United States Patent [191
`Dumble
`
`US005190972A
`[11] Patent Number:
`[45] Date of Patent:
`
`5,190,972
`Mar. 2, 1993
`
`[54] METHOD OF COMBATTING
`CY CLOSPORINE ORGAN TOXICITY WITH
`PROSTAGLANDIN ANALOGS
`[75] Inventor: Lynette J. Dumble, Victoria,
`Australia
`
`[73] Assignee:
`
`The University of Melbourne,
`Melbourne, Australia
`[21] Appl. No.: 825,669
`[22] Filed:
`Jan. 27, 1992
`
`[51] Int. Cl.5 ............................................ .. A61K 31/35
`
`[52] U.S. Cl. . . . . . . . . . . . . . . . . .
`
`. . . . .. 514/454; 514/569
`
`[58] Field of Search .............................. .. 514/569, 454
`
`[56]
`
`References Cited
`U.S. PATENT DOCUMENTS
`
`4,095,036 6/1978 Yankee ...................... .. 560/121
`4,205,178 5/1980 Axen
`560/121
`.
`4,239,778 12/ 1980 Venton et
`424/305
`4,306,075 12/1981 Aristoff ....... ..
`560/56
`4,977,174 12/1990 Stein et a1. ........................ .Y's14/3s2
`
`V FOREIGN PATENT DOCUMENTS
`
`0347243 12/1989 European Pat. Off. .
`
`OTHER PUBLICATIONS
`R. Klein et al., in RU 486 Misconceptions, Myths and
`Morals (Spinifex Press, Australia) (Aug. 1991), pp.
`82-85, 146-147.
`J. Thomson, “Production of Severe Atheroma In A
`Transplanted Human Heart,” The Lancet (Nov. 1969),
`pp. 1088, 1090, 1092.
`Progress in Medicinal Chemistry, vol. 21 (G. Ellis and G.
`
`West Eds.) (Elsevier Science Publishers, B.V.), pp.
`238-279 (1984).
`F. Miihlbacher et al., Transplantation Proceedings XIX,
`No. 5, 4162-4263 (1987).
`N. Redgrave et a1., Transplantation Proceedings 23, No'.
`1 (Feb), 346-347 (1991).
`‘
`'
`L. Bowes et al., Transplantation Proceedings 21, No. 5,
`3769-3770 (1989).
`'
`M. Moran et al., New Eng. J. Med. 332, No. 17,
`1183-1188 (1990).
`R. Griepp et :11, Surgery 81, No. 3, 262-269 (1977).
`C. Leithner et al., Prostaglandins 22, No. 5, 783-788
`(1981).
`A. Mirisklavos et 81., Journal of Applied Cardiology 1,
`109-123 (1986).
`S. Teraoka et al., Transplantation Proceedings XIX, No.
`1, 2115-2119 (1987).
`B. Uretsky et a1., Therapy and Prevention-Cardiac
`Transplantation 76, No. 4, 827-834 (1987).
`B. Whittle and S. Moncada, Platelets and Vascular Oc
`clusion, 72, No. 6, 1219-1225 (1985).
`J. Wiederkehr et al., Aust. N. Z. J. Surg. 60, 121-124
`(1990).
`Primary Examiner-Marianne M. Cintins
`Assistant Examiner-John Peabody
`Attorney, Agent, or Firm—Bell, Seltzer, Park & Gibson
`[57]
`ABSTRACT
`The present invention is concerned with methods for
`the treatment of medical conditions with benzindene
`prostaglandins. Medical conditions which may be
`treated by the methods of the present invention include
`transplant rejection and atherosclerosis.
`
`7 Claims, No Drawings
`
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`

`

`1
`
`METHOD OF COMBA'ITING CY CLOSPORINE
`ORGAN TOXICITY WITH PROSTAGLANDIN
`ANALOGS
`
`FIELD OF THE INVENTION
`The present invention relates to therapeutic methods,
`in particular combating transplant rejection and treating
`atherosclerosis with certain benzindene prostaglandins.
`BACKGROUND OF THE INVENTION
`cyclosporine (CyA; formerly called cyclosporin A)
`‘ is a cyclic peptide produced by the fungus Tolypo
`cladium in?atum. CyA is an immunosupressant adminis
`tered to human alloqeneic transplant recipients or
`human subjects undergoing treatment for an autoim
`mune disease such as myasthenia gravis. A problem
`with CyA, however, is its organ toxicity. The major
`toxic side-effect of CyA is nephrotoxicity, but hepato
`toxicity and cardiotoxicity have also been noted.
