CA2093361C - Porphyrin derivative - Google Patents

Abstract

Photochemotherapeutic agents and method for making same. Lipophilic and amphiphilic compounds which are amide de-rivatives of porphyrins displaying at least one of photosensitizing, radio-pharmaceutical and immunological activity and having water solubilizing groups attached thereto by bonds selected from amide bonds and peptide bonds, for selective accumulation in malignant tissue in a living body.

Description

PORPAYRIN DERIVATIVES
Field of Invention This invention relates to a novel concept and technique for the chemical modification of lipophilic and amphiphilic drugs to increase their selective accumulation within malignant tissues, to a novel class of compounds having photochemo-therapeutic properties, to a method of making same, and to a method for treating tumours in patients.
Cross reference to related application This invention is related to U.S. Patent No.
5, 079, 262, issued 7 January 1992 in the names of James Kennedy, Roy Pottier and Robert L. Reid, filed 28 July 1989.
Background of Invention Every cytotoxic agent used in the treatment of cancer causes damage to normal as well as malignant cells. The maximum therapeutically useful dose is limited by toxicity to essential normal tissues, and in many cases it is not possible to give a dose large enough to destroy the cancer without also killing the patient. However, if the concentration of the cytotoxic drug could be increased in the malignant tissues only, then the therapeutic effectiveness of the drug would be increased without a ~~~~J~~
corresponding increase in toxicity.
Many types of malignant tumors develop zones in which the blood supply is inadequate, resulting in poorly nourished and hypoxic cells and areas of necrosis. The malignant cells in hypoxic zones normally are quiescent, but many retain the capacity to proliferate without limit or control and will do so if given a more adequate supply of oxygen and nutrients. 'However, such cells often are relatively resistant to both radiation therapy and chemotherapy. Hypoxic cells are relatively resistant to radiation therapy that involves X-rays or gamma-rays, since molecular oxygen is,required for some of the radiochemical processes that eventually lead to cell death. Also, hypoxic or poorly nourished cells normally are not in the praliferative cycle, and resting cells are relatively resistant also to the many chemotherapeutic agents Whose toxicity is restricted primarily to cells that are proliferating. Finally, since oxygen, nutrients, and drugs all reach cells by diffusion from adjacent capillaries,.
cells that are hypoxic or poorly nourished because they are too far from the nearest capillary may not be exposed to an adequate dose of chemotherapeutic agent. Failure to kill hypoxic or poorly nourished cells during attempts to eradicate a malignant tumor (by radiotherapy, chemotherapy, ~1~~"~~~
a.

2~~~3~1 or any other form of therapy) may be followed by tumor recurrence.
Thus, there is need for therapy that is effective against the hypoxic cells of malignant tumors.
Photodynamic therapy is an experimental form of treatment for cancer. It involves the localized or systemic administration of a photosensitizing compound or a metabolic precursor thereof, followed by exposure of the malignant tissue and adjacent normal tissues to photoactivating light.
The tissue specificity of the resultant phototoxic damage is determined largely (though not entirely) by the relative concentrations of the photosensitizer in each tissue at the time of its exposure to the photoactivating light.
Following systemic administration, certain derivatives of porphyrins, phthalocyanines, and chlorins accumulate preferentially Within malignant tissues. At present the proprietary preparation of hematoporphyrin derivatives known under the tradename "Photofrin II" is undergoing clinical evaluation for the treatment of carcinomas involving the bladder, esophagus, lung, brain, and other anatomical sites.
In addition, 5-aminolevulinic acid, a precursor of protoporphyrin IX in the biosynthetic pathway for heme, is now being used to selectively induce photosensitizing y . .~ ,n d/ ~ ~ ~ ~ ~ ~~i -: v :~
~I ~ Y C~ r concentrations of protoporphyrin IX in basal cell carcinomas and squamous cell carcinomas as described in U.S. Patent No. 5, 079, 262, supra. In suitable clinical circumstances, both "Photofrin II" and protoporphyrin IX
induced by 5-aminolevulinic acid show a clinically useful degree of specificity for malignant tissues. However, not all porphyrins, phthalocyanines, or chlorins accumulate preferentially in tumors.
Injections of hematoporphyrin derivatives such as, but not limited to, Photofrin II cause a clinically significant degree of skin photosensitization that persists for at least two weeks and sometimes for as long as four months. During this photosensitive period the patient must avoid exposure to.sunlight, even sunlight that has been filtered through window glass. Clearly this deleterious side effect causes considerable inconvenience to patients and severely limits the clinical usefulness of photodynamic therapy.
Like most drugs, photochemotherapeutic agents usually enter malignant tissues by diffusion from capillaries. As a result, zones of tissue that are poorly supplied with capillaries will be exposed to relatively low concentrations of the compound, perhaps too low to be therapeutically effective, unless the compound has a special affinity for hypoxic or necrotic tissue. The primary mechanism by which 2fl9336~.
most photosensitizers kill cells requires effective contact between a molecule of photosensitizes and a molecule of oxygen. The probability that enough such contacts will take place within hypoxic tissue will be reduced if the concentration of the photosensitizes is low, but will increase if the concentration of the photosensitizes in the hypoxic tissue is increased. Attempting to do so simply by increasing the dose of photosensitizes that is administered may produce too high a concentration in vital non-malignant tissues. However, if the photosensitizes had a significant degree of affinity or specificity for hypoxic tissues, it would accumulate preferentially in such tissues.
Thus, there is a need for better photochemotherapeutic agents that are cleared rapidly from normal tissues and especially skin, and ones that are effective in the hypoxic areas of tumors.
obiect of Invention It is, therefore, an object of the present invention to provide novel photochemotherapeutic ag~.nts of the porphyrin and chlorin type substituted with functionalized alkyl amide groups, which are good tissue photosensitizers, accumulate preferentially in necrotic and/or hypoxic areas of malignant tumors, show low systemic toxicity, clear rapidly from skin ..e 4 ~ i" ,r~ ~ ~'° ~e..
oy ii n ii. r.

WO 92/06097 PCT/CA9~/00346 ~~D93yb..l and most other normal tissues, and which show some degree of anti tumor activity even in the dark.
It is another object of the invention to provide novel lipophilic and amphiphilic drugs with appropriate water solubilizing groups linked thereto by bonds that can be cleaved readily by one or more of the proteases that are present in the extra-cellular f luid of many types of malignant tissues, which accumulate selectively in malignant tissues.
Brief Statement of Invention By one aspect of this invention there are provided compounds of the formula (ZNHCO)nX wherein:
Z is selected from mono-, di-, and tri- hydroxyalkyl and mono-, di-, and tri- halogenoalkyl;
X is selected from substituted tetrapyroles in which the substituent is at least one of the group consisting of methyl, ethyl, vinyl, hydroxyethyl, alkoxyethyl, carboxymethyl, carboxyethyl, Z-substituted propylamide,.
phenyl and Z-substituted phenylamide, and .
n is an integer from 1 to 8.
By another aspect of this invention there is provided a method for treating malignant tissue abnormalities in a ~/~~ww 2~~~~3~~
patient comprising administering to said patient an effective amount of a lipophilic or amphiphilic compound having water solubilizing groups attached thereto by amide bonds and exposing said tissue abnormality to light within the photoactivating spectrum of said lipophilic compound.
By yet another aspect of this invention there is provided a method for treating malignant tissue abnormalities in a patient comprising administering to said patient an effective amount of a lipophilic or amphiphilic compound having water solubilizing groups attached thereto by bonds cleavable by a protease present in said malignant tissue, so as to cause selective accumulation of said compound in said malignant tissue.
Detailed Description of Preferred Embodiments The novel compounds of the present invention can be characterized by the general formula (ZNHCO)nX where Z = mono-, di- or tri- hydroxyalkyl or mono-, di- or tri- halogenoalkyl, X = substituted tetrapyrole, wherein the substituent group or groups are selected from methyl, ethyl, vinyl, hydroxyethyl, alkoxyethyl, carboxymethyl, carboxyethyl, Z-substituted propylamide, phenyl and Z-substituted phenylamide, and n is an integer from 1 to 8.
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2i~9336~
Preferred compounds may be derived from commercially available hematoporphyrin IX dihydrochloride HPIX.2HC1 (Roussel, France) or mesoporphyrin IX dihydrochloride ~IX.2HC1 (Aldrich Chemical Co. U.S.A.) or deuteroporphyrin IX dimsthyl ester (Aldrich Chemical Co. U.S.A.) according to the following reaction scheme:
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WO 92/06097 PCf/CA91 /00346 20~3~~~;.
HPMEEA HPEEEA HPPEEA HPHEEA HPBEEA HPiEEA

#2 ~ ,#2 ~ ~2 ~ ,#2 ~ ~2 ~ ,~2 ~' 1 HPIX HPMEME HPEEEE HPPEPE HPHEHE HPBEBE HPIEIE

.2HC1 ~'3 ~ ~3 ~ #3 ~ ~ ,#3 HPME HPEE HPPE HPHE

#4 ~ ~4 ~ ~4 ~ #4 HPMEFE HPEEFE HPPEFE HPHEFE

,~5 ~ ,~5 ~ ~5 ~ #5 HPMEDA HPEEDA HPPEDA HPHEDA

or ~ or ~ or ~ or HPMETA HPEETA HPPETA HPHETA

or or or or HPMF~tA HPEERA HPPERA HPHERA

#4 #5 MPIX MPFE MPDA or MPRA
or MPTA

.2HC1 ,~2 MPME MPEA

,# 2 DPME DPEA
where HPMEME HPIX dimethyl ether dimethyl ester HP1~EA HPIX dimethyl ether diethanolamide HP~ HPIX dimethyl ether HPt~FE HPIX dimethyl ether di(2,2,2-trifluoruethyl) ester FIPt~DA HPIX dimethyl ether di(bie(hydroxymethyl)methanamide) HPMETA HPIX dimethyl ether di(tris(hydroxymethyl)methanamide) HPt~RA HPIX dimethyl ether dialkylamide HPEEEE HPIX diethyl ether diethyl ester HPEEEA HPIX diethyl ether diethsnolamide ~ ,.~ ...~a r ~.. . a -~-~.r~'' ~' v ~ 6 ~ 1 ~ ~ ~ ~ '~t c.''. iL l .

WO 92/06097 PCT/CA91l00346 HPEE HPIX diethyl ether HPEEFE HPZX diethyl ether di(2,2,2-trifluoroethyl) ester HPEEDA HPIX diethyl ether di(bis(hydroxymethyl)methanamide) HPEETA HPIX diethyl ether di(tris(hydroxymethyl)methanamide) HPEERA HPIX diethyl ether dialkylamide HPPEPE HPIX di-n-propyl ether di-n-propyl ester HPPEEA HPIX di-n-propyl ether diethanolamide HPPE HPIX di-n-propyl ether HPPEFE HPIX di-n-propyl ether di(2,2,2-trifluoroethyl) ester HPPEDA HPIP di-n-propyl ether di(bis(hydroxymethyl)methanamide) HPPETA HPIX di-n-propyl ether~di(tris(hydroxymethyl)methanamide) BPPERA HPIX di-n-propyl ether dialkylamide BPf~BE BBIX di(2-hydroxyethyl)ether di(2-hydroxyethyl eater) BPHEEA HPIX di(2-hydroxyethyl)ether diethanolamide CBE HPIX di(2-hydroxyethyl)ether HPBEFE HPIX di(2-hydroxyethyl)ether di(2,2,2-trifluoroethyl)ester B'PBEDA IiPIX di(2-hydroxyethyl)ether di(bie(hydroxymethyl)methanamide) IiPBETA HPIX di(2-hydroxyethyl)ether di(trie(hydroxymethyl)methanamide) HP~RA FIPIX di(2-hydroxyethyl)ether dialkylamide HPBEBE HPIX dibutyl ether dibutyl ester HPBEEA 13PIX dibutyl ether diethanolamide BpiEiE HPIX diieobutyl ether diisobutyl ester IiPiEEA HPIX diiaobutyl ether diethanolamide MPItA MPIX diethanolemide DPEA DPIX diethanolamide MPH'E MPIX di(2,2,2-trifluoroethyl) ester MPDA MPIX di(bie(hydroxymethyl)methanamide) MPTA MPIX di(trie(hydroxymethyl)methanamide) MPRA MPIX dialkylamide R: for general X-substituted alkyl groups The five reactions illustrated above may be summarized as follows:
Reaction ~'1 HPIX + R-OH > HPRERE
~~~~ r ~ ~~~ ~~~~~~

m Reaction #2 HPRERE + H2N-CH2CH2-OH > HPREEA
ioo-iae~c Reaction #3 THF/KOHa (2M) HPRERE q > HPRE
ref lux 3 hours + stand overnight Reaction ~4 HPRE > HPREFE
gaseous HC1 Reaction #5 DMF
HPREFE + R-NH2 > HPRERA
80-95°C
It will be appreciated,~however, that other natural and synthetic porphyrins and chlorins may also be used. Such other compounds include:
Coproporphyrins and their esters Deuteroporphyrins and their esters Hematoporphyrin and their esters Hematoporphyrins dialkyl ethers and their esters Mesoporphyrins and their eaters Protoporphyrina and their esters Oroporphyrina and their esters Chlorin e6 and its derivatives Pentacarboxyporphyrin I & III and their esters Hexacarboxyporphyrin I & III (both isomers) and their esters Heptacarboxyporphyrin I & III and their esters zu~'~rb~

5,10,15,20-tetra(y-carboxyphenyl)porphin and its esters 5,10,15-tri(y-carboxyphenyl)-20-phenylporphin and its esters 5,10-di(y-carboxyphenyl)-15,20-diphenylporphin and its esters 5,15-di(y-carboxyphenyl)-10,20-diphenylporphin and its esters 5-(y-carboxyphenyl)-10,15,20-triphenylporphin and its esters where y = 2 to 4 .1s2 and the chlorins (reduced porphyrins) corresponding to all of the porphyrins listed above.
Example 1 Production of Hematouorphyrin di-n-propel ether di-n-probvl ester(HPPEPE).
ig; 1.67 mmol hematoporphyrin IX dihydrochloride (Roussel, France) was dissolved in an excess of a 10%
solution of concentrated sulfuric acid in 25 ml of n-propanol. The reaction mixture was refluxed for three hours to produce mostly HPPEPE Which was extracted from tl~e reaction mixture in a standard manner with ethyl ether. The residue obtained was chromatographed on silica gel with a mixture of ethyl ether and petroleum ether to afford pure HPPEPE.
r i~ w~'' : ~ $. ~

WO 92/06097 PCf/CA91/00346 ~~93301 HO-C-CH=CH= C:1~ O
\ \
'\ C~~i C-OH
NH N
O ~ V H
HO-C-CH=CH~ ~ ~ / I' ~ CHI
CH=CH7 frOH
~i ~ O
O
Pentacacboiypocphy rin I
O
O Cllr-G=OH
D HO-C~~7 O
G-CH t.0-O-~ \ ~ \ t ~~i G.pH
O ~~~[~~ //\
HO-C-CH.CH, v ~ .~CH_ He.~racarboxypor ph~-rir.=

cH; ~oH o Hoo HO-C-CHI ~ HH ' ~~'C'OH
O
HO-_ . HOOC~ 'COOH
O
Heptacarboxyporphprin I ~.10,:~.20-tetrafy-carbo:cyphenyl)porphin HOO HOOCn ~COOH
5.10,15-trify-earboxyphenyl)-20-phenylporphin ~.10-dify-ca:boxyphenyl)-15,20-diphen5~lporphir HOO
I
I
~.1~-di(y-carboryphenpl)-10.20-diphenytporphin~-~Y-car~oxy-phenl1-10,1.20-triphenylporphi c Y,. E.

2.~~933G1 c-off u,, 'w ~ \ c~crt; c-o~
NN
N
~7 \ ( / ~ ~J
HO-C
O O
O
Coproporphyrin I Coproporphyrin III ester CN, ~,~ Cyt, -OCH~ hO-i O
Deuteroporphyrin IX ester Hematoporphyrin IX
c NO-C
O
Hematoporphy rin It Walicy I ether . '!esoporphyrin I1 ,R = allcyl CH=CHZ CHI 0 0 \ ~ HO-C-CH=CN= ~i C'OH

CH_C~ O
NH N'~ ~ ~ CH
HO-C-Cit CH
C-OH

y =
H J

H

~ \

N H o H H

CHI ~ ~ / ~ C' 7 HO'C-~:CHt \ / ~i O-OH
~ /

CH~O-C-CHZCHZCHzCHZ C-OCH~ Ho-c-Cry cH7cH=-~-off p O p 0 Protoporphyrin (irooorphyrin Il ester t a 1 ~ ~ L w.7a 6~~ ~, 15 ~~~~J~~i ExamQle 2 Production of hematoporphyrin IX di-n-propvl ether diethanolamide IHPPEEAI.
1g; 1.30 mmol of HPPEPE was dissolved in 2-aminoethanol (10 ml; 166 mmol) and heated at 100-120°C for three hours to give HFPEEA which was extracted from the reaction mixture by precipitation in cold water, followed by centrifugation.
The residue obtained was chromatographed on deactivated silica gel with a mixture of chloroform and methanol. After evaporating the solvent, the product was dissolved in a small amount of ethanol and precipitated in a mixture of ethyl ether - petroleum ether for final purification. Pure crystalline HPPEEA was recovered, in 76% yield from HPIX, by centrifugation.
Example 3 Production of hematoporphYrin di-n-propyl ether ~ PE).
HPPEPE (1g; 1.30 mmol) was dissolved in a small amount of tetrahydrofuran (THF). 100 ml of 2 M aqueous potassium hydroxide was added and refluxed for three hours before standing in the dark overnight. The solvent was evaporated and the remaining alkaline solution was adjusted to pH 5.5 with hydrochloric acid, thus precipitating HPPE, which was recovered by centrifugation and washed 3 times with distilled water.
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WO 92/06097 PC1'/CA91/00346 Example 4 Production of mesoporphyrin dif2,2,2-trifluorethvl) ester IMPFE).
100mg; 0.156 mmol of mesoporphyrin IX dihydrochloride (Aldrich Chemical Co.) was dissolved in 20 ml; 275 mmoles of trifluoroethanol. The solution was then saturated with gaseous hydrochloric acid and allowed to stand overnight in the dark. 10 ml of benzene Was then added and the mixture evaporated to dryness, producing di(2,2,2-trifluoroethyl) ester (MPFE).
Example 5 Production of mesonorphvrin diltrisfhydrox~methvl) methanamidel ~MPTA).
A solution of 2-amino 2-(hydroxymethyl)-1,3-propanediol (500mg, 4.13 mmol) in hot (80-95°C) dimethylfarmamide (DMF) (15m1) was added to the MPFE prepared in Example 4. The temperature was maintained between 80 and 95°C for two hours and led to the formation of MPTA. The reaction mixture was cooled in ice, inducing the precipitation of excess amine which was' then removed by vacuum filtration. After evaporation of the remaining liquid, the residue was dissolved in a small amount of methanol and added to about 50 ml of ethyl ether in which MPTA precipitated and Was recovered by centrifugation.
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WO 92/06097 PCT/CA91 /0034b All compounds synthesized and used for biological testing were checked for purity using thin layer chromatography (TLC) and elemental~analyses. All compounds showed only one spot on TLC plates eluted with two different solvent systems. Their elemental analyses were identical to calculated values for C,H,N within 0.3%. The structures of the compounds were determined by infrared, 1H NMR and mass spectra.
It is known that as malignant tumors enlarge from a single cell to a palpable nodule, their growth pattern is such that certain areas of tumor develop an inadequate blood supply. The cells in such zones~are both poorly nourished and hypoxic. Some of these cells die, but others merely reduce their metabolic activity to a basal level. Such cells are relatively resistant to destruction by X-rays and gamma-rays, since (i) molecular oxygen is required for some of the radiation chemistry that can cause DNA damage and cell death, and (ii) quiescent cells are relatively resistant to radiation damage. Hypoxic and poorly nourished cells tend to be resistant to many typsa of chemotherapeutic agents also. Chemotherapeutic agents usually enter tissues via the blood, and malignant cells whose blood supply is inadequate may not receive a lethal dose. In addition the 5~~~ d ~ ~ Y~ I ~ ~r~"~r~.

is toxicity of many common chemotherapeutic agents is restricted primarily to cells that are in cell cycle.
Consequently, malignant cells that are poorly nourished and/or hypoxic may survive courses of radiotherapy and/or chemotherapy that otherwise might have been curative. Such surviving cells may proliferate subsequently to cause a recurrence of the cancer.' Thus, a drug which shows sufficient preferential toxicity for hypoxic cells may be given in doses that should kill the hypoxic cells in tumors without causing unacceptable toxicity to the normally-oxygenated cells of nonmalignant tissues. Such a drug might not be curative if given as the sole therapy, since only some of the cells in tumors are hypoxic but it would be a very useful adjunct to radiotherapy and/or chemotherapy, since these tend to kill well oxygenated cells preferentially. For example, certain vitro-containing compounds accumulate preferentially in hypoxic tissues where they cause preferential toxicity for the hypoxic cells.
It has been found that compounds of the present invention show a strong tendency to accumulate preferentially in the necrotic and/or hypoxic areas of malignant tissues. Thus, it is reasonable to expect that such photosensitizers are preferentially effective against .w.
~ r..y ~ ~ ~1 d~

19 ~~~ Ji5~7 hypoxic cells and thus, under appropriate circumstances, may be an adjunct to radiation therapy or chemotherapy for local control of malignant tumors that otherwise would be incurable. For example, squamous cell carcinomas of the head and neck, which usually contain a relatively high percentage of necrotic material may be treated. Such cancers usually respond poorly to chemotherapy, and they sometimes recur even after treatment with a tissue tolerance dose (maximum safe dose) of ionizing radiation. Since cancers of this type are quite common, even a small improvement in the local control rate would be of benefit to a significant number of patients.
Phototoxic damage to hypoxic tissue can be increased either by increasing the tissue concentration of oxygen or by increasing the concentration of photosensitizer. The former is not very effective but as the photosensitizers of the present invention tend to accumulate in necrotic/hypoxic tissue they selectively increase the intensity of the phototoxic reaction in such tissues.
Examu,~e 6 (B6D2)F1 mice bearing subcutaneous transitional cell carcinoma FCB were each injected intraperitoneally with 10 mg per kg body weight every other day for l0 days, for a h~~~~~ . 5 rm .a ~ ~ L % ~ r l .v ~ ~. d it ~. C.

2~93~6~. Zo total dose of 50 mg per kg body weight of a compound selected from: MPEA, MPDA, MPTA, HPMEEA, HPEEEA, HPPEEA and DPEA as defined herein above. The mice were then killed and their tissues examined for porphyrin fluorescence under W
light 48 hours following the final injection, All of the compounds tested showed strong fluorescence in the necrotic areas of the tumor with little or no fluorescence in the adjacent healthy tumor. Significant fluorescence was also observed in the pancreas, but no fluorescence was observed in the skin, bowel, skeletal muscle, lungs, heart, thymus, liver, spleen or kidneys. Tissues that contained the modified mesoporphyrins MPEA, MPDA, and MPTA which showed porphyrin fluorescence at autopsy continued to do so for at least several weeks of storage in buffered formalin, but tissues containing HPPEEA or HPEEEA completely lost their original porphyrin fluorescence while stored in buffered formalin in the dark.
Example 7 ' The procedures of Example 6 were repeated on (B6D2)F1 mice bearing Lewis lung carcinoma. 100 mg of HPPEEA per kg body weight was injected over a 10 day period. At autopsy, intense porphyrin fluorescence was observed in the necrotic areas of the tumors with very little fluorescence in other locations except the pancreas.
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2~~~33~1 From the results of examples 6 and 7 it may be concluded that the unusual tissue specificity observed with the seven porphyrins tested is associated with the presence of an amide linkage between. the~propionic acids of the porphyrin and an alcohol-containing solubilizing group. It will be appreciated that mesoporphyrin cH.cH. cN~
'!esopocphTr!n I1 has' two propionic acid groups but shows very poor solubility in water within the physiological pH range. When alcohol groups are added, via an amide bond, modified porphyrin molecules having a range of solubilities can be produced viz MPEA (mesoporphyrin diethanolamide) has 2 added alcohol groups. MPDA (mesoporphyrin di(bis(hydroxymethyl) methan-amide)) has 4 added alcohol groups and MPTA (mesoporphyrin di(tris(hydroxymethyl)methanamide)) has 6 added alcohol groups. Hematoporphyrin t;o_~o _ , . o nw -~a ~ A I1~ P
A ~ n ~

WO 92/06097 PC1'/CA91 /003~t6 '~ ~ 2 2 is somewhat more soluble in water, within the physiological pH range, than is mesoporphyrin. Modifications to the hydroxyethyl groups to form HPMEEA (hematoporphyrin dimethylether diethanol-amide), HPEEEA (hematoporphyrin diethylether diethanol-amide) and HPPEEA (hematoporphyrin di-n propyl ether diethanolamide~) resulted in compounds having a range of solubilities. Without wishing to be bound by this theory, it appears that lipophilic and ampiphilic drugs, such as porphyries or chlorins, can be linked via amide or appropriate polypeptide chains to appropriate water solubilizing groups and that such compound drugs will tend to accumulate in malignant tissues.
Although various proteases are present in the blood and extracellular fluid of non-malignant tissues, such proteases normally show little activity. However, malignant cells often release proteolytic enzymes and may induce the release or activation of such enzymes by normal cells. Activated protease enzymes are often present in the necrotic areas of malignant tissues, derived from the dead cells and from the phagocytes that have been attempting to liquefy them. In the absence of active transport, drugs must dissolve in the plasma membrane, which is mostly lipid, in order to enter a cell. Thus, drugs whose lipid/water partition ratio is high (lipophilic drugs) tend to enter cells more readily than .A .~." ~ r. ~ ~ e-a.,~.~ ~. .- p.....
- ~: w. .~, ~9 a. ~.
.~ a a a .~.~ r ~.. ..o a r :~ i.. i WO 92/0609% PCT/CA91/0034b r, .; ~ /~
v~ t~' eJ L) '~
those whose partition ratio is low (hydrophilic drugs).
Therefore, if a lipophilic drug is converted into a hydrophilic compound by linking it with a solubilizing group via an amide bond or an appropriate polypeptide chain, such a compound will not enter cells in normal tissues as readily as the original drug. In the extrace11u1ar =iuia in malignant tissues at least some of, the hydrophilic compound will be converted back to the lipophilic form of the drug by protease-mediated cleavage of the amide or peptide bonds, with consequent increased solubility in the plasma membranes of the cells. Clearly, while the present invention has been specifically illustrated by means of modified hematoporphyrins, mesoporphyrins, and deuteroporphyrins the principles described above may be applied to virtually any lipaphilic drug to which solubilizing groups are added via an amide bond or appropriate polypeptide chain.
Example 8 Dark toxicity of HPPEEA
Eight (B6D2)F1 mice were maintained in the dark and were intraperitoneally injected with HPPEEA at a dose rate of 10 mg per kg body weight on each of three successive days. There was no apparent toxicity.
The experiment was repeated on a second set of mice ~~~~ ~ ~ 1~~~~~ ~~t~~'~.

WO 92/0609 i PCT/CA91 /00346 into which 10 mg/kg body weight HPPEEA was injected daily for six successive days. There was no apparent toxicity.
The experiment was repeated on a third set of mice into which 100 mg/kg body weight HPPEEA was injected daily for three days. There was definite toxicity, but no deaths.
Examble 9 ~zerapeutic effects in the dark.
HPPEEA at a concentration of l.Omg HPPEEA per ml of 10%
DMSO in serum was injected intravenously into Skh:HR-1 mice bearing well-developed Adenocarcinoma 755 (a) in ascites form, or (b) as a solid tumor growing within the muscles of the thigh; and also into Skh:HR-1 mice bearing advanced subcutaneous tumors of FCB transitional cell carcinoma of the bladder. The dose in each case was l0mg of HPPEEA per kg of body weight. The mice Were maintained in dim light following the HPPEEA injection, to minimize the possibility of ~photodynamic effects. In each case a small proportion of the injected mice showed rapid and complete regression of the cancer, such regression being maintained until termination of the experiment 10 weeks later. Cancers in control mice normally grew progressively. However, please note that the particular tumor/mouse combination used is not completely histocompatible and therefore may provide an unusually sensitive detection system for anti-tumor activity z~~»~~
of HPPEEA in the dark.
Example l0 Phototoxicity and Therapeutic Effectiveness of HPPEEA.
Lewis Lung Carcinoma was transplanted subcutaneously into the flank of Skh:HR-1 hairless mice, and allowed to grow until the tumor was approximately 10 mm in diameter. A
dose of lOmg HPPEEA per kg of body weight was then injected intraperitoneally. One day later., the tumor and adjacent normal skin was exposed to a dose of 50 mWhr/cm2 photoactivating light (wavelengths greater than 600 nm) at an intensity of 200 mW/cm2.
Immediately following exposure to the photoactivating light, the tumor was noted to have changed colour, and there was obvious edema within the tumor. Within the next 24 hours, the skin covering the centre of the tumor became necrotic but the skin immediately adjacent showed only mild phototoxic damage. Since both areas of skin received similar doses of photoactivating ligh~~, HPPEEA appears to have a clinically useful degree of tissue specificity.
Six days after exposure to the pl~iotoactivating light, there was no evidence of residual tumor:.
~~~~~6~~?~

