Methods for high energy phototherapeutics

Methods of treating and imaging diseased tissue having the steps of administering a radiosensitizer agent proximate to or into the diseased tissue; and treating the diseased tissue with x-rays or other ionizing radiation so as to activate the radiosensitizer agent in the diseased tissue. Preferably, the radiosensitizer agent is a halogenated xanthene.

BACKGROUND OF THE INVENTION
 The present invention is directed to high energy phototherapeutic agents,
 or specifically to radiosensitizing and methods of treating and imaging
 using such phototherapeutic or radiosensitizer agents. More specifically,
 the treating and imaging is of diseased tissue, such as tumors,
 particularly cancerous tumors.
 Diseased tissue or tumors, such as those for cancer, are often treated
 using ionizing radiation, in a process known as radiation therapy.
 Radiation therapy (which typically uses electromagnetic radiation with
 energies of 1 keV or higher) for cancer typically works by attacking
 rapidly growing cells with highly penetrating ionizing radiation. Use of
 such radiation is attractive due to its ability to penetrate deeply into
 tissue, especially when diseased tissue is, or is located within, bone or
 other dense or opaque structures. Unfortunately, using rapid growth as the
 sole targeting criterion does not limit the effects of such treatment to
 cancer cells.
 As a result, improvements have been made in the methods for delivery of the
 ionizing radiation to the site of the cancerous tumor so as to limit the
 effects of such radiation to the general area of the cancerous tumor.
 However, since healthy tissue and cancerous tissue typically have a
 similar biological response to radiation, a need exists to improve the
 potency of (or biological response to) the delivered radiation within and
 in the vicinity of the tumor, while not affecting the surrounding healthy
 tissue.
 As an alternative to the use of ionizing radiation, photodynamic therapy
 (PDI) has been developed and shows considerable promise for treatment of a
 variety of cancers. Photodynamic therapy is the combination of a
 photosensitive agent with site-specific illumination (using non-ionizing,
 optical radiation) to produce a therapeutic response in diseased tissue,
 such as a tumor. In PDT, a preferential concentration of photosensitizer
 is to be located in the diseased tissue, either through natural processes
 or via localized application, and not in the healthy surrounding tissue.
 This provides an additional level of tissue specificity relative to that
 achievable through standard radiation therapy since PDT is effective only
 when a photosensitizer is present in tissue. As a result, damage to
 surrounding, healthy tissue can be avoided by controlling the distribution
 of agent. Unfortunately, when using conventional methods for the
 illumination step in PDT (1) the light required for such treatment is
 unable to penetrate deeply into tissue, and (2) the physician has minimal
 spatial control of the treatment site. This is particularly troublesome
 whenever the diseased tissue or tumor is deeply seated or located within
 bone or other opaque structures. Some of the inventors of the present
 invention have been able to resolve many of these problems for PDT, as
 shown in commonly-assigned U.S. Pat. No. 5,829,448.
 Others, however, have focused their efforts on developing agents that are
 sensitized or activated by the ionizing radiation mentioned above.
 Potentially, the use of such radiation would enable treatment of more
 deeply seated diseased tissue than that possible with optical radiation.
 The agents used with such radiation are known as radiosensitizers. It is
 also desirable to achieve preferential concentration of the
 radiosensitizer in the diseased tissue, either through natural processes
 or via localized application, so as to provide additional specificity
 relative to that achievable through standard radiation therapy. The
 desired result is for radiation to become more efficacious when the
 radiosensitizer is present in tissue, so that less radiation is needed to
 treat the lesion tumor or other diseased tissue, and accordingly,
 potential damage to surrounding healthy tissue, resulting from collateral
 exposure to the radiation, is reduced. Hence, safety and efficacy would
 then be improved.
 The ultimate success or failure of the radiosensitizer approach depends on:
 (1) therapeutic performance of agents, and (2) disease specificity in the
 site of activation. Currently used agents and targeting approaches,
 however, have had unacceptable results in each of these categories.
