Journal of Photochemistry

and Photobiology, B: Biology, 6 (1990) 259-274

NEW TRENDS IN PHOTOBIOLOGY CHLORINS AS PHOTOSENSITIZERS lMEDICINE

259

(Invited Review) IN BIOLOGY AND

JOHN D. SPIKES Department

of Bido~,

University of Utah, Salt Lake City, VT 84112 (U.S.A.)

(Received January 26, 1990; accepted February 5, 1990)

Keywords. Aspartyl chlorin e,, bonellin, chlorin e,, chlorins, conjugated chlorins, linked chlorin-porphyrins, photodynamic therapy, photosensitization, tumors.

The photodynamic therapy (PDT) of tumors involves ilhunination of the tumorous area following the administration of a tumor-localizing photodynamic sensitizer. Hematoporphyrin derivative (HPD) and PhotoiYin II (a purified form of HPD), the main sensitizers used clinically for PDT to date, are complex mixtures of porphyrins; furthermore, these preparations absorb light very poorly in the red region of the spectrum (wavelengths greater than 600 nm) where light penetration into mammalian tissues is greatest. Thus there is considerable interest in identifying new sensitizers that localize more effectively in tumors, absorb more strongly at longer wavelengths and can be prepared in high purity. Much of this interest has been directed towards chlorins (reduced porphyrins), which typically absorb strongly in the red. This review summarizes research that has been carried out on selected types of chlorins, some of which may have important applications as sensitizers for PDT.

1. Introduction

There has been a large increase in research on photochemotherapy during the last decade [ 11. In this modality, the patient is given an appropriate photosensitizing drug prior to light treatment. Much of the recent interest in photochemotherapy has centered on the treatment of tumors (so-called photodynamic therapy (PDT)). PDT offers the possibility of double selectivity in the treatment of solid tumors by administration of a photosensitizer that is selectively retained in the tumor, followed by localized illumination of the tumorous area. Several thousand patients have been treated by PDT over loll-1344/90/$3.50

0 EIsevier Sequoia/Printed in The Netherlands

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the last decade, with generally favorable results for certain types of tumors [2,3]. Most clinical trials have utilized hematoporphyrin derivative (HPD), a complex mixture of porphyrins, as the photosensitizer; a somewhat purified HPD, Photofrin II, has also been used. HPD absorbs poorly in the red region where tissue penetration by light is great. As a result, several other types of sensitizers have been examined that absorb more strongly and at longer wavelengths in the red, in particular the phthalocyanines and naphthalocyanines, various types of chlorins, verdins and different kinds of porphyrin derivatives. There are several recent general reviews of these “second generation” sensitizers for PDT [4-S]. Chlorins are reduced porphyrins. For example, hydrogenation of one of the pyrrole double bonds in the simplest porphyrin, porphine, gives the parent molecule, chlorin, a dihydroporphyrin (Pig. l(A)). Bacteriochlorins are tetrahydroporphyrins. Chlorins typically have a strongly absorbing band in the 640-700 run range in the red, depending on the substituents on the macrocycle. The absorption properties can be altered by chelated metal ions. This can be compared with the very weak absorption peaks of HPD and many other porphyrins at approximately 630 nm. Space does not permit the discussion of all categories of chlorins in this review; the emphasis is on bonellin, chlorin e, and some of its derivatives, purpurins and some miscellaneous chlorins and chlorin conjugates. Chlorophylls, chlorophyllides, chlorophyllins, pheophytins and pheophorbides, all of which are formally chlorins, are not discussed since they have recently been reviewed elsewhere [91. The chemical nature, physical properties (solubility, absorption and emission spectra), photophysics and photosensitizing behavior (in solution and in biological systems at the subcellular, cellular and organism levels) are reviewed for each example or category of chlorin. Finally, the possible application of chlorins as photosensitizers in PDT is examined.

