CA2257564A1 - Use of green porphyrins in the manufacture of a medicament in the treatment of atherosclerosis without purposeful irradiation of the porphyrin derivatives - Google Patents

Abstract

A method to prevent or inhibit the development of arterial plaque, comprising the steps of: (a) administering to a subject an amount of a green porphyrin compound effective to prevent or inhibit said development; and (b) allowing the inhibition to occur in the absence of irradiation with light absorbed by the green porphyrin; or a pharmaceutical composition for preventing or inhibiting the development of arterial plaque. The composition comprises: (a) an amount of a green porphyrin compound effective to prevent or inhibit the development when administered to a subject in need of such treatment, even in the absence of irradiaton with light absorbed by said green porphyrin; and (b) a pharmaceutically acceptable excipient. The administration of the green porphyrin need not be accompanied by any purposeful irradiation with light.

Description

USE OF GREEN PORPllYR~S lN THE MANUFACIURE OF A MEDICAMENT IN THE TREAT-MENT OF ATHEROSCLEROSIS WlTHOUT PURPOSEFUL IRRADIATION OF THE PORPHYRIN
DERIVATIVES
BACKGROUND OF THE rNVENTION
Field of the Invention The present invention relates to the I~talmel~t offatty dcposits in - atherosclerotic blood vessels. In particular, the invention relates to the prevention or inhibition of arterial plaque by ndr..~ sl~ I ing a green porphyrin compound. Unlike standard photodynamic protocols, no purposeful irradiation with light is needed to effect the desired inhibition.

Description of the Related Art Atherosclerosis is an arterial disease in which fatty sU~sl~n~eC (lipids) accum~ te in the intima or iMer media, the i~ ellllost n,c..l~-di.es cnco~ Ac~ the lumen of the artery. The resulting lesions are referred to as atheroscle.~,t.c plaque.
15 Clinical symptoms finally occur because the growing mass ofthe ath.,.oscle;otic plaque gradually reduces blood flow through the involved artery, thereby cG.--pr~,l.u~;.,g the tissue or organ function distal to it. A~,c~scle.osis and its contrlicstion~ such as myocardial ;.~a., lion, stroke, and ~ helal vascular dicPa~s remain major causes of morbidity and mortality in the Western World. Coronary heart disease alone has been 2 0 reported to account for over half a million deaths in the United States annually.
The fim~ l Iesion of alherosclc~ os;s is the atherc,-..atous or l'.brofutly plaque, which causes na. ~ u~. ing of the artery, p- ~.I;sposes to thrombosis, c~lcifies, Ieads to we~l~P.n~ of the muscle, and causes aneurysmal di1ation. Alh~ oscl~,rotic plaques are appro~imqtely rounded, raised lesions, usually off-white to white in color 2 5 superficially, and perhaps a c~ , in ~ t~ ~ . The center of larger plaques may exude a yellow, grumous fluid. A typical ceîlular plaque is shown in Figure 3 and consists of:
1. a fibrous cap composed mostly of smooth muscle cells with a few leukocytes and relatively dense co~ c~ e tissue, which co.lt~ns 3 0 elastin, cc"a~P~ fibrils, proteoglycans, and b~P~e~t n.c.l~ e;
2. a cellular area beneath and to the side of the cap CQn';~l;i.Q of ~ mixtllrP
of macrophages, smooth muscle cel}s, and T Iymphocytes; and WO 97/4a393 PCT/CA97/00393 3. a deeper "necrotic core" containing cellular debris~ extracelh-l~r lipid droplets, and cholesterol crystals Munro et al., "Biology of Disease; The Pathogenesis of Atherosclerosis:
Atherogenesis and Inflammation", Labora~ory InvesJi~ation 58:3, 249-SO ( 1988).
Left unchec~ed, the formation of plaque can result in the complete occlusion of the artery and severe clinical consequences. For example, when complicated, the lesion becol-,es a calcified fibrous plaque col~tai~,i,.g various degrees of necrosis, thrombosis and ulceration. With increasing necrosis and aGcum~ tion of cell debris, the arterial wall prog~ ~ss;vely weakens, and rupture of the intima can occur. causing 0 aneurysm and hemorrhage. Arterial emboli can form when r, . g".~ C of plaque dislodge into the lumen. Stenosis and i-npair~d organ filnction result from gradual occlusion as plaques thicken and thrombi form.
The l, ea~l.e.,l of atherosclerosis generally focuses on the care of patients suffering from atherosclerotic complications and typically follows one of four basic approaches: (1) the dice~eed vascular segments may be replaced with prosthetic or natural grafts, going as far as whole heart ~,~ns,vlant~tion, (2) drugs, such asantiarrhythmic agents, ~lticoael~lqnts and plasma lipid lowering agents, may be admini~tered to enable the patient to live with the conditions; (3) the plaque may be physically reduced in size by the use of a balloon catheter in angioplasty; or (4) 2 0 photodynamic therapy may be used which couples the adminictration of photosensitive agent and subsequent irradiation with light to excite the photose,.s;l;,er, thusproducing a cytotoxic effect. Spears, U.S. Patent Nos. 4,512,762, issued 23 April 1985 and U.S. Patent No. 4,566,636 issues 25 March 1986.
In photodynamic therapy, the photosen~;ti7ers used are capable of localizing in 2 5 ~nqli~nDnt cells, either by natural tendency or because they have been intentionally targeted to a specific type of tissue, or both. When irradiated, they may be capable of fluorescing and, thus, may be useful in dia~nostic method~ related to detecting tarBet tissue. However, even more i~npo,l~-lly~ the photosensiti7er has the capacity, when irradiated with light at a wavclel)glh which the compound absorbs~ of causing a 3 o cytotoxic effect against whatever cells or other tissue in which the photosenciti7~r has SUBSTIT~TE SHEET (RULE 26) WO 97/48393 PCTtCA97100393 localized. Although not yet definitively established, it is thought that this cytotoxic effect is due to the formation of singlet oxygen upon irradiation.
In most photodynamic therapy protocols, a method must be found for the irradiating light to reach the targeted tissue where the photose.~ i,er has been5 localized This is particularly difficult when the wavelength of irradiation that is required to activate the cornro~n~ is in the range of about 630 nm, a wavelength that is readily absorbed by natural chromophores in blood and other surrounding tissues. In one technique, the patient must be catheterized with a light-emitting catheter being inserted into the ~iice~ed artery or other vessel so that the light-emitting portion of the 10 catheter is adjacent to the atherosclerotic plaque For example, a light e.,u~ling balloon catheter may be used to di~place light-opaque blood between the external balloonsurface and the atherosclerotic plaque by inflation of the balloon. Altematively, a fonn of"liquid light" may be injected into the vascular tree such that the "liquid light", which mixes freely with blood or a blood replacement, perfilses the ~liceAced artery.
Spears, U.S. Patent No. 4,512,762.
In another method, to keep dosages low and decrease the patient's sensitization to light, a ~ ftl ~ ~d group of "green porphyrins" have made it possible to conduct photodynamic therapy with light having a wavelength range outside of that normally strongly absorbed by the blood or other norrnal tissues, specifically around 670-780 2 0 n n. In addition to providing an effective in vivo treatment at lower concentrations and reducing h~,el~ers;~i~ity of non-target tissues, a greater depth of pel,el~alion by the irr~diqtin~ light is also usually acll ~ied. R~ se these photosens;tiG~;,a appear green in color rather than red, they have been nicL ~ ed "green porphyrins." It is known that green po~phyrins can be used to detect and treat athel oscle. olic plaques in a photodynamic therapy protocol. See, for eY~mple, Levy et al., U.S. Patent No.
5,399,583 issued 21 March 1995 (column 2, lines 14-15); Levy et al., U.S. Patent No.

