZ, H.. BAKER, H., and V ~ ~ Z I N A , C. 1975.
`Rapamycin (AY-22,989), a new antifungal anti-
`biotic.: in vitro and in vivo studies. 15th Interscience
`Conference on antimicrobial agents and chemo-
`therapy, abstract 26.
`VBZINA, C., KUDELSKI, A., and SEHGAL, S. N. 1975.
`Kapatnycin (AY-22,989), a new antifungal anti-
`biotic. I. Taxonomy of the producing streptomycete
`and isolation of the active principle. J. Antibiot. 28,
`721-726.
`WALZ, D. T., DIMARTINO, M. J., and MISHER, A. 1971.
`Adjuvant-induced arthritis in rats. 11. Drug effects
`on physiologic, biochemical and
`immunologic
`parameters. J. Pharmacol. Exp. Ther. 179, 223-23 1.
`WAX. J., WINDER, C. V., TESSMAN, D. K., and
`STEPHENS, M. B. 1974 Comparative activities, toler-
`ances and safety of non-steroidal anti-inflammatory
`agents in rats. J. Pharm. Exp. Ther. 192, 172-178.
`
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