Claims (12)

We claim:

1. A compound of the formula (ZNHCO)n X wherein Z is an amino alcohol less the amino group and is selected from mono-, di- and tri-hydroxyalkyl, or is selected from mono-, di- and tri-halogenoalkyl, X is selected from substituted tetrapyrroles in which the substituent is at least one of the group consisting of methyl, ethyl vinyl, hydroxyethyl, alkoxyethyl, carboxymethyl, carboxyethyl, Z-substituted propylamide, phenyl, and Z-substituted phenylamide, and n is an integer from 1 to 8.

2. The compound of claim 1 wherein X is selected from hematoporphyrin, mesoporphyrin, deuteroporphyrin, coproporphyrin, protoporphyrin, uroporphyrin, pentacarboxyporphrin, hexacarboxyporphyrin, heptacarboxyporphyrin, 5,10,15,20-tetra(y-carboxyphenyl) porphin, 5,10,15-tri(y-carboxyphenyl)-20-phenyl porphin, 5,10-di(y-carboxyphenyl)-15,20-diphenyl porphin, 5,15-di(y-carboxyphenyl)-10,20-diphenylporphin, and 5-(y-carboxyphenyl)-10,15,20-triphenyl porphin, where y is an integer from 2 to 4; and corresponding chlorins, and esters and derivatives thereof.

3. The compound of claim 2, selected from the group consisting of:
HPIX dimethylether di-2-hydroxyethylamide HPIX diethylether di-2-hydroxyethylamide HPIX dipropylether di-2-hydroxyethylamide HPIX dibutylether di-2-hydroxyethylamide HPIX diisobutylether di-2-hydroxyethylamide DPIX di-2-hydroxyethylamide MPIX di-2-hydroxyethylamide HPIX dimethylether di-2-halogenoethylamide HPIX diethylether di-2-halogenoethylamide HPIX dipropylether di-2-halogeneothylamide HPIX dibutylether di-2-halogenoethylamide HPIX diisobutylether di-2-halogeneothylamide DPIX di-2-halogeneothylamide MPIX di-2-halogenoethylamide MPIX di(bis(halogenomethyl)methanamide) MPIX di(tris(halogenomethyl)methanamide) HPIX dimethyl ether diethanolamide HPIX dimethyl ether di(bis(hydroxymethyl)methanamide) HPIX dimethyl ether di(tris(hydroxymethyl)methanamide) HPIX dimethyl ether dialkylamide HPIX diethyl ether diethanolamide HPIX diethylether di(bis(hydroxymethyl)methanamide) HPIX diethyl ether di(tris(hydroxymethyl)methanamide) HPIX diethylether dialkylamide HPIX di-n-propyl ether diethanolamide HPIP di-n-propyl ether di(bis(hydroxymethyl)methanamide) HPIX di-n-propyl ether di(tris(hydroxymethyl)methanamide) HPIX di-n-propyl ether dialkylamide HPIX di(2-hydroxyethyl)ether diethanolamide HPIX di(2-hydroxyethyl)ether di(bis(hydroxymethyl)methanamide) HPIX di(2-hydroxyethyl)ether di(tris(hydroxymethyl)methanamide) HPIX di(2-hydroxyethyl)ether dialkylamide HPIX dibutyl ether diethanolamide HPIX diisobutyl ether diethanolamide MPIX diethanolamide DPIX diethanolamide MPIX di(bis(hydroxymethyl)methanamide) MPIX di(tris(hydroxymethyl)methanamide) MPIX dialkylamide and the corresponding chlorins, wherein HPIX refers to hematoporphyrin IX, MPIX refers to mesoporphyrin IX, and DPIX refers to deuteroporphyrin IX.

