CA2144189A1 - Prevention of stenosis - Google Patents

Abstract

Stenosis, particularly restenosis associated with angioplasty or cardiac bypass operations, is treated by administering a therapeutically effective amount of mycophenolate mofetil, or a pharmaceutically acceptable salt or derivative thereof.

Description

W O 94/12184 2~ q P~rUS92/09932 USE OF MYCOPHENOLIC ACID, MYCOPHENOLATE MOFETIL OF DERIVATE THEREOF TO
INHIBIT STENOSIS

Field of the Invention The pre~3ent invention relate~3 to method~ of preventing ~tenosi~
following ~urgical treatment, particularly the prevention of re~teno~i~
following angiopla~ty through the A~` i n i~tration of mycophenolic acid or a related r~ _und, particularly mycophenolate mofetil.

Back~round Information Mycophenolic acid i8 a weakly-active antibiotic found in the fermentation broth of Pennicillium brevicompactum. Compound~ relating to mycophenolic acid, and their uses in the treatment of inflammatory disease~, autoimmune ~i~eA~es, viral diseases, cancer, and/or for the p-evenlion of allograft rejection, are disclosed in U.S. Patent~ No~. 4,686,234; 4,725,622;
4,727,069; 4,748,173; 4,753,935; 4,786,637; 4,808,592; 4,861,776; 4,868,153;
4,948,793; 4,952,579; 4,959,387; and 4,922,467, all incorporated herein by reference.
Mycophenolic acid, mycophenolate mofetil, or a phA -reuticaly acceptable salt or derivative thereof have the one of the following general ~tructure~.
A ~ und of Formula 1:

~CH--3X 1C CH2) q ~

and the rh~ -~eutically acceptable ~alts thereof, where:
Rl is H or lower alkyl having 1 to 6 carbon atoms;
R2 is H, lower alkyl having 1 to 6 carbon atoms or ph~nyl-4-CO2R3, in which R3 i~ H, lower alkyl havLng 1 to 6 carbon atoms or a phn -reutically acceptable cation;
R~ and R5 are each independently H or lower alkyl having 1 to 6 carbon atom~;
X~ and Y~ are each independently O or S; and ~ i~ an integer of 1-6.
OR
A compound of Formula 2:

W O.94tl2i84 ~ f ~ 2- PCT~US92/09932 t~4~89 U C.

and the phA ~ceutically acceptable salt~ thereof, where:
A iB oxygen or ~ulfur;
R~ elected from the group con~Lsting of:
A~ O Q H
H, -CR2, -C(CH2)qCO2R3 and -C=C-QI, in which:
A~ is oxygen or ~ulfur; J
q is an integer from 0-6;
R2 is alkyl, haloalkyl or -NR4R5, where:
R4 and R5 are independently H, alkyl, haloalkyl, cycloalkyl, phenyl optionally monosub~tituted with halogen, hydroxy, carboxy, chlorocarbonyl, sulfonylamino, nitro, cyano, phenyl, alkyl, acyl, alkoxycarbonyl, acylamino, dialkylamino or dialkyl; inoethoxycarbonyl, phenyl opt;on~lly di3ubstituted with hydroxy, carboxy, nitro or alkyl, or benzyl optionally ~ub~tituted with dialkylamino;
R3 is H, alkyl or a phA -~eutically acceptable cation;
Q and Q~ are independently H or -CO2R3; and Z~ i~ selected from the group con~isting of: IH-tetrazolyl, -CH2OH, -CHO, 2 5 -CN, -C(O)A2R5 and -C ( O ) NR,R" in which:
A2 is oxygen or ~ulfur;
R5 i~ H, alkyl, alkenyl, cycloalkyl, optionally 3ub~tituted phenyl, optionally ~ub~tituted benzyl or a ph~ -ceutically acceptable cation; and R, and R, are independently H, alkyl or cycloalkyl, or R7 and taken together are -(CH2)2O(CH2)2-, -(CH2)4, or -(CH2)5-;
with the proviso that R, and R5 cannot beoth be H i~ A and A2 are oxygen.
OR
A c _:~nd of Fo- ~a A:
o O-Z CH3 ~CH--3 - O- CH2- CH2~/--\0 wherein Z i~ hy~ ogen or -C(O)R, where R i~ lower alkyl or aryl, and the ph~ -~eutically acceptable ~alts thereof.
OR
A compound of Formula I:

W O 94/12184 21~ 418 9 PCT ~S92~9932 !4 ' ~ =e, e ~
,~
o O~ Z CH3 ~ O-CCHz~ - N3 wherein:
m i~ an integer from two to four;
Z i~ t3elected from Fo laP (a), (b), (c), or (d), af3 followf3:
10 (a) o - clR 1 J

in which:
Rl i~ hydrogen, alkyl having f3even or more carbon atoms including cycloalkyl 3uch af3 adamantyl, or -NR2R3, where R2 i~3 hydrogen or lower alkyl, and R3 i~ hydrogen, lower alkyl, -phenyl-4-CO2R2 or a pha -~eutically acceptable cation;
(b) -CR4, in which:
R4 is hyd~oyen~ alkyl, aryl or -NR2R3;
(c) - CC CH7) n~ Co2R5, in which:
n i~ an integer from zero~to ~ix, and R5 i~ hyd~oyan~ lower alkyl, or a ph~ -~eutically acceptable cation;
(d) Rb -C CH R7, in which:
R5 and R7 are independently hyd ogen or -Co2R5; and Y i~ lower alkylene of four to f3iX carbon atomt3, or lower alkylene of three to five carbon atom~ and one member that i~ -O-, -S- or ~N-R8 where R~ i~ hydrogen or alkyl of one to five carbon atomn.
OR
A compound of Formula II:

WO 94/12184 PCTrUS92/09932 -21~4189 o o_z1 CH3 ~C H 3-- C- O- C C H2) m- N3 wherein:
m is an integer from two to four;
Zl L~ hydrogen or -C(o)R9, where R9 is lower alkyl or aryl; and Y~ is lower alkylene of four to ~ix carbon atoms, or lower alkylene of three to five carbon atom~ and one member that i~ -0-, -S-, or ,N--Ra where R~ i~ hydrogen or alkyl of one to five carbon atom~; and the Fh~ ~eutieally aceeptable salt~ thereof;
exeept that when m is two, yl does not include -(CH2)2-0-(CH2)2-.

Mycophenolate mofetil, the morpholinoethyl ester of mycophenolic aeid, i~ de~eribed in U.S. Patent No. 4,753,935 (previously ineorporated by reference)~ and ha~ the ch ic~l name morpholinoethyl E-6-(1,3-dihydro-4-hydLo~y ~ Lhoxy-7-methyl-3-oxo-5-i~oben7ofuranyl)-4-methyl-4-h~YDno~te. It ha~ been shown effective in ~ ~venLing allograft rejeetion, includLng chronic allograft rejection.
Steno~is i~ a narrowing of the lumen of a blood ves~el eau~ed by the thi e~ning of a blood ves~el wall, involving - 1~Y interaction~ beL._en the cell~ of the ves~el wall (conneetive ti~ue cell~, e3pecially smooth mu~cle cells) and eireulating blood el~ ~ts, with eon~equent restrietion of blood flow. Steno~i~ ha~ been a~30eiated with in3ult to the endothelial lining or underlying layer~ of the ve~sel-wall, typieally during a surgical proeedure (e.g., in plaeing ~utures through the blood ves~el wall a~ in by-pa~ ~urgery, and during angioplasty whether by balloon, laser or otherwi~e). Angioplasty involve~ the removal of ob~truetions (e.g., plaque) and recult~ in the wi~ning of eonstrieted blood ve~els, i.e., a treatment for stQnosis; the proeedure often entail~ an in~ult to the endo~h~ l lining or underlying layer~, which trigger~ an early va~cular cell proliferation, e~pecially of smooth mu~cle eell~ (one of the eell type~ e~ponding to the in~ult) and other eonneetive ti~ue eells, and eauseR a thic~ning of the vessel wall with a eorresponding narrowing of the lumen, ealled re~teno~is.
It ha~ long been sought to provide a treatment for preventing stenosi~
or re~tenosi~ following 3urgieal procedure~, and ha~ now, nurpri~ingly, been di~eovered, that ~uch treatment can be effeeted by the ~;n;~tration of an effective amount of mycophenolic acid or a related compound, partieularly mycophenolate mofetil.

