Photoactivatable drugs

TITS - April 1987 [Vol. 81

Hagan Bayiey, Francis Gasparro and Richard Edelson Formany yeurs, photoactivatable drugs have been used to treat skin diseases. Recently, a variety ofnew light-mediated therapies have been devc!oped and some are being tested clinically. Hagan Bayiey and colleagues describe this approach which has special potentiai for the treatment of cancer, including internal tumors, modification of the immune system, and the controlled local reiease of virtually any pharmacological agent. A surprising variety of diseases can be treated with light. Systematic studies date back to the 19th Century, and in 1903 Niels Finsen was awarded a Nobel Prize in Medicine and Physiology for the treatment of skin lesions including cutaneous tuberculosis with artificial ultraviolet radiation. Modern medicine prescribes light for several maladies, for example jaundice in new-boms, acne vulgtis, psoriasis, vitamin D deficiency and even depression. Lasers are now used routinely in surgery for ablation, coagulation and welding of tissues. From the early days it was recognized that dmgs and light might be used together to produce completely new therapeutic tools. Indeed the ancient Egyptians ate a weed, Ammi majus, and then exposed themselves to sunlight to treat depigmented patches of skin (vitiligo)‘. The active constituents of this plant are now known to be psoralens. Around the turn of the century, several studies were performed OILthe effects of light and forei molecules on living tissue Rn. Some of these were of clinical relevance. For example, Von Tappenier and Jesionek, in 1903, used the photosensitizing dye eosin and light to treat skin cancers. These early studies were not pursued vigorously although a great deal of information on phototoxicity was obtained in the years prior to the 1950s when a dramatic revival of phototherapy began with the toxicological and clinical study by Aaron Lemer and his associates, of pure E-methoxypsoralen and ultraviolet light for HagnnBayley is Assistant Investigator at the

Howard Hughes Medical Institute, Columbia Univcn&f, New York. Francis Gaspawo is Research /sso&ai and Rirhard Edelson is ProfessorQ~GChairman in the Department of Dermatology, Yale University, New HaveE, CT, USA. @ 1987. Uwier

Publications.

Cambridge

the treatment of vitiligo4. This revival has continued with the introduction of tar extracts or psoralens5 to treat psoriasis, later, psoralens for cutaneous T-cell lymphoma6, and recently hematoporphyrins to treat various forms of cancer7. Advantages and potential applicationsofphotoactivatable dws Photoactivatable drugs possess a number of desirable attributes as therapeutic agents. In essence, they mediate the controlled, local generation of potent biologically active molecules by the following means: a Photoactivatabre drugs may be activated at specific locations, for example at a tumor site or within a particular organ. While the actions of some conventional drugs may be localized fairly well (e.g. by application to the skin, or bjj local or intravenous injection), photochemical activation can be extraordinarily accurate and precise. The direction of a laser &earn can be exactly controll& and its diameter can be reduced far below that of a cell, a property which has already been used effectively in microsurgery. Other physical means, such as heat or magnetism, have been used to activate or localize the action of drugs but none has the precision of light. l Many of the species that can be generated by light (including ex-

cited states, singlet oxygen [‘O,], and free radicals) are very short lived. The mean distances that small reactive intermediates will diffuse in water before they are inactivated by decay to the ground state or by chemical reaction with solvent are given in Table I. These are maximum distances because the local viscosity of the tissues is certainly greater than that of water and a high concentration of molecules that might bind, inactivate or react with these intermediates is present. Clearly then the potential exists to create drug molecules whose active forms are confined to a small tumor, a single cell, or even an organelle within a cell. More stringent’control over the site of action of cytotoxic drugs would be particularly welcome in cancer chemotherapy where treatment is often limited by damage to organs distant from the tumor site: for example, the bone-marrow, intestines and skin during the conventional chemotherapy of breast cancer. l Extremely reactive species may be generated photochemically. The high energy of an ultraviolet or near ultraviolet photon (e.g. 350 nm: 340 kJ mol-r) is sufficient to break many chemical bonds (250-420 kJ mol-’ in organic molecules). As a consequence, photogenerated molecules can induce covalent and therefore permanent changes of their targets within the cell: usually proteins or DNA. Even molecules generated with relatively low energy radiation may be highly reactive. For example, a reactive excited state of molecular oxygen, singlet oxygen (‘Oz), lies only 90 kJ mol-l above the triplet ground state, enabling sensitizers absorbing at >600 nm to efficiently catalyse its formation. Permanent modification rather than reversible binding to a target has been a goal of several conventional therapeutic strategies