4,920,143 issued 24 April 1990 (column 10, lines 58-59); Levy et al., U.S. Patent No.

5,095,030 issued 10 March 1992 (column 2, lines 8-9 and column 15, lines 29-30); and ~ Levy et al., U.S. Patent No. 5,171,749 issued 15 Dece"llJel 1992 (column 2, lines 12-3 o l 3 and column 18, lines I -4 and 35-47).

SUBSTITUTE SHEEr (RULE 26) It has also been found that green porphyrins can exert certain "dark effects"~
which take place without any purposeful irradiation at all with photoactivating light.
For example, it has been found that administration of a green porphyrin following a vascular intervention procedure, such as angioplasty, can si~ r.c~ y reduce the 5 proliferation of smooth muscle cells that typically builds up in the intima to cause "restenosis" or "intimal hyperplasia". See Vincent et al.~ U.S. Patent No. 5,422,362 issued 6 June 1995. Moreover, it has been found that the irnrnune response to a specific antigen may be mod~ ted and that the intercellular communication that results in thrombosis or blood clot forrnation in the bloodstream by may be i,~e.r~,.,d with by 10 a~ ni~tering green porphyrins in the absence of light~ as described in co-pending Application Serial No. 08/374,158 filed 13 January 1995.
However, until now, the use of green porphyrins to treat fatty atherosclerotic plaques has been limited to a photodynamic therapy protocol. which requires that the ...;n sl, ation of the photosen.citi7~r be followed by the irradiation of the inner 5 "~e..lb,~lle of the blood vessel with light having a wavelength capable of stimul~tinE the green porphyrin to produce a cytotoxic effect. It would thus be advantageous to provide a ll~at~ to inhibit or prevent arterial plaque that would co"lpl-:ely avoid the neces~ for speci~li7~d light e, Itin~ devices. It has now been discovered that green porphyrins may be used to prevent or inhibit the development of arterial plaque 2 0 without having purposeful light exposure of any kind.

SUMMARY OF THE INVENTION
According to the present invention, there has been developed a method to prevent or inhibit the de~lop...mt of arterial plaque, CO,l"., is;"g the steps of:
2 5 a. administering to a subject an amount of a green porphyrin compound effective to prevent or inhibit said development; and b. allowing said inhibition to occur in the absence of irradiation with light absorbed by said green porphyrin.
The invention also concerns a phar -ceutir~' composition for preventing or inhibiting 3 o the development of arterial plaque. The composition comprises:

SUBSTITUTE SHEEl- (RULE 26) .

a. an amount of a green porphyrin compound effective to prevent or inhibit said development when ~r~minict~red to a subject in ~ need of such ~ e-.h even in the absence of irradiation with light absorbed by said Gp; and b. a pharm~ceutic~lly acceptable excipient.
As a result of the invention, the ~ alion of the green porphyrin, surprisingly, need not be accomp~nied by irradiation with light absorbed by the green porphyrin administered. Rather, the lle~l-.lenl is typically p~.rol~--ed without purposeful irradiation with light of any type.

