United States Patent [19]
`Dreyfuss et al.
`
`lllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllll
`5,116,816
`May 26, 1992
`
`1150051 16816A
`[11] Patent Number:
`[45] Date of Patent:
`
`[54] CYCLOSPORIN PEPTOLIDES HAVING AN
`a-HYDROXYCARBOXYLIC ACID AT
`POSITION 8
`
`[75] Inventors: Michael M. Dreyfuss; Max H.
`Schreier, both of Basel; Hans
`Tscherter, Allschwil; all of
`Switzerland
`
`[73] Assignee: Sandoz Ltd., Basel, Switzerland
`
`[21] Appl. NO.: 209,680
`
`[22] Filed:
`
`Jun. 20, 1988
`
`Foreign Application Priority Data
`[30]
`Jun. 19, 1987 [CH] Switzerland ....................... .. 2317/87
`Jul. 2. 1987 [CH] Switzerland ....................... .. 2517/87
`
`[51] Int. c1.5 ..................... .. A61K 37/02; c071< 7/50;
`C07K 7/54; CO7K 11/02
`[52] us. c1. .................................... .. 514/11; 514/885:
`530/317; 530/323; 530/335; 530/338; 530/345;
`435/711; 435/254
`[58] Field of Search .................. .. 530/323, 317; 514/9.
`514/11,.885
`
`[56]
`
`References Cited
`US. PATENT DOCUMENTS
`
`7
`
`4,220.641 9/1980 Traber et al. ....................... .. 514/11
`4.384396 5/1983 Bollinger et al. .
`.... .. 530/321
`4,649,047 3/1987 Kaswan .............. ..'. .............. .. 514/11
`
`OTHER PUBLICATIONS
`Rudinger, Peptide Hormones, Parsons (Ed), U. Park
`Press, Baltimore, pp. 1-7 (1976).
`Twentyrnan et al., Br. J. Cancer, vol. 56, pp. 55-57
`(1987).
`Primary Examiner-Christina Chan
`Attorney. Agent, or Firm—-Gera1d D. Sharkin; Robert S.
`Honor; Thomas O. McGovern
`[57]
`ABSTRACT
`Cyclic peptolides having the structure of a cyclosporin
`in which one amide linkage is replaced by an ester link
`age are obtained by fermentation of fungal strains of the
`genus Cylindrotrichum Bonorden, or by cyclization of
`a hydroxy-undecapeptide. The cyclic peptolides have
`immunosuppressive, anti-inflammatory and anti-para
`sitic properties.
`
`9 Claims, N0 Drawings
`
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`

`
`1
`
`5,116,816
`
`CY CLOSPORIN PEPTOLIDES HAVING AN
`a-HYDROXYCARBOXYLIC ACID AT POSITION 8
`
`This invention relates to novel cyclic peptolides use
`ful as pharmaceuticals.
`The term peptolide is used herein to mean a natural or
`synthetic compound consisting of a-hydroxy and a
`amino acids joined together by both amide and ester
`linkages. Thus the structure obtained by replacing an
`amide linkage by an ester linkage in a peptide is a pepto
`lide.
`An important class of peptides is the cyclosporins,
`which are characterised by a cyclic structure, normally
`comprising 11 amino acid residues, one of which is the
`N-Methyl-(4R)-4-but-2E-en~l-yl-4-methyl-(L)-threonyl
`residue, designated MeBmt, or a derivative thereof.
`Many cyclosporins have pharmacological properties,
`particularly immunosuppressive and antiinflammatory
`properties. The ?rst cyclosporin to be isolated was the
`naturally occurring fungal metabolite cyclosporin A,
`(Ciclosporin) sold commercially under the registered
`Trade Mark Sand Immune
`This compound has the
`structure indicated in formula I
`
`2
`-continued
`CH3
`\
`
`CH3
`
`MeBmtl-Ig
`
`15
`
`The preferred cyclic peptolides according to the
`invention have the structure shown in formula II
`
`20
`
`MeBmt-Abu-Sar-MeLeu-Val-MeLeu-Ala-(D)Ala—MeLeu-MeLeu-MeVal
`10
`ll
`l
`2
`3
`4
`5
`6
`7
`8
`9
`
`F
`
`I 6
`
`‘l
`
`(For a complete list of abbreviations used herein. see
`Table II)
`‘By convention, the amino-acid residues of cyclospo
`rins are have the (L) con?guration unless otherwise
`shown; thus in formula I the alanine at position 8 has the
`i (D) con?guration. The symbol Me before the abbrevia
`tion for an amino acid signifies that the amino acid
`residue is N-methylated on the nitrogen in the amide
`linkage.