TABLE I. Mean diffusion paths of short-lived intermediates in water Haittife of intermediate

Example

Mean dlffusion

- 1 ns

Mary singlet excited states

- 1 nm, e%entially immobile

-lP

Singlet C&(-- 5 *;

- 33 nm. little diffusion between

path

organelles -1ms

Many triplet excited states, free radicals

- 1 pm, limited intracellular diffusion

>ls

Reactive, ground state photoproducts

> 33 pm, diffusion to neighboring cells

0165 - 6147/ 871$~l2~W

TIPS - April 1987 [Vol. 81 such as the chemical modification of DNA by mustards in cancer chemotherapy and the use of active site directed inhibitors and suicide substrates to inhibit enzymes; for exarnp!p, renicillinase in resistant bacteria. The potency of such agents is demonstrably greater than that of drugs that bind reversibly. l Finally, the dose of a photoactivatable drug may be controlled after it has been administered. This control may be at several levels. The timing of administration might be critical, as in the exposure during surgery of the area surrounding an excised tumor to a phototoxic agent intended to destroy residual tumor or micrometastases. The duration and strength of the dose might also be controlled by manipulating the exposure to light, and only with a drug activated by physical means might different doses be given at different sites. In keeping with the properties described above, photoactivatable drugs are being developed mainIy as cytotoxic agents, both for cancer chemotherapy and also for modifying the immune system. The latter includes immunosuppression by destroying lymphocytes and the induction of specific immunologic reactions against damaged malignant cells (see below). More speculatively, light might also be used for the local release of a wide variety of drugs, or even to inactivate drugs as they exit perfused limbs or organs. Accessibility of the tissues to light The tissues are penetrated quite well by visible light. The optical penetration depth (the distance to which 37% (l/e) of the light reaches) at 630 nm has been determined for several tissues and ranges from l-4 mm (Ref. 7). Unfortunately the attenuation increases as a steep inverse function of the wavelength, due to both scattering and absorption and near ultraviolet light (UVA: 32& 400 nm) penetrates no more than the depth of a few cells making it useful only for superficial treatments, for example of the skin or of a thin layer of blood. The diversity of photoactivatable drugs Photoactivatabie drugs may be divided into at least five classes. In

139 the first two (viz. photosensitizers and molecules that damage DNA) are cytotoxic drugs that are currently in use clinically or under intensive investigation in experimental animals. The others are in the early stages of development or merely speculative.

Photosensitizers

Photosensitizers are molecules, usually polycyclic aromatic hydrocarbons (e.g. pyrene) or organic dyes (e.g. methylene blue or acridine orange: Fig. la), whose presence renders an organism susceptible to damage by light. The action of most sensitizers is dependent upon the presence of molecular oxygen and it is believed that they function largely by catalysing the conversion by light of ground-state triplet O2 to l4, an extremely reactive molecule capable of modifying virtually any component of a biological system, including lipids, proteins and nucleic acids. Because the lifetime of ‘02 is short (approximately 5~) even in the absence of reactants (the mean diffusion path in water is approximately 0.2 um), it is likely that the site of action of a photosensitizer is largely determined by its intracellular location, e.g. a photosensitizer that binds tightly to DNA would be likely to bring about genetic damage, while a lipidsoluble photosensitizer would bring about membrane damage. For example, Oseroff and colleagues have recently examined phototoxic cationic dyes that are selectively retained by the mitochondria of carcinoma cells. The immediate damage caused by such agents is to mitochondria, followed by slow lysis of the piasma membrane*. An interesting possibility would be to administer precursors that are metabolized to photosensitizers. This idea has already been used successfully to make photoactivatahle herbicides by Rebeiz and his coworkers’,l’. The most fully tested drug among the photosensitizers is the photocytotoxic hematoporphyrin derivative (Fig. lb), which is under inten;Gve investigation as a tool for cancer chemotherapy (see below).

