WO2007149951A2

WO2007149951A2 - Systems and methods for use of luminescent compounds in disease treatment and medical imaging

Info

Publication number: WO2007149951A2
Application number: PCT/US2007/071717
Authority: WO
Inventors: Howard Bell; Joseph Friedberg; Victor Belov; Josh Collins
Original assignee: Sunstone Inc.
Priority date: 2006-06-20
Filing date: 2007-06-21
Publication date: 2007-12-27
Also published as: WO2007149951A3

Abstract

A system for treating a living being is disclosed including at least one photosensitizing agent for application to diseased tissue that is responsive to visible or infra-red light stimulation to generate species toxic to the diseased tissue; a luminescent compound that is excitable by tissue-penetrating radiation to produce visible or infra-red light to stimulate the photosensitizing agent to generate the toxic species; and a non-invasive or minimally invasive source of the tissue-penetrating radiation to excite the luminescent compound.

Description

SYSTEMS AND METHODS FOR USE OF LUMINESCENT COMPOUNDS IN DISEASE TREATMENT AND MEDICAL IMAGING

CROSS-REFERENCE TO RELATED APPLICATIONS [0001] The present application claims priority benefit under 35 U.S.C. §119(e) of U.S. Provisional Application Nos. 60/776,016 filed June 20, 2006 and 60/805,437 filed June 21, 2006, the disclosures of both of which are incorporated by reference.

FIELD OF INVENTION [0002] This invention relates to the medical use of rare earth element compounds that emit visible or infra red light when excited by infra red light or x-rays.

BACKGROUND OF THE INVENTION [0003] Photodynamic Therapy (PDT) is an approved method to treat a number of different human cancers. Successful treatment with PDT depends on the use of 1) light excitation, 2) a sensitizer dye, and 3) presence of molecular oxygen. Usually the treatment is accomplished by first administering photosensitizer dye (hematoporphyrin derivative, HPD) to the patient and after a period of time has elapsed — usually 48-72 hours — the tumor is treated with a light source — usually a laser light. Studies have shown that HPD is cleared more slowly from malignant tissue. Furthermore, HPD has the added advantage of having an absorbance peak above wavelength above 600 nm (Tissue readily absorbs light of an energy less than 600 nm). Light energy of wavelength above 600 nm provides the advantage of greater tissue penetration and hence greater probability to effectively treat malignant cells that are deeper than a few millimeters.

[0004] After having administered the HPD, light administration is delayed so that the cytocidal effects of PDT will not result in normal tissue toxicity. The source of light is usually a laser because more efficient use of the energy can be made to selectively excite the photosensitizer dye. Once the photosensitizer dye absorbs energy and is excited to the excited singlet state, there is a systems crossover such that an excited triplet state of the photosensitizer is achieved. The triplet photosensitizer can then relax back to ground state by exchanging spin energy with triplet state molecular oxygen. The result is that ground state triplet oxygen is excited to the reactive excited singlet state. Singlet oxygen is the final common path by which PDT results in tumor kill. [0005] A serious limitation of PDT is the problem associated with tissue penetration. Although advances have been made in the use of PDT for treatment of cancer, the treatment modality will be substantially improved when and if light of much greater wavelength can be used, i.e. infrared excitation. [0006] In an ovarian cancer model, a one-time treatment with PDT resulted in an approximately 15% cure rate, whereas treatment daily for four days resulted in an approximately 90% cure rate. Unfortunately, in patients undergoing PDT for treatment of intrathoracic and intraperitoneal cancer, major surgical procedures for four straight days would pose an insurmountable stress to the patient. Therefore, if a material could be used that would provide the opportunity to treat the patients at least 4 succes sive days , then the entire modality of PDT for treatment of intraactivity cancers may be advanced considerably

[0007] Compounds are known that emit visible light when irradiated with infra-red light. Suitable phosphor host materials include: sodium yttrium fluoride (NaYF₄ ), lanthanum fluoride (LaF₃), lanthanum oxysulfide, yttrium oxysulfide, yttrium fluoride (YF₃), yttrium gallate, yttrium aluminum garnet, gadolinium fluoride (GdF₃ ), barium yttrium fluoride (BaYFs, BaY₂Fg), and gadolinium oxysulfide. Suitable activator couples are selected from: ytterbium/erbium, ytterbium/thulium, and ytterbium/holmium. Other activator couples suitable for up-conversion may be used. By combination of these host materials with the activator couples, at least three phosphors with at least three different emission spectra (red, green, and blue visible light) are provided. Generally, the absorber is ytterbium and the emitting center can be selected from: erbium, holmium, terbium, and thulium; however, other up-converting phosphors of the invention may contain other absorbers and/or emitters.