BRIEF DESCRIPT~ON OF THE DRAWINGS
The present invention will be more clearly understood by referring to the following drawings, in which:
Fig. l shows the forrnulas of typical green porphyrins useful in the method and 5 composition of the invention.
Fig. 2 shows the formulas of four particularly prefel, t;d embodiments of ~he green porphyrins of the invention, BPD-DA, BPD-DB, BPD-MA and BPD-MB.
Fig. 3 . el" e3~ s a cross-sectional view of an ~ll.c(osclerotic plaque.

Atherosclerosis tends to involve large and medium-sized mllcc~ r arteries.
Most commonly affected are the aorta and the iliac, femoral, corona-y, carotid, basilar, vertebral and cerebral arteries.
As used herein, "atheroscl~r-)lic arterial plaque" is defined as a 2 5 pathophysiological pheor.,~,.)on which is ~-~o,l,Dr,ied by the proliferation of fatty or lipid deposits within the intima or the iMer media, the innermost layers enco.~.r~cs;i-g the lumen of the artery.
As shown in the cross-section scl~e-~lat;c of Figure 3, an atherosclerotic plaque occurs within the intima l or the media 2 (medial smooth muscle cells not shown), 3 o both of which extend over the adventitia 3, the outer coat of the artery. An overlaying endothelial layer overlies the plaque. The fibrom~cc~lat "cap" portion of the plaque SUBSTITUTE SHEET ~RULE 26) . , . . ~ , . .

conla...s smooth muscle cells 4, macrophages 5, and collagen bands 8. Underneath this is the "core" with ~Un~?nt extrs~cellul~r lipid droplets 9, "foam" cells whose cytoplasm is filled with lipid droplets, cholesterol crystals 10, and necrotic cellular debris. Macrophages 5 are widely distributed, and Iymphocytes 6 can be found in the 5 cap and to the sides of the core. Adventitial capillaries 7 gravitate toward the intimal lesion. Munro et al., "Biology of Disease; The Pathogenesis of Atherosclerosis.
Atherogenesis and Illnallllllaliol~ ~ Lc~boralory Inves~i~at10n 58:3, 249-50 ( 1988).

The Green Porphyrins The green porphyrins useful in the method of the invention are described in detail in Levy et al., U.S. Patent No. 5,1 î1,749 issued 15 December 1992, which is incorporated herein by ~ ~fe~ ence. "Green porphyrins" refer to porphyrin derivatives obtained by reacting a po~lJhr~in nucleus with an alkyne in a Diels-Alder type reaction to obtain a monohydrobe.~opo- ~ yrin. Typically, green porphyrins are sel~cted from 15 a group of porphyrin derivatives obtah~ed by Diels-Alder reactions of acetylene derivatives with protoporphyrin under conditions that promote r. a-tion at only one of the two available conju~,~ e d, nonaromatic diene structures present in the protoporphyrin-IX ring system (rings A and B).
Several structures of typical green porphyrins are shown in Fig. I . The Diels-2 o Alder reaction initially results in the formation of a cyclohPx~:ene -- referred to herein as "hyd- ube.~o" -- fused to the A or B pyrrolic ring, as shown in formulas I and 2.
Real I A~ge.~ of the 7C system in the h~Y7 ' ~ ring results in the formation of compounds of fonnulas 3 and 4, and reduction provides compounds of formulas ~ and 6. These compounds are shown in formulas 1-6 with hydrogen occupying the internal 2 5 ring nitrogens. However, it is to be understood that the met~l~ted forrns, in which a cation replaces one or both of these h~/d- ùg~ns~ can also be used. The pl epa- ~ion of the green porphyrin compounds useful in this invention is described in detail in U.S.
Patent No. 5,095,030, which is incorporated herein by reference.
For conve . e ,ce, an abbreviation ofthe terrn hydromonobenzopol~hyrin 3 0 derivative--"BPD"--is gcue. ally used to refer to compounds of formulas 3 and 4 of ~ig.
I . Compounds of the formulas 3 and 4 and mixtures thereof are particularly preft. ~ ~d.