2t~189 W O 94/12184 PCTrUS92/09932 , ~i ,,.;
SUMMARY OF THE lNv~ ON
One aspect of the pre~ent invention concern~ u~e of a therapeutically effective amount of mycophenolic acid or mycophenolate mofetil, or a ph~ ~ceutically acceptable salt or derivative thereof for the treatment of stenosis.
Another aspect of the present invention concerns use of a therapeutically effective amount of ycophenolic acid or mycophenolate mofetil, or a ph~ ~ceutically acceptable salt or derivative thereof for performing angioplasty or by-pass surgery including prophylactic 0 A(' i ni gtration-Still another aspect of the present invention concern~ inhibitingintimal va~cular proliferation, especially of smooth muscle cells following an insult to a blood vessel wall, by ~t' ;n;stering a proliferation inhibitory amount of mycophenolic acid or mycophenolate mofetil, or a pharmaceutically acceptable salt or derivative thereof.
Still another aspect of the present invention is a ph~ ~ce~ltical composition for the treatment of stenosis compri~ing a ph~ -ceutically acceptable non-toxic excipient and a therapeutically effective amount of mycophenolic acid, mycophenolate mofetil, or a phA ~ceutically acceptable salt or derivative thereof.
In a preferred aspect of the present invention, mycophenolate mofetil, or a ph~ -ceutically acceptabla salt thereof, is orally ~l` ; n i ~tered to p ~v~nL ~tQnosis or restenosis following angioplasty or a cardiac by-pass surgical procedure.
DETAILED DESCRIPTION OF THE lNv~lION

Definitions and General Parameters The following definitions are set forth to illustrate and define the I--ning and scope of the variou~ tQrms used to describe the invention herein.
As u~ed herein, the term "alkyl" refers to a fully saturated monovalent radical con~ining only carbon and hydrogen, and which may be a cyclic, branched or straight chain radical. Thi~ term is further exemplified by r~ic~l~ nuch as methyl, ethyl, t-butyl, pentyl, heptyl, pivalyl, cyclopentyl, and cyclohexyl.
The term lower alkyl" refers to a monovalent alkyl radical of one to ~ix carbon atoms. This term iB further ~7 ~lified by ~uch radicals as methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, i-butyl (or 2-methylpropyl), isoamyl, pentyl and i~o~e..Lyl~
The term alkylene" refer~ to a fully saturated divalent radical cont~i n; ng only carbon and hydrogen, and which may be a branched or straight chain radical. This term is further exemplified by radicals such as methylene, ethylene, n pro~ylene~ t-butylene, i-pentylene, and n-heptylene.
The term "alkoxy" refers to the group -OR wherein R is lower alkyl a~7 herein defined.
The term "aryl refers to a substituted or unsub~tituted monovalent -W O 94/12184 PCTrUS92/09932 -2 1 g 4 1 8 9 -6-un~aturated aromatic carbocyclic radical having a ~ingle ring (e.g., phenyl) or two con~en~ed ring~ (e.g " naphthyl~.
The term "acyl~ refer~ to a radical baned on an organic acid, e.g., -C(O)RI where Rl i8 alkyl or aryl.
As u~ed herein, the term "halo" refer~ to fluoro, bromo, chloro and iodo.
I~olation and purification of the compounds~and intermediates de~cribed herein can be effected, if de~ired, by any ~u $àble separation or purification procedure ~uch a~, for example, filtration, ex~raction, cry~tallization, column chromatography, thin-layer chromatogr~phy or thick-layer chromatography, or a combination of the~e ~.ocedures.
A "phA -~eutically acceptable ~alt" may be any ~alt derived from an inorganic or organic acid. The term "ph~ -ceutically acceptable anion"
refer~ to the anion of ~uch ~alts. The ~alt and the anion are cho~en not to be biologically or otherwi~e unde~irable. These Qalt~ are formed with inorganic acid~ ~uch a~ hydrochloric acid, hydrobromic acid, ~ulfuric acid (giving the culfate and bi~ulfate salts), nitric acid, phosphoric acid and the like, and organic acid~ ~uch ac acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, malic acid, malonic acid, succinic acid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, c; nnr ; C acid, -n~elic acid, methane~ulfonic acid, ethan~ulfonic acid, p-toluenesulfonic acid, ~alicylic acid and the like.
As uced herein, the term "treatment" or "treating" means any trcatment of a ~;~eAne in a mammal, including:
(i) pr~v~nting the ~;P~-~e, that is, cau~ing the c~;n;cnl symptom~ of the ~i~ea~e not to develop;
(ii) inhibiting the ~i~e~e, that is, arre~ting the development of clinical ~ymptoms; and/or (iii) relieving the dicea~e, that i~, cau~ing the regres~ion of clinical symptoms.
A~ uced herein, the term~ "effective amount" or "therapeutically ~ffective amount" means a do~age sufficient to provide treatment for the A i ~eA Qe state being treated. Thi~ will vary ~pen~; n~ on the patient, the ~;~eAne and the treatment being effected.
As usQd herein, the term "stenosi~" chould be read to includQ
"re~teno~is," except to th~ extent that the context or specific description indicate~ the contrary.
A~ used herein, the term "derivative" mean~ a compound based upon the ~tructure of mycophenolic acid bearing a cub~tituent for -OH on the 4-poRition of the bicyclic ring and/or on the carboxylic acid of the side chain, a~
dencribed in U.S. Patent~ Nos. 4,686,234; 4,725,622; 4,727,069~ 4,748,173 4,753,935; 4,786,637; 4,808,592; 4,861,776; 4,868,153; 4,948,7g3; 4,952,579 4,959,387; and 4,922,467, all previou~ly inco.~orated herein by rcference (e.g., the groups Z and -(CH2)n-N Y in U.S. Patent No. 4,748,173).

~ W O 94/12184 214 418 9 PCTrUS92/09932 - ; ~
Sources Of The ComPounds UQed In The Method~ Of The Invention Mycophenolic acid i~ available, for example, from Sigma Chemical Company, of St. Loui~, Mi~ouri.
Mycophenolate mofetil, or morpholinoethyl E-6-tl,3-dihydro-4-hydroxy-6-methoxy-7-methyl-3-oxo-5-isobenzofuranyl)-4-methyl-4-hexenoate, can be made, formulated and administered a~ described in U.S. Patent No. 4,753,935, previously incorporated herein by reference.
The phA -ceutically acceptable salts or derivatives of mycophenolic acid and mycophenolate mofetil can be made, formulated and ~ i ni qtered a~
de~cribed in U.S. Patentc No~. 4,686,234; 4,725,622; 4,727,069; 4,748,173;
4,753,935; 4,786,637; 4,808,592; 4,861,776; 4,868,153; 4,948,793; 4,952,579;
4,959,387; and 4,922,467, all previously incorporated herein by reference.

Utilitv, Testinq and ~mi n~ ~tration General UtilitY
The compound~ u~ed in the methods of the pre~ent invention inhibit proliferating cellc, including ~mooth muscle cells, acting through the inhibition of inosine monophGa~hate dehydrogen~e and the consequential depletion of deoAy~uano~ine triphosphatQ, which i~ required for DNA synthesi~
and cell proliferation. The compound~ are u~eful for p a~en~ing proliferative responses to vA~culAr injury, e.g., ~tenosis following an insult to a blood ves~el wall.
In particular, mycophenolic acid, ycophenolate mofetil, or a pharmaceutically acceptable salt or derivative thereof, iB A~' ; n i stered to apatient y.ophylactically and/or following a surgical p.ocedure a~ociated with injury to the endothelium or underlying layers of a blood vessel wall (e.g., a pLocedure involving removal of, or damage to, endothelial cells). The c_ _und~ u~ed in tha method~ of the pre~ent invention do not require the co-~' ini ~tration of another active agent for efficacy, although such additional active agents may be employed.