BRIEF SUMMARY OF THE INVENTION [0008] A system for treating a living being is disclosed including: at least one photosensitizing agent for application to diseased tissue that is responsive to visible or infra-red light stimulation to generate species toxic to the diseased tissue; a luminescent compound that is excitable by tissue-penetrating radiation to produce visible or infra-red light to stimulate the photosensitizing agent to produce the toxic species; and a non-invasive or minimally invasive source of tissue- penetrating radiation for exciting the luminescent compound. [0009] The tissue-penetrating radiation can be infra red or x-ray radiation. High energy radiation such as x-ray radiation is capable of exciting luminescent compounds anywhere within a living being from an external source. Infra red radiation may or may not be applied from an external source, depending upon the location of the tissue to be treated. When the infra red radiation cannot be applied from an external source, a minimally invasive means of delivery is used to deliver the infra red radiation within excitation proximity of the luminescent compound.

[0010] The species that is toxic to the diseased tissue is typically singlet oxygen. The singlet oxygen is preferably generated from molecular oxygen in the diseased tissue to which the photosensitizing agent is applied. [0011] A method for treating a living using the system of the present invention is also disclosed. At least one photosensitizing agent is introduced into the body of the living being in proximity to diseased tissue, wherein the photosensitizing agent generates species toxic to the diseased tissue in response to stimulation by visible or infra red light. A luminescent compound is introduced into the body of the living being, wherein the luminescent compound is excitable by tissue-penetrating radiation to produce visible or infra-red light and the luminescent compound is in sufficient proximity to the photosensitizing agent to stimulate the production of the toxic species. Tissue-penetrating radiation for exciting the luminescent compound is then applied thereto from a source that is within minimally invasive or non-invasive excitation proximity of the luminescent compound.

[0012] The present invention also provides systems and methods for enhancing the diagnostic utility of x-ray radiation. Accordingly, a diagnostic system for imaging an internal portion of the body of a living being is also disclosed. The system includes a luminescent compound that is introduced into the body for residence at a location to be imaged. The luminescent compound is excitable by x-rays from outside the body to produce visible or infra-red light. X-rays are transmitted into the body to excite the luminescent compound and an imaging device sensitive to visible or infra-red light is used to locate the portion of the body where the luminescent compound is resident by the infra-red or visible light produced by the luminescent compound.

[0013] A method for high resolution tissue imaging is also disclosed. The method includes the steps of labeling a tissue to be imaged with a luminescent compound, where the compound is excitable by x-rays to produce visible or infra-red light. The luminescent compound is coupled to probes that bind specifically to biological markers on the tissue. Exciting the luminescent compound with x-rays causes it to emit infra-red or visible light and the infra-red or visible light is converted to a visible image.

BRIEF DESCRIPTION OF SEVERAL VIEWS OF THE DRAWINGS [0014] The invention will be described in conjunction with the following drawings in which like reference numerals designate like elements and wherein:

[0015] Fig. 1 a drawing of a human brain with a tumor to be treated according to an embodiment of the invention;

[0016] Fig. 2 is a block diagram of an exemplary process for treating a tumor with a photosensitive material illuminated by a luminescent material that is excitable with x-rays;

[0017] Fig. 3. is a block diagram of an exemplary process for imaging tissue with a probe- bound luminescent material;

[0018] Fig. 4 depicts the results of ex vivo PDT treatment of lung cancer cells using photofrin photosensitizer dye;

[0019] Fig.5 depicts a mouse into which 30 nm erbium and ytterbium doped gadolinium oxysulfide in PBS was injected into the pleural cavity intra-thoracically and externally illuminated with 810 nm infrared wavelengths (5 mW cm power);

[0020] Fig. 6 depicts the result of in vivo treatment of mesothelioma tumors in mice, wherein illumination with IR and nanophosphors resulted in minimal delay in growth while addition of the photosensitizer resulted in PDT induced killing; and

[0021] Fig.7 depicts the survival analysis of the mice treated in Fig. 6.

