This invention relates to new preparations which are valuable for photodetection and photochemotherapy of tumors and malignant tissues. The active compounds of these therapeutic preparations are mono-, di- or polyamides of an amino alcohol and a cyclic tetrapyrrole containing at least one carboxyl group. The general structure of these active compounds is EQU (ZNHCO).sub.n X wherein Z is the amino alcohol molecule less the amino group, X is the tetrapyrrole molecule less a carboxyl group, and "n" is an integer from 1 to 8.

FIELD OF THE INVENTION 
This invention relates to a novel class of compounds which have 
photochemotherapeutic properties and are utilizable for photodetection and 
photochemotherapy of tumors and malignant tissues. 
BACKGROUND OF THE INVENTION 
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 a proprietary 
preparation of hematoporphyrin derivatives known under the tradename 
"Photofrin.RTM. 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 concentrations of protoporphyrin IX 
in basal cell carcinomas and squamous cell carcinomas as described in U.S. 
patent application Ser. No. 386,414 which is assigned to the present 
assignee. In suitable clinical circumstances, both "Photofrin.RTM. 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.RTM. 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. 
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 types 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 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 
non-malignant 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 nitro-containing compounds accumulate preferentially in hypoxic 
tissues where they cause preferential toxicity for the hypoxic cells. 
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 most photosensitizers kill 
cells require effective contact between a molecule of photosensitizers 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 
photosensitizer is low, but will increase if the concentration of the 
photosensitizer in the hypoxic tissue is increased. Attempting to do so 
simply by increasing the dose of photosensitizer that is administered may 
produce too high a concentration in vital non-malignant tissues. However, 
if the photosensitizer 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. 
OBJECT OF INVENTION 
It is, therefore, an object of the present invention to provide novel 
photochemotherapeutic agents of the porphyrin and chlorin type substituted 
with hydroxylated residues via amide bonds, which are good tissue 
photosensitizers, accumulate preferentially in malignant tissues, 
especially in necrotic and/or hypoxic areas, show low systemic toxicity, 
clear rapidly from skin and most other normal tissues. 
Another object is to provide chemotherapeutic agents of the above type 
which exhibit at least some degree of anti-tumor activity even in the 
dark. 
BRIEF STATEMENT OF THE INVENTION 
By one aspect, of this invention there are provided compounds of the 
formula 
EQU (ZNHCO).sub.n X 
wherein: 
Z is selected from mono-, di-, and polyhydroxyalkyl residues with the 
provisio that Z does not include a carboxyl group; 
X is selected from substituted tetrapyrroles in which the substituent is at 
least one of the group consisting of methyl, ethyl, vinyl, hydroxyethyl, 
alkoxyethyl, methylcarboxy, ethylcarboxy, Z-substituted propylamide, 
phenyl and (ZNHCO).sub.n -substituted phenylamide, and n is an integer 
from 1 to 8. 
In a preferred aspect of this invention the tetrapyrrole is either derived 
by various procedures from naturally-occurring tetrapyrroles or 
synthesized by coupling of suitably substituted pyrroles and 
benzaldehydes. These cyclic tetrapyrroles have the following basic ring 
structure 
##STR1## 
and also include perhydro-, e.g., dihydro- and tetrahydro-derivatives of 
the said ring structure. 
By another aspect of this invention, there is provided a method for 
detection and treatment of malignant tissue abnormalties in a patient 
comprising administering to said patient an effective amount of 
tetrapyrrole derivatives of amino alcohols described herein before and 
exposing said tissue abnormality to light within the photoactivating 
spectrum of said tetrapyrrole derivatives. 
DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS 
The compounds of the present invention are designated as derivatives of the 
tetrapyrrole for convenience. Hence, the terms "tetrapyrrole" and 
"porphyrin" are used here to designate compounds of the cyclic structure 
shown herein before and their corresponding perhydro derivatives. Another 
characteristic of the present new compounds is that they all bear at least 
one residue which includes one amide bond by which an hydroxylated residue 
is linked to the tetrapyrrole part. 