 The therapeutic performance of a radiosensitizer is primarily a function of
 enhanced absorption of the applied radiation dose in sensitized tissues
 relative to that in non-sensitized tissues. This differential absorption
 is commonly effected by use of agents having a high absorption
 cross-section for a particular type of radiation (such as x-rays). For
 example, metal or halogen atoms are often used, either in atomic form or
 incorporated into a molecular carrier, due to their high x-ray
 cross-section. Absorption of x-rays by such atoms appears to lead to
 secondary radiative emissions, ionization, and other chemical or physical
 processes that increase the localized cytotoxicity of the applied energy
 (i.e., radiation-induced cell death, or "light cytotoxicity").
 However, a high light cytotoxicity is not enough to make an agent an
 acceptable agent. The agents must also have a negligible effect when
 energy is not applied (i.e., have a low toxicity in the absence of
 radiation, or "dark cytotoxicity"). Unfortunately, many agents presently
 under investigation as radiosensitizers have the disadvantage of either:
 (a) a relatively high dark cytotoxicity or (b) a low light
 (cytotoxicity)-to-dark cytotoxicity ratio which limits their effectiveness
 and acceptability. Agents having a high light-to-dark cytotoxicity ratio
 are desirable because they (1) can be safely used over a range of dosages,
 (2) will exhibit improved efficacy at the treatment site (due to the
 compatibility with use at higher dosages as a consequence of their
 relative safety), and (3) will be better tolerated throughout the
 patient's body.
 An additional problem with many current radiosensitizers is that the agent
 does not achieve significant preferential concentration in tumors.
 Specifically, most radiosensitizer targeting has been based on physical
 targeting, such as diffusion into tumors through leaky neurovasculature,
 which ultimately succeed or fail based on permeability of the tumor to
 agents that are aqueously soluble or are in a suspension formulation. As a
 result, large doses of the agent typically need to be administered, either
 locally or systemically, so as to saturate all tissues, hopefully reaching
 a therapeutic level in the desired treatment region or target. After such
 agent administration, a patient has to wait a clearance time of hours to
 days to allow excess agent to hopefully clear from healthy living tissues
 surrounding the desired treatment site. Thereafter, irradiation of
 residual agent at the treatment site hopefully produces the desired
 cytotoxic effect in the diseased tissue. This approach may unfortunately
 also damage healthy surrounding tissue by undesired but unavoidable
 activation of residual agent still present in the healthy surrounding
 tissue. One approach to solving this problem is to couple the
 radiosensitizer with a moiety capable of providing improved biotargetting
 of the diseased tissue. This, however, has proven to be very difficult to
 achieve.
 It would also be highly desirable if the radiosensitizer could be used to
 improve identification of target size, location and depth so that the
 therapeutic radiation could be more precisely delivered to the target,
 such as a cancerous tumor. Combined diagnostic use (as a contrast agent)
 and therapeutic use (as a radiosensitizer) of the agent would reduce risk
 to the patient by (1) reducing the number of required procedures necessary
 for diagnosis and treatment, (2) reducing the overall diagnosis and
 treatment time, and (3) reducing cost.
 Accordingly, one object of the present invention is to develop new
 radiosensitizers that have one or more of the following characteristics:
 (1) improved light-to-dark cytotoxicity ratio; (2) improved accumulation
 of agent into diseased tissue with strong contrast between diseased and
 healthy tissue; (3) rapid clearance from normal tissue; and (4) capability
 of combined imaging and therapy. Further desirable characteristics include
 low agent cost, and significant regulatory history (so as to facilitate
 acceptance by the regulatory and medical communities).
 SUMMARY OF THE INVENTION
 The present invention is directed to a radiosensitizer agent for treatment
 of diseased tissue using radiosensitization or ionizing radiation
 comprising a halogenated xanthene. Preferably, the halogenated xanthene is
 Rose Bengal or its derivative.
 In a further embodiment of the present invention, the radiosensitizer agent
 also acts as an imaging contrast agent.
 The present invention is also directed to a radiosensitizer agent for
 treatment of diseased tissue using radiosensitization or ionizing
 radiation wherein the agent exhibits a preference for concentration in
 biologically sensitive structures in tissue, such as, for example,
 cellular membranes. Preferably, the agent biologically or chemically
 targets the biologically sensitive structures.