4. A pharmaceutical composition comprising a therapeutically effective amount of a compound of the formula (ZNHCO)n X wherein Z is an amino alcohol less the amino group and is selected from mono-, di- and tri-hydroxyalkyl, or is selected from mono-, di- and tri-halogenoalkyl, X is selected from substituted tetrapyrroles in which the substituent is at least one of the group consisting of methyl, ethyl vinyl, hydroxyethyl, alkoxyethyl, carboxymethyl, carboxyethyl, Z-substituted propylamide, phenyl, and Z-substituted phenylamide, and n is an integer from 1 to 8, and a pharmaceutically acceptable carrier therefor.

5. The pharmaceutical composition of claim 4 wherein X is selected from hematoporphyrin, mesoporphyrin, deuteroporphyrin, coproporphyrin, protoporphyrin, uroporphyrin, pentacarboxyporphrin, hexacarboxyporphyrin, heptacarboxyporphyrin, 5,10,15,20-tetra(y-carboxyphenyl) porphin, 5,10,15-tri(y-carboxyphenyl)-20-phenyl porphin, 5,10-di(y-carboxyphenyl)-15,20-diphenyl porphin, 5,15-di(y-carboxyphenyl)-10,20-diphenylporphin, and 5-(y-carboxyphenyl)-10,15,20-triphenyl porphin, where y is an integer from 2 to 4; and corresponding chlorins, and esters and derivatives thereof.

6. The pharmaceutical composition of claim 4, wherein the compound is selected from:
HPIX dimethylether di-2-hydroxyethylamide HPIX diethylether di-2-hydroxyethylamide HPIX dipropylether di-2-hydroxyethylamide HPIX dibutylether di-2-hydroxyethylamide HPIX diisobutylether di-2-hydroxyethylamide DPIX di-2-hydroxyethylamide MPIX di-2-hydroxyethylamide HPIX dimethylether di-2-halogenoethylamide HPIX diethylether di-2-halogenoethylamide HPIX dipropylether di-2-halogeneothylamide HPIX dibutylether di-2-halogenoethylamide HPIX diisobutylether di-2-halogeneothylamide DPIX di-2-halogeneothylmide MPIX di-2-halogenoethylamide MPIX di(bis(halogenomethyl)methanamide) MPIX di(tris(halogenomethyl)methanamide) HPIX dimethyl ether diethanolamide HPIX dimethyl ether di(bis(hydroxymethyl)methanamide) HPIX dimethyl ether di(tris(hydroxymethyl)methanamide) HPIX dimethyl ether dialkylamide HPIX diethyl ether diethanolamide HPIX diethylether di(bis(hydroxymethyl)methanamide) HPIX diethyl ether di(tris(hydroxymethyl)methanamide) HPIX diethylether dialkylamide HPIX di-n-propyl ether diethanolamide HPIP di-n-propyl ether di(bis(hydroxymethyl)methanamide) HPIX di-n-propyl ether di(tris(hydroxymethyl)methanamide) HPIX di-n-propyl ether dialkylamide HPIX di(2-hydroxyethyl)ether diethanolamide HPIX di(2-hydroxyethyl)ether di(bis(hydroxymethyl)methanamide) HPIX di(2-hydroxyethyl)ether di(tris(hydroxymethyl)methanamide) HPIX di(2-hydroxyethyl)ether dialkylamide HPIX dibutyl ether diethanolamide HPIX diisobutyl ether diethanolamide MPIX diethanolamide DPIX diethanolamide MPIX di(bis(hydroxymethyl)methanamide) MPIX di(tris(hydroxymethyl)methanamide) MPIX dialkylamide and the corresponding chlorins, wherein HPIX refers to hematoporphyrin IX, MPIX refers to mesoporphyrin IX, and DPIX refers to deuteroporphyrin IX.

7. Use of a compound of the formula (ZNHCO)n X for detecting malignant tissue abnormalities in a subject, wherein Z is an amino alcohol less the amino group and is selected from mono-, di- and tri-hydroxyalkyl, or is selected from mono-, di- and tri-halogenoalkyl, X is selected from substituted tetrapyrroles in which the substituent is at least one of the group consisting of methyl, ethyl vinyl, hydroxyethyl, alkoxyethyl, carboxymethyl, carboxyethyl, Z-substituted propylamide, phenyl, and Z-substituted phenylamide, and n is an integer from 1 to 8.

8. The use of claim 7, wherein X is selected from hematoporphyrin, mesoporphyrin, deuteroporphyrin, coproporphyrin, protoporphyrin, uroporphyrin, pentacarboxyporphrin, hexacarboxyporphyrin, heptacarboxyporphyrin, 5,10,15,20-tetra(y-carboxyphenyl) porphin, 5,10,15-tri(y-carboxyphenyl)-20-phenyl porphin, 5,10-di(y-carboxyphenyl)-15,20-diphenyl porphin, 5,15-di(y-carboxyphenyl)-10,20-diphenylporphin, and 5-(y-carboxyphenyl)-10,15,20-triphenyl porphin, where y is an integer from 2 to 4; and corresponding chlorins, and esters and derivatives thereof.