Testinq In vitro activity for treating ~teno~is iB demon~trated by inhibiting the proliferation of smooth mu~cle cells. Thi~ iB established by the human arterial ~mooth mu~cle cell proliferation a~ay. Human smooth mu~cle cells are grown in culturQ. A test group iB treated with the te~t compound added at selected concentration~ in fre~h media. Both groups receive 2~Ci tritiated thy i ~; n~ (~HTdR), a radioisotope label. After 24 hours, the cells areharvested and the amount of label incorporated into DNA iB counted by scintillation; this iB compared for the test and control groups, the amount being proportional to cell proliferation. Inhibition of smooth mu~cle proliferation i~ e~tabli~hed when the te~t group ha~ a lower radioisotope count than the control group. The concentration~ of test compound required to inhibit proliferation by 50~ (the IC~), and to inhLbit proliferation by more W O 94/12184 PCTrUS92/09932 -21 4~1~9 -8-than 95% are determined.
In vivo activity for treating stenosis is demon~trated in a rat model for arterial ~teno~iu. A test group is treated with the test compound, ~tarting 6 day~ before and continuing for 14 days afrer injury to the left carotid artery~ the tent group i~ compared to a control group receiving vehicle without the te~t compound. Injury is achieved by a gentle perfu~ion of air through a lO mm long section of the left artery. The right artery is left intact. Arterial cross-~ections (lO ~m) are taken from both the left and right arterie~ of each subject, and the area of the ves~el wall (endothelium, intima, media) i~ measured. The amount of vascular proliferatLon is calculated by subtracting the mean area of the Lntact, right carotLd artery from the mean area of the injured, left carotid artery. Reduction in va~cular proliferation i~ e~tabli~hed when the te~t group ~how~ len~ proliferation than the control group.
A~mini~tration Mycophsnolic acid, mycophenolate mofetLl, and the phA ~ceutically acceptable salts and derivatives thereof, can be ~ i ni ~tered via any of the accepted mode~ and formulation~ for agents ~erving ~imilar utilities, e.g., a~
described in U.S. Patents Nos. 4,753,935 and 4,922,467, previou~ly incGL~orated herein by reference.
A ' ini ~tration can be, for example, orally, na~ally, parenterally or topically, in thQ form of solid, semi-~olid, lyophi I i7cd powder, or liquid do3age forms, such a~ for example, tablets, suppositories, pill~, cap~ule~, powders, ~olutionq, suspen~ion~, emulHions, cream~, lotions, aerosols, oin~ , g~ls, or the like, pr~ferably in unit dosage form~ suitable for ~imple A~' ini ~tration of preci~e dOE-328. The compo~itions will include a convantional ph~ -~eutical carrier or excipient and an active ~c r_und (mycophenolic acid, mycophenolatQ mofetil, or a ph~ ~ceutically acceptable 3alt or derivative thereof) andj in addition, may include other medicinal agent~, ph~ ~ceutical agent~ carrier~, adjuvants, etc.
Generally, the c~ ~_L-'~ are ~ ini~tered in a therapeutically effective amount, i.e., a do~age ~ufficient to effect treatment, which will vary depen~ing on the individual and condition being treated. In the ~.~s~-t invention, the therap~uLically effective amount inhibit~ cellular proliferative re~pon~e to vascular injury. Preferably, a pla~ma concentration of about 0.3 ~M to lO.0 ~M, mo3t preferably about l.0 ~M ia thec~QeuLically effective; thi~ is a proliferation inhibitory amount.
The preferred manner of ~ ini~tration, for the condition~ detailed above, i~ oral u~ing a convenicnt daily dosage regimen which can be ad~unted according to the degree of affliction. For ~uch oral ~,` i ni ~tration, a phA -ceutically acceptable, non-toxic composition i~ formed by the incorporation of any of the normally employed ~xcipienta, such a3, for example, ph~ -ceutical grade~ of mannitol, lactoQe, starch, magne~ium ~tearate, sodium ~accharine, talcum, cellulo~e, glucose, gelatin, Qucro~e, magnesium carbonate and the like. such compo~ition~ take the form of W O 94/12184 21 gg189 PCTrUS92/09932 _ g _ (, ~ -~olutionq, su~pensions, tablets, pill~, cap~ule~, powders, suGtained release formulations and the like. -Preferably the compositions will take the form of a pill or tablet andthu~ the composition will contaLn, along with the active ingredient, a diluent ~uch as lactose, ~ucro~e, dicalcium pho~phate, and the like; a disintegrant such a~ starch or derivatives thereof; a lubricant such a~ magnesium stearate and the like; and a binder such as starch, gum acacia, polyvinylpyrrolidone, gelatin, cellulo~e and derivative~ thereof, and the like.
Liquid ph~ o~eutically ~ ;ni~terable compositions can, for example, be prepared by dis~olving, dispersing, etc. an active compound (about 0.5% to about 20~), as described above, and optional ph~ -ceutical adjuvants in a carrier, such as, for example, water, saline, aqueous dextrose, glycerol, ethanol and the like, to thereby form a solution or ~uspension.
If desired, the phA -ceutical composition to be ~ ini~tered may also contain minor amounts of non-toxic ~UYi 1 i A~y ~ubstances such as wetting or emulsifying agents, pH buffering agents and the like, such as for example, so~i acetate, sorbitan monolaurate, triethanolamine oleate, etc.
Actual methods of preparing such dosage forms arc known, or will be apparent, to thosQ skilled in thi~ art; for example, ~ee Reminaton~s Pharmaceutical Sciences, 16th Ed., ~Mack Publl shi ng Company, Easton, Pennsylvania, 1980). The composition to be ~ inintered will, in any event, contain a guantity of the active compound(~l in a ph~ -~eutically effective amount for relLef of the particular condition being treated when ~ ; ni ntered in accordance with the te~ah;ng~ of this invention.
A th~rapeutically effective daily oral dose is from a~ low as 0.02 mg/kg to about 100 mg/kg of body weight, preferably from about 25 mg/kg to about 60 mg/kg. lntravenous dose~ are comparable. Mycophanolate mofetil i~
A-' ini~tered for plavenLing allograft rejection in oral dosages of 2.0, 3.0, 3.5 and 4.0 grams per day, correspon~ng to a daily dosage from about 25 mg/kg to about 60 mg/kg, ~epQn~ing upon the patient and the allograft being treated.
Similar dosing regimens are effective in the methods of treatment of the present invention.
EXAMPLES

The following preparations and examples are given to enable those skilled in the art to more clearly unde~-and and to practice the pre~ent invention. They ~hould not be considQred a~ limiting the scope of the invention, but merely as being illustrative and ~eplsseQ~tive thereof.

This example illustrate3 the preparation of a representative ph~ ~ceutical formulation for oral ~ ini~tration containing an active compound, e.g., mycophenolic acid, mychophenolate mofetil, or a pharmaceutically acceptable salt or derLvative thereof, e.g., morpholinoethyl E-6-(l~3-dihydro-4-hydroxy 6 -thoxy-7-methyl-3-oxo-5-inohen70furanyl)-4-WO 9~/12184 PCTrUS92/09932 ~
21~4~8g -10-~ methyl-4-hexenoate hydrochloride.
Quantity per Inaredient~ CaD~ule, mqs.
Active - sund 200 5 lacto~e, spray-dried 148 magne~ium ~tearate - 2 The above ingredient~ are mixed and introduced into a hard-~hell gelatin capsule.

This example illustraten the preparation of another representative ph~ -~eutical formulation for oral a~in;Rtration contA;n;ng an active ~ , e.g., mycophenolic acid, mychophenolate mofetil, or a FhA -reutically acceptable salt or derivative thereof, e.g., morpholinoethyl E-6-(1,3-dihydro-4-hydLG~y 6 -Lhoxy-7-methyl-3-oxo-s-;soben~ofuranyl)-4 lS methyl-4 hF~oAte hydrochloride.
Quantity per Inaredients CaP~ule, mas.
Active - au ' 400 cornstarch 50 20 lactose 145 magnesium stearate 5 ThQ above $ngredient3 are mixed intimately and pressed into ningle scored tablet-.

This example illustrates th~ preparation of a representative phA -~eutical fc l~tion cont~ining an active - _~n~ e.g., mycophenolic acid, mych~hen~late mofetil, or a ph~ ~eutically acceptable ~alt or derivativo thereof, e.g., morpholinoethyl E-6-(l,3-dihydro-4-hyd~o~y 6 methoxy-7-methyl-3 ~o 5 ;Qoben~ofuranyl)-4-methyl-4-hFYeno~te hydrochloride.
Inaredients Active _ _ ' l.0 g.
fumaric acid 0.5 g.
sodium chlorLde 2.0 g.
35 methyl paraben O.l g.
granulated ~ugar 25.5 g.
sorbitol (70~ solution) 12.85 g.
Veegum R (Vanderbilt Co.) l.0 g.
flavoring 0.035 ml 40 coloring~ 0.5 mg distilled water q.s. to lO0 ml 214~189 ~ W O 94/12184 PCTrUS92/09932 --11-- , . .. .

Det~ ;n~tion of In Vitro Activity Utilizing Human Arterial Smooth Muscle Proliferation As~ay Human ~mooth muscle cell~, AG11545 (obtained from Coriell Cell Repository) were plated at low concentration (2.5 x 104 cells/ml) in 24-well plates ~Dulbecco'~ modified Eagle's medium contA;n;ng 10% fetal calf serum).
The cells were allowed to grow for 48 hours. Fresh media contA;n;ng mycophenolic acid (0.01 ~M, 0.1 ~M, 1.0 ~M and 10 ~M) was then added (except to the control wells). A label of 2~Ci ~TdR/well wa~ also added. The cell~
were allowed to grow for 24 hours, and then harvested using TCA precipitation.
The amount of label incorporated into DNA was counted by scintillation, and compared for test and control wells. From the results, the in vitro concentration of y~o~henolic acid effectlve for reducing smooth muscle cell proliferation by 50~ (IC~), and by more than 95%, were dete ; n~d.
When tested by thi~ method, mycophenolic acid had an IC~ of about 0.3 ~M. Concentration~ of 1.0 ~M inhibited smooth mu~cle cell proliferation by more than 95~. This is predictive that mycophenolic acid is useful for inhibiting stenosi~ or resteno~is in human patient~ undergoing surgical ocedure~.
By following the same p.ocedurQ and ~ubstituting ~h~ ~reutically acceptable salt or non e~er derivative of ~cophenolic acid for y~oQhenolic acid, there i~ obtained similar activity in reducing smooth muscle cell proliferation. Thi~ in predictive that the ~ha -~eutically acceptable ~Altn and non-ester derivative~ of yco~henolic acid are useful for inhibiting ~tenosis or restenosis in human patientff undergoing surgical p.ocedure3.
Mycophenolic acid concentration~ of 0.3 to 10 ~M are readily attainable in humans treated with da$1y oral doses of about 25 to about 60 mg/kg of mycophenolate mofetil. This i~ predictive that ycoph~nolate mofetil, it~
phA -reutically acceptable ~alt~ and the Qster derivatives of ycophenolic acid are useful for inhibiting Atenosis or re~tenosis in human patients undergoing surgical p-ocedure~.

F~MPLE 5 Dete in~tion of In Vivo Activity Utilizing The Rat Arterial Stenosis Model Test Materials Mycophenolate mofetil i~ Qu~pended in SSV, a vehicle con~i~ting of 0.5%
~odium ca.bo~y ~hylcellulone, 0.9% NaCl, 0.4% Tween 80, and 0.9~ benzyl alcohol in water.
Animal~
Male Sprague-Dawley rats (Crl, CD~ (SD) BR), 3-4 months old and weighing 350+25 grams, were used. The animal~ were hou~ed individually and fed W O 94/12184 PCTnJS92/09932 ~2 1 ~ 12-~tandard rodent chow.
Treatment Re~imen An experimental group of 30 rat~ wa~ treated orally with a daily doae of 30 mg/kg of mycophenolate mofetil, divided into 2 equal do~e~, given 6 hour~
apart. Treatments began 6 days before and continued for 14 days after Lnjurinq the carotid artery to induce neointimal proliferation. A control group of 30 rat~ wa~ ~imilarly treated with t~e SSV vehicle alone.
ArterLal IniurY Model The left carotid artery of each rat wa~ injured u~ing the technique de~cribed by Fishman, JA, et al., "Endothelial regeneration in the rat carotid artery and the ~ignificance of endothelial denudation in the pathognneoi~ of myointimal thickening," Lab Invest., 1975, 32:339-351.
Each animal wa~ ane~thetized by Al' i n; stering ip a mixture of 58%
ketamine (Fort Dodge Laboratories, Iowa) 60 mg/kg, and 42% xylazine (Lloyd Laboratories, Iowa) 10 mg/kg. A 10 mm long nection of the distal left common carotid artery wa~ then ex~o~cd. Silk tie~ were po~itioned and loo~ely ligated at each end of the e~o~ed artery. A puncture ~ite wa3 created near each ligature, using a 30-gauge needle attached to a ~yringe cont~;n;ng Tyrode'~ ~olution (Sigma). Next, Tyrode's ~olution was perfu~ed through the~e orifice~ to rin~e blood from the i~olated ve~el. Air wa~ then gently perfused through the ~ame orifices at a rate of 25 ml/min for 3 minutea, to injure the artery. The puncture site~ were allowed to clot, the ligature~
were ~ ~ved, and the wound wa3 clo~ed. Thi~ ~cocedure produced endothelial denudation as well a~ ~ome dinruption to the underlying intima and media of the ve~el wall.
Fourteen day3 after injury, the animal~ were sacrificed. Both the left and right carotid arteries were .~ ~ved to 10~ fn -lin and then to 30~
~ucrose, three day~ before mounting and ~coring for neointimal proliferation.
Analv~i~ of Neointimal Proliferation The recovered arterie~ were cut into quarter~ and the 4 ~ t~
: ~e~ together in a ~ingle block of Optimum Cutting Temperature (O.T.C.) ~ Lle~, Tn~;Ana). From the block, 15 arterial cross nections, each 10 ~m thLck, were ~liced with a microtome/cryo~tat (Miles, In~iAnA) at -20C
and than ~taLned wLth hematoxylin-eoain. The ~tained arterial cro~ ections were projocted through a microncope onto a digitizing board connected to a computer p~oy ~' with SigmaScan (Jandel Scientific, Corte ~adera, CA).
U~ing an electronic pen to draw around the projected inner and outer arterial wall, the mean cros~-~ectional area of both the left (injured) and right (control) arteries were computed electronically from ~ection~ l, 4, 7, 10, and 13 of the 15 arterial cro~-sections. The final area of ~mooth mu~cle proliferation for each rat wa~ calculated by ~ubtracting the mean area of the control artery from that of the injured artery. All ~coring wa~ done blindly, with the inve~tigator unaware of whether the cron~-~ection came from a te~t or control rat, or from an injured or intact artery.
~5 21~189 W O 94/12184 PCTrUS92/09932 Statistical Analy~is Both a parametric unpaired t-tent and a nonparametric Mann Whitney U
te~t were used to compare the mycophenolate mofetil-treated group to the SSV
vehicle-treated control group, to determine whether there wa~ significant reduction in mean leaion size.
~e~ults ~ Myco~henolate mofetil at 30 mg/kg markedly reduced the va~cularproliferativQ response to air-perfusion-induced vQ~sel wall injury at Day 14 after injury, yiving the following mean cro~ ~ectional areas:
Iniured ArterY Intact Artery ~hanolate mofetil 1772 + 295 mm2 1176 _ 103 mm2 SSV vehicle control 2289 + 532 mm2 1186 + 69 mm2 giving a mean area of vascular proliferation (subtracting area of in~ured artery from area of intact artery) of:
Va~cular Proliferation Mycophenolate mofetil 546 + 280 mm2 SSV vehicle control 1103 ~ 533 mm2 co,,~o~in7 to a 51~ reduction in neointi -1 proliferation (p c 0.05~.

Conclusion Mico~h^~late mofetil inhibits neointimal proliferation by 51~ following vascular injury in vivo. Thi~ ia predictiv~ that mycophenolat~ mofetil i3 useful for inhibiting stenosis or re~tQnoais in human patients undergoing surgical ~ ocedures.

By r~peating the ~ocedure and aubstituting mycoph~n~lic acid or a rh~ -~eutically -cceptahle ~alt or derivativQ thereof, or a rha -ceutically acceptabl~ salt or derLvativ~ oi ~-o~he-lolatQ mofetil, for ~coph~nolate mofetil, similar inhibition of neointimal proliferation i~ obs~ved.
MycOph~nOlic acid and the r~ eutically z~cept~hle salts and derivative~
thereof, and th~ ph~ utically ~cspt~hl~ salt- and derivatives of ~co~henolat- mofetil, ar- useful for ~nhibiting ~teno~i~ or ~ enosi~ in human patient~ unde~oing surgical p~ocedu~s.

While the ~ esel.L invQntion has been de~cribed with reference to the spQcific ~ rLs thQreof, it should be underatood by tho~e skilled in the art that variou~ ch~nges may be made and equivalent~ may be substituted without d~parting from the true spirit and scope of the invention. In addition, many modifications may be madQ to adapt a particular situation, material, compo~ition of matter, proce~s, proceaa ~tep or step~, to the objective, apirit and scope of the ~, senL invention. All auch modifications are intended to be within the scope of the claim~ app~nded hareto.

Claims (15)

WHAT IS CLAIMED IS:

1. Use of a therapeutically effective amount of mycophenolic acid, mycophenolate mofetil, or a pharmaceutically acceptable salt or derivative thereof to inhibit stenosis in a mammal in need thereof.

2. The use of Claim 1 comprising administering mycophenolic acid, mycophenolate mofetil, or a pharmaceutically acceptable salt or derivative thereof, to an angioplasty or cardiac by-pass patient.

3. The use of Claim 2 comprising administering mycophenolic acid, mycophenolate mofetil, or a pharmaceutically acceptable salt or derivative thereof, prophylactic to the angioplasty or cardiac by-pass procedure.

4. The use of Claim 3 comprising the administration of mycophenolate mofetil or a pharmaceutically acceptable salt thereof.

5. The use of Claim 1 consisting of the administration of mycophenolic acid, mycophenolate mofetil, or a pharmaceutically acceptable salt or derivative thereof.

6. The use of Claim 1 consisting of the administration of mycophenolic acid, mycophenolate mofetil, or a pharmaceutically acceptable salt thereof.

7. Use of a proliferation inhibitory amount of mycophenolic acid, mycophenolate mofetil, or a pharmaceutically acceptable salt or derivative thereof to inhibit the proliferation of vascular cells following an insult to a vessel wall in a mammal in need thereof.

8. The use of Claim 7 comprising inhibiting the proliferation of smooth muscle cells.

9. The use of Claim 7 wherein said proliferation inhibitory amount is a plasma concentration of about 0.3 µM to about 10.0 µM.

10. The use of Claim 9 comprising the oral administration of mycophenolate mofetil in a therapeutically effective amount to give a proliferation inhibitory amount of mycophenolic acid.

11. The use of Claim 10 comprising the oral administration of about 25 mg/kg to about 60 mg/kg of mycophenolate mofetil.

12. Use of a therapeutically effective amount of mycophenolate mofetil, or a pharmaceutically acceptable salt thereof for treatment to inhibit stenosis in a mammal.

13. The use of Claim 12 wherein said therapeutically effective amount is about 25 mg/kg/day to about 60 mg/kg/day.

14. The use of Claim 13 wherein mycophenolate mofetil or a pharmaceutically acceptable salt thereof is administered orally.

15. A pharmaceutical composition for the treatment of stenosis comprising a pharmaceutically acceptable non-toxic excipient and a therapeutically effective amount of mycophenolic acid, mycophenolate mofetil, or a pharmaceutically acceptable salt or derivative thereof