DETAILED DESCRIPTION OF THE INVENTION

[0022] PDT is a light based cancer treatment where a photosensitizing drug is activated with a visible light. PDT is executed by administering the photosensitizing drugs (which are non-toxic and inactive without light) intravenously and/or locally and illuminating them with visible light.

The light energy is captured by the photosensitizing drugs, which then transfer that energy to produce toxic species, typically by transferring that energy to molecular oxygen, generating excited quantum states of oxygen that effect PDT. It is an extremely effective cancer treatment and works by several mechanisms: direct cell kill, induced cell death (apoptosis), destruction of neovascularization supplying a tumor, induction local inflammation response to tumor, systemic immune response to tumor. [0023] The greatest limitation to employing PDT is light delivery, as the activating wavelengths of visible light only penetrate tissue for several millimeters. It is not currently possible, therefore, to treat a tumor with PDT if it is not accessible for having a light shined directly on it. Treatment of lung cancer, for instance, is essentially limited to treating endobronchial tumors that can be visualized through a bronchoscope. [0024] Luminescent materials that emit infra red or visible radiation upon x-ray stimulation are known as cathodoluminescent materials. Examples of cathodoluminescent materials suitable for use in the present invention are described in U.S. Patent Application Serial No. 11/494, 157, the disclosure of which is incorporated by reference. Specific examples of cathodoluminescent materials include Calcium Tungstate (CaWO₄), Gadolinium Oxysulphide (Gd₂O₂S), Yttrium Oxide:Terbium (Yt₂O₃ITb). Other examples include Gadolinium Oxysulphide: Europium (Gd₂O₂SiEu); Lanthanam Oxysulphide: Europium (La₂O₂S: Eu); and Gadolinium Oxysulphide: Promethium, Cerium, Fluorine.

[0025] These can be formed by a high temperature combustion synthesis technique, such as is disclosed by U.S. Provisional Patent Application Serial No. 60/721,917, the disclosure of which is incorporated herein by reference. All references cited herein are fully incorporated by reference. Other infra red excitable luminescent materials are disclosed in this application including Er₂O₃ and Y₂O₃. One type of luminescent material generally has the structure L₂O₃, wherein L represents one or more rare earth elements of the lanthanide series. Luminescent materials suitable for use in the present invention may contain a plurality of L₂O₃ compounds. Essentially any luminescent material that can be excited by wavelengths that are not absorbed by water or hemoglobin and emit wavelengths that excite a photosensitizer dye can be used. Other processes for forming exemplary luminescent materials include Sol-Gel Processes; Airogel Processes; Xerogel Processes; Co-precipitation; Solution Processes; Spray Pyrolysis Processes; Spray Flame Pyrolysis Processes; Chemical Vapor Synthesis Processes; Emulsion Liquid Membrane Methods; and Hydrothermal Processes.

[0026] The luminescent material is suspended in a pharmaceutically acceptable carrier separately or in combination with a photosensitizer dye. The luminescent material and dye are selected so that the luminescent material emits a visible wavelength that excites the photosensitizer dye.

[0027] In embodiments requiring injection, the luminescent material is preferably in the form of particles less than 150 nm in size to preclude capillary obstruction. The particles are more preferably less than 100 nm in size, even more preferably less than 50 nm in size and most preferably between about 5 and about 30 nm. The particles may be coated to form a stable suspension. Available coatings and their application to particles are well known to those of ordinary skill in the art. In other delivery embodiments where the luminescent material is applied, for example by aerosolization or direct application to the chest cavity, larger particles can be used.

[0028] Compositions containing the luminescent material, and optional photosensitizer dye, may be presented in forms permitting administration by the most suitable route. The invention also relates to administering such compositions to a patient in need thereof. These compositions may be prepared according to the customary methods, using one or more pharmaceutically acceptable adjuvants or excipients. The adjuvants comprise, inter alia, diluents, sterile aqueous media and the various non-toxic organic solvents. The compositions may be presented in the form of solutions or suspensions than can be injected or administered to a treatment site following exposure of the treatment site via surgical means, for example.