9. The use of claim 7, wherein the compound is selected from:
HPIX dimethylether di-2-hydroxyethylamide HPIX diethylether di-2-hydroxyethylamide HPIX dipropylether di-2-hydroxyethylamide HPIX dibutylether di-2-hydroxyethylamide HPIX diisobutylether di-2-hydroxyethylamide DPIX di-2-hydroxyethylamide MPIX di-2-hydroxyethylamide HPIX dimethylether di-2-halogenoethylamide HPIX diethylether di-2-halogenoethylamide HPIX dipropylether di-2-halogeneothylamide HPIX dibutylether di-2-halogenoethylamide HPIX diisobutylether di-2-halogeneothylamide DPIX di-2-halogeneothylamide MPIX di-2-halogenoethylamide MPIX di (bis(halogenomethyl)methanamide) MPIX di(tris(halogenomethyl)methanamide) HPIX dimethyl ether diethanolamide HPIX dimethyl ether di(bis(hydroxymethyl)methanamide) HPIX dimethyl ether di(tris(hydroxymethyl)methanamide) HPIX dimethyl ether dialkylamide HPIX diethyl ether diethanolamide HPIX diethylether di(bis(hydroxymethyl)methanamide) HPIX diethyl ether di(tris(hydroxymethyl)methanamide) HPIX diethylether dialkylamide HPIX di-n-propyl ether diethanolamide HPIP di-n-propyl ether di(bis(hydroxymethyl)methanamide) HPIX di-n-propyl ether di(tris(hydroxymethyl)methanamide) HPIX di-n-propyl ether dialkylamide HPIX di(2-hydroxyethyl)ether diethanolamide HPIX di(2-hydroxyethyl)ether di(bis(hydroxymethyl)methanamide) HPIX di(2-hydroxyethyl)ether di(tris(hydroxymethyl)methanamide) HPIX di(2-hydroxyethyl)ether dialkylamide HPIX dibutyl ether diethanolamide HPIX diisobutyl ether diethanalamide MPIX diethanolamide DPIX diethanolamide MPIX di(bis(hydroxymethyl)methanamide) MPIX di(tris(hydroxymethyl)methanamide) MPIX dialkylamide and the corresponding chlorins, wherein HPIX refers to hematoporphyrin IX, MPIX refers to mesoporphyrin IX, and DPIX refers to deuteroporphyrin IX.

10. Use of a compound of the formula (ZNHCO)n X as an anti-tumour agent, wherein Z is an amino alcohol less the amino group and is selected from rnono-, di- and tri-hydroxyalkyl, or is selected from mono-, di- and tri-halogenoalkyl, X is selected from substituted tetrapyrroles in which the substituent is at least one of the group consisting of methyl, ethyl vinyl, hydroxyethyl, alkoxyethyl, carboxymethyl, carboxyethyl, Z-substituted propylamide, phenyl, and Z-substituted phenylamide, and n is an integer from 1 to 8.

11. The use of claim 10, wherein X is selected from hematoporphyrin, mesoporphyrin, deuteroporphyrin, coproporphyrin, protoporphyrin, uroporphyrin, pentacarboxyporphrin, hexacarboxyporphyrin, heptacarboxyporphyrin, 5,10,15,20-tetra(y-carboxyphenyl) porphin, 5,10,15-tri(y-carboxyphenyl)-20-phenyl porphin, 5,10-di(y-carboxyphenyl)-15,20-diphenyl porphin, 5,15-di(y-carboxyphenyl)-10,20-diphenylporphin, and 5-(y-carboxyphenyl)-10,15,20-triphenyl porphin, where y is an integer from 2 to 4; and corresponding chlorins, and esters and derivatives thereof.

12. The use of claim 10, wherein the compound is selected from:
HPIX dimethylether di-2-hydroxyethylamide HPIX diethylether di-2-hydroxyethylamide HPIX dipropylether di-2-hydroxyethylamide HPIX dibutylether di-2-hydroxyethylamide HPIX diisobutylether di-2-hydroxyethylamide DPIX di-2-hydroxyethylamide MPIX di-2-hydroxyethylamide HPIX dimethylether di-2-halogenoethylamide HPIX diethylether di-2-halogenoethylamide HPIX dipropylether di-2-halogeneothylamide HPIX dibutylether di-2-halogenoethylamide HPIX diisobutylether di-2-halogeneothylamide DPIX di-2-halogeneothylamide MPIX di-2-halogenoethylamide MPIX di(bis(halogenomethyl)methanamide) MPIX di(tris(halogenomethyl)methanamide) HPIX dimethyl ether diethanolamide HPIX dimethyl ether di(bis(hydroxymethyl)methanamide) HPIX dimethyl ether di(tris(hydroxymethyl)methanamide) HPIX dimethyl ether dialkylamide HPIX diethyl ether diethanolamide HPIX diethylether di(bis(hydroxymethyl)methanamide) HPIX diethyl ether di(tris(hydroxymethyl)methanamide) HPIX diethylether dialkylamide HPIX di-n-propyl ether diethanolamide HPIP di-n-propyl ether di(bis(hydroxymethyl)methanamide) HPIX di-n-propyl ether di(tris(hydroxymethyl)methanamide) HPIX di-n-propyl ether dialkylamide HPIX di(2-hydroxyethyl)ether diethanolamide HPIX di(2-hydroxyethyl)ether di(bis(hydroxymethyl)methanamide) HPIX di(2-hydroxyethyl)ether di(tris(hydroxymethyl)methanamide) HPIX di(2-hydroxyethyl)ether dialkylamide HPIX dibutyl ether diethanolamide HPIX diisobutyl ether diethanolamide MPIX diethanolamide DPIX diethanolamide MPIX di(bis(hydroxymethyl)methanamide) MPIX di(tris(hydroxymethyl)methanamide) MPIX dialkylamide and the corresponding chlorins, wherein HPIX refers to hematoporphyrin IX, MPIX refers to mesoporphyrin IX, and DPIX refers to deuteroporphyrin IX.