`U.S. Pat. No. 4,306,075 describes novel benzindene
`prostaglandins which produce various pharmacological
`responses, such as inhibition of platelet aggregation,
`reduction of gastric secretion, and bronchodilation. It is
`indicated that the compounds have useful application as
`anti-thrombotic agents, anti-ulcer agents, and anti
`asthma agents. There is no indication that these com
`pounds may be used to combat tissue transplant rejec
`tion.
`European Patent Application 347243 of AS. Tade
`palli et al. discloses fused-ring prostaglandin derivatives
`for treating or preventing pulmonary hypertension and
`for diagnosing primary pulmonary hypertension pa
`tients who have active pulmonary vasoconstriction (see
`also US. Pat. No. 5,028,628).
`The present invention is based on our ongoing re
`search into cyclosporine therapies.
`SUMMARY OF THE INVENTION
`A ?rst aspect of the present invention is a method for
`the treatment of a medical condition selected from the
`group consisting of transplant rejection and atheroscler
`osis in a subject in need of one or more of such treat
`ments, comprising administering to said subject a thera
`peutically effective amount of a compound of formula
`(I):
`
`20
`
`30
`
`5,190,972
`2
`or a physiologically acceptable salt or acid derivative
`thereof (i.e., an “active compound”).
`A second aspect of the present invention is a method
`of combatting transplant rejection in a subject in need of
`such treatment. The method comprises concurrently
`administering the subject an effective transplant-rejec
`tion combatting amount of cyclosporine and a com
`pound of formula (I) as given above, or a physiologi
`cally acceptable salt or acid derivative thereof, in an
`amount effective to enhance the activity of the cyclos
`porine.
`A third aspect of the present invention is a method of
`combatting cyclosporine organ toxicity in a subject in
`need of such treatment. The method comprises adminis
`tering said subject an effective cyclosporine organ tox
`icity-combatting amount of a compound of formula (I)
`as given above or of a physiologically acceptable salt or
`acid derivative thereof.
`A fourth aspect of the present invention is a method
`of combatting atherosclerosis (e.g., heart transplant
`atherosclerosis) in a subject in need of such treatment.
`The method comprises administering said subject an
`effective atherosclerosis-combatting amount of a com
`pound of formula (I) as given above or of a physiologi
`cally acceptable salt or acid derivative thereof.
`Further aspects of the present invention include the
`use of a compound of formula (I), or a pharmaceutically
`acceptable salt or acid derivative thereof, for the manu
`facture of a medicament for combatting transplant re
`jection, for combatting cyclosporine organ toxicity, and
`for combatting atherosclerosis.
`A further aspect of the present invention is a pharma
`ceutical formulation comprising cyclosporine in an ef
`fective irnmunosupressive amount and a compound of
`formula (I) above, or a physiologically acceptable salt
`or acid derivative thereof, in an amount effective to (a)
`enhance the activity of cyclosporine, (b) combat cyclos
`porine organ toxicity or (c) both, together in a physio
`logically acceptable carrier.
`The present invention also provides a method of
`combatting hyperlipidemia (e.g., cyclosporine im
`munosupression-induced hyperlipidemia) in a subject in
`need of such treatment. The method comprises adminis
`tering said subject an effective hyperlipidemia-combat
`ting amount of a compound of formula (I) as given
`above or of a physiologically acceptable salt or acid
`derivative thereof. Also provided is the use of a com
`pound of formula (I) as given above or of a physiologi
`cally acceptable salt or acid derivative thereof for the
`preparation of a medicament for combatting hyperlipid
`emia.
`The benzindene prostaglandins described above have
`a surprising level of potency in the various therapeutic
`methods referred to herein. As an advantageous conse
`quence, dosage levels may be kept within a low range
`(as hereinafter described) when compared to the admin
`istration levels of other prostaglandin compound
`5/ analogues. The prostaglandin analogues of this inven
`tion do not possess the well known side effects which
`exist with other prostaglandin compounds/analogues.