 Further, the present invention is directed to a method for treating
 diseased tissue.
 One embodiment of the method of the present invention includes the steps of
 administering a radiosensitizer agent, preferably a halogenated xanthene,
 a portion of radiosensitizer agent being retained in diseased tissue; and
 treating the diseased tissue with x-rays or other ionizing radiation to
 activate the radiosensitizer agent in the diseased tissue.
 A further embodiment of the method of the present invention includes the
 step of imaging a patient using the radiosensitizer agent to identify the
 diseased tissue.

DETAILED DESCRIPTION OF THE PRESENTLY PREFERRED EMBODIMENTS
 The present invention is directed to agents that can efficiently interact
 with x-rays or other types of ionizing radiation to produce a beneficial
 biological response and to methods of treatment and imaging using such
 agents.
 The inventors of the present invention have discovered that radio dense
 agents, such as the halogenated xanthenes discussed infra, which exhibit a
 preference for concentration in cellular membranes and other key
 components and structures of diseased tissue, will exhibit additional
 therapeutic dose enhancement over that possible with previously known
 agents or enhancement mechanisms. This additional dose enhancement is a
 consequence of increased radiosensitization yield of such agents owing to
 improved proximity of such agents, upon interaction with diseased tissue,
 to sensitive structures during irradiation and subsequent
 radiosensitization. Specifically, most radiosensitizers function by
 absorbing highly-penetrating energy (which in itself has little direct
 interaction with tissue), and then releasing this energy in a
 less-penetrating, more cytotoxic form (such as lower-energy re-emission)
 that is capable of interacting primarily only with proximal,
 biologically-sensitive structures or materials (such as cellular membranes
 and genetic material).
 Thus, any radiodense agent, such as halogenated xanthenes, that exhibits
 chemical or biological targeting to such biologically-sensitive structures
 or materials, and which thereby becomes substantially concentrated in
 areas in physical proximity to such structures or materials, will increase
 the overall efficiency of radiosensitization (i.e. conversion of
 high-energy stimulating excitation into localized cytotoxic effects). This
 yield enhancement results from the increased probability that
 proximally-released energy will interact favorably with the sensitive
 target (before annihilating or otherwise dissipating in an inefficacious
 manner) whenever the agent responsible for such re-emission is
 concentrated as close as possible to such a target. Stated in simple
 terms, the released energy, having a short mean free path, will have a
 higher probability of interacting with the target if it is emitted from an
 agent located closer to the target.
 Such approaches to radiosensitization enhancement are not taught in the
 prior art, which are based primarily on permeability-based targeting. In
 contrast, targeting as taught by the present invention uses the superior
 approach based on chemical or biological targeting. This type of targeting
 can be effected by chemical partitioning of the agent at, near or into the
 target (for example, using an agent that partitions into cell walls, such
 as Rose Bengal discussed infra, the chemical structure of which is
 illustrated in FIG. 1a), by controlled agent delivery at, near or into the
 target (for example by encapsulation of an agent, such as Rose Bengal,
 into a delivery vehicle, such as a micelle, nanoparticle, or liposome,
 that interacts preferentially with a target site, such as cell walls, and
 may adhere, fuse, combine, or otherwise interact in such a way that agent
 is delivered to the target), or by physically increasing local
 concentration of agent at, near or into the target, for example by
 localized delivery via injection, flooding, or spraying.
 Preferably, these agents have a large x-ray cross-section, a high
 light-to-dark cytotoxicity ratio, a preference for accumulation in
 diseased tissue, low agent cost, rapid clearance from normal tissue, and a
 significant regulatory history (so as to facilitate acceptance by the
 regulatory and medical communities).
 Applicants have discovered a class of agents that fits this criteria and is
 preferably used in the present invention. These agents are referred to as
 halogenated xanthenes and are illustrated in FIG. 1b, where the symbols X,
 Y, and Z represent various elements present at the designated positions,
 and the symbols R.sup.1 and R.sup.2 represent various functionalities
 present at the designated positions. Chemical and physical properties
 (such as the chemical constituents at positions X, Y, and Z and the
 functionalities R.sup.1 and R.sup.2, along with molecular weight) of
 representative halogenated xanthenes are summarized in attached Table 1.