Thus, the present invention concerns porphyrin or perhydroporphyrin 
derivatives of amino alcohols, in which the porphyrin chromophore and the 
hydroxylated residues are linked by amide bonds. This amide bond involves 
the amino group of the amino alcohol and a carboxyl group attached to the 
porphyrin. 
The tetrapyrroles employed in the present invention to form the aforesaid 
amide bond include two major classes which are both well-known to those 
skilled in the art, i.e. 1) the carboxy-containing tetrapyrroles and their 
perhydro analogues derived by various means from natural porphyrins and 2) 
the carboxy-containing meso-tetraphenylporphins and their 
perhydroanalogues. Exemplary tetrapyrroles valuable for the preparation of 
the compounds contemplated by the present invention are listed in Table I. 
The amino alcohols employed in the present invention to form the aforesaid 
amide bond include mono-, di- or polyhydroxylated acyclic or cyclic, 
primary or secondary amines. Hence, these amino alcohols show three 
characteristics: 1) they contain, of course, at least one hydroxyl group; 
2) they 
TABLE I 
__________________________________________________________________________ 
Tetrapyrrole 2 3 5 7 8 10 12 13 15 17 18 20 
__________________________________________________________________________ 
Coproporphyrin I PO Me H PO Me H PO Me H PO Me H 
Coproporphyrin III 
PO Me H PO Me H PO Me H Me PO H 
Deuteroporphyrin IX 
PO Me H Me H H Me H H Me PO H 
Hematoporphyrin IX 
PO Me H EO Me H EO Me H Me PO H 
Hematoporphyrin IX dialkylethers 
PO Me H EOE Me H EOE Me H Me PO H 
Mesoporphyrin IX PO Me H Et Me H Et Me H Me PO H 
Protoporphyrin IX PO Me H V Me H V Me H Me PO H 
Uroporphyrin IX PO AO H PO AO H PO AO H AO PO H 
Chlorine.sub.6 (16-17-dihydro) 
CO Me H Et Me H V Me H Me, H 
PO, 
H 
Pentacarboxyporphyrin I 
PO Me H PO Me H PO Me H PO AO H 
Pentacarboxyporphyrin III 
PO Me H PO Me H PO Me H AO PO H 
Hexacarboxyporphyrin I 
PO Me H PO AO H PO Me H PO AO H 
Hexacarboxyporphyrin III 
PO Me H PO Me H PO AO H PO AO H 
Heptacarboxyporphyrin I 
PO Me H PO AO H PO AO H PO AO H 
Tetracarboxyphenylporphyrin 
H H .phi.-CO 
H H .phi.-CO 
H H .phi.-CO 
H H .phi.-CO 
Tricarboxyphenyl-phenylporphin 
H H .phi.-CO 
H H .phi.-CO 
H H .phi.-CO 
H H .phi. 
Dicarboxyphenyl-diphenylporphin-S 
H H .phi.-CO 
H H .phi. 
H H .phi.-CO 
H H .phi. 
Dicarboxyphenyl-diphenylporphin-A 
H H .phi.-CO 
H H .phi.-CO 
H H .phi. 
H H .phi. 
Carboxyphenyl-triphenylporphin 
H H .phi.-CO 
H H .phi. 
H H .phi. 
H H .phi. 
__________________________________________________________________________ 
CO = --CO.sub.2 H 
AO = --CH.sub.2 CO.sub.2 H 
PO = --CH.sub.2 CH.sub.2 CO.sub.2 H 
EO = --CH(OH)CH.sub.3 
EOE = --CH(OR)CH.sub.3, wherein R = alkyl 
Me = --CH.sub.3 
Et = --CH.sub.2 CH.sub.3 
V = --CH.dbd.CH.sub.2 
.phi. = Benzene ring do not contain a carboxyl group; 3) their amino 
group is available to form the aforesaid amide bond with a carboxyl group 
of the selected tetrapyrrole. Thus, various amino alcohols are valuable 
for the present invention including 2-aminoethanol, 
2-amino-1,3-propanediol, 2-amino-2-(hydroxymethyl)-1,3-propanediol, 
D-glucosamine and similar such amino alcohols. 