SUBST~rUTE S~IEEl (RULE 26) WO 97/483g3 PCT/CA97/00393 As shown in Fig. I, R', R2, R and R4 are non-interfering substituents that do not appreciably affect the activity of the compound in the method and composition of the invention. More specifically, the term ~'non-interfering substituents" is used to mean substinl~ntc that do not destroy the ability ofthe green porphyrin to prevent or inhibit arterial plaque. For the compounds of Figs. I and 2, generally, R' and R2 are each, independently, moderately electron-withdrawing substituents or any other activating substit~nts that are not s~fficie~t~y electron-withdrawing to result in the Diels-Alder reaction proceeding with both A and B rings, rather than only one.
Examples of suitable R' and R2 groups include carbalkoxy (2-6C), alkyl ( l -6C) 0 sulfonyl or aryl (6-10C) sulfonyl, aryl (6-10C), cyano, and -CONRsCO- where R5 is aryl (6-l 0C) or alkyl (l -6). One of Rl and R2 may also be hydrogen, so long as the other is an electron-withdrawing substituent of sufficient strength to f~ilit~te the Diels-Alder reaction. Most cc,l~u..ol~ly, R' and R2 are carbalkoxy groups, preferably methyl or ethyl carboxy esters. Pl efe, I ~d compounds are those in which R~ and R2 are 5 the same and are carbalkoxy, particularly carboethoxy.
As used herein, the term "carboxy" is, as conventionally defined, -COOH, while "carbalkoxy" re~,rese.,~ -COOR where R is alkyl. "Carboxyalkyl" refers to the substituent -R'-COOH where R' is alkylene. "Carbalkoxyalkyl" refers to -R'-COOR
where R' and R are alkylene and alkyl r~spec~ ely. "Alkyl" generally represents a 2 0 saturated straight or b.al-clled chain hydrocarbyl moiety of l -6 carbon atoms, such as methyl, n-hexyl, 2-methylpentyl, t-butyl, n-propyl, and so forth. "Alkylene" is the same as "alkyl" except that the group is divalent rather than monovalent. "Aryl"represents a phenyl group, optionally c~bstin~ted with 1-3 s-~bstituents, which may be independently selected from the group consisting of halo, such as fluoro, chloro, 25 bromo or iodo; lower alkyl (I~C); and lower alkoxy (]~C) "A~yl" or "alkyl sulfonyl" groups have the forrnula -SO2R where R is alkyl or aryl as defined above.
R3 is indep~ndently a cl)-carboxyalkyl group (2-6C~ or a salt, amide, ester or acylhydrazone thereof, or is alkyl (1-6C). Pl~r~,.ably, R3 is 2-carboxyethyl or the alkyl ester thereof, and R4 is vinyl. These ç~bod;~-e~ , however, are p- ere, ~ ed because of 3 o the availability of native porphyrins, rather than being m~rld~ted by considerations of biological efficacy. As shown in Fig. 1, adducts forrned by the reaction of R'-C-C-R2 SUBSTITUTE SHEET (RULE 26) .

with a protoporphyrin-IX ring system (where R3 is a protected form of 2-carboxyethyl~
such as 2-carbometho~cyethyl or 2-carboethoxyethyL and R4 is -CH=CH2) are compounds of the formulas I and 2. Compounds of formula I result from the addition to the A ring, and compounds of formula 2 result from the addition to the B ring.
Convenient starting materials for the green porphyrin compounds of the invention include the naturally-occurring porphyrins where R~ is either -CH2CH2COOH or -CH2CHRCOOR where R is alkyl ( I -6C). However, the exact nature of R3, unless it conlah,s a 7c-bond conjug~ed to ring ~I-bond, is ordinarily not relevant to the progress of the Diels-Alder reaction or to the effectiveness of the 0 resulting product. R3 can thus be any one of a wide variety of groups such as, for example, lower alkyl ( I -4C), and ~-carboxyalkyl (2-6C) and the esters and amides thereof. The R3 substituent may also be sukstituted with halogen, such as fluoro, chJoro, bromo or iodo, or with other nomeactive sukstitupntc When R~ is CH2CHRCOOR, it has been found advantageous to hydrolyze, or partially hydrolyze, the esterified carboxy group. Typically, the hydrolysis at the R3-position conv~nie :~ 'y occurs at a much faster rate than that of the ester groups of R' or R2. Further, the solubility and biodistribution characteristics of the resulting compounds are more desirable than those of the unhydrolyzed forrn. Hydrolysis results in the diacid or monoacid products (or their salts).
2 0 In compounds of formulas I and 2, R4 is usually -C~I=CH2, at least initially, but this vinyl group is readily derivatized to other embodim~ntc of Rq by the addition to, or oxidation of, the vinyl ring s~bstit~lPrlt of ring B or A in forrnula I or 2 1 es~,e~lh/ely.
Thus, R4 can be any one of a wide variety of s~lbstituents that are ~onr;ctent with that forrned by a &cile addition reaction. For ~ n;-~le, an exçmrl~ry addition reagent may be of the forrn HX where H is added to the carbon q/~ CÇnt to the ring to provide an R4-position having the forrnula:

Thus, in one embodiment, one of the added substituents is a hydrogen, and the other one is selected from the group consisting of hydrogen; halo such as fluoro~