 While many of the halogenated xanthenes are highly soluble in aqueous
 solution, in general all demonstrate a preference for selective
 partitioning into hydrophobic environments, such as within cell membranes.
 In general, halogenated xanthenes are characterized by a low dark
 cytotoxicity and chemical properties that are substantially unaffected by
 the local chemical environment or the attachment of functional derivatives
 at positions R.sup.1 and R.sup.2. Moreover, the halogenated xanthenes will
 target some tumors or other diseased tissues based on their inherent
 selective partitioning properties.
 A specific example of a halogenated xanthene is Rose Bengal
 (4,5,6,7-tetrachloro-2',4',5',7'-tetraiodofluorescein; see 10 in FIG. 1a).
 In particular, Rose Bengal has been found to accumulate preferentially in
 (i.e. target) some tumors and other diseased tissues. Moreover, Rose
 Bengal has other desirable characteristics such as a negligible dark
 cytotoxicity, relatively low cost, the ability to clear rapidly from the
 body, and a partially established regulatory history. Furthermore, the
 inventors have found that the special chemical properties of Rose Bengal
 allow it to be dissolved in aqueous solution at high concentrations while
 retaining a significant preference for hydrophobic environments, such as
 within cell membranes.
 The present inventors have also discovered that the facility with which the
 halogenated xanthenes target specific tissues or other sites can be
 optimized by attachment of specific functional derivatives at positions
 R.sup.1 and R.sup.2, so as to change the chemical partitioning or
 biological activity of the agent. For example, attachment of one targeting
 moiety or more at positions R.sup.1 or R.sup.2 can be used to improve
 targeting to specific tissues, such as cancerous tumor tissues or sites of
 localized infection. These targeting moieties include DNA, RNA, amino
 acids, proteins, antibodies, ligands, haptens, carbohydrate receptors or
 complexing agents, lipid receptors or complexing agents, protein receptors
 or complexing agents, chelators, encapsulating vehicles, short- or
 long-chain aliphatic or aromatic hydrocarbons, including those containing
 aldehydes, ketones, alcohols, esters, amides, amines, nitriles, azides, or
 other hydrophilic or hydrophobic moieties.
 An example of this feature would be to combine Rose Bengal with a lipid (at
 position R.sup.1, via esterification, so as to increase the lipophilicity
 of Rose Bengal, and thereby modify its targeting properties in a patient.
 Such a modified agent could be administered directly as a micelle
 suspension, or delivered in conjunction with a delivery vehicle, such as a
 surfactant, and would exhibit increased targeting to tumor cells. Suitable
 formulations of such an agent include topical creams and lotions, and
 liquids for intravenous or parenteral injection.
 FIG. 4 demonstrates that strong absorption for the halogens of the
 halogenated xanthenes occurs well below the energies used for standard
 diagnostic or therapeutic x-ray devices, which generally use energies
 greater than 30 keV. In fact, the halogen content of the halogenated
 xanthenes makes this class of agent potent x-ray absorbers, and thus
 highly suitable as radiosensitizers. Further, since x-ray cross-section
 increases substantially in the order F&lt;Cl&lt;Br&lt;I, it is preferred
 that those halogenated xanthenes with a large content of I or Br be used
 for x-ray sensitization. Furthermore, tests indicate that the presence of
 I or Br yields enhanced sensitization relative to that possible with other
 halogens. Therefore, as shown in Table 1, Tetrabromoerythrosin, Rose
 Bengal, Phloxine B, Erythrosin B, and Eosin Y have larger x-ray
 cross-sections than Solvent Red or Eosin B as a consequence of respective
 differences in halogen content, and thereby are preferred for use as x-ray
 sensitizing agents. More preferably, the high iodine content of Rose
 Bengal and its derivatives and the additional bromine substitution of
 4,5,6,7-Tetrabromoerythrosin and its derivatives, makes these agents the
 most preferred x-ray sensitizing agents of this class.
 Accordingly, in a preferred embodiment of the present invention, at least
 one halogenated xanthene is used as an x-ray sensitizer or radiosensitizer
 agent for treatment of diseased tissue using radiosensitization. Prior to
 radiosensitization, the agent can be administered orally, systemically
 (e.g. by an injection), or topically, in a manner well known in the art.
 In a further preferred embodiment of the present invention, Rose Bengal or
 its derivatives or 4,5,6,7-Tetrabromoerythrosin or its derivatives is the
 radiosensitizer agent. It is also preferred that x-rays or other ionizing
 radiation with energy .gtoreq.approximately 1 keV and .ltoreq.1000 MeV be
 used to activate the agent. Preferably, the agent is activated using
 x-rays having an energy in excess of 30 keV.
 Applicants have also discovered that halogenated xanthenes can be used as
 an imaging contrast agent for x-ray or other ionizing radiation imaging,
 such as CAT scan, fluorography or other related procedures. In particular,
 the inventors have discovered that halogenated xanthenes are particularly
 proficient as imaging contrast agents because of their large x-ray
 cross-sections and because their chemical structure, which has a high
 electron density due to their significant halogen content, renders them
 opaque to x-rays or other ionizing radiation used for imaging. For
 example, Rose Bengal is highly opaque to the x-rays used for CAT scan or
 normal x-ray imaging. FIGS. 2 and 3 illustrate the opaqueness of Rose
 Bengal versus standard x-ray contrast agents and a control. These figures
 are drawings of actual pictures of experiments done by the inventors of
 the present invention. For example, the CAT scan image of test tubes
 containing various solutions shown in FIG. 2 demonstrates that iodine (350
 mgI/mL in aqueous base), Rose Bengal (225 mg halogen/mL in saline), and
 Omnipaque.TM. (350 mgI/mL Iohexol) have similar x-ray densities.
 Furthermore, these densities are dramatically greater than that of a
 control (saline). A CAT scan image of various dilutions of these same
 solutions (held in wells in a 96-well sample plate) illustrated in the
 drawing in FIG. 3 further demonstrates that Rose Bengal shows comparable
 response to that of the standard x-ray contrast agents across a range of
 concentrations.
 Accordingly, it is a further preferred embodiment of the present invention
 to use at least one halogenated xanthene agent as an imaging contrast
 agent for x-ray or ionization radiation based imaging and detection of
 diseased tissue, and then treat the detected diseased tissue by
 radiosensitization of the residual agent present in such tissue.
 This description has been offered for illustrative purposes only and is not
 intended to limit the invention of this application, which is defined in
 the claims below. For example, it will be clear to those of ordinary skill
 in the art that the targeting described herein for the specific example of
 the halogenated xanthenes can be adapted or otherwise applied to other
 radiodense materials, including conventional radiosensitizers.
 What is claimed as new and desired to be protected by letters patent is set
 forth in the appended claims:
 TABLE I
 Physical Properties of Example Halogenated Xanthenes:
 Substitution
 Compound X Y Z R.sup.1 R.sup.2 MW (g)
 Fluorescein H H H Na Na 376
 4',5'-Dichlorofluorescein Cl H H Na Na 445
 2',7'-Dichlorofluorescein H Cl H Na Na 445
 4,5,6,7-Tetrachlorofluorescein H H Cl H H 470
 2',4',5',7'- Cl Cl H Na Na 514
 Tetrachlorofluorescein
 Dibromofluorescein Br H H Na Na 534
 Solvent Red 72 H Br H H H 490
 Diiodofluorescein I H H Na Na 628
 Eosin B NO.sub.2 Br H Na Na 624
 Eosin Y Br Br H Na Na 692
 Ethyl Eosin Br Br H C.sub.2 H.sub.5 K 714
 Erythrosin B I I H Na Na 880
 Phloxine B Br Br Cl Na Na 830
 Rose Bengal I I Cl Na Na 1018
 4,5,6,7-Tetrabromoerythrosin I I Br Na Na 1195