United States Patent [19]
`Ondeyka et al.
`
`[54] LIPOPHILIC MACROLIDE USEFUL AS AN
`IMMUNOSUPPRESSAro..'T
`Inventors: John Ondeyiul, Fanwood; Otto
`HenseDS, Red Bank; Jerrold Liesch,
`Princeton Junction. all of N.J.
`
`[75]
`
`[73] Assignee: Merck" Co., Inc., Rahway. N.J.
`[21] Appl. No.: 690,407
`[22] Filed:
`Apr. 23, 1991
`[51]
`Int. Cl.s ................... A61K 31/395; C07D 491/16
`[52] U.s. Cl •.................................... 514/291; 540/456;
`540/452
`[58] Field of Search ................ 540/546. 542; 514/291.
`514/63
`
`[561
`
`References Cited
`U.S. PATENT DOCUMENTS
`3,929,992 1211975 Sehgal et aI ........................ 424/124
`4,316,885 211982 Rakhit ................................... 546/90
`4,401,653 8/1983 Eng ..................................... 424/114
`4,650,803 3/1987 Stella et al. ......................... 514/491
`
`OTHER PUBLICATIONS
`H. Baker et aI., J. Antibiotics, vol. 31 (6) pp. 539-545.
`J. P. Devlin and K. D. Hargrave. Tetrahedron, vol. 45
`(14). pp. 4327-4369 (1989).
`C. P. Eng et aI., J. Antibiotics, vol. 37 (10) pp.
`1231-1237 (1984).
`J. A. Findlay and L. Radics, Can. J. Chern., vol. 58, pp.
`579-590 (1980).
`J. A. Findlay et aI., Can. J. Chern., vol. 60, pp. 2046
`(1982).
`M. W. Harding et aI., Nature. vol. 341, pp. 758-760
`(1989).
`S. N. Sehgal et al.. J. Antibiotics, vol. 28 (10), pp.
`727-732 (1975).
`S. N. Sehgal et al., J. Antibiotics. vol. 36 (4), pp.
`351-354 (1983).
`K. Singh et aI .• J. Antibiotics, vol. 32 (6), pp. 630-645
`(1979).
`.
`
`111111111111111111111111111111111111111111111111111111111111111111111111111
`USOO5091389A
`[11] Patent Number:
`[45] Date of Patent:
`
`5,091,389
`Feb. 25, 1992
`
`S. M. Stepkowski et al., Transplantation Proc., vol. 23
`(1), pp. 507-508 (1991).
`M. J. Tocci, J. Immunology, vol. 143 (2). pp. 718-726
`(1989).
`C. Vezina, J. Antibiotics, vol. 28 (10), pp. 721-726
`(1975).
`
`Primary Examiner-Robert T. Bond
`Attorney, Agent, or Firm-Curtis C. Panzer; Hesna J.
`Pfeiffer
`
`ABSTRACf
`[57]
`Disclosed is a novel lipophilic macrolide of assigned
`Formula I:
`
`OH
`
`OOCH'
`QH3C ..... r····/· "
`1'"
`
`o
`
`II
`o
`
`N
`
`HO
`
`The compound of assigned Formula I is an analog of
`rapamycin which has activity as an antifungaI agent and
`as an immunosuppressant.
`
`3 Claims, 1 Drawing Sheet
`
`NOVARTIS EXHIBIT 2041
`Par v Novartis, IPR 2016-00084
`Page 1 of 6
`
`

`
`u.s. Patent
`
`Feb. 25, 1992
`
`5,091,389
`
`NOVARTIS EXHIBIT 2041
`Par v Novartis, IPR 2016-00084
`Page 2 of 6
`
`

`
`1
`
`5,091,389
`
`LIPOPHILIC MACROLIDE USEFUL AS AN
`IMMUNOSUPPRESSANT
`
`BACKGROUND OF THE INVENTION
`This invention relates to macrolides having activity
`as an antifungal agent and as an immunosuppressant.
`In particular, this invention relates to analogs of the
`compound rapamycin, which is a compound of the
`following formula:
`
`2
`expected to share a broad range of utilities as immuno(cid:173)
`suppressive agents. CycJosporin A, FK-506, rapamycin
`and analogs thereof find utility in the prevention of
`rejection or organ and bone marrow transplants; and in
`5 the treatment of psoriasis, and a number of autoimmune
`disorders such as type 1 diabetes mellitus, multiple scle(cid:173)
`rosis, autoimmune uveitis, and rheumatoid arthritis.