CA 02257564 l99X-12-03 chloro, bromo or iodo; hydroxy; lower alkoxy; amino; amide, sulfllydryl~ or an organosulfide. For example. the Markovnikov addition of water provides a substituent structure analogous to a hemaloporphynn ring system at the relevant ring. The vinyl group can also be oxidized to obtain~ as a substituent in the R4-position, -CH20H~ -CHO, or COOH or its salts or esters The addition or oxidation products can themselves also be substituted if the added substituents are functional leaving groups. For example, when Br is a substituent, it may be replaced by such moieties as -OH, -OR where R is alkyl (1-6C) as described above, halo, -NH2. -NHR_ -NR2 and the like 0 Thus, in general, R~ ,ep~ese,ll~ any substitll~nts to which the vinyl group -CH=CH2 is readily converted by cleavage or addition, and further substituents formed by the reaction of leaving good groups with additional rnoieties. Preferably, however, R~ is vinyl (-CH=CH2); -CHoR4 where R4 is H or alkyl ( I -6C), optionally substituted with a hydrophilic substin~ellt such as -CH20H; -CHO; -CooR4 such as COOH or -COOCH3; -CH(oR4 )CH3 such as -CH(OH)CH3 or -CH(OCH3)CH3;
-CH(oR4 )CH20R4; -CH(oH~CH2o~;-cH(sR4 )CH3 such as -CH(SCH3)CH, and the ~isulfi~e thereof; -C~(NR4 )CH3; -CH(CN)CH3; -CH(pyridinium bromide)CH3;
-CH(CooR4 )CH3; -CH(CooCR4 )CH3; -CH2(halo)C~13 such as -CHBrCH3; or -CH(halo)CH2(halo). Alternatively, R4 can be an organic group of less than 12 carbon 2 0 atoms resulting from the direct or indirect derivatization of vinyl. Or R4 can provide additional porphyrin or porphyrin-related ring systems, such as a group containing ~om 1-3 tetrapyrrole-type nuclei of the formula -L-P, as defined below. Those compounds in which R~ is -CH=CH2~ -CH(OH)CH3, -CH(halo)CH3, or a group C~UI~;QiQ~ 1-3 tetrapyrrole-type nuclei of the forrnula -L-P, as defined below, are 2 5 p~ef~., ed.
As used herein, the term "tetrapyrrole-type nucleus" r~pres~n~s a four-ring system of the skeleton:

WO 97/48393 PCT/C~97/00393 Me - Me ~IH N

(~ N H N -- ~
Me~ 1 . I Me or a salt, ester, amide, or acylhydl~20ne thereof, which is highly conjugated. It includes the porphyrin system, which is in effect a completely conjng~ted system, the chlorin system, which is in effect a dihydro fonn of the porphyrin; and the reduced 5 chlorin system, which is a tetrahydro form of the conjugated porphyrin system. When "porphyrin" is specified, the completely conjugated system is in-~ic~ed. Green porphyrins are effectively a dihydro forrn of the porphyrin system.
In one embodiment, the substituent R4 ineludes at least one additional tetrapyrrole-type nucleus. The resulting compounds of the invention are dimers or 0 oligomers in which at least one of the tetrapyrrole-type ring systems is a green porphyrin. Linkage between the green porphyrin moiety at the R4-position to an ~ldi~io~l tetrapyrrole-type ring system may be by an ether, amine or vinyl linkage.
Porphyrin ring systems having two available s~lbstitllent positions (in both A and B
rings) corresponding to R4 can be additionally derivatized, as explained below.
When R4 is "-L-P," -L- is selected from the group cuns.~i.. g of (a) -fH-O-CH-, 20 (b) -CHNHCH-, (c) -CH=CH-CH-, (d) -CH-CH=CH-, (e) =CH-C-CH-, and Il I

SU~STITUTE SHEET (RULE 26) WO 97/48393 PCT/CAg7tO0393 (f ) -CH- -C-CH=;

CH3 o and P is a porphyrin structure or a second green porphyrin of the formulas l-6 shown in Fig. l, except that any second R4 group is replaced by L above.
(It is also understood that, when -L- is of the fonnula (e) or (fl shown above, the ring system to which the double bond is attached will have a resonance system 0 corresponding to ~ N ~