Moleculesthat damge DNA

Photosensitizers that bind preferentially to double-stranded DNA, such as the acridine dyes, probably act primarily by causing

damage at this site. In addition, there exist two other classes of photoactivatable drugs that act on DNA: the psoralens and certain metal complexes of the bleomycins. The psoralens (Fig. Id) occur naturally in plants and may act as light-activated insecticides*“~ll. Members of this family of planar tricyclic molecules intercaIate into double-stranded DNA in the dark. Upon photoactivation with near UV light they form adducts with pyrimidines, primarily thymidine. The initially formed monoadducts may further react, again upon absorbing near UV photons, to form interstrand crosslinks’*. Light fhrxes that do not harm cells in the absence of psoraIens kill cells in the presence of low levels of these drugs. For example, the proliferation of human T lymphocytes after stimulation with mitogens can be inhibited by more than 90% after incubation with S-methoxypsoralen (100 n ml-*) and UVA at 3 J cm-Q (Ref. 13). Several synthetic psoralens have been tested for activity and the water-soluble derivative aminomethyl-45’,8-trimethylpsoralen was found to be especially potentl*. As described here, psoralens have been used to treat vitiligo, psoriasis and leukemia, and they are under development as modifiers of the immune systemi5. They have also been used to inactivate virus particles for vaccine production16. Bleomycin (Fig. le) has been used in cancer chemotherapy for many years. The drug arts by degrading DNA in tumor cells and its action depends on the presence of metal ions, moiecular oxygen, and a reducing agent. Under physiological conditions the main form is an iron (II) complex, which can induce the removal of bases as well as single-strand and doublestrand breaks. Recently, Meares and colleaguesI have synthesized bleomycin derivatives that require light for activity. When irradiated with DNA at 366nm, several cobalt complexes induce base loss and single-strand breaks in the absence of oxygen or reductants. These complexes had previously been shown to concentrate in the nuclei of certain tumor cells. In an attempt to make more stable metal complexes that might be activated with visible light, a ruthenium (II)

TIPS -April

140 complex was recently prepared”, which required both light and oxygen for activity. Both this and the cobalt complexes have been reported to crosslink DNA, as well as nick it. As yet, these compounds have not been used clinkally. Morespeculativednrgsfor pkototherapy Several other ways of using light to activate drugs can be envisioned: l Photoactivatable site-directed agonists and antagonists (photoaffinity reagents) might be used to

activate or inactivate key proteins by covalent attachment to their binding sites. Photoaffinity reagents have been used for many years to identify and cnaracterize the proteins that bind biological ligands I9. For example, on irradiatic> at 350 nm ICYP-diazirine (see Fig. If) loses molecular nitrogen to form a highly reactive carbene that attaches to p-adrenergic receptors20. l Prodrugs that break down or isomerize on irradiation might be used to generate high local concentrations of active molecules. Ex-

1987 [Vol. 81

amples from the biochemical literature include the photogeneration of an acetylcholine receptor agonist by cis-tmns isomerization of an azo compound2’, the manipulation of ion concentrations by photosensitive chelators (Fig. lg), and the photochemical release of nucleotides (Fig. lh). Similar principles might be used to inactivate drugs. 0 Finally, drugs might be released locally from macromoiccular carriers using light. Photosensitive liposomes have been devised that will release their contents on

Photoaffinity reagent (f) GYP - Diazirine

0-Cl+-CH-CH2-NH-C

I

CH3 I -CH2-NH

Becomes covalently attached to padrenergic receptors upon phoiolysis

I CH3 NC

Hoocc\n2 N-CH2 /

HOOCCH2

The cis form of the molecule binds Zn2+ but the transform does not.

On photolysis the o-nitrobenzyl protecting group detaches and CAMP is released.

Photoisomeriwble lipid

The trans form will form lipid vesicles but the cis form will not.

“s(cH,),~

o~N~N-~OfCH2~~-;~CHa~~

Fig. 1. Pholaactiva~able dNgs

lmM,

M- et al. WW

Patent 4359i37

SCiem 214, 71F-72;t Morad, M.

et al. (1993) Nature 304,635-639;

Voorhees, J. J. and Wieranga, W. (1990) US

TIPS - April 2987 [Vol. 81

141

irradiation (Ref. 22, Fig. li). It would also be a relatively simple matter to attach drugs to protein carriers through linkers that are cleaved by light. Drugs released from carriers would be particularly useful for achieving ‘two-fold specificity’ (see below). The pharmacology of these reagents has not yet been evaluated.

psoriasis with psoralen and WA. The molecular basis of at least the latter can be envisioned; the genetic material of the proliferating cells is so badly damaged that they can no longer replicate. In the treatment for vitiligo, melanocytes appear to be stimulated in the. areas of depigmentation. A more recent development has been the treatment of the leukemic phase of cutaneous T-cell lymphoma (CTCL) with psoralen and UVA23. After oral administration of &methoxypsoralen, a patient’s blood is fractionated by leuko-