[0029] The choice of vehicle and the luminescent material in the vehicle are generally determined in accordance with the solubility and chemical properties of the product, the particular mode of administration and the provisions to be observed in pharmaceutical practice. When aqueous suspensions are used they may contain emulsifying agents or agents which facilitate suspension. Diluents such as sucrose, ethanol, polyols such as polyethylene glycol, propylene glycol and glycerol, and chloroform or mixtures thereof may also be used.

[0030] For parenteral administration, emulsions, suspensions or solutions of the luminescent material in vegetable oil, for example sesame oil, groundnut oil or olive oil, or aqueous-organic solutions such as water and propylene glycol, injectable organic esters such as ethyl oleate, as well as sterile aqueous solutions of the luminescent material, are used. The injectable forms must be fluid to the extent that it can be easily syringed, and proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Dispersion can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils.

[0031] Sterile injectable solutions are prepared by incorporating the luminescent material with an optional amount of a photosensitizer dye in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above.

[0032] The coated luminescent particles may be coupled to a ligand for binding the particle to the tissue to be illuminated. Alternatively, the luminescent particles may be directly administered to the treatment site. Binding ligands are readily identified by the ordinarily skilled artisan.

[0033] Exemplary photosensitizer dyes for use in the present invention include:

Porfimer sodium

[0034] Porfimer sodium is a complex mixture of oligomeric ester and ethers of hematoporphyrin. The drug is intravenously administered at a dose of 0.5-2mg/kg, and light exposure is carried out 24-72 hours later. Light activation at the 630-nm peak is used clinically because of its deeper tissue penetration. The main disadvantage of porfimer sodium is prolonged cutaneous photosensitivity, which lasts for 4-6 weeks or even several months because of its relatively slow rate of clearance from the skin.

5 -Aminolevulinic acid (ALA)

[0035] ALA is the first intermediate in the biosynthetic pathway of heme. When given in excess, ALA drives heme synthesis until intracellular iron is depleted; this process leads to the accumulation of a heme precursor protoporphyrin IX (PpIX) in the cells. PpIX is a moderately potent photosensitizer with an optimum absorption peak at 635 nm. Because it is relatively short-lived, any photosensitivity resolves within 24 hours after treatment. Moreover, because of its low molecular weight, ALA can be topically applied to the skin lesions, eliminating generalized photosensitivity. PpIX can be detected 4 hours after topical application of ALA. [0036] The standard procedure of topical ALA-PDT for skin lesions involves the application of 5-20% ALA in an oil-in- water emulsion with or without occlusion. Depending on the diagnosis, the lesions are exposed to either blue light or light at 630-635 nm 3-6 hours after application.

Benzoporphyrin derivative monoacid ring A (BPD-MA)

[0037] BPD-MA is a chlorin synthesized from protoporphyrin. It is a hydrophobic molecule with an absorption peak at 690 nm. This activating wavelength provides deeper tissue penetration compared with applications at 630-635 nm as used with ALA. Peak tissue levels are reached 3 hours after injection, with 50-60% tissue clearing in 48 hours. Cutaneous photosensitivity usually lasts less than 1 week. Trials with BPD-MA for age-related macular degeneration, skin cancer, and psoriasis are ongoing.

Tin ethyl etiopurpurin (SnET2)

[0038] SnET2 is a synthetic chlorophyll analogue with an absorption peak at 660 nm. The drug dose is usually 1-1.6 mg/kg, and light treatment is given 24-72 hours after injection. SnET2 has been successfully used for the treatment of basal cell carcinoma (BCC), Bowen disease (BD), Kaposi sarcoma, and cutaneous metastases.

[0039] Other suitable compounds include 5,10,15,20-tetrakis(m-hydroxyphenyl) chlorin (Foscan®); 2-devinyl-2-(l'-hexyloxy)ethyl-pyropheophorbidea (HPPH); Lutetium Texaphyrin (Lutex) and Benzoporphyrin Derivative (BPD).