,, /~
in the ring to which the double bond is ~tt~che~l as shown.) The hydro-monobenzopo-~Jh~rins that directly result from the Diels-Alder reaction described above can also be iso"~e. i~ed to the BPD compounds of formulas 3 and 4 of Fig. l . The d~pictions of compounds 3 and 4 in Fig. l do not show the relative position of the exocyclic methyl group (ring A of formula 3 and ring B of formula 4) with respect to the R2 s~bstitu~nt. Either isomer is available. Compounds of formulas 3 and 4 are particularly p- efe., ed in the methods and CO""~G ,;~ions of the invention.
2 o In addition, the Diels-Alder products can be selectively reduced by treating with hydrogen in the p, esence of a catalyst, such as pq~ ivm on charcoal, to give the saturated ring analogs, shown as forrnulas 5 and 6 in Fig. I, which correspond to the rei~ecli~e Diels-Alder products of rings A and B. The de~c, i~tion set forth above with respect to the compounds offormulas I and 2 conc~i.".ng deriva~ tiort by conversion 2 5 of the r~m~ining vinyl substituent (R~) and with respect to the variability of R3 applies as well to the compounds of formulas 3, 4, 5 and 6.
P. t;fe,.ed embodiments of the green porphyrins of the invention are those in which the Diels-Alder product is I ~,a, ~ an~,ed and partially hydrolyzed. Even more p.~l~".,d are the compounds offormulas 3 and 4 (BPD's) in which the carbalkoxy 3 o groups in the R3-positions have also been hydrolyzed or partially hydrolyzed.
SUBSTlTUTE SHEET ~RULE 26) , CA 02257564 l998-l2-03 WO g7/483g3 PCI'/CA97/003g3 Compounds of the invention that contain -COOH may be pl ~pared as either the free acid or in the form of salts with organic or inorganic bases.
Fig. 2 shows four particularly prcfe.l ed compounds of the invention covered by formulas 3 and 4, which are collectively d~si~ted as benzoporphyrin derivatives, i.e.
BPD-DA, BPD-DB, BPD-MA and BPD-MB. These are hydrolyzed or partially hydrolyzed forms of the l ~a. ~ ~nged products of formula 3 and 4, wherein one or both of the protected carboxyl groups of R3 have been hydrolyzed. The ester groups at R' and R2 hydrolyze relatively slowly, so that conversion to the forrns shown in Fig. 2 is easily effected. The most pr~,~e--~,d ofthese green porphyrin compounds is BPD-MA.
0 In Fi~. 2, R3 is -CH2CH2CooR3 where R3 varies by individual compound Spe~ ;r.c~lly, in BPD-DA. R~ and R2 are carbalkoxy, R3 is hydrogen, and derivatization is at ring A. BPD-DB is the corresponding compound with derivatization at ring B.
BPD-MA .~,pres~,.t~ the partially hydrolyzed forrn of BPD-DA, and BPD-MB
, ep~cs~,nls the partially hydrolyzed form of BPD-DB. Thus, in these latter compounds, Rl and R2 are carbalkoxy, onê R~ is hydrogen, and the other R3 is alkyl (I-6C).
The compounds of formulas BPD-MA and BPD-MB may be homogeneous, in which only the C ring carbalkoxyethyl or only the D ring carbalkoxyethyl would be hydrolyzed, or may be mixtures of the C and D ring substit~ent hydrolyzates. In addition, mixtures of any two or more of BPD-MA, 2 0 -MB, -DA and -DB may be used in the methods of and compositions of the invention.
It should be noted that many of the compounds of Fig. I contain at least one chiral center and, thus, may exist as optical isomers. The method of the invention can use compounds having both configurations of the chiral carhons, whether the compounds are supplied as isolates of a single stt, ~oisG~ ,r or are rnixtures of enantiomers and/or diaste.eonle,~. Separation of mixtures of diaste-,,on,er~ may be effected by any conventional means. Mixtures of enantiomers may be separated by any of the usual te~hni~uec~ such as by r ~ 7 cting them with optically active p, ~a~lions and sepa.~ting the resulting diastereon,c~.
It should further be noted that the reaction products may be unse,tJal aled 3 0 mixtures of A and B ring additions, e.g., mixtures of forrnulas l and 2 or 3 and 4 or 5 SU8STITUTE SHEET (RULE 26) .. . .

wo 97/48393 PCr/CA97/00393 and 6. Either the separated forms, e.g . forrnula 3 alone or 4 alone, or mixtures in any ratio, may be used in the methods and compositions of the invention.
Further still, dimeric forms of the green porphyrin and dimeric or m--ltimeric forms of green porphyrin/porphyrin combinations can be used. The dimers and 5 oligomeric compounds of the invention can be prepared using reactions analogous to those for di.--e.i~aeion and oligomerization of porphyrins per se. The green porphyrins or green porphyrin/porphyrin linkages can be made directly, or porphyrins may becoupled~ followed by a Diels-Alder reaction of either or both tenninal porphyrins to convert them to the co, les,~onding green porphyrins.

Ad.l.,nisllalion of Green Porphyrin Compound:
In the method of the invention, the subject is administered an amount of the green porphyrin compound or a mixture of green porphyrin compounds in one or several dosages. The dosage depen~lc upon the purity and chemical forrn of the green 15 porphyrin and the deBree of a~so. IJtion expected. Suitable ~mo~nt~ per dose are typically greater than about 0.5 mg/kg of body weight, pl cf,.ably in the range of frorn about 0.5 to 5.0 mg/kg per dose, and most preferably is about 0.5 to 2.0 mg/kg per dose. These dosage ranges are intended to be su~&estive and should not nececc~ ;ly be considered as lirniting, since the individual reactions of particular subjects will vary.
2 o Adjustment of the dosage ranges in accordance with individual variations is routine anlong practitioners.
Similarly, no single protocol appears to be des;,l~'e for all cases at this time.
However, typical protocols will include either a single dose or an initial dose followed by 1-4 a~ditionql doses at weekly, biweekly or monthly intenrals. A particularly2 5 pref~. ~ed protocol calls for 3 doses about two weeks apart. Again, these protocols are not intended to be limiting in view of the wide variation perm~tted in protocol design.
The green porphyrin compounds of the invention may be administered as a single compound, preferably BPD-MA, or as a mixture of various green porphyrins.Suitable fonnulations include those ap~ropiiate for systemic ~n~inis~ration~ including 30 p.e,)dralions for injection. transmucosal or Ir~nsde...-al administration, or oral ad~ ation. A particularly pl ef,. I ed means of forrnul~ting the green porphyrin of WO 97148393 PCrICA97/00393 the invention for this use is in the form of a solution or as liposomes suitable for injection into a p~liph~.al vein. The green porphyrin may be included within theliposomes, attached to their surface, or both. Suitable methods for pre~al i"g liposomes are well-known in the art. The inclusion of green porphyrin compounds in such preparation is dea~lilJed, for example, in Allison et al., U.S. Patent No 5,214~036 issued 25 May 1993 and Desai et al.~ co-pending application Serial No. 08/489,850 filed l3 June l 995, both of which are incorporated herein by refe,ence.
When injected intravenously, the solution or liposome formulation diss~n,h~ates throughout the vascular system and thus comes into direct contact with the arterial 0 plaques being targeted, where the green porphyrin is selectively absorl,e,d, with peak conc~."l ation typically occurring within about 24 hours. The pl ~scl-ce of green porphyrin in the plaque may be detected within a few hours after injection and may persist for as long as several days to two weeks.
In one embodiment, the absorption of green porphyrins by alh~.~,r,.alous plaques may be enh~nced by binding to the green porphyrin some antibody specific to a component of the plaque. Monoclonal antibodies may be particularly useful due totheir eAI, ~,n,e .I,e~ ;r.c;~y. Con,l)onc.lts of plaque that can serve as a.~ligcn c targets include elastic cl~,.n~ s, collagen, and lipid co~ctit~Pntc.
In the methods of the invention, the green porphyrin compounds, forrnulated into pharrn~~eut~ co,nposilions, are ~rnini~tered without the need to irradiate invasively at the site of the potential or growing arterial plaque with light that is absorbed by the green porphyrin. The expression "in the absence of irradiation with light absorbed by the green porphyrin" is int~n(led to mean that no such deliberate irradiation is 7(in-inict~red The phrase does not, of course, exclude inadvertent, 2 5 coincidPnt~l, or normal exposure of the affected tissues to ambient light.
The following examples are intPnded to illustrate but not to limit the invention.