Clinical examples TreatmetitofCTCLwithpsoralen As mentioned earlier, two of the earliest photochemotherapies were the treatment of vitiligo and

phoresis and the white cells are passed through a plastic cassette in which they are irradiated as a thin (1 mm) layer. In a recent clinical trial more than 70% of the patients responded to this therapy. In more than 20% of the cases a very high reduction in skin infiltration was observed far exceeding the response for classical therapies. These results suggest that the treatment is doing more than simply destroying circulating malignant T lymphocytes. It is possible that the reinfusion of dying cells is inducing an immuno-

(b) Dihematoporphyrin ether

(a) Acrldlneorange

A photosensitizer that binds to DNA.

(c) PyreneconteiningHpld CH3(CH2)~&OOCH2 I 2 c o -! -OCH&H&(CH3)3 I

Proposed structure of the active constituent 0: hematoporphyrin derivative.

0’ For forming phototoxic liposomes

Moleculesthat damage DNA (d) g - Methoxypsoralen

(e) BleomycinA,

CLgCXo OCH3

The metal free form

TIPS - April 1987 [Vol. 81

142

Phototoxic liposomes-directed at selected cells twofold specificity in drug delivery by using liiht to activate a drug in a defined region and a monodonal antibodv to direct the drug to a subset of cells within that region. We chose human T lymphocyka as a convenient but clinically relevant prepamtion on which to test the idea’ . Liposomes were constructed containing pyrene which had previously been used to render low-density lipoprotein pattides phototoxic. The pyrene was covalentiy attached to the lipid bilayer (Tig. 1~). A monodonal antibody BE3 was attached to the surfaces of these liposomes, also c~vakndy. This antibody binds to approximately9O%oftheTceIlsin human peripheral blood lymphocytes and rematns fully active after attachment to limes. Liposomes are a vematik method for delivering dru$; they can carry both hydrophilic motecuks (trapped in the intravesicular space) and. as here, hydrophobic molecules (attached to the lipid bilayer). In both cases, a large number of drug molecules can be introduced into a cell per attached antibody. The iiposomes probably enter the cell by vtor-mediated endocytosis. Lymphoq+es were incubated with theliposomesandthenirradiatedwith W (3204UU nm). The integrity of the

wehave examined

cells

was

then

assayed

in

Antibody covalently attached to lipid head group

Liposome is internalized by endocystosis and ends up in a

attached antibody molecules. Each liposome contains

( ‘4) is evolved resulting in eventual destruction of the

three

complementary ways: their viability was monitoted over three days, the uptake of radioactive thymidine into DNA was measured after stimulation with the mitogen PI-LAphytuhemagglutinin and finally the ratio of T cells to B cells in the remaining cells was recorded. The results indicated that the majority of the T cells hadbeen killed while the B ceJls had been spared. This was confirmed in studies using transformed T- and B-cdllines. Controlexperimentsshowedthatthedamage was light-dependent and that the effect could not be mediated bv an irrelevant antibodv. While the intention of these &periments was to demonstrate the feasibility of using two-fold specificity in drug delivery the method as presented may have practical applications, for example in the depletion of subsets of T lymphocytes in various immunologic

logic response in which unaffected malignant cells are attacked and destroyedr5=.

Trials with hematoporphyrin derivative (PWD) for cancer chemotherapy are far advanced, particuktr!y for lung, bladder and skin tumors7. Patients ingest HF’D several days before tumors are irradiated with light of 630 run,

diseases induding malignancies, autoimmune diseases and immune deficiencies. It is not dear whether Bposomes, even small ones, will cross the microvasculature in sufficient numbers to be useful in treating solid tumors. Therefore we and others (see the text) dre experimenting with smaller carriers. DIL,,,, ReINellLC3 1 Yemul, S., Berger, C., Estabrook,A., Edelson,R. andBayley,H. (1985) Ann. N.Y. Acad. Sci. 446,403-414 2 Yemul, S., Berger,C., Estabrook,A.,Suarez,S.,Edelson,R. and Bayley,H. (1987) Proc. NatZ Acnd. Sci. USA 84,245-250 3 Ostm, M. J. (1987) Sci. Atn. Ian. 256,102-111

usually administered from a laser source via fiber optics. When large tumors are encountered the termini of the fibers are placed within the tumor. Over 2000 patients have been treated with an overall response rate of 70%: 80% for early stage disease. The active component of HPD (acetylated and base treated hematoporphyrin) is probably a porphyrin dimer, dihematoporphyrin ether (Fig. lb). HF’D is taken up by

most tissues but selectively retained in tumors. The drug may not act primarily on malignant cells; tumor tissue appears to be destroyed through lack of oxygen after rapid damage to the local microvasculature. A special advantage of HPD is that small tmnors containing the drug can be visualized by their red fluorescence in blue light. However, there are some problems. For example, the drug-is retained by