`The present invention provides a cyclic peptolide
`which has the structure of a cyclosporin in which one
`amide linkage is replaced by an ester linkage.
`Preferably the cyclic peptolide is composed of one
`MeBmt residue or a derivative thereof, 9 other a-amino
`acid residues and one a-hydroxyacid residue, which is
`preferably located at position 8.
`Preferred derivatives of MeBmt are the 8'hydroxy
`derivative (8’-OHMeBmt) and the saturated dihydro
`derivative MeBmtI-IZ, having the structures shown be
`low;
`
`35
`
`45
`
`in which
`W is MeBmt. 8’-OHMeBmt or MeBmtI-Ig,
`X is Sar or Gly,
`Y is MeLeu or Leu,
`Z is Leu, Ile or Val,
`and A is the residue of an a-hydroxycarboxylic acid,
`preferably of formula III
`
`R
`
`111
`
`55
`
`More preferably in formula III R is isopropyl, so that
`A represents
`
`CH3
`
`.CH3
`
`(Ill-I
`
`-O—Cl-l—CO--.
`
`65
`
`the residue of a-hydroxy isovaleric acid, abbreviated
`Hiv. The most preferred compound according to the
`invention is that in which, in formula II, W is MeBmt, X
`is Sar, Y is MeLeu, Z is Leu, and A is (D)l-Iiv. This may
`be represented by the full structural formula shown in
`formula IV~
`
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`
`5,116,816
`
`or by using the now conventional nomenclature for
`cyclosporins, based upon the structure of Ciclosporin
`(cyclosporin A) shown in formula 1. This is done by
`listing in order each residue in the molecule which
`differs from that found in Ciclosporin, and adding the
`term “Ciclosporin". Thus the compound of formula IV
`may be represented as
`(Thr)3(Leu)5(D-Hiv)5(Leullo-ciclosporin.
`
`30
`
`35
`
`55
`
`that is. Ciclosporin in which Thr replaces Abu in posi
`tion 2, Leu replaces Val in position 5, (D)Hiv replaces
`(D)Ala in position 8 and Len replaces MeLeu in posi
`tion 10, the other residues being identical with those in 40
`Ciclosporin.
`The cyclic peptides according to the invention may
`be produced by cultivating a producing microorganism
`strain in a nutrient medium. Preferred microorganisms
`are fungal strains of the genus Cylindrotrichum Bo- 45
`norden, in particular the strain NRRL 18230, which
`produces cyclic peptolides of formula II.
`The strain has been isolated from a leaf sample from
`Waldenburg in the Swiss Jura, and a viable culture of
`the strain was deposited on Jun. 17, 1987 at the US
`50
`Department of Agriculture (North Central Region,
`Northern Regional Research Centre), Peoria, 111. and
`was given the reference number NRRL 18230. The
`culture may also be obtained from Sandoz Ltd., Basle,
`Switzerland.
`The fungal strain NRRRL 18230 is a hyphomycete
`and when incubated at 2l°—24° C. on 2% malt extract/a
`gar (=MA; 2% malt extract, 0.4% yeast extract, 2%
`agar in demineralized water) produces aseptate or fre
`quently l-septate bacilliform hyaline conidia, 6-15u X 1
`5-2.7}; (mostly 9.5—l3.5p.) large.
`The conidiogenic cells are generally cylindrical and
`have a conspicuous colarette; some cells appear sym
`podial-polyphialidic. According to the identi?cation
`key of M. B. Elles (Dematiaceous Hyphomycetes;
`Commonwealth Mycological Institute, Kew, Surrey,
`England, 1971), the strain may best be classi?ed in the
`genus Cylindrotrichum Bonorden.
`
`60
`
`65
`
`The fungal strain NRRL 18230 grows relatively
`slowly and after 10 days incubation at a temperature of
`21° C. forms colonies of 4-7 mm diameter with a vel
`vety grey aerial mycelium. The optimum growth tem
`perature is between 18° C. and 27° C., and above 33° C.
`no growth occurs. The branched and septate aerial
`mycelium of colonies cultivated on MA at 21° C. is
`generally 1.5-3.5}; (usually 2-3p.) wide; in the substrate
`mycelium hyphae of up to 5.5p. width can be observed.
`The invention also provides fermentation broths ob
`tained by cultivation of a strain of the fungal genus
`Cylindrotrichum Bonorden. The novel strain NRRL
`18230 may be cultivated by an aerobic surface or im
`mersion process at suitable temperature in a variety of
`nutrient media containing the nutrients and minerals in
`usuable form.
`Thus, the medium should contain an assimilatable
`source of carbon and optionally mineral salts and
`growth factors. All of these constituents may be added
`in the form of well-de?ned simple compounds or in the
`form of complex mixtures obtained from biological
`sources. Cultivation is carried out according to conven
`tional methods, and the cyclic peptolides formed during
`the fermentation may ?nally be isolated from the cul
`ture medium by the use of known chromatographic
`methods. The cyclic peptolides of the invention may
`also be obtained by the cultivation of variant or mutant
`strains obtained by selection or by the effect of muta
`tion-inducing agents e.g. U.V. light, X-rays or chemical
`mutagens on NRRL 18230 or other strains of Cylindro
`trichum Bonorden.
`The cyclic peptolides of the present invention may
`also be prepared by synthetic or semi-synthetic meth
`ods, for example by the cyclisation of a linear peptolide
`or a linear peptide having an —OH terminal group in
`place of an —NHZ terminal; or by the replacement of an
`amide linkage in a natural, synthetic or semi-synthetic
`cyclosporin with an ester linkage.
`The total synthesis of the preferred compounds of
`formula 11 may be carried out in a manner analogous to
`the total synthesis of cyclosporin A and analogues as
`described for example in European Patent 34 567 or by
`
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`

`
`5
`R. Wenger in Transplantation Proceedings, vol. XV pp.
`2230-2241 (1983). According to this method the C
`protected heptapeptide having the formula V
`
`5,116,816
`
`where R is as de?ned above.
`Preferably the group R‘ is selected from the groups
`
`in which 82 is the benzyl group and W, X. Y and Z are
`as de?ned above is ?rst prepared. and this is then re
`acted with a tetrapeptide corresponding to the sequence
`8 through 11.
`This tetrapeptide, of formula VI
`
`0
`
`HO-A-MeLeu-—Leu—-MeVal
`8
`9
`l0
`11
`
`VI
`
`15
`
`contains three normal peptide bonds‘ but has an O-ter
`minal in place of an N-terminal since the residue at
`position 8 is derived from an a-hydroxy acid rather than
`from an a-aminoacid.
`The tetrapeptide may be prepared according to the
`scheme shown in the following flow sheet:
`
`tBuSi(Cl-I3)g—-. The preferred compounds of formula
`VII may be obtained by reacting the a-hydroxy acid, in
`carbonyl-protected from, e.g. as the benzyl ester, with
`dihydrofuran, ethoxyethylene, t-butyldimethylchlorosi
`lane or chlorodimethyl ether respectively.
`Reaction of the COOH-protected heptapeptide V
`with the hydroxy tetrapeptide VI, in OH protected
`form, gives rise to a linear hydroxy undecapeptide of
`formula VII having the sequence 8 through 7.
`
`10
`
`ll
`
`1
`
`2
`
`3
`
`4
`
`5
`
`e
`
`7
`
`VIII
`
`Finally cyclisation of this linear hydroxypeptide may
`be carried out by removing the protecting groups by
`acidic and basic hydrolysis and coupling residue 8 to 7
`with the formation of an ester linkage. The coupling
`reaction is preferably carried out in methylene chloride
`using a carbodiimide reagent for example N-ethyl-N’
`(3-dimethylamino)propyl carbodiimide.
`The heptapeptide of formula V and the tetrapeptide
`of formula VI may also be obtained by controlled hy
`drolysis of cyclic peptolides of formula II obtained from
`fermentation. This treatment with trifluoroacetic acid at
`low temperature cleaves the bond between residues 11
`and l to give a linear undecapeptolide containing resi
`dues l (N-terminal) through 11 (C-terminal), with an
`ester linkage at 7-8. Alkaline hydrolysis gives the 1-7
`heptapeptide and the 8-11 hydroxytetrapeptide. Semi
`synthetic cyclic peptolides may then be produced for
`example by reacting the hydroxytetrapeptide produced
`in this way with a synthetic heptapeptide, or vice versa,
`and cyclising the linear product.
`For the purposes of the cyclisation reaction the pep
`tide may if desired be in O-protected form, that is, the
`hydroxy groups in the l-MeBmt or derivative thereof,
`and/or in the 2-threonine residue may bear protecting
`groups, as described in European Patent 34 567. Such
`O-protecting groups are then removed subsequent to
`ring closure by standard methods. Removal of for ex
`ample a benzyl group by hydrogenation will also lead to
`the hydrogenation of MeBmt to MeBmtl-Ig, and in any
`case initially-produced cyclic peptolides containing a
`MeBmt residue at position 1 may be converted to the
`corresponding MeBmtHg compound by hydrogenation.
`Accordingly the invention provides a process for the
`production of a cyclic peptolide of formula II, stated
`above, which process comprises
`a) removing the O-protecting groups from a cyclic
`peptolide of formula II in O-protected form;
`b) cyclising a straight chain hydroxy-endecapeptide of
`formula VIII, in unprotected form or O-protected on
`one or both of residues 1 and 2, and, when required,
`carrying out process step (a); and, when desired
`
`35
`
`50
`
`55
`
`in which BOC is the N-protecting group t-butyloxycar
`bonyl and R’ is a suitable O-protecting group. Thus the
`reagent represented above as R’—A—OI-I is an Ol-l
`protected a-hydroxycarboxylic acid, which when A is
`of formula III, has the formula VII
`
`65
`
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`

`
`5,116,816
`7
`c) hydrogenating a cyclic peptolide of formula 11 thus
`obtained wherein W is MeBmt to obtain the corre
`sponding cyclic peptolide wherein W is MeBmtllg.
`The cyclic peptolides of the invention exhibit phar
`macological activity and are, therefore, useful as phar
`maceuticals. In particular, the compounds show immu
`nosuppressant, anti-inflammatory and anti-parasitic ac
`tivity in the following tests:
`
`8
`IN VIVO MODELS (4-9)
`4. Formation of plaque forming cells (humoral immune
`response)
`Female rats (OFA) are immunised by the i.v. injec
`tion of(1 X 108) sheep erythrocytes (SRBC) and treated
`on three consecutive days with the drugs under investi
`gation. Spleen cell suspensions are prepared 6 days after
`immunisation and l><l06 lympocytes are plated onto
`soft agar in the presence of indicator cells (SRBC) and
`complement. Lysis of the indicator cells due to secre
`tion of speci?c antibody and presence of complement
`yields plaques. The number of plaques per plate are
`counted and corrected for the number of plaques per
`spleen.
`[N. K. Jerne & A. A. Nordin Science 140:405 (1969);
`N. K. Jerne, A. A. Nordin & C. Henry (1963) In: "Cell
`Bound Antibodies”, B. Amos & H. Koprowski, Eds,
`Wistar Inst. Press, Philadelphia pp. 109-125 (1963)].
`The compounds according to the invention produce
`this effect in the rat when given orally at an ED50 of
`approx. 6.0-8.0 mg/kg.
`5. Localised graft-versus-host-reaction
`Spleen cells (1x107) from 6 week old female Wis
`tar/Furth (WF) rats are injected subcutaneously on day
`0 into the left hind-paw of female (F344XWF)FI rats
`weighing about 100 g. Animals are treated for 4 consec
`utive days and the popliteal lymph nodes are removed
`and weighed on day 7. The difference in weight be
`tween the two lymph nodes is taken as the parameter
`for evaluating the reaction.
`[W. L. Ford, W. Burr & M. Simonsen Transplanta
`tion 10:258-266 (1970)]
`The compounds of the invention have an oral EDjQII‘l
`this test of approx. 20-30 mg/kg.
`
`40
`
`65
`
`6. Freund‘s Adjuvant Arthritis
`OFA and Wistar rats (male or female, 150 g body
`weight) are injected i.c. at the base of the tail or in the
`hind paw with 0.1 ml of mineral oil containing 0.6 mg of
`lyophilized heat-killed Mycobacterium smegmatis.
`Treatment is started on day 14, when the secondary
`in?ammation is well developed (days 14-20). At the end
`of the experiment, the swelling of the joints is measured
`by means of a micro-caliper. ED50 is the oral dose in
`mg/kg orally which reduces the swelling (primary or
`secondary) to half of that of the controls. For the com
`pounds of the invention the oral ED50 is up to 30
`mg/kg.
`[C. A. Winter & G. W. Nuss Arthritis and Rheuma
`tism 9:394-404 (1966)]
`7. Kidney allograft reaction in the rat
`One kidney from a female Fisher 344 rat is trans
`planted onto the renal vessel of a unilaterally (left side)
`nephrectomized Wistar/Furth recipient rat using an
`end-to-end anastomosis. Ureteric anastomosis is also
`end-to-end. Treatment commences on the day of trans
`plantation and is continued for 14 days. A contralateral
`nephrectomy is done seven days after transplantation,
`leaving the recipient relying on the performance of the
`donor kidney. Survival of the recipient is taken as the
`parameter for a functional graft.
`[P. C. Hiestand, et al Immunology 55 249-255 ( 1985)]
`The compounds of the invention are effective in the
`rat at an oral ED50 of from 6 to approx. 9 mg/kg.
`
`5
`
`25
`
`IN VITRO MODELS (l-3)
`l. Interleukin 2 (IL-2) inhibition
`Interleukin 2 is induced by mitogen stimulation of
`mouse spleen cells. Forty eight hour supernatants are
`collected and assayed for their content of IL-2 by use of
`a IL-Z-dependent cell line (CTLL). The growth of
`these cells is assayed after 48 hours by use of an enzy
`matic assay which measures mitochondrial activity.
`[T. Mosmann J. Immunol. Methods 65:55-63 (1983)]
`The compounds of the invention have an inhibitory
`effect at concentrations from IC50 0.01 to approx. 0.1
`ug/ml.
`2. Proliferative Response of Lymphocytes to
`Allogeneic Stimulation
`Murine Mixed Lymphocyte Reaction (MLR)
`Spleen cells (O.5>< 106) from Balb/c mice (female,
`8-10 weeks) are co-incubated for 5 days with 0.5><1O6
`irradiated (2000 rads) or mitomycin C treated spleen
`cells from CBA mice (female, 8-10 weeks). The irradi
`ated allogeneic cells induce a proliferative response in
`the Balb c spleen cells which can be measured by la
`beled precursor incorporation into the DNA. Since the
`stimulator cells are irradiated (or mitomycin C treated)
`they do not respond to the Balb/c cells with prolifera
`tion but do retain their antigenicity.
`[T. Meo “Immunological Methods", L. Lefkovits
`and B. Pernis, Eds., Academic Press, N.Y. pp. 227-239
`(1979)]
`The compounds of the. invention have an inhibitory
`effect at concentrations of from IC50=0.000l to approx.
`0.001 ug/ml.
`3. Primary Humoral Immune Response to Sheep Red
`Blood Cells in vitro (Mishell-Dutton Assay)
`Mouse spleen cells (OFI, female, 8-10 weeks, 1 X107)
`are co-cultured with sheep erythrocytes (SRBC,
`3 X 107) for 3 days in 1 ml final volume in 24 well plates.
`Lymphocytes are harvested, washed and plated at a
`density of l X 106 cells onto soft agar with fresh antigen
`(SRBC). Complement (guinea pig serum) is added after
`a 60-90 minute incubation period and incubation is
`continued for another 60 minutes after which the test is
`evaluated by counting the plaques under the micro
`scope. During the 3 day incubation, the lymphocytes
`are sensitized to the antigen (SRBC). When incubated
`with antigen again, B-lymphocytes secrete speci?c anti
`body which binds to the antigen in the vicinity of the
`secretory lymphocyte. Addition of complement causes
`lysis of the antibody-coated erythrocytes yielding a
`plaque. Each plaque represents a single antibody-pro
`ducing cell.
`[R. I. Mishell & R. W. Dutton J. Exp. Med.
`126:423-442 (1967)]
`The suppression of plaque-forming cells is observed
`at concentrations of compound according to the inven
`tion of from IC5Q0.01 to approx. 0.1 ug/m].
`
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`

`
`9
`8. UV Erythema test
`The test is carried out on female albino guinea pigs.