2. Bonellin Bonellin, a green chlorin unrelated to chlorophyll, is found in tissues of the marine echiuroid worm, BonelZia viridti; its structure is shown in Fig. l(B) [lo]. Several monopeptide conjugates of bonellin have also been isolated from the organism, including the valine (major component), isoleucine, leucine and alloisoleucine derivatives [ 111. The copper complex also occurs in the worm, and both the copper and zinc complexes have been synthesized [12-141. It was shown in 1955 [15] that the ability of crude bonellin preparations to cause the cytolysis of echinoid cells is markedly enhanced in the light. Since this early work, a number of studies have been made of the photophysical and photosensitizing properties of bonellin. Bonellin in ethanol has a strong Soret absorption band at 388 run; it also has a major absorption band at 639 run in the red [ 141. In ethanol, the pigment shows a single fluorescence emission band at 643 nm [ 14,161. The zinc complex

261

P

M

Bonellin

Chlorin

“MT” Chlorin g, (Ce6)

(CH,),CO-ASP

Mono-L-Aspartyl chlorines (Ace61

J-c D

H

H

M

A Purpurin

P

Mesochlorin

Fig. 1. Structures of representative chlorins. Possible isomers are not shown. Abbreviations used: E, -CH,CH,; M, -CH,; P, -(CH&COOH; V, -CH=CH,.

of bonellin is fluorescent, but not the copper derivative [12]. Illumination generates bonellin triplets with a high yield; the triplet lifetime is approximately 430 ps. The bonellin triplet is efficiently quenched by ground state oxygen and benzoquinone; the latter process probably involves an electron transfer mechanism. Copper bonellin has a much shorter triplet lifetime (less than 20 ns) [14]. These effects of copper on the fluorescence and the triplet lifetime are simikw to those observed with porphyrins [ 171. Illumination of linolenic acid and cholesterol in the presence of bonellin results in the peroxidation of these substrates, as measured by the formation of malondialdehyde-type photoproducts. The free radical scavengers, acetyl-

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homocysteine-thiolactone and meclofenoxate, inhibit these photoperoxidation reactions [ 181. Bonellin inhibits the growth of a number of species of terrestrial and marine bacteria in the dark; with some, but not all, species, the inhibition is increased by light. Protoplasts of Bacillus subtilis are rapidly lysed on illumination in the presence of bonellin, but not those of Micrococcus lysodeikticus [ 191. Sea urchin sperm illuminated in the presence of bonellin lose their fertilizing capability, show a decreased oxygen consumption, become swollen and immotile, and die. Sea urchin eggs treated in .the same way are no longer fertilizable. Photodynamic treatment of normal eggs with bonellin at various times after fertilization results in abnormal embryonic development. Bonellin has little effect on sea urchin gametes in the dark [ 131. Glutathione protects sea urchin eggs to some extent against bonellin-sensitized photodamage [20]. Illumination of young larvae of the ascidian, Cionu, in the presence of bonellin rapidly and irreversibly stops their motility. Analysis of tail muscle from treated larvae shows that myosin molecules are cross-linked by the photodynamic treatment. Frog muscle myofibrils show the same behavior. With the treatment used, there is no damage to muscle mitochondria 1211. Human erythrocytes swell and then hemolyse on illumination in the presence of bonellin; the rate of hemolysis is decreased in the absence of oxygen. The action spectrum for this process shows peaks at approximately 390 and 630 nm, corresponding to the absorption peaks of the sensitizer. Copper bonellin does not sensitize photohemolysis, as would be expected from its very short triplet lifetime as described above. If bonellin is illuminated and then added to the erythrocytes, no hemolysis occurs. Benzoquinone, which efficiently quenches bonellin triplets, inhibits hemolysis, as do the singlet oxygen quenchers, Aocopherol and p-carotene [ 13). With He-Ne laser illumination (632.8 run), bonellin sensitizes the photohemolysis of human erythrocytes more efficiently than HPD [22]. Membrane proteins are cross-linked [20] and membrane lipids are peroxidized [ 181 in erythrocyte ghosts on ilhnnination in the presence of bonellin; photoperoxidation is decreased by free radical quenchers. With illumination in the 610-660 run range, bonellin sensitizes the photodynamic killing of Sarcoma-180 tumor cells in culture ten times more efficiently than mesoporphyrin. Both of these sensitizers are taken up equally well by the cells, and both appear to accumulate in cellular regions with the same dielectric constant, perhaps at an interface between hydrophobic and hydrophilic regions of cell membranes. Thus the greater photosensitizing efficiency of bonellin probably results from its much stronger absorption in the red [ 161. Bonellin, injected intraperitoneally into mice with implanted Lewis lung tumors, does not localize detectably in the tumors [23]. 3. Chlorin e, and its derivatives 3.1. Chlarin e, Chlorin e, (Ce6) (Fig. l(C), sometimes termed phytochlorin, is derived from chlorophyll a. It is essentially monomeric in dilute solution in both