`Advantages arise from the co-administration of cy
`closporine and the benzindene prostaglandins of this
`invention. cyclosporine is nephrotoxic, cardiotoxic and
`hepatotoxic. According to the methods of this inven
`tion, less cyclosporine may be administered to a patient
`when compared with traditional therapies, when the
`cyclosporine is co-administered or administered in con
`cert with the benzindene prostaglandins (i.e., the active
`
`no2c\
`
`/x
`
`'
`
`(CHM:
`
`Y
`
`Q/\/ R
`
`no
`
`5,,
`
`(I)
`
`55
`
`wherein:
`a is an integer of from 1 to 3;
`X and Y, which may be the same or different, are se
`lected from —O— and --CH2—;
`R is --(CH2)5Rl wherein R1 is hydrogen or methyl, or
`65
`R is cyclohexyl, or R is —CH(CH3)CH1C!CCH3;
`and
`the dotted line represents an optional double bond;
`
`WATSON LABORATORIES, INC. , IPR2017-01621, Ex. 1020, p. 2 of 7
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`

`

`5
`
`F1
`
`_
`
`20
`
`35
`
`45
`
`50
`
`55
`
`(II)
`
`5,190,972
`4
`3
`9-Deoxy-2',9a-methano-3.oxa-4,S,6-trinor-3,7-(l‘,3’
`compounds) of this invention due to the synergistic
`interphenylene)- l 5-cyclohexylprostaglandin
`effect between benzindene prostaglandins and cyclos
`(Compound C);
`porine in immune suppression.
`9-Deoxy-2',9a.-methano-3-oxa-4,5,6-trinor-3,7-(1’,3’
`DETAILED DESCRIPTION OF THE
`interphenylene)-20-methylprostaglandin F1 (Com
`INVENTION
`pound D); and
`(l5S,l6RS)-9-Deoxy-2',9a-methano-l6-methyl-3-oxa
`Subjects to be treated by the methods of the present
`18, l 8,19,l9-tetradehydro-4,5,6-tiinor-3,7-(l’,3'-inter- _
`invention are typically human subjects, such as trans
`phenylene)-prostaglandin F1 (Compound E).
`plant recipients or subjects undergoing treatment for an
`The compounds of the present invention may be
`autoimmune disease such as myasthenia gravis. Trans
`prepared in accordance with known techniques, such as
`plant recipients may be recipients of kidney, liver, heart,
`methods the same as or analogous to those described in
`heart-lung, bone-marrow, and cornea transplants. The
`U.S. Pat. No. 4,306,075.
`organ transplant tissue itself is typically human in on'— .
`The amount of a compound of formula (I), or a physi
`gin, but may also be from another species such as the 15
`ologically acceptable salt or acid derivative thereof,
`rhesus monkey. Where the compound of formula (I) is
`which is required in a medication according to the in
`administered to combat cyclosporine organ toxicity, it
`vention to achieve the desired effect will depend on a
`may be administered to combat either nephrotoxicity,
`number of factors, in particular the speci?c application,
`or hepatotoxicity, but the principle use currently con
`the nature of the particular compound used, the mode
`templated is in combating nephrotoxicity.
`‘
`of administration, and the condition of the patient. In
`Used alone (and in combination with cyclosporine),
`general, a daily dose per patient is in the range 25 pg to
`the benzindene prostaglandin analogues (i.e., active
`250 mg; typically from 0.5 pg to 2.5 mg, preferably
`compounds) described herein reduce the severity of
`from 7 pg to 285 pg, per day per kilogram bodyweight.
`rabbit heart transplant atherosclerosis. Since athero
`For example, an intravenous dose in the range of 0.5 pg
`sclerosis, rather than rejection, is the predominant cause 25
`to 1.5 mg per kilogram bodyweight per day may conve
`of patient death in adult and paediatric heart transplant
`niently be administered as an infusion of from 0.5 ng to
`recipients, the benzindene compounds are useful in pre
`1.0 pg per kilogram bodyweight per minute. Infusion
`venting this lesion in these patients. Further, since the
`?uids suitable for this purpose contain, for example,
`active compounds described herein are believed to ne
`' from 10 ng to 10 pg per milliliter. Ampoules for injec
`gate the hyperlipidemia induced by cyclosporine im- 30
`tion contain, for example, from 0.1 pg to 1.0 mg and
`munosupression, they are also contemplated as useful in
`orally administrable unit dose formulations, such as
`combatting hyperlipidemia in the general population.
`tablets or capsules, contain, for example, from 0.1 to 100
`Preferred compounds of formula (I) having particu
`mg, typically from 1 to 50 mg. In the case of physiologi
`larly desirable properties include those wherein X is
`cally acceptable salts, the weights indicated above refer
`—O—; Y is —CH;—; and R is --(CH2)4CI-I3.
`to the weight of the active compound ion,’ that is, the‘
`The term “acid derivative” is used herein to describe
`ion derived from the compound of formula (I).