[0040] Treatment methods are essentially conventional and are similar to existing photodynamic therapy methods, but for the introduction of infra red light and x-ray excitable luminescent materials . A combination of either infra red or x-ray radiation and visible light may be administered to excite the photosensitizer dye by directly exposing the dye to visible light and stimulating the luminescent material to emit additional visible light to further excite the dye. In the alternative, the infra red or x-ray radiation may be applied without co-administration of visible light. The light is applied for a length of time effective to kill tumor cells. In order to activate the PDT material, the peak absorption frequency of the PDT material must substantially overlap with the emission frequency of the luminescent compound.

[0041] X-ray radiation may be applied from an external source in all circumstances. The use of a solely external infra red radiation source will depend upon the location of the diseased tissue. When the diseased tissue is in a location too deep for externally applied infra red radiation to penetrate through overlying tissue, the infra red radiation is delivered to the diseased tissue by minimally invasive means.

[0042] For purposes of the present invention, a minimally invasive means of delivery is defined as means for the delivery of infra red radiation through a body cavity opening or incision that is too small for the infra red radiation to be delivered directly from the source therethrough in quantities sufficient to stimulate the luminescent material to produce sufficient photons for the generation of an effective amount of species toxic to the diseased tissue by the photosensitizing agent. For example, infra red radiation may be delivered by means of a fiber optic diffuser (Pioneer Optics Co., Bloomfield, CT).

[0043] Delivery of infra red light by means of a fiber optic diffuser to a luminescent material to generate visible wavelength photons for stimulation of a photosensitizing agent is still more efficient than using the fiber optic diffuser to deliver stimulation wavelengths directly to the photosensitizing agent. Visible wavelengths do not travel as efficiently through fiber optic diffusers and are absorbed by tissues between the end of the optical fiber and the photosensitizing agent. When infra red radiation is used, it is not necessary to directly contact the photosensitizing agent with the optical fiber.

[0044] . Fig. 4 depicts a mouse into which 30 Nm erbium and ytterbium doped gadolinium oxysulfide in PBSwas injected into the plural cavity intra-thoracically and externally illuminated with 810 nm infrared wavelengths (5 mW cm power). Fig. 2 shows visible wavelengths emitted by the erbium and ytterbium doped gadolinium oxysulfide and visible from the exterior of the mouse, demonstrating that it is possible to excite a luminescent material with infra red radiation from a non-invasive external source.

[0045] Exemplary diseased tissue treatment areas include localized cancers such as mesothelioma, malignant pleural effusions, malignant ascites, ovarian cancers, breast cancers, etc. These tumors can be treated by local application of infra red or x-ray stimulated phosphors

(slurry or poudrage) and using infra red light or x-rays to activate the phosphor to generate light for PDT. Depending on synergy with radiation, strategies will be devised to yield the optimal effect of the two modalities. In addition, phosphors can be delivered endoscopically to such areas as: the lung, the pancreas, the liver, the bladder, the kidneys, the spinal canal, the brain and the intestines. Tumors that concentrate gadolinium

[0046] Gadolinium is one of the luminescent materials suitable for use with the present invention. Gadolinium is used as an MRI contrast agent because it tends to accumulate in brain tumors and other malignancies. In an embodiment, luminescent phosphors can be intravenously introduced to a patients with a gadolinium avid tumor, like brain cancers, thereby "delivering the light source" to the target tumor. With reference to Figure 1, a brain 10 is shown with a tumor 20. A gadolinium-based compound 30 is shown collecting at the tumor 20. With reference to Figure2, an exemplary process is shown for treating the tumor 30. At step 200, a gadolinium- based luminescent compound is introduced into the patient. The compound, which has an affinity for certain types of tissue, including brain tumors is allowed to collect at the tumor site at step 210. At step 220, an MRI image is made to determine whether the gadolinium-based compound has collected at the tumor site. Once collection of the compound at the tumor site is confirmed, a photosensitive compound is introduced into the tumor area a step 230. At step 240, x-rays are applied to excite the luminescent compound which produces light to activate the photosensitive compound, which will destroy the tumor tissue.

[0047] Exemplary embodiments of non-cancer applications include treatment of closed space infections and emphysema.

Closed space infections:

[0048] It is known to kill a pathogenic fungus with PDT. With the increase in antibiotic resistant organisms and an increase in the population of immuno-suppressed patients, these types of infections are on the rise. In an exemplary application, a catheter can be used to aspirate the abscess and then deliver the sensitizer and a suspension of the phosