throbber
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
`
`Can. J. Physiol. Pharmacol. Downloaded from www.nrcresearchpress.com by UNIV CHICAGO on 06/26/15
`
`For personal use only.
`
`000001
`
`

`

`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
`
`Can. J. Physiol. Pharmacol. Downloaded from www.nrcresearchpress.com by UNIV CHICAGO on 06/26/15
`
`For personal use only.
`
`000002
`
`

`

`50
`
`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.
`
`Can. J. Physiol. Pharmacol. Downloaded from www.nrcresearchpress.com by UNIV CHICAGO on 06/26/15
`
`For personal use only.
`
`000003
`
`

`

`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.
`
`Can. J. Physiol. Pharmacol. Downloaded from www.nrcresearchpress.com by UNIV CHICAGO on 06/26/15
`
`For personal use only.
`
`000004
`
`

This document is available on Docket Alarm but you must sign up to view it.


Or .

Accessing this document will incur an additional charge of $.

After purchase, you can access this document again without charge.

Accept $ Charge
throbber

Still Working On It

This document is taking longer than usual to download. This can happen if we need to contact the court directly to obtain the document and their servers are running slowly.

Give it another minute or two to complete, and then try the refresh button.

throbber

A few More Minutes ... Still Working

It can take up to 5 minutes for us to download a document if the court servers are running slowly.

Thank you for your continued patience.

This document could not be displayed.

We could not find this document within its docket. Please go back to the docket page and check the link. If that does not work, go back to the docket and refresh it to pull the newest information.

Your account does not support viewing this document.

You need a Paid Account to view this document. Click here to change your account type.

Your account does not support viewing this document.

Set your membership status to view this document.

With a Docket Alarm membership, you'll get a whole lot more, including:

  • Up-to-date information for this case.
  • Email alerts whenever there is an update.
  • Full text search for other cases.
  • Get email alerts whenever a new case matches your search.

Become a Member

One Moment Please

The filing “” is large (MB) and is being downloaded.

Please refresh this page in a few minutes to see if the filing has been downloaded. The filing will also be emailed to you when the download completes.

Your document is on its way!

If you do not receive the document in five minutes, contact support at support@docketalarm.com.

Sealed Document

We are unable to display this document, it may be under a court ordered seal.

If you have proper credentials to access the file, you may proceed directly to the court's system using your government issued username and password.


Access Government Site

We are redirecting you
to a mobile optimized page.





Document Unreadable or Corrupt

Refresh this Document
Go to the Docket

We are unable to display this document.

Refresh this Document
Go to the Docket