263

ethanol and phosphate buffer; in buffer at pH 7.4 it has a major Soret peak at approximately 402 nm and a strong red peak at approximately 654 nm [24]. Ce6 has fluorescence maxima at 675 and 735 nm in both ethanol and buffer; the fluorescence lifetimes are approximately 5.3 and 4.5 ns in ethanol and buffer respectively [25,26]. Illuminated Ce6 in organic solvents and DzO efficiently generates singlet oxygen as measured by a luminescence technique (271; comparisons of singlet oxygen generation in several homogeneous and heterogeneous systems have been made [28]. The quantum yield for singlet oxygen photoproduction with Ce6 is 0.7 [29]. On ilhunination in buffer, Ce6 gives a good yield of a long-lived triplet with a lifetime of approximately 760 p. The triplet is efficiently quenched by ground state oxygen and benzoquinone; the latter process appears to involve electron transfer. Ce6 sensitizes the photo-oxidation of cysteine, histidine, methionine, tyrosine, guanosine and thymine, and the photodynamic inactivation of the enzyme, lysozyme, as efficiently as good porphyrin photosensitizers. The quantum yields for these photo-oxidations are the same with illumination at the Soret band and at the long wavelength absorption peak [ 241. Both bovine and human serum albumins strongly bind Ce6 in a 1: 1 ratio [ 301. Ce6 is reduced on illumination in the presence of an ascorbate-pyridine mixture [ 311. The photoreduction of methyl viologen and methyl red with mercaptoethanol as the electron donor is sensitized by Ce6; it also sensitizes the Bavoprotein-mediated photoreduction of NADP [ 321. The spectral and energy properties of Ce6 are not altered by binding to human serum albumin, and the bound chlorin still efficiently sensitizes the photoreduction of methyl viologen in the presence of ascorbate [33]. The chlorin binds more strongly to human high density lipoprotein than to serum albumin [34]. Ce6 incorporated into egg yolk lecithin small unilamellar liposomes is present as monomers in a rigidly fixed position at the boundary between the polar and non-polar parts of the phospholipid membrane [35]. The CeG-sensitized photodamage to bacteria with various defects in their DNA repair systems has been examined [36]. Oxygen consumption and oxidative phosphorylation by isolated rat liver mitochondria are photodynamically inhibited with Ce6 [37]. Protein components of the membranes of human erythrocytes and erythrocyte ghosts bind Ce6 in a monomeric form. Illumination of the cells in the presence of oxygen results in hemolysis [38,39]. Proteins in the treated membranes are cross-linked, and fatty acid peroxidation products accumulate. Scavenger studies show that membrane damage is mediated primarily by singlet oxygen; however, superoxide may also be involved [40-42]. Murine leukemia L1210 cells bind Ce6 at both plasma membrane and cytoplasmic sites [34]. The distribution of Ce6 in HeLa cells has been examined using fluorescence microscopy [43]. Mouse ascites MH134 hepatoma cells also bind Ce6. On illumination, the cells lose the ability to cause tumors when implanted in mice. The illuminated cells show a swollen cytoplasm, gain water and sodium ions, but lose magnesium and potassium ions. Furthermore, they have a decreased ATP activity and a decreased rate of DNA synthesis. DLl 10 leukemia and CBMT mammary