`approx. 150 g wt. The animals are shaved on both ?anks
`using a Braun micro razor, without causing skin irrita
`tion. For each test substance ?ve animals are subjected
`to a de?ned intensity of UV radiation for 10 seconds on
`each of four areas of skin (10 mm diameter). Immedi
`ately afterwards 50 microliters of a solution of the test
`substance in ethanol/propylene glycol/dimethylaceta
`mide (19:19z2 v/v) is applied to two of the irradiated
`areas on each animal and 50 microliters of the solvent
`mixture to the other two as controls. Four hours after
`application, the degree of erythema is estimated visu
`ally.
`{Raake, W. Arzneim.-Forsch. 34(1) No. 4 (1984)]
`The compounds of the invention are effective at a
`concentration of from l~to 10%.
`
`15
`
`5,116,816
`10
`pound employed, the host, the mode of administration
`and the nature and severity of the condition being
`treated. However. in general, satisfactory results in
`animals are indicated to be obtained at daily dosages
`from about 1 mg/kg to about 200 mg/kg body weight.
`In larger mammals, for example humans, an indicated
`daily dosage is in the range from about 70 to about 3000
`mg of a compound according to the invention conve
`niently administered in divided doses up to four times a
`day.
`The compounds of the invention may be administered
`by any conventional route, in particularly orally, for
`example in the form of tablets or capsules, parenterally
`in the form of injectable solutions or suspensions or
`topically in the form of a lotion, cream or gel.
`The compound of Example 2 : (Thr)2 (Leu)5 (D
`Hiv)8 (Leu)l°-Ciclosporin is the preferred compound. It
`has, for example, been determined that this compound is
`effective at a concentration of 5% in the UV-erythema
`test in the mouse (test method 8 above) as compared to
`a concentration of 2.5% for indomethacin. It is there
`fore indicated that for topical use in treatment of in?am
`matory skin conditions, the compound of Example 2
`may be administered to larger mammals, for example
`humans, by similar modes of administration at corre
`spondingly higher dosages than conventionally em
`ployed with indomethacin.
`It has also been determined that the compound of
`Example 2 has an ED50 value of approx. 25 mg/kg in
`the treatment of localized graft versus host reaction. It
`is therefore indicated that for this indication the com
`pound of Example 2 may be administered orally at daily
`dosages of from 1400 mg to 2100 mg to larger mammals,
`for example humans.
`The present invention also provides pharmaceutical
`compostions containing a compound according to the
`invention in association with at least one pharmaceuti
`cal carrier or diluent. Such‘ composition may be manu
`factured in conventional manner. Unit dosage forms
`contain, for example, from about 20 mg to about 1500
`mg of a compound according to the invention.
`The following Examples, in which all temperatures
`are in degrees Centigrade, illustrate the invention:
`
`EXAMPLE 1
`Culture of strain NRRL 18230 in Erlenmeyer ?ask
`Starting cultures of the strain NRRL 18230 are incu
`bated at 21° for 14 days on MA in slants. A preculture
`is then produced by homogenising the complete con
`tents of one starting culture under sterile conditions,
`transfering to a 500 ml Erlenmeyer ?ask containing 200
`ml nutrient solution M (2% malt extract, 0.4% yeast
`extract in demineralized water), and incubating on a
`rotating shaker at 200 rpm for 10 days at 21".
`Intermediate cultures are then obtained by transfer
`ring 20 ml of the preculture to a 500 ml Erlenmeyer
`?ask containing 200 ml of nutrient solution M and incu
`bating on a rotary shaker at 200 rpm for 7 days at 21".
`Finally for a production culture, 100 Erlenmeyer
`?asks (500 ml) each containing 200 ml of nutrient solu
`tion M are each inocculated with 20 ml of the intermedi
`ate cultures. The ?asks are incubated at 21° in a rotary
`shaker at 200 rpm. After 10 days the 20 liters of fermen
`t-ation broth are combined for the extraction of the
`product.
`
`50
`
`55
`
`65
`
`3O
`
`35
`
`45
`
`9. Anti-malaria test
`Mice (OFI, male) are infected i.p. on day 0 with 0.2
`ml of a suspension of erythrocytes containing 107 cells
`parasitized by Plasmodium berghei (Strain NK 65). The
`test substance is administered s.c. at varying dosages
`using 5 to 10 dose/mice. The survival time is recorded.
`and the minimum effective dose (MED) calculated by
`comparison of survival time with that for untreated
`controls. For controls. survival time is approx. 7 days.
`The MED is the dosage at which survival time is dou
`bled.
`[L. Rane in “Chemotherapy and Drug Resistance in
`Malaria, ed. W. Peters, Academic Press, New York,
`(1979)]
`.
`In view of their immunosuppressive activity, the
`compounds are therefore useful for the prophylaxis and
`treatment of conditions and diseases requiring a sup
`pression of the immune response, for example in the
`treatment of autoimmune diseases, the prevention of
`tissue rejection in transplantation, e. g. bone marrow and
`kidney transplantation. Speci?c autoimmune diseases,
`for which the compounds of the invention may be used
`include aplastic anaemia, pure red cell anaemia. idio
`pathic thrombocytopenia, systemic lupus erythemato
`sis, polychondritis, sceleroderma, Wegner’s granuloma
`tosis, dermatomyositis, chronic active hepatitis, myas
`thenia gravis, Steven-Johnson syndrome, psoriasis, idio
`pathic sprue, Crohn’s disease, Graves’ opthalmopathy,
`sarcoidosis, multiple sclerosis, primary biliary cirrhosis,
`primary juvenile diabetes, posterior uveitis, interstitial
`pulmonary ?brosis and psoriatic arthritis.
`Because of their antiin?ammatory properties the
`compounds of the invention are useful in the treatment
`of inflammatory conditions, particularly in?ammatory
`states with an aetiology which includes an autoimmune
`component, for example the treatment of arthritis and
`rheumatic diseases such as chronic progressive arthritis.
`In view of their anti-parasitic activity, the compounds
`are also useful for the treatment of parasitic disease, for
`example schistosomiasis, ?lariasis, leishmaniasis, coc
`cidioidomycosis and, in particular, malaria.
`The compounds of the invention are also useful in the
`treatment of certain skin diseases and conditions, which
`include, in addition to those already mentioned above,
`alopecia areata, urticaria, vasculitis, erythema, atopic
`dermatitis, eczema, cutanous eosinophilia and angi
`oderrna.
`For these indications, the appropriate dosage will, of
`course, vary depending upon for example the com
`
`NOVARTIS EXHIBIT 2008
`Par v Novartis, IPR 2016-00084
`Page 6 of 8
`
`

`
`5,116,816
`
`12
`
`-continued
`
`Example
`
`Rf. value
`
`3
`
`4
`
`5
`
`6
`
`7
`
`0.308
`
`0.337
`
`0.263
`
`0.242
`
`0.179
`
`11
`EXAMPLE 2
`Isolation of (Thr)2 (Leu)5 (D-I-Iiv)8 (Leu)1°-Cic1osporin
`The 20 liters of culture medium obtained from Exam
`ple 1 are subjected to high-shear mixing in a rod mixer
`(Lutz, Wertheim, Germany) to break up the cells, then
`extracted three times with 20 liters of ethyl acetate. The
`60 liters of organic phase are combined, dried over
`anhydrous sodium sulphate and evaporated to dryness
`under vacuum, giving a residue of 17.4 g.
`The residue is dissolved in 80 ml methanol and chro
`matographed on a 90 mm diameter column containing
`1300 g SEPHADEX LH-20 (Pharmacia Fine Chemi
`cals AB, Uppsala, Sweden). Fractions of 100 ml are
`taken, whereby (Thr)2 (Leu)5 (D-l-liv)8 (Leu)1°-Ciclos
`porin appears in fractions 22-30. These are combined
`and evaporated under vacuum to give 8400 mg of a
`light coloured solid foam. This residue is dissolved in
`wet ethyl acetate and chromatographed on a 55 mm 20
`diameter column containing 500 g KIESELGEL
`(Merck) of particle size 0.04-0.063 mm. The desired
`product is detected in fractions 9-l4 (100 ml fraction
`size). Evaporation gives a pale yellow residue (4200
`mg). Treatment with decolorizing charcoal (Merck) in
`diethyl ether and ?ltration through a thin layer of talc
`gives (Thr)2 (Leu)5 (D-l-liv)8 (Leu)1°-Ciclosporin as a
`white powder, recrystallized from ether m.p. l63°-l64°
`(decomp.);
`la]D1°= -223° (C=l in cHcn).