`C14 alkyl esters and amides, including amides wherein
`“Concurrently administering” means the compound
`the nitrogen is optionally substituted by one or two C14
`of formula (I) and the cyclosporine are administered to
`alkyl groups.
`the subject either (a) simultaneously in time (optionally
`The present invention also includes bioprecursors or
`by formulating the two together in a common carrier),
`“pro-drugs” of the above-de?ned compounds, that is,
`or (b) at different times during the course of a common
`compounds which are converted in vivo to compounds
`treatment treatment schedule. In the latter case, the two
`of formula (I) or pharmaceutically active derivatives
`compounds are administered sufficiently close in time to
`achieve the intended effect.
`Cyclosporine may be administered in a manner and
`' amount as is conventionally practiced. See, e.g., Good
`man and Gilman’s The Pharmacological Basis of Thera
`peutics, 1299 (7th ed. 1985). The speci?c dosage will
`depend on the condition being treated, the state of the
`subject, and the route of administration, but will typi
`cally be from about 1 to 20 milligrams per kilogram of
`subject body weight daily, or more preferably from
`about 1 to 15 milligrams per kilograms body weight
`daily. For example, cyclosporine may be provided as an
`oral solution of 100 mg/ml with 12.5% alcohol, and for
`intraveneous administration as a solution‘ of 50 mg/mL,
`with 33% alcohol and 650 mg of polyoxyethlated castor
`oil. For a transplantation subject a typical oral dose is 10
`to 15 mg/kg daily, starting a few hours before trans
`plantation and continuing for l to 2 weeks, with the
`dosage then being gradually reduced to a maintenance
`level of 5 to 10 mg/kg daily. When administered intra
`veneously, CyA may be given as a dilute solution of 50
`mg per 20 to 100 ml of normal saline solution or 5%
`dextrose in water, by slow infusion over a period of 2 to
`6 hours. The intraveneous dose is typically one third of
`the oral dose. An adrenocorticosteroid such as predni
`
`and pharrnaceutically acceptable salts and acid deriva
`tives thereof.
`Other compounds useful for practicing the present 65
`invention include:
`9-Deoxy-2',9a-methano-3~oxa-4,5,6-trinor-3,7-(l’,3'
`interphenylene)-prostaglandin F1 (Compound B);
`
`thereof. '
`
`'
`
`A particularly preferred compound of formula (I)
`above is 9-deoxy-2',9a-methano-3-oxa-4,5,6-trinor-3,7
`(l’,3’-interphenylene)- l 3, l4-dihydro-prostaglandinF1
`(Compound A), which has the structure of formula (II):
`
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`

`

`20
`
`25
`
`30
`
`5
`sone is optionally administered with the CyA, as is
`known in the art.
`The present invention extends to nonphysiologically
`acceptable salts of the compounds of formula (I) which
`may be used in the preparation of the pharmacologi
`cally active compounds of the invention. The physio
`logically acceptable salts of compounds of formula (I)
`‘include salts derived from bases. Base salts include am
`monium salts, alkali metal salts such as those of sodium
`and potassium, alkaline earth metal salts such as those of
`calcium and magnesium, salts with organic bases such
`as dicyclohexylamine and N-methyl-D-glucamine, and
`salts with amino acids such as arginine and lysine.
`Quaternary ammonium salts can be formed, for exam
`ple, by reaction with lower alkyl halides, such as
`methyl, ethyl, propyl, and butyl chlorides, bromides,
`and iodides, with dialkyl sulphates, with long chain
`halides, such as decyl, lauryl, myristyl, and stearyl chlo
`rides, bromides, and iodides, and with aralkyl halides,
`such as benzyl and phenethyl bromides.
`In the manufacture of a medicament according to the
`invention, hereinafter referred to as a “formulation,”
`the compounds of formula (I) and the physiologically
`acceptable salts thereof, or the acid derivatives of either
`thereof (hereinafter referred to as the “active com
`pound") are typically admixed with, inter alia, an ac
`ceptable carrier. The carrier must, of course, be accept
`able in the sense of being compatible with any other
`ingredients in the formulation and must not be deleteri
`ous to the patient. The carrier may be a solid or a liquid,
`or both, and is preferably formulated with the com
`pound as a unit-dose formulation, for example, a tablet,
`which may contain from 0.5% to 95% by weight of the
`active compound. One or more active compounds may
`be incorporated in the formulations of the invention,
`which may be prepared by any of the well known tech
`niques of pharmacy consisting essentially of admixing
`the components, optionally including one or more ac
`cessory ingredients such as cyclosporine and (option
`ally) an adrenocorticosteroid such as prednisone.