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cancer cells are photoinactivated with Ce6 [44,45]. Ce6 also sensitizes the photokilling of human HT-29 colon carcinoma cells [ 461 and murine L12 10 leukemia cells [34]. Fluorescence measurements show that human NHIK 3025 cells take up Ce6 in a monomeric form. Treated cells are killed on ihumination, and 1,3-diphenylisobenzofuran (which rapidly reacts with singlet oxygen) incorporated into the cells is quickly destroyed [47]. The action spectrum for cell killing very closely resembles the fluorescence excitation spectrum of Ce6 in the cells [48]. Sodium Ce6 is somewhat effective as a sensitizer for the PDT of ascites tumors in mice [49]. Hepatomas in rats accumulate Ce6 to higher concentrations than normal liver tissue [ 501. Ce6 is not retained very selectively by C3H mouse mammary tumors [47]. However, it does localize somewhat in FM3A mouse mammary carcinomas, attaining a maximum concentration a few hours after injection; it is an effective sensitizer for the PDT of this type of tumor [ 51,521. Photodynamic treatment of Ehrlich ascites carcinoma with Ce6 increases the number of polyploid cells; also, the rate of lightinduced cytolysis increases in the terminal stage of tumor development ]531. 3.2. Derivatives of chlorin e, Illumination of mono-L-aspartyl chlorin e6 (ACe6) (Pig. l(D)) in buffered aqueous solution generates singlet oxygen with a quantum efficiency of approximately 0.48; the efficiency with diaspartyl Ce6 is approximately 0.20. Both chlorins efficiently sensitize the photo-oxidation of furfuryl alcohol, imidazole and tryptophan [54,55]. The physical, photophysical and solution photosensitizing properties of Ace6 are rather similar to those described above for Ce6 [24]. In studies with human blood plasma fractions, Ace6 is found to bind more strongly to plasma proteins than to lipoproteins [34]. Ace6 is rather sensitive to photodegradation, especially in the presence of serum proteins and when in cells 1561. Since it is somewhat more polar than Ce6, Ace6 partitions largely into cytoplasmic loci in murine leukemia L12 10 cells [34]. Both Ace6 and diaspartyl Ce6 penetrate into kangaroo rat PTK2 epithelial cells. Both derivatives sensitize the photodynamic killing of CHOKl Chinese hamster ovary cells with a dye laser at 664 run, with Ace6 being the more effective (571. Mammalian cells take up Ace6 by endocytosis. The chlorin appears to localize in the lysosomes of the cells, and sensitizes damage to the lysosomes on illumination. Hydrolytic enzymes appear to degrade Ace6 in the cells [58-601. However, the Ace6 peptide bond is not hydrolyzed when the chlorin is incorporated into mouse leukemia L1210 cells or Pane 02 murine tumors [ 341. The efficiency of the photodynamic killing of mammalian cells by Ace6 is much less than would be expected on the basis of its efficiency of singlet oxygen generation; this may result from degradation of the chlorin in cells [54,55]. The photodynamic treatment of mouse leukemia L12 10 cells in vitro with Ace6 inhibits membrane transport processes, decreases the ATP pool size and kills the cells; however, the efficiency is less than that observed with the more hydrophobic Ce6 [34].

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A number of Ce6 derivatives, including ACe6, diaspartyl Ce6, monoseryl Ce6 and other amino acid derivatives, are retained better in tumors and sensitize the photodynamic destruction of tumors better than Ce6 itself. These derivatives have been examined in several animal tumor models including the Buffalo rat with implanted Morris hepatomas 7777 and 5123TC and DBA/2Ha Ros-d + Ha mice with SMT-F tumors [ 61-631. Intravenously injected Ace6 shows a high degree of selective retention in m-KSA kidney sarcomas in mice. Wee days after injection, the tumors contain five times as much sensitizer as other organs, as measured by an excimer dye laser fluorescence technique [64]. Ace6 efficiently sensitizes the PDT of EMT-6 sarcomas in BALB/c mice; with appropriate sensitizer and light dosages, 100% cures are obtained. Therapeutic dosages of ACe6, unlike most of the porphyrins used in studies of PDT, cause very little skin photosensitization in mice and guinea pigs; this would be of great advantage in the clinical situation [56,65 1. Starting within 1 h after PDT with ACe6, there is a progressive destruction of the subendothelial zone of the tumor vasculature, with fragmentation of the fiber elements and collagen. Plasma proteins and erythrocytes then extravasate into the subendothelial zone and around the tumor cells. Tumor destruction appears to result from photosensitized effects on the vasculature rather than directly on the tumor cells [ 661. Ce6 is an extremely effective sensitizer for the PDT of BA mammary carcinomas in mice; optimal cure results when the tumors are illuminated at 4-7 h after introduction of the sensitizer. The chlorin is rapidly cleared from the animals, so that little effect is observed with ilhunination at 24 h. This would be advantageous in the treatment of patients [67,68]. Hexadecyl Ce6 is reported to have a significant phototumoricidal activity [ 61.

4. Purpurins A number of purpurins and their metallo derivatives have recently been synthesized in high purity and are attracting considerable interest as candidate sensitizers for the PDT of tumors [4,5 1. Purpurins are formally chlorins, since they have one reduced pyrrole group. Those currently under study also have a fused five-membered isocyclic ring, as shown in Fig. l(E). Like many other chlorins, purpurins have a strong absorption band in the red region of the spectrum in the 630-7 15 run range. Photophysical measurements have been made on two derivatives: purpurin NT2 (an octaethylpurpurin with R1 =CH&H3, Rz =CH2CH3, RS=C02CH2CH3, Fig. l(E)) and ZnET2 (a zinc(I1) etiopurpurin with R1 = CH3, R2 = CH2CH3,R3 = CO&H&Ha, Fig. l(E)) [69, 701. NT2 and ZnET2 have red absorption bands at 695 and 665 nm respectively. On illumination, both give good yields of triplets with lifetimes greater than 100 +s which are efficiently quenched by ground state oxygen to give singlet oxygen with quantum yields of 0.70 and 0.60 respectively. Prolonged ilhunination of purpurins can lead to oxidative cleavage of the isocyclic ring to form the corresponding formyl chlorins. Another purpurin

266

derivative, SnETZCla, absorbs at 659 nm with an extinction coefficient five times greater than the red peak of Photofrin II [ 711. The purpurins are insoluble in aqueous buffer. However, they can be administered to animals as an emulsion in Cremophor EL (a polyoxyethylene derivative of hydrogenated castor oil) or incorporated into liposomes. SnET2C12 sensitizes the efficient in vitro photodynamic kilhng of AY2 7 rat bladder cells [ 711. Metal-free NT2 partitions into a very hydrophobic environment in mouse leukemia LI210 cells in vitro, and sensitizes photodamage to DNA synthesis, but not to membrane transport. In contrast, SnET2C12 localizes in a somewhat more hydrophilic location in the cells, perhaps at a membrane interface, and sensitizes photodamage to membrane transport processes [ 721. A number of studies have been made of the PDT of transplantable FANFT-induced tumors in Fischer rats using a series of free base and metallopurpurins (silver, nickel, tin, zinc), synthesized as described in ref. 73. Sensitizers, in Cremophor EL emulsions, are introduced intravenously via the tail vein. Most of the free base purpurins and the tin and zinc metallopurpurins sensitize extensive hemorrhagic tumor necrosis; however, the nickel derivative has no effect, and the silver purpurin has only a slight sensitizing effect. Octaethylpurpurin and etiopurpurin sensitize signiflcant tumor regression and, in some cases, complete cures; the tin and zinc derivatives are even more effective [73, 741. The relative effectiveness of some of the better purpurin sensitizers is in the order SnET2 > SnNT2 >ZnET2 >ZnNT2. The maximum therapeutic effect with SnET2 is obtained with illumination of the tumor at 24 h after injection of the sensitizer [75]. Normal animal tissues, including the rat footpad and jejunum and the skin of hairless mice, show much less photosensitivity following treatment with therapeutic levels of SnET2 than with HPD [75, 761. The effect of PDT with SnET2 (introduced in a Cremophor EL emulsion or incorporated into liposomes) on blood flow to transplantable FANFI’ bladder tumors implanted in rats has been examin ed using the radioactive microsphere technique. As with HPD and phthalocyanines, such treatment causes a rapid decrease in blood flow to the tumor. Systematic heparinization of the rats prior to illumination of the tumors has no effect on the decrease in blood flow produced [77,78].

5. Miscellaneous

types of chlorins

Illumination of the synthetic compounds meso-tetraphenylisobacteriochlorin and meso-tetraphenylbacteriochlorin gives good yields of triplet states; the triplet lifetime for the latter chlorin is approximately 140 w [ 791. The photophysical and photodynamic properties of a dicarboxylic chlorin derived from hydroxyethylvinyldeuteroporphyrin have been examined. This chlorin has an enhanced absorption at 660 run; on flash illumination, a good yield of a triplet state with a lifetime of approximately 87 E.CS is produced. As

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measured by a luminescence technique, the sensitizer produces singlet oxygen as efficiently as good porphyrin sensitizers [80]. Bacteriochlorin a sensitizes the photo-oxidation of histidine, tryptophan, guanosine and dithiothreitol;these reactions are inhibited by azide and enhanced by DzO, suggesting the involvement of singlet oxygen. This chlorin also efficiently sensitizes the photodynamic killing of L929 cells in culture [Bl 1. Chlorin p,, a tricarboxylic chlorin derived from chlorophyll, has a red absorption band at 654 nm in aqueous btier. With white light, the chlorin sensitizes the photo-oxidation of histidine at approximately the same rate as hematoporphyrin. With illumination peaking at 650 nm from red fluorescent lamps, the chlorin is several orders of magnitude more efficient in sensitizing photodamage to human fibroblasts in culture than hematoporphyrin [ 821. Mesochlorin (Pig. l(F)), a dicarboxylic hydrophobic chlorin analog of mesoporphyrin, binds preferentially to high density and low density lipoproteins rather than to albumin in human blood plasma fractions. It localizes primarily in the membranes in cells. With murine leukemia L1210 cells, mesochlorin is an effective sensitizer for photodynamic damage to plasma membranes and mitochondria, as well as for cell killing [ 341. Several new reduced hydroxyporphyrins soluble in polar solvents (the ortho, m&a and pm-u isomers of 5,10,15,20-tetra(hydroxyphenyl)chlorin and 5,10,15,2O-tetra(m-hydroxyphenyl)bacteriochlorin) have recently been described as possible sensitizers for PDT. The chlorins have a good absorption in the red at approximately 650 run, and the bacteriochlorin absorbs at 735 nm in methanol (741 nm in serum). All four compounds show good activity as sensitizers for the photonecrosis of PC6 plasma cell tumors in mice; all are more potent than the corresponding porphyrins [83]. Some benzochlorins with absorption peaks in the 675-682 nm range have recently been prepared and tested as possible sensitizers for the PDT of tumors. These compounds are insoluble in aqueous buffer and are therefore administered as emulsions. The benzochlorins are better sensitizers for the PDT of a bladder tumor model in rats than HPD. A sulfonated, more hydrophilic benzochlorin derivative, is less effective than the non-sulfonated counterpart; a tin derivative is more effective than the metal-free compound [84]. Finally, a number of synthetic bacteriochlorins prepared from porphyrindiones are effective sensitizers for the PDT of a bladder tumor model in rats. Some of these derivatives absorb as far in the red as 738 nm [85]. Another new sensitizer, benzoporphyrin derivative, is formally a chlorin, since one of the pyrrole rings in the molecule is reduced. The derivative, as synthesized from protoporphyrin, is a mixture of two isomers and a monoacid and diacid; as the name implies, it has a fused six-membered isocyclic ring. In solution in aqueous buffer, the derivative has an absorbance at its red peak (approximately 690 run) that is approximately four times greater than the absorbance of the red peak of Photofrin II at 623 run. All of the components of benzoporphyrin derivative are potent photosensitizers for the photodynamic killing of a number of types of normal and cancer cells in vitro [86-881. All four of the derivatives are as active as Photofrin

268

II as photosensitizers for the photodynamic destruction of M-l rhabdomyosarcomas in mice [87].

6. Linked chlorin-porphyrin

systems

Most clinical studies of the PDT of tumors have utilized a complex mixture termed HPD as the sensitizer. The tumor-localizing fraction of this mixture is variously regarded as a dihematoporphyrin ether or ester or as an oligomer containing seven or eight hematoporphyrin rings [23,89]. One approach to the development of long wavelength absorbing sensitizers for PDT has been to esterify a chlorin molecule to a hematoporphyrin molecule. Bonellin-hematoporphyrin and mesochlorin-hematoporphyrin preparations show both the weak porphyrin absorbance at approximately 630 run and the much stronger chlorin absorbance at approximately 640 run. In vitro, the mixed esters are five times more effective as sensitizers for the photodynamic killing of L1210 mouse leukemia cells than dihematoporphyrin ester, even though approximately equal amounts of the various sensitizers are accumulated by the cells. Lntraperitoneally injected hematoporphyrin, bonellin and mesochlorin are not taken up detectably by Lewis lung tumors implanted in mice; however, signiiicant amounts of the dihematoporphyrin ester and the mesochlorin-hematoporphyrin ester are accumulated [23]. Another approach to the development of better red absorbing photosensitizers for PDT has been to convert one of the porphyrin residues in the oligomeric hematoporphyrin derived from HPD to a chlorin. This gives a preparation with a tenfold increased absorption at 660 nm. The quantum yield of singlet oxygen generation by the chlorin derivative on illumination is the same as that of the starting porphyrin oligomer [ 89,901. A chlorin-porphyrin derivative has also been prepared by linking a chlorin derived from hematoporphyrin to hematoporphyrin 1901.

7. Chlorins

conjugated

to site-specific

agents

In general, tumors do not accumulate Ce6 very well. Thus several techniques have been developed in attempts to deliver Ce6, as well as other sensitizers, to specific types of tumors in larger concentrations and with greater selectivity. In one approach, the selective in vitro photodynamic destruction of HPB-ALL human T-cell leukemia cells has been obtained using a Ce6 derivative (CeG-monoethylenediamine monoamide) conjugated covalently through a dextran spacer to a monoclonal antibody directed towards the leukemia cells as the sensitizer. The conjugate does not appreciably sensitize the photodynamic lolling of other cell types. Cell photokilling is enhanced in DzO. Free Ce6 derivative does not sensitize cell photokilling, even at a concentration 100 times greater than that present in the conjugate experiments. Binding the Ce6 derivative to the antibody does not change

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its absorption spectrum or its efficiency of generating singlet oxygen on ilhunination [29]. More recently, the Ce6 derivative has been conjugated to a variety of monoclonal antibodies via a polyglutamic acid spacer. The appropriate conjugates show selective phototoxicity in vivo towards target leukemia cells, human bladder and ovarian carcinoma cells and mouse lymphoma cells. Subcutaneous fibrosarcomas in mice injected directly with a conjugate directed towards the tumor cells are reduced to a non-measurable size 14 days following illumination with a laser at 656 nm. There is no photodamage to the overlying skin. In contrast, untreated tumors and tumors treated similarly with the free chlorin derivative or buffer continue to increase in size [91,92]. Water-soluble synthetic polymers have been studied extensively as carriers for the targeted delivery of drugs [93]. Recently, these polymers have been examined as carriers for the delivery of photosensitizers in the PDT of tumors. Synthetic copolymers of A%(2-hydroxypropyl)methacrylamide, with galactosamine-terminated side-chains and covalently bound Ce6, target to asialoglycoprotein receptors on human hepatoma cells and are then taken up by pinocytosis; the treated cells are killed on subsequent ilhnnination. Non-targetable copolymers with bound Ce6 are less phototoxic [ 941. CeG-copolymer conjugates, with covalently bound rabbit polyclonal anti-Thy 1.2 antibodies as targeting moieties, sensitize the photokilling of mouse splenocytes more efficiently than a similar non-targetable conjugate without antibody. Bound Ce6 shows much less dark toxicity to the cells than the free chlorin [95,96]. In vitro, bovine trabecular meshwork cells (the most active phagocytic cells in the anterior chamber of the eye) rapidly phagocytose 1.0 pm latex microspheres with Ce6 monoethylenediamine monoamide conjugated to the microsphere surface. Subsequent illumination at 664 nm kills the cells. Unirradiated cells and cells incubated with an equivalent concentration of the free chlorin derivative are not photosensitive [97].

Acknowledgments

The preparation of this review and the original work described were supported in part by American Cancer Society Grant No. PDT-259, the Utah Laser Institute and the DOD/SD10 Medical Free Electron Laser Program.

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