Fy~rnrle l: DoseVa~ialions This study was pc. ~o, med to assess the effect of liposomal BPD-MA
3 o verteporfin on the development of aort.ic fatty nlaqlle in rAhhits fed a high fAt diet Twenty test animals were selected and divided into four groups. Thro~lghout the WO 97/48393 PCI'/CA97/003g3 study, all animals were maintained on a diet consisting of re-pelleted rabbit chow con~ining 7% corn oil and 1.5% cholesterol Three of the groups were dosed with either 0.5, I.0 or 2.0 mg/kg of BPD-MA at two weeks and at six weeks after the start of the high fat diet. The fourth group of animals received no BPD-MA at all and thus 5 served as a control group. All test animals were necrops;ed at 7 weeks, and their aortas were discected out and fixed for macroscopic and mi~;loscopic ~Y~ tion by a certified veterinary pathologist. Histopathology was carried out on formalin-fixed sections using standard haematoxylin and eosin staining techniquçs. Data from the five individual animals in each test group was averaged to obtain the values for the group.
In the macroscopic asses~-~.cnl, whole rabbit aortas of about 15 cm in length were exarnined for the severity and extent of plaque. The results are s~ u-,a,ized below in Table I

Table I
Extent of Affected % Of Drug Dose Tissues2 Affected Group (mg/kg) Severity~ (cm! Tissues 0 2.2 7.0 46.7 2 0.5 l.8 2.5 16.~
3 l.0 1.3 4.8 32.0 4 2.0 1 .2 0.9 6. 1 5'Severity: 0 = No plaque.
I = Small linear plaques (3 mm or less).
2 = Confluent areas of plaque involving less than l/2 of the arterial circu~ ,noe.
3 = Confluent areas of plaque involving more than l /2 of the arterial 2 o circu~ rence.
2Extent: The total length ofthe aorta in linear cm that was affected by plaque.
Where several separate areas were involved, the lengths affected were added to give a total value.

2 5 A section of rabbit aorta app~u~i"lately I cm from the aortic bifilrcation was taken for rnicroscopic e ~ ;on. This particular portion of the aorta was s~lected because it ~ ~ . .... . .

CA 02257564 l998-l2-03 is generally considered to best, e~" ~sent overall plaque development. The results are sunun.~ ed below in Table 2.

Table 2 No. Sections Drug DoseSeverity of Cont~ining Group (mg/kg) Plaquel Aortic Plaque 0 1.0 4/5 2 0.5 0.8 3/5 3 1.0 0.2 1/5 4 2.0 0 015 'Severity: 0 = Normal aorta.
I = One or more sub-endothelial fatty plaques with less than 30% ofthe aortic section involved.
2 = Sub-endothelial fatty plaques with 30 - 60% of the section affected.
3 = Sub-endothP~ fatty plaques with greater than 60% of the section 10 involved. Both the severity of plaque and the extent of plaque-affected tissue appeared to decrease with increasing doses of BPD-MA. These effects were observed both macroscopically and mi ~ oscop;cally. In both cases, the greatest effect was seen in the arumals that received 2 0 mg/kg of the green po 15 F- -: ,rle 2: Variation of Number of Doses In this study, two groups of test animals (n=6 per group) were dosed with 2 mg/kg of BPD-MA at either two weeks after the start of the high fat diet dcscl il,ed above in F- - , le I or at two, four and six weeks a~er the start of the diet. A third group served as a control group and, thus, received no BPD-MA. All animals were 2 o nc.,rops;ed at 8 weeks, and the rabbit aortas were dissected out and fixed as described above in Example 1.
Macroscopic e~ ;on was carried out on aortas ranging from about 13.0 to 17. j cm in length. The results are prescnted below in Table 3:

W097/~ 93 PCT/CA97nO393 Tab,le 3 Time of Drug Dose from Start of Diet Extent of % of Affected GroupDrug Dose(weeks) Severity2Affected Tissue2 Tissue 0 0 2.2 5.0 30 6 2 20 2 1.7 25 178 3 20 2,4,6 1.2 09 5 9 ' and 2: See Table 1 Sections of rabbit aorta were also taken for microscopic examin~ion at 5 approximately 1, 2 and 3 cm from the aortic bifurcation. The l-cm section v~asconsidered to be co,.",aral)le to the section taken in Example 1 The results aresumrnarized below in Tablè 4:
Table 4 No. Sections Time of Drug Severity of Co,.~ ng Drug Dose Dose from Start Plaque' (Sec~ions Aortic Plaque Group (mg/kg)of Diet (weeks) 1.2.~ cm) (Sections 1,2~3) 0 0 1.3, 1.7~ 2.2 416, S/6, 5/6 2 2.0 2 08, 1.0,2.0 4~6, 4/6, 515 3 2.0 2,4,6 07, 1.3,2.0 2t6, 5/6, 516 'See Table 2.

The results from both the macroscopic and microscopic e~ ..;n ~ions indicated a decrease in the severity and extent of aortic plaque a~Ler a single dose of BPD-MA
six weeks before the end of the study. A~er three repeated doses of drug at two-week intervals, however, the decreases apl)eared to be even more pronol~nced

Claims (33)

We claim:

1. A method to prevent or inhibit the development of arterial plaque, said method comprising:
a. administering to a subject in one or more doses an amount of a green porphyrin compound effective to prevent or inhibit said development; and b. allowing said inhibition to occur in the absence of purposeful irradiation with light absorbed by said green porphyrin.

2. The method of claim I wherein said administering is conducted by administering from one to five doses.

3. The method of claim 2 wherein said administering is conducted by administering one dose.

4. The method of claim 2 wherein said administering is conducted by administering three doses.

5. The method of claim 1 wherein said effective amount is greater than 0. 5 mg/kg in each dose.

6. The method of claim 5 wherein said effective amount is from 0.5 to 5.0 mg/kg in each dose.

7. The method of claim 5 wherein said effective amount is from 0.5 to 2.0 mg/kg in each dose.

8. The method of claim 1 wherein said green porphyrin is administered in a liposomal formulation.

9. The method of claim 1 wherein said green porphyrin is selected from the group consisting of wherein R1, R2, R3 and R4 are non-interfering substituents \

10. The method of claim 9 wherein R1 and R2 are independently carbomethoxy or carboethoxy.

11. The method of claim 9 wherein each R3 is --CH2CH2COOH
or a salt, amide, ester or acyl hydrazone thereof.

12. The method of claim 9 wherein said porphyrin has the formula 3 or 4.

13. The method of claim 12 wherein said green porphyrin is selected from the group consisting of BPD-DA, BPD-DB, BPD-MA and BPD-MB.

14. The method of claim 13 wherein said green porphyrin is BPD-MA.

15. The method of any one of claims 1-14 wherein said treatment occurring in the absence of purposeful irradiation with light is conducted under conditions of exposure to ambient light.

16. The use of a composition comprising a green porphyrin compound effective to prevent or inhibit development of arterial plaque when administered to a subject in need of such treatment, even in the absence of purposeful irradiation with light absorbed by said green porphyrin, for the manufacture of a medicament for preventing or inhibiting the development of arterial plaque.

17. The use of claim 16 wherein the medicament comprises a unit dosage amount of said green porphyrin compound which is greater than 0.5 mg per kg body weight of said subject.

18. The use of claim 17 wherein said unit dosage is from 0.5 to 5.0 mg per kg body weight of said subject.

19. The use of claim 17 wherein said unit dosage is from 0.5 to 2.0 mg per kg body weight of said subject.

20. The use of claim 16 wherein said composition is a liposomal formulation.

21. The use of claim 16 wherein said green porphyrin is selected from the group consisting of:

wherein R1, R2, R3 and R4 are non-interfering substituents.

22. The use of claim 21 wherein R1 and R2 are independently carbomethoxy or carboethoxy.

23. The use of claim 21 wherein each R3 is --CH2CH2COOH or a salt, amide, ester or acyl hydrazone thereof.

24. The use of claim 21 wherein green porphyrin has the formula 3 or 4.

25. The use of claim 24 wherein said green porphyrin is selected from the group consisting of BPD-3A, BPD-DB, PBD-MA and BPD-MB.

26. The use of claim 25 wherein said green porphyrin is BPD-MA.

27. The use of any one of claims 16-26 wherein the medicament comprises a pharmaceutically acceptable excipient.

28. The use of any one of claims 16-27 wherein the composition is effective to prevent or inhibit development of arterial plaque when administered to said subject under conditions of exposure to ambient light.

29. A method to prevent or inhibit the development of arterial plaque, said method comprising: administering a medicament manufactured in accordance with any one of claims 16-28 in one or several doses.

30. The method of claim 29 wherein said medicament is administered in one to five doses.

31. The method of claim 30 wherein said medicament is administered in one dose.

32. The method of claim 30 wherein said medicament is administered in three doses.

33. The method of any one of claims 28-32 wherein said administration is conducted under condition of exposure to ambient light.