UPS - April 1987 [Vol. 81 the patient for several weeks during which exposure to sunlight must be avoided, and the drug is cc;lcentratdd in the normal liver, spleen, and kidney making it difficult to treat tumors in the vicinity of these organs. Two-fold specificity in drug delivery If a photoactivatable drug could be directed towards a subset of cells within a target area, extraordinary selective toxicity might be achieved. Such an approach would be valuable in several instances. For example, in cancer therapy micrometastases in organs accessible to fiber optics (such as the lung or bladder) might be eliminated without damage to surrounding tissue, or in modifying the immune system a selected class of lymphocytes might be removed from the blood by extracorporeal irradiation. Preliminary investigations of two-fold specificity have been undertaken by several groups. Mosley and colleagues first demonstrated that cells bearing the low-density lipoprotein receptor could be killed by a modified lipoprotein in which the neutral lipids had been extracted and replaced by a cholesteryl ester containing the photosensitizing pyrenyl group**. In an interesting variation on the theme, they also showed that a photoprotective azo dye could be introduced into cells by the same means=. Conceivably, such an agent could be used to protect normal cells from photosensitizers that are taken up systemically. A mom general method is needed if this approach is to be applied to a variety of tumors and to the modification of the immune system. Antibodies, hormones and lectins are generally useful targeting agents. Antibodies can, in principle, be used to deliver drugs to any class of cells. Monoclonal antibody technology or the use of synthetic peptides as antigens can provide highly specific antibodies in abundance. Levy and coworkers have used hematoporphyrin linked to antibodies to destroy tumor cells bearing myosarcoma or leukemia associated antigens in vitro26s27. They also reported preliminary experiments in which the survival time of mice injected subcutane-

143 ously with myosarcoma cells was increased after subsequent injection with a hematoporphyrinantibody conjugate and whole body irradiation with visible lighp6. A similar approach has been taken by Oseroff and colleagues who treated normal and lqukemic T lymphocytes in vitro2*‘L’. They first linked a porphyrin to a dextran carrier, and then linked the carrier to the antibody to increase the rati; of drug to targeting agent. In preliminary experiments, we have shown that aminomethyl4,5’,8-trimethylpsoralen covalently attached to insulin is phototoxic towards T lymphocytes that have been stimulated to proliferate in vitro30. It had earlier been shown that stimulated T lymphocytes express insulin receptors that are rapidly internalized upon binding ligand. We have also used phototoxic liposomes coupled to monoclonal antibodies to kill T lymphocytes. By using liposomes as carriers numerous drug molecules may be transferred into a cell for each antibody molecule that binds to the cell surface (see Box). Prospects In conclusion, the investigation of photoactivatable drugs is an active area of research from which several useful therapies have already been derived. More will certainly be forthcoming if important design criteria are followed including, activation at the longest possible wavelengths (to prevent tissue damage and give the deepest light penetration), rapid clearance (to avoid problems with subsequent exposure to sunlight), the production of shortlived active species (so that they do not diffuse from the sites at which they are generated) and, if carriers are used, the use of the smallest possible (for better access to the tissues). The application of these drugs will be aided by computer guided irradiation eqlpment. References 1 El Mofty, A. M. (1948) I. Egypt. Med. Assoc. 31.651-665 2 Spikes, J. D. and Straight, R. (1967) Avvv. Rev. Pkvs. Ckem. 18.409-436 3 Tappenier, H. von and Jesionek, D. R. (1903) Munch. Med. Wocksckr. 50, 2042-2844 4 Lemer, A. B., Denton, C. R. and ~ikpakick, T. 8. (1953) Dennnfol. 20, 299-314

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Parrish. 1. baum, L. N. Engl. /. 6 Gilchrest,

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Radiufiov Okcofogy (Peters, L. J., ed.),

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