`Additional indications are discussed infra.
`
`10
`
`SUMMARY OF THE INVENTION
`This invention relates to a compound of assigned
`Formula I:
`
`15
`
`20
`
`25
`
`30
`
`which is useful as an antifungal agent and is useful in the
`suppression of the immune response.
`As early as 1975, raparnycin was identified as an anti- 35
`fungal antibiotic harvested from a Streptomyces hygro(cid:173)
`scopic us culture, which culture was isolated from an
`Easter Island soil sample. See Vezina et aI., J. Antibiot.
`28,721-726 (1975); and U.S. Pat. No. 3,929,992, which
`issued to Sehgal, et. a!. Dec. 30, 1975. The ability of this 40
`compound to inhibit the immune response was first
`described by Martel, R. et aI., Can. J. Physiol. Phar(cid:173)
`macol., 55, 48-51 (1977). In this work, the authors show
`the utility of this compound in inhibiting the response to
`allergic encephalomyelitis, adjuvant-induced arthritis 45
`and antibody production in rats. More recently, CaIne,
`R. Y. et aI., has shown rapamycin to be immunosuppres(cid:173)
`sive in rats given heterotopic heart allografts. CaIne, R.
`Y. et al., Lancet vol. 2, p. 227 (1989). Equally important,
`less toxicity was said to be experienced than would be SO
`anticipated with FK-S06 (U.S. Pat. No. 4,894,366, as(cid:173)
`signed to Fujisawa, which issued on Jan. 16, 1990), with
`which rapamycin shares some structural features.
`More recently, rapamycin has been shown to be use-
`ful in combination therapy with Cyclosporin A. This 55
`combination has the advantage of reducing the amount
`of Cyclosporin A
`required
`to produce
`its
`im(cid:173)
`munosupressive effect, such as in heart, kidney, bowel,
`pancreas or other transplantation, and thereby effec(cid:173)
`tively reducing the nephrotoxicity inherent in treatment 60
`with Cyclosporin A. See Stepkowski, S. M. et aI.,
`Transplantation Proceedings, vol. 23, pp 507-508
`(1991).
`As appreciated by those of skill in the art, and as
`exemplified by Harding, M. W. et aI., Nature, vol. 341, 6S
`p. 758-760(1989) and Devlin, J. P. and Hargrave, K. D.
`Tetrahedron, vol. 45, p. 4327-4369 (1989), CycJosporin
`A, FK-S06, rapamycin, and analogs thereof, can be
`
`which compound is a useful antifungal agent and immu(cid:173)
`nosuppressive agent.
`
`BRIEF DESCRIPTION OF THE DRAWINGS
`FIG. 1 is the 400 MHz 'H-NMR spectrum of the
`compound of assigned Formula I recorded in CD2Cb.
`
`bET AILED DESCRIPTION OF THE
`INVENTION
`This invention relates to a compound of Formula I,
`
`OH
`
`OOC"'
`H3C ..... r .. ·· .. ··
`.....
`1· .. · ..
`
`o
`
`II
`o
`
`Q
`
`N
`
`. ......... .
`
`The compound of Formula I may also be described as
`29-desmethyl rapamycin. The invention also relates to
`
`NOVARTIS EXHIBIT 2041
`Par v Novartis, IPR 2016-00084
`Page 3 of 6
`
`

`
`5,091,389
`
`.
`
`3
`substantially pure compound of assigned Formula I.
`For purposes of this specification substantially pure
`shall designate a purity in excess of 98% and free of
`rapamycin.
`This invention also relates to pharmaceutical compo(cid:173)
`sitions for inducing immunosuppression in a subject in
`need of such treatment, comprising: administration of a
`therapeuticalIy effective amount of 29-desmethyl rapa-
`mycin.
`In view of its immunosuppressive activity, 29- 10
`desmethyl rapamycin is useful for the prophylaxis and
`treatment of diseases and conditions requiring a reduc(cid:173)
`tion of the immune response. Thus it may be used to
`suppress the proliferation of lymphocytes and im(cid:173)
`munocytes, e.g. in treatment of autoimmune diseases or 15
`in preventing the rejection of transplants e.g. skin, lung,
`heart, heart-lung, bone-marrow, kidney, spleen and
`corneal transplants.
`Specific auto-immune diseases for which the com(cid:173)
`pound of formula I is useful includes all of those for 20
`which treatment with cyclosporin A and/or FK-506
`has been proposed or used, for example, aplastic anae(cid:173)
`mia, pure red celI anaemia, isopathic thrombocytopa(cid:173)
`enia, systemic lupus erythematosus, polychondritis, 25
`scleroderma, Wegener granulomatosis, chronic active
`hepatitis, myasthenia gravis, psoriasis, Steven-Johnston
`syndrome, idiopathic sprue, Crohn's disease, Graves
`opthalmopathy, sarcoidosis, multiple sclerosis, primary
`biliary cirrhosis, primary juvenile diabetes, uveitis pos- 30
`terior, interstitial lung fibrosis and psoriatic arthritis as
`welI as insulin-dependent diabetes mellitus, nephrotic
`syndrome and AIDS.
`This invention also relates to a pharmaceutical com(cid:173)
`positions for inducing immunosuppresion in a subject in 35
`need of such treatment, comprising a therapeutically
`effective amount of Cyclosporin A and 29-desmethyl
`rapamycin.
`This invention also relates to a method of inducing
`immunosuppression in a subject in need of such treat- 40
`ment, comprising administration of a therapeutically
`effective amount of 29-desmethyl rapamycin.
`The compound of assigned Formula I can be conve(cid:173)
`niently prepared by fermentation of a culture of Strep(cid:173)
`tomyces hygroscopicus such as NRRL 5491, which 45
`strain can be obtained from the culture collection at the
`National Center for Agricultural Utilization Research,
`USDA, ARS, Peoria, Ill. NRRL 5491 is also available
`from the American Type Culture Collection, Rockville,
`Md. as as A TCC 29253. This organism, and procedures 50
`for its cultivation are described in Vezina et al., J. An(cid:173)
`tibiot. 28, 721-726 (1975); Sehgal et al J. Antibiot. 28,
`727-732, and U.S. Pat. No. 3,929,992; said references
`being hereby incorporated by reference.
`As appreciated by those of skill in the art, microor- 55
`ganisms for production of 29-desmethyl rapamycin may
`include other natural or artificial mutants or variants
`derived from the described culture. The artificial pro(cid:173)
`duction of mutant strains may be achieved by physical
`or chemical mutagens, for example, ultraviolet irradia- 60
`tion or N-nitrosoguanidine treatment and the like. Re(cid:173)
`combinant DNA techniques such as protoplast fusion,
`plasmid incorporation, gene transfer and the like are
`also envisioned.
`In general cultivation of NRRL 5491 can be carried 65
`out by conventional aerobic fermentation of suitable
`nutrient media which contain sources of assimilable
`carbon, nitrogen and inorganic salts.
`
`4
`In general; many carbohydrates such as glucose, mal(cid:173)
`tose, mannose,. sucrose, starch, glycerin, millet jelly,
`molasses, soy bean and the like can be used as sources of
`assimilable carbon. Sources of assimilable nitrogen in-
`5 cludes such materials as yeast and casein hydrolysates,
`primary yeast, yeast extracts, cottonseed flour, soybean
`solids, wheat germ, meat extracts, peptone, corn steep
`liquor, and ammonium salts. The inorganic salt nutri-
`ents which can be incorporated in the culture medium
`are the customary salts yielding sodium, iron, magne(cid:173)
`sium, potassium, cobalt, phosphate and the like. In gen-
`eral, of course, the techniques employed wiII be chosen
`having regard to industrial efficiency. The nutrient
`media described herein are merely illustrative of the
`wide variety of media that may be employed and are not
`intended to be limiting.
`The fermentation has been carried out at tempera-
`tures ranging from about 22° C. to 32" C.; however, for"
`optimum results it is preferable to conduct the fermenta(cid:173)
`tion at about 28° C. The pH of the medium is controlled
`at about pH 6-7 by the use of suitable organic or inor-
`ganic buffers incorporated into the fermentation me(cid:173)
`dium or by periodic addition of a base. Good yields of
`29-desmethyl rapamycin can be achieved within 48 to
`72 hours. Variation of the medium or the microorgan(cid:173)
`ism will alter the yield of the compound of 29-
`desmethyl rapamycin and/or its rate of production. The
`preferred media compositions are set forth in the exam(cid:173)
`ples.
`Specific examples of fermentation isolation and re(cid:173)
`covery conditions we have found to be advantageous
`are provided in the Examples Section below.
`As stated above, in view of its immunosuppressive
`activity, 29-desmethyl rapamycin is useful for the pro(cid:173)
`phylaxis and treatment of diseases and conditions re(cid:173)
`quiring a reduction of the immune response. Thus they
`may be used to suppress the proliferation of lympho(cid:173)
`cytes and immunocytes, e.g. in treatment of autoim(cid:173)
`mune diseases or in preventing the rejection of trans(cid:173)
`plants e.g. skin, lung, heart, heart-lung, bone-marrow,
`kidney, spleen and corneal transplants.
`Specific auto-immune diseases for which the 29-
`desmethyl rapamycin are useful include all of those for
`which treatment with cyclosporin and FK-506 have
`been proposed or used, for example, aplastic anaemia,
`pure red cell anaemia, isopathic thrombocytopaenia,
`systemic lupus erythematosus, polychondritis, sclero(cid:173)
`derma, Wegener granulomatosis, chronic active hepati(cid:173)
`tis, myasthenia gravis, psoriasis, Steven-Johnston syn(cid:173)
`drome, idiopathic sprue, Crohn's diseases, Graves op(cid:173)
`thalmopathy, sarcoidosis, multiple sclerosis, primary
`biliary cirrhosis, primary juvenile diabetes, uveitis pos(cid:173)
`terior, interstitial lung fibrosis and psoriatic arthritis as
`well as insulin-dependent diabetes mellitus, nephrotic
`syndrome and AIDS.
`Moreover, the compound of assigned Formula I can
`be used in combination therapy with Cyclosporin A as
`discussed in Stepkowski, S. M. et al., Transplantation
`Proceedings, vol. 23, pp 507-508 (1991), which is
`bereby incorporated by reference.
`In addition the compound of assigned Formula I can
`be used as an antifungal agent.
`For all these uses the dosage will, of course, vary
`depending on the compound employed, mode of admin(cid:173)
`istration and treatment desired. However, in general,
`satisfactory results are obtained when administered at a
`daily dosage of from about 1 mg to about 200 mg per kg
`animal body weight, conveniently given in divided
`
`NOVARTIS EXHIBIT 2041
`Par v Novartis, IPR 2016-00084
`Page 4 of 6
`
`

`
`5,091,389
`
`6
`rapamycin was not reversed by exogenous IL-2 (50
`units/ml).
`
`EXAMPLE 1
`PRODUCTION OF 29-DESMETHYL
`RAPAMYCIN
`
`5
`doses 2 to 4 times a day or in sustained release form. For
`the larger mammals, the total daily dosage is in the
`range from about 50 to about 5000 mg, and dosage
`forms suitable for oral mg (e.g. 25-300 mg) of the com(cid:173)
`pounds admixed with a solid or liquid pharmaceutical 5
`carrier or diluent.
`The present invention also provides a pharmaceutical
`composition comprising a compound of formula I such
`29-desmethyl rapamycin is produced from fermenta-
`tion of Streptomyces Hydroscopicus NRRL 5491. The
`as in association with a pharmaceutical carrier or dilu-
`ent.
`10 ~ train was developed through four stages as follow-
`mg:
`Such compositions may be in the form of, for exam-
`a) fIrst stage is B flask (250 ml unbaffied Erlenmeyer
`pIe, a solution, a tablet or a capsule and in ointments
`especially for the treatment of psoriasis.
`flask) with 40 ml of seed medium as: yeast extract
`F1DCO 20 gil, HY-CASE SF 20 gil, cerelose 20 gil,
`29-desmethyl rapamycin may be administered by any
`conventional route, in particular in accordance with 15 potassium nitrate 2 gil, POLYGLYCOL (as antifoam)
`0.3 mill and trace elements mix as: FeS04 6H20 0.025
`means currently practiced in relation to administration
`of cycIosporin, in particular via intravenous infusion,
`gil, NaCI 0.5 gil, MgS04 7H20 0.5 gil, MnS04 H20
`0.005 gil, ZnS04 7H20 om gil, eaCh 2H20 0.02 gil,
`e.g. in the case of organ transplant, pre- and immedi-
`sterilized as 121° C. for 25 min. is inoculated with 0.3 mI
`ately post-transplant, as welI as during episodes of gas-
`trointestinal disturbance which might otherwise impair 20 of suspended in sterile water spore inoculum, and incu-
`absorption, or orally, e.g. in the form of an oral solution.
`bated at 28° C. for 72 hours on the 220 rpm shaker.
`b) second stage of seed in C flask (2000 mI unbaffied
`Biological activity as a immunosupressant can be
`measured in terms of inhibition of T -celI proliferation.
`Erlenmeyer flask) with 500 ml seed medium is inocu-
`lated with 7.5 mI of ftrSt stage and incubated at 28° C.
`T -celI proliferation was measured in mouse T -celI
`cultures stimulated with ionomycin plus phorbol myris- 25 for 48 hours on 220 rpm shaker.
`c) third stage of seed is cultivated in 300 liters (75
`tate acetate (PMA). Spleen celI suspensions from
`C57Bl/6 mice were prepared and separated on nylon
`gallons) stainless steel agitated fermenter with tempera-
`wool columns. The recovered T -celIs were suspended
`ture, pH and DO control. Fermenter with 180 liters of
`at 1()6 cellslml in complete culture medium with addi-
`seed medium previously sterilized at 121° C. for 20 min.
`tion of ionomycin (250 nglml) and PMA (10 ng/ml). 30 is inoculated with three C flasks of second stage seed
`(0.8% inoculum) and grow at 27° C. for 68 hours.
`The cell suspension was immediately distributed in 96
`well-flat bottom microculture plates at 200 JLl/well.
`.
`Control medium or various concentrations of test com-
`ProductIon stage is run in 800 liters (200 gallons)
`stainless steel agitated fermenter equipped with auto(cid:173)
`pound were added in triplicate wells at 20 JLl/well.
`matic temperature, air flow, back pressure, pH and
`Parallel cultures were set up with exogenous IL-2 (50 35 dissolved oxygen controllers. All fermenters are
`units/ml). The plates were incubated as 37° C. in a
`charged with 500 liters of an aqueous production me-
`humidified atmosphere of 5% C02-95% air for 44
`hours. The cultures were then pulsed with tritiated-
`dium consisting of the following ingredients: cerelose
`thymidine (2 uCilwelI) for an additional 4 hour period
`20 gil, NUTRISOY 30 gil, glycerol 20 gil, L-lysine 4
`and cells were collected on fiber glass filters u~ing a 40 gil, ~onium sulfate. 5 gil potassiu~ t;>hosphate
`muitisample harvester. Incorporated radioactivity was
`monobasIC 2.5 gil, potassIUm phosphate dlbaslc 2.5 gil,
`measured in aBET APLA TE COUNTER (PHAR-
`·and POL YGLYCOL P-2000 (as antifoam agent) 2
`MACIA/LKB, Piscataway, N.J.) and the mean count
`mIll. The media are sterilized at 121° C. for 25 min.,
`per minute (cpm) values of triplicate samples calculated.
`cooled .to 27° C. and the pH is. adjusted. with sodium
`The percent inhibition of proliferation was calculated
`hydrOXIde to 6.5 before the seed mtroductlOn to produc-
`according to the formula:
`45 tion medium. All batches are inoculated with 25 liters
`(5% inoculum) of third stage seed and 88 hours of fer(cid:173)
`mentation cycle is controlled at temperature 27° C., pH
`6-7 and 50% of oxygen saturation.
`
`% Inhib. = 100 _ mean cpm experimental
`mean cpm control medIUm x 100
`
`This assay is described in detail in Dumont, F. J. et al, J. 50
`Immuno!. (1990) 144:251
`
`INHIBITION OF T CELL PROLIFERATION
`STIMULATED WITH IONOMYCIN + PMA BY
`29·DESMETHYL RAPAMYCIN
`29-desmethyl-rapamycin
`Percent Inhibition
`Concentration (JLM)
`of Proliferation
`
`11.2
`1.2
`0.12
`0.01
`0.001
`
`88
`S9
`49
`32
`7
`
`29-desmethyI-rapamycin was found to inhibit the
`proliferation of mouse T cells stimulated with 65
`ionomycin+PMA. Under the same conditions, 1.1 JLM
`rapamycin inhibited the proliferation by 65%. As for
`rapamycin, the inhibitory activity of 29-desmethyl-
`
`EXAMPLE 2
`Isolation of 29-Desmethyl Rapamycin from
`Fermentation Broth
`330 gallons of fermentation broth was dewatered via
`55 a WESTF ~LIA decanter. The product was extracted
`into approximately 250 gallons of methanol from the
`cell cream to yield approximately 253 gallons of metha(cid:173)
`nol extract. The cell cream was again extracted into
`about 75 gallons of methanol to yield about 78 gallons of
`60 extract. The methanol extracts were combined and
`concentrated partially in a vacuum evaporator to 85
`gallons. The extract was then washed twice with hex(cid:173)
`ane (80 gallons each wash) and further concentrated to
`25 gallons. The product in the concentrated methanol
`solution was extracted into ethyl acetate via two extrac(cid:173)
`tions. The ethyl acetate extracts (approximately 16 gal-
`lons each) were combined and concentrated to 5 gal(cid:173)
`lons. Precipitates of impurities that formed during con-
`
`NOVARTIS EXHIBIT 2041
`Par v Novartis, IPR 2016-00084
`Page 5 of 6
`
`

`
`5,091,389
`
`8
`IHNMR
`The IH NMR spectrum of cuts 23-25 in CD2Ci2 is
`shown in FIG. 1. The spectrum was recorded at 400
`MHz on a VARIAN XL400 NMR spectrometer at 21·
`C. Chemical shifts are shown in ppm relative to TMS at
`zero ppm using the solvent peak at 5.32 ppm as the
`internal standard.
`What is claimed is:
`1. A compound of Formula I,
`
`7
`centration were filtered off. Filtrate was loaded onto a
`20-gallon silica gel column, which had been equilibrated
`with 30/70% acetonelhexane solution. The column
`was then eluted with 10 bed volumes of 30/70% aceto(cid:173)
`nelhexane solution, and fractions of 5 gallons in size 5
`were collected. The cut richest in rapamcyn like com(cid:173)
`pound (e.g. cuts 12 to 18) were then concentrated fur(cid:173)
`ther.
`The combined rich cuts containing about 12 G of
`rapamycin in 3 liters of ethyl acetate was charged to a 10
`30 gallon silica gel column (GRACE silica) and eluted
`with 3:1, hexane:acetone. 36 5-gallon cuts were taken
`and rapamycin found in cuts 11-19. Cuts were analyzed
`by silica TLC and HPLC. WHA TMAN silica gel 60 15
`TLC plates were used with a 95:5, methylene chloride:(cid:173)
`methanol solvent system and visulization was by UV or
`iodine staining. WHA TMAN ODS-3 analytical column
`was used with a SPECTRA PHYSICS 8700 pumping
`system at 40 C using methanol:water (8-2) at a flow rate 20
`of 1.5 min/ml monitoured at 277 nM. The retention time
`was 8.5 min. Four related minor components were de(cid:173)
`tected in cuts 9-10 and 16-20 based on uv ratio by
`HPLC. Cuts 16-20 were dissolved in etoaclhex/ace
`and charged to a 2 liter silica gel column in 3; 1 hexane- 25
`:acetone. Cuts 36-46 contained rapamycin while cuts
`47-50 contained some minor components as well as cuts
`51-5Z. Cuts 51-52 (0.5 g) was dissolved in 3 ml metha(cid:173)
`nol and 1 ml charged to a 25 cc X 22 mm DS-3 column
`and eluted with methanol:water (8-2) at 7 mllmin and 7 30
`ml cuts collected. This was repeated twice. Cuts 23-25,
`from all 3 chromatographies, contained compound 1 (25
`mg). This material was subjected to NMR and MS
`studies as well as biological assay.
`
`FAB-MS
`Cuts 23-25 was found to have a molecular weight of
`899 as determined by FAB-MS (observed (M+Na) at
`m/z 922, and in the lithium spiked spectrum (M + Li) at 40
`m/z 906. In contrast to rapamycin, the m/z 541 ion is
`absent in the EI spectrum; but a new ion is observed at
`m/z 527.
`
`or a pharmaceutically acceptable salt thereof.
`2. A pharmaceutical compositions for inducing im-
`35 munosuppression in a subject in need of such treatment,
`comprising:
`a pharmaceutical carrier and a therapeutically effec(cid:173)
`tive amount of compound according to claim 1.
`3. A method of inducing immunosuppression in a
`subject in need of such treatment, comprising adminis(cid:173)
`tration to said subject a non toxic therapeutically effec(cid:173)
`tive amount of compound according to claim 1.
`• • • • •
`
`45
`
`50
`
`55
`
`60
`
`65
`
`NOVARTIS EXHIBIT 2041
`Par v Novartis, IPR 2016-00084
`Page 6 of 6