`EXAMPLES 3-7
`By modi?cation of the above chromatographic tech
`niques, the following compounds may be isolated in
`minor amounts from the production fermentation broth: 35
`
`Ex. No.
`3
`4
`5
`6
`7
`
`Compound
`(Tim2 (D-HMS (Leu)‘O-Ciclosporin
`(Thr)2 (lle)5 (D-l-liv)8 (Lemm-Ciclosporin
`(Tm)2 (Leu)4 (1.6!!)5 (D-Hi\')8 (Leu)1°-Ciclosporin
`(Thr)2 (Gly)3 (Leu)5 (D-l-liv)s (Leu)]O-Ciclosporin
`(8'-OHMeBm1)1 (T1611 (I.€U)5 (D-HMS
`(Lemw-Ciclosporin
`
`The compounds have the properties shown in Table
`
`v
`
`40
`
`45
`
`I:
`
`Com-
`pound
`of Ex.
`No.
`
`TABLE I
`
`formula
`
`MW MW
`calc
`found‘ mp °C.
`
`3
`4
`5
`6
`7
`
`164 (dec)
`C63H112N10014 1233.6 1233.9
`C64H1|4N1o0|4 1247.7 1247.6 151-153 (dec)
`C631-1112N1o014 1233.6 1233.6
`166-168 (dec)
`C63I-l11gN10O14 1233.6 1233.6
`140-143 (dec)
`C64H|44N10O15 1263.7 1263
`15s (sinters)
`'
`195 (dec)
`
`‘by FAB (Fast Atomic Bombardment) mass spectrometry
`
`[Q1132o
`(c =
`in
`MeOH
`
`50
`
`~184"
`-1s0°
`-174° 55
`-154"
`-174‘
`
`EXAMPLE 8
`Synthesis of (Thr)2 (Leu)5 (D-Hiv)8 (Leu)10-Cicl0sporin
`by cyclisation
`
`At room temperature, 2.4 g (2.29 mmol of the unpro
`tected hydroxy-undecapeptide
`
`HO-(D)Hiv-MeLeu-Leu-MeVal-MeBmt-Thr-Sar
`MeLeu-Leu-MeLeu-Ala-Ol-l.
`
`0.84 g (6.88 mmol) of 4-dimethylaminopyridine and 0.66
`g (1.5 equiv, 3.44 mmol) of N-ethyl-N-(3~dime
`thylamino)propyl carbodiimide are dissolved in 145 ml
`methylene chloride and reacted for three days with
`stirring and exclusion of moisture. The resulting solu
`tion is diluted with 300 ml of methylene chlorde, shaken
`with 100 ml of Water aci?fied to pH 2 with 1N HCl,
`filtered through talc and evaporated. The residue is
`chromatographed on 100 g of silica gel using 10%
`M8OH/CH3C13, to give the title compound (2.55 g,
`89%). Identity with the compound of Example 2 is
`established by NMR spectroscopy and [011020 (~220",
`c=1 in CHCl3).
`
`TABLE II
`
`Abbreviations
`
`a-aminobutyric acid
`alanine
`t-butyloxycarbonyl
`benzyl
`glycine
`a-hydroxyisovaleric acid
`isoleucine
`
`lencine
`N-methyl-(4R)-4;but-2E-en-l-yl-4-methyl-(L)
`threonine
`N-methyl-(4R)-4-but-l-yl-4-methyl-(L)-threonine
`
`Abu
`Ala
`BOC
`Bz
`Gly
`Hiv
`lle
`
`Leu
`MeBmt
`
`MeBmtH;
`
`Thin Layer Chromatography
`Thin layer plates Merck: Silica gel
`Carrier system: CH2Cl2-7% CH3OH
`Length of run: 120 mm
`Colouring with iodine vapour
`
`60
`
`65
`
`Sar
`Thr
`Val
`
`sarcosine
`threonine
`valine
`
`Example
`2
`
`Rl' value
`0.358
`
`We claim:
`1. A cyclosporin of formula ll
`
`NOVARTIS EXHIBIT 2008
`Par v Novartis, IPR 2016-00084
`Page 7 of 8
`
`

`
`NOVARTIS EXHIBIT 2008
`Par v Novartis, IPR 2016-00084
`Page 8 of 8