Preferred compounds may be derived from commercially available 
tetrapyrroles selected from hematoporphyrin IX dihydrochloride, 
mesoporphyrin IX dihydrochloride, protoporphyrin IX dihydrochloride or 
deuteroporphyrin IX dimethylester. Hence, the preferred tetrapyrroles from 
which the new compounds are derived are those wherein at least two 
carboxyl groups or lower alkyl esters are present in the tetrapyrrole, 
preferably asymmetrically attached to the porphyrin ring system. These 
preferred tetrapyrroles are represented by the formula 
##STR2## 
wherein 
Me: methyl 
X: H, vinyl, ethyl, acetyl, formyl, hydroxyethyl, alkoxyethyl 
Y: H, vinyl, ethyl, acetyl, formyl, hydroxyethyl, alkoxyethyl 
PO: propionic acid 
The present new compounds are prepared by usual amidification procedures 
between the selected amino alcohol and the carboxyl containing 
tetrapyrrole. These procedures are well known to those skilled in the art. 
They involve any amide-forming derivative of the tetrapyrrole carboxylic 
acid, e.g. lower alkyl esters, activated esters, acyl chlorides, 
anhydrides and mixed anhydrides. 
The preferred preparative procedures use lower alkyl ester and activated 
(trifluoro acetate) ester. The reactants are mixed, in a suitable solvent 
when necessary. Heating up to 90.degree. C. can be used. Examples of 
detailed procedures are to be found below. Unreacted tetrapyrrole and 
side-products are separated from the amide derivatives by chromatographic 
techniques and crystallization. 
Agents valuable for photodetection or phototherapy should have the 
following properties 
1) be non-toxic, in the dark, to normal tissue, at dosage requested for 
photodetection or phototherapy; 
2) accumulate selectively within neoplastic tissue, i.e. clear rapidly from 
normal tissue and be retained by malignant tissue; 
3) emit detectable fluorescence when illuminated with light 
(photodetection); 
4) be photoactivated by illumination with light in the red spectral region 
and deactivate by a process that exert a cell killing effect 
(phototherapy). 
The new compounds of this invention show all the properties listed herein 
before and present an improvement relative to other porphyrins actually 
used for photodynamic therapy. 
The present compounds are apparently non-toxic at therapeutic dosages. 
Dosage levels up to 100 mg/kg body weight caused no mortality of test 
animals. 
It has been shown by various methods that the compounds contemplated by the 
present invention accumulate selectively within the tumors. In vivo 
pharmacokinetics experiments monitored with fluorescence equipment 
demonstrate that, at any time after administration of the compounds to 
test animals, the malignant tissues fluoresce at least twice as much as 
the normal tissue, this fluorescence being typical of the present 
compounds. When the animals were sacrificed, all of the compounds tested 
showed strong fluorescence in the tumor with no fluorescence in the 
adjacent healthy tissue as well as in the skin, bowel, skeletal muscle, 
lungs, heart, thymus, liver, spleen or kidneys. Of all the organs observed 
only the pancreas showed significant but low fluorescence intensity. 
The present compounds can be photoactivated by illumination with light in 
the red spectral region so as to exert a cell killing effect. This 
property has been shown by in vitro as well as in vivo experiments. 
Hemolysis of erythrocytes upon irradiation in the presence of a 
photosensitizer is a method used by those skilled in the art to test the 
potency of photosensitizing agents to cause the death of cells by 
irradiation. Experiments made according to the said technique with the 
present compounds demonstrate that they produce higher level of 
photohemolysis, at lower dose, than the compounds actually used for 
photodynamic therapy. 
Experiments were conducted in mice transplanted with a carcinoma. The 
present compounds were administered to the mice and the region of the 
tumor was irradiated with light of wavelength greater than 600 nm. Several 
days after exposure to the photoactivating light, there was no evidence of 
residual tumor. 
An interesting property of the present compounds is their strong tendency 
to accumulate preferentially in the necrotic and/or hypoxic areas of 
malignant tissues, as demonstrated by fluorescence in vivo experiments on 
test animals bearing carcinomas. 
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 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.