`The formulations of the invention include those suit
`able for oral, rectal, topical, buccal (e.g., sublingual),
`parenteral (e.g., subcutaneous, intramuscular, intrader
`mal, or intravenous) and transdermal administration,
`although the most suitable route in any given case will
`depend on the nature and severity of the condition
`being treated and on the nature of the particular active
`compound which is being used.
`Formulations suitable for oral administration may be
`presented in discrete units, such as capsules, cachets,
`lozenges, or tablets, each containing a predetermined
`amount of the active compound; as a powder or gran
`ules; as a solution or a suspension in an aqueous or non
`aqueous liquid; or as an oil-in-water or water-in-oil
`emulsion. Such formulations may be prepared by any
`55
`suitable method of pharmacy which includes the step of
`bringing into association the active compound and a
`suitable carrier (which may contain one or more acces
`sory ingredients as noted above). In general, the formu
`lations of the invention are prepared by uniformly and
`intimately admixing the active compound with a liquid
`or finely divided solid carrier, or both, and then, if
`necessary, shaping the resulting mixture. For example, a
`tablet may be prepared by compressing or moulding a
`powder or granules containing the active compound,
`optionally with one or more accessory ingredients.
`Compressed tablets may be prepared by compressing, in
`a suitable machine, the compound in a free-?owing
`
`5,190,972
`6
`form, such as a powder or granules optionally mixed
`with a binder, lubricant, inert diluent, and/or surface
`active/dispersing agent(s). Moulded tablets may be
`made by moulding, in a suitable machine, the powdered
`compound moistened with an inert liquid binder.
`Formulations suitable for buccal (sub-lingual) admin
`istration include lozenges comprising the active com
`pound in a flavoured base, usually sucrose and acacia or
`tragacanth; and pastilles comprising the compound in
`an inert base such as gelatin and glycerin or sucrose and
`acacia.
`Formulations of the present invention suitable for
`parenteral administration conveniently comprise sterile
`aqueous preparations of the active compound, which
`preparations are preferably isotonic with the blood of
`the intended recipient. These preparations are prefera
`bly administered intravenously, although administra
`tion may also be effected by means of subcutaneous,
`intramuscular, or intradermal injection. Such prepara
`tions may conveniently be prepared by admixing the
`compound with water or a glycine buffer and rendering
`the resulting solution sterile and isotonic with the blood.
`lnjectable formulations according to the invention gen
`erally contain from 0.1 to 5% w/v of active compound
`and are administered at a rate of 0.1 ml/min/kg.
`Formulations suitable for rectal administration are
`preferably presented as unit dose suppositories. These
`may be prepared by admixing the active compound
`with one or more conventional solid carriers, for exam
`ple, cocoa butter, and then shaping the resulting mix
`ture.
`Formulations suitable for topical application to to the
`skin preferably take the form of an ointment, cream,
`lotion, paste, gel, spray, aerosol, or oil. Carriers which
`may be used include vaseline, lanoline, polyethylene
`glycols, alcohols, and combinations of two or more
`thereof. The active compound is generally present at a
`concentration of from 0.1 to 15% w/w, for example,
`from 0.5 to 2% w/w.
`Formulations suitable for transdermal administration
`may be presented as discrete patches adapted to remain
`in intimate contact with the epidermis of the recipient
`for a prolonged period of time. Such patches suitably
`contain the active compound as an optionally buffered
`aqueous solution of, for example, 0.1 to 0.2 M concen
`tration with respect to the said active compound. _
`Formulations suitable for transdermal administration
`may also be delivered by iontophoresis (see, for exam
`ple, Pharmaceutical Research 3 (6), 318, (1986)) and
`typically take the form of an optionally buffered aque
`ous solution of the active compound. Suitable formula
`tions comprise citrate or bis tris buffer (pH 6) or
`ethanol/water and contain from 0.1 to 0.2 M active
`ingredient.
`The present invention is explained in greater detail in
`the following Examples.
`EXAMPLE 1
`In Vitro Comparison of the Immunosuppressive
`Potential of Synthetic Prostaglandin Analogues
`Heparinised blood was obtained from ?ve healthy
`donors without history of previous blood transfusion,
`pregnancy, or organ transplantation.
`Sandimmun (Sandoz Ltd. Basel, Switzerland) Cy
`closporine (CyA) at 50 mg/mL was diluted in physio
`logical saline to concentrations of 1.0-0.001 ug/mL.
`
`50
`
`60
`
`65
`
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`5,190,972
`8
`7
`Prostaglandin (PG) analogues PGE], PGEZ, PGFza,
`PGFza. The inhibition induced by CyA and PGI; com
`and PGI; were supplied by the Upjohn Company (Kal
`binations indicates a synergy that parallels that ob
`served between CyA and the PGE analogues. The 50%
`amazoo, Mich.) in the form of l5-(s)-l5-methyl PGE1,
`16,16-dimethyl PGEZ, 16,16-dirnethyl PGFZa, and 9
`level was obtained with CyA at 0.01 u/mL and P61; at
`deoxy-2,9a-methano-3-oxa-4,5,6-trinor-3,7-(l',3'-inter
`0.01 ug/mL.
`phenylene)-13,14’-dihydroprostaglandin d A). Each
`was provided in methyl acetate at 10 mg/mL that was
`subsequently diluted, ?rst with ethyl alcohol to a con
`centration of 1 mg/mL and thereafter with physiolog
`ical saline to the concentration range required for the
`study, 10.0-0.001 ug/mL.
`Responder lymphocytes were isolated from the
`healthy donor heparinised blood by density gradient
`separation and suspended in glutamine, antibiotic-sup
`plemented RPMI medium containing 10% autologous
`responder lymphocyte donor serum at a concentration
`of 1X106 cells/mL. The stimulator population con
`sisted of Epstein-Barr virus (EBV)-transformed lym
`phoblastoid cells that were irradiated (130 Gy) before
`suspension also at 1><106 cells/mL in glutamine, antibi
`otic-supplemented RPMI medium that contained 10%
`autologous responder lymphocyte donor serum.
`Equal volumes (0.1 mL) of responder and stimulator
`cells were incubated in triplicate wells of microtitre
`trays with each of the following:
`1. 0.05 mL of medium
`2. 0.025 mL of medium plus 0.025 mL of serial dilu
`tions of CyA or PGE1, PGEz, PGFza, or PGI;;
`3. 0.025 mL of serial dilutions of CyA plus 0.025 mL
`of serial dilutions of PGE], PGEZ, PGFza, 0, p012.
`30
`Following incubation at 37' C. (5% CO2) for 4 days,
`1.0 mCi of tritiated thymidine was added to all cultures
`before further incubation for 6-8 hours, after which
`cells were automatically harvested (Skatron, Liebyen,
`Norway) on Titertek ?lter paper (Flow Laboratories,
`Irvine, Scotland) that was placed in vials containing
`liquid scintillation ?uid for counting in a Beckman beta
`counter (Beckman, Brea, CA). Triplicate MLC re
`sponses from responder-, stimulator-, and drug-contain
`ing cultures were compared with triplicate responses
`from cultures containing 0.05 mL of medium as a substi
`tute for the appropriate drugs to maintain constant cul- .
`ture volume, in order to determine the minimum con
`centrations of CyA, PGE], PGEZ, PGFza, and PGI;
`which alone induced 50% MLC inhibition and the mini
`mum concentrations of CyA plus PGE1, PGEZ, PGFza,
`or PGIZ which induced the same degree of inhibition.
`The percent MLC inhibition induced by CyA and the
`PG analogues as single agents is shown in Table 1. The
`most potent MLC suppression, on a milligram for milli
`gram basis, was obtained from CyA and P61; which
`each induced 50% inhibition at a minimum concentra
`tion of 0.1 rig/m1... The PGE1 and PGE; analogues
`induced the same degree of inhibition from 10 times
`greater concentrations, 1.0 ug/mL, while 50% inhibi
`tion due to PGFZa was only achieved with a concentra
`tion of 10.0 ug/mL.
`-
`Table 2 shows the effect of CyA combined with each
`of the PG analogues on the MLC response. The inhibi
`tion induced by CyA and the PGE analogues indicates
`a similar degree of synergy from CyA and PGE, or
`CyA and PGE; with the 50% level achieved with'CyA
`at 0.01 ug/mL and PGE1 or PGE; at 0.01 ug/mL. CyA
`and PGFza suppression of the MLC indicates that the
`combined action has no influence further than that ex
`65
`erted when each was added alone to cultures as 50%
`MLC suppression was observed only from cultures that
`contained at least 0.1 ug/mL CyA or 10.0 ug/mL
`
`These-results indicate that each of the PG analogues
`has the potential to suppress the in vitro lymphoid re
`sponse to alloantigens. However, the concentrations of
`each analogue that are required to suppress that re
`sponse differ signi?cantly, with PGI; being 10 times
`more effective than PGE; and PGEZ, and the PGE
`analogues being a further 10 times more effective than
`the PGFZal analogue which showed no indication of
`synergistic interaction with CyA.
`In combination with CyA, the PGIZ, PGE1 and
`PGE; analogues promote even greater immunosuppres
`sive activity. Fifty percent suppression of the MLC was
`obtained with CyA at 0.01 ug/mL when combined with
`P612 at 0.01 ug/mL, which is a 10-fold decrease in the
`concentration of each that was necessary to achieve the
`same degree of suppression when either was used alone.
`Similarly, CyA and PGE] or PGE; combinations are
`effective in the induction of 50% MLC suppression
`with a 10-fold decrease in the concentration of CyA but
`a l00-fold decrease in PGE concentration. These results
`suggest that the PGE and PGIZ derivatives may have
`CyA sparing potential.
`TABLE 2
`Suppression of MLC Responses by CyA
`in Combination with PG Analogues
`PG Analogue
`CyA Concentration (Hg/mL)‘
`(pg/mL)
`0.01
`0.1
`1.0
`10.0
`
`TABLE 1
`Suppression of MLC Responses by
`CyA, PGE1. PGEZ, PGFga and PGIZ
`Concentration gg/mL'
`10.0
`0.01
`0.1
`1.0
`0.001
`Agent
`84
`25
`52
`72
`l 1
`CyA
`61
`18
`36
`56
`5
`P65]
`58
`18
`32
`51
`. 6
`FOB;
`50
`4
`16
`29
`0
`PGFM
`77
`22
`51
`61
`10
`Prostacyclin
`‘Mean % (mean calculated from % decreases obtained in MLC: of ?ve hmlthy,
`nonaenaitiaed donors) decrease of cpm in drug-containing cultures compared with
`cpm (mean cpm in drug-free cultures = 29 414) in drug-free cultures.
`
`20
`
`40
`
`45
`
`B1
`
`E;
`
`F2,‘
`
`12
`
`0.001
`
`0.01 '
`0.1
`1.0
`10.0
`0.01
`0.1
`1.0
`10.0
`0.01
`0.1
`1.0
`10.0
`0.01
`0.1
`1.0
`10.0
`
`22
`20
`59
`62
`23
`26
`58
`60
`14
`14
`31
`58
`27
`68
`81
`86
`
`52
`51
`62
`67
`49
`52
`56
`71
`25
`24
`30
`65
`5a
`76
`85
`8B
`
`64
`64
`70
`72
`55
`5a
`67
`72
`55
`55
`5a
`69
`75
`36
`85
`85
`
`74
`74
`79
`82
`63
`66
`so
`as
`70
`72
`76
`79
`a6
`a5
`a5
`86
`
`86
`a3
`84
`85
`68
`70
`as
`86
`86
`86
`85
`a6
`87
`a5
`88
`86
`
`' EXAMPLE 2
`Synergistic Prolongation of Rabbit Renal Allograft
`Survival
`This study was performed to assess the effectiveness
`of Compound A as an immunosuppressive agent in a
`rabbit renal allograft model.
`
`WATSON LABORATORIES, INC. , IPR2017-01621, Ex. 1020, p. 5 of 7
`
`

`

`5,190,972
`Left orthotopic renal transplantation was performed
`from New Zealand White rabbit donors into Anglo
`lop-ear recipients. See D. Francis et al., Aust. NZ. J.
`Surgery 60, 45, 1990. Contralateral nephrectomy and
`graft biopsy were performed on the ?rst postoperative
`day. Each recipient was subsequently dependent on its
`transplant for renal function. Serum creatinine (sCr)
`was measured every 48 hours and animals were culled
`once sCr had risen above 1.2 mMoles/l, taken as an
`arbitrary end-point representing graft failure. Graft
`biopsies were performed weekly and post-mortem to
`ensure that graft loss was secondary ,to rejection. Stan
`dard haematoxylin and eosin and Masson’s trichrome
`stains were performed.
`Compound A was provided as a powder which was
`dissolved in absolute ethanol to yield a 1 mg/ml solu
`tion. 50 ug/kg/day was administered subcutaneously
`for seven days commencing perioperatively. Ethanol
`(50 ul/kg/day) was administered in vehicle-only con
`trol animals. CyA (Sandimmun, Sandoz Ltd. Switzer
`land) was administered intravenously as a single periop
`erative dosage of 20 or 5 mg/kg. Blood CyA levels
`were measured 24 hours post-dosage (Abbott TDX
`fluorescence immunoassay).
`The transplant groups and their survival results are
`shown in Table 3. Compound A prolonged allograft
`survival over that of untreated animals, but this survival
`prolongation was not signi?cantly different from that
`observed with ethanol vehicle alone. However, Com
`pound A and CyA synergistically enhanced graft sur
`vival, whereas CyA and ethanol did not. No CyA-spar
`ing effect was demonstrated when Compound A and 5
`mg/kg of CyA, which did not prolong graft survival
`alone, were administered together. Histological assess
`ment revealed that the pattern of graft in?ltrate during
`acute rejection was not different between groups. Cy
`
`10
`
`20
`
`25
`
`35
`
`10
`TABLE 3-continued
`Survival of Renal Allografts in Rabbits Treated
`with Compound A. Ethanol Vehicle and/or CyA
`
`Graft Survival (d)
`
`‘ Median
`Graft
`Survival
`(d)
`
`12.5
`ll
`
`Group‘ Treatment
`CyA (5 ms/ks)
`8, 11, 12, 13, 18, 18
`Ethanol
`Ethanol + CyA 10, 10, 11, ll, 14, 19
`(20 Ins/ks)
`
`7
`B
`
`EXAMPLE 3
`Prevention of Acute Cyclosporine (CyA)
`Nephrotoxicity in Rabbits
`New Zealand White rabbits were studied in three
`treatment groups: (1) CyA alone; (2) CyA and Com
`pound A; and (3) CyA and ethanol vehicle.
`CyA (Sandimmun, Sandoz Ltd. Switzerland) was
`administered by slow intravenous injection, 100
`mg/kg/day, for seven days. Compound A was pro
`vided as a powder which was dissolved in absolute
`ethanol to yield a 1 mg/ml solution. 50 ug/kg/day was
`administered subcutaneously for seven days. Ethanol
`(50 ul/kg/day) was administered in vehicle-only con
`trol animals. Blood CyA levels were measured 24 hours
`following completion of a seven days treatment (Abbott
`TDX ?uorescence immunoassay).
`Serum creatinine (Cr) and urea were measured prior
`to commencement of treatment (day 0) and 24 hours
`following treatment (day 7). Day 7 renal biopsies were
`stained with haematoxylin and eosin and Masson’s tri
`chrome. Results are shown in Table 4.
`TABLE 4
`Serum creatinine and urea in Rabbits Treated
`with High Dose CyA 1- Compound A or Ethanol
`Mean
`Mean
`Mean
`Cr/Urea Day Cr/Urea Day
`Cr/Urea
`0 (mMoles/l) 7 (mMoles/l)
`Change
`nos/6.5
`0.1l/13.5
`+0.06"/+7"
`
`Mean CyA
`Level
`(ng/ml)
`860
`
`0.08/59
`
`0.07/5.7
`
`—0.01“/-0.2b
`
`713
`
`0.07/9
`
`0.14/26
`
`+0.07"/+17b
`
`607
`
`Group Treatment
`1
`CyA
`(n = 4)
`CyA +
`Compound A
`(n = 4)
`CyA +
`Ethanol
`(n = 4)
`
`2
`
`3
`
`‘Group 1 or 3 vs 2, P < 05;
`‘Group 1 or 3 vs 2, P < 05 (Mann-Whitney U test).
`
`closporine levels were not signi?cantly different be
`tween treatment groups (270 ng/ml, median group 3;
`264 ng/ml, median group 4).
`TABLE 3
`Survival ‘of Renal Allografts in Rabbits Treated
`with Compound A, Ethanol Vehicle and/or CyA
`Median
`Graft
`Survival
`(d)
`9.5
`13.5
`16.5
`26
`
`Graft Survival (d)
`Group‘ Treatment
`7, 9, 9, 10, 10, 13
`1
`mil
`13, 13, 13, 14, 15, 21
`2
`Compound A
`3
`CyA (20 mg/kg) 9, 12, 13, 20, 26, 38
`4
`Compound A + 20, 23, 26, 26, 31, 35, 43
`CyA (20 Ina/ks)
`7, 8, 10, 10, 11, l2, l5
`CyA (5 mg/kg)
`Compound A + 13, 14, 14, 15, l7, 19, 27
`
`5
`6
`
`l0
`15
`
`60
`
`Consistent nephrotoxicity was observed in rabbits
`treated with 100 mg/kg/day of CyA. However, treat
`5 ment was associated with 60% mortality and 7-10%
`5
`weight loss. Irrespective of the treatment group, Com
`pound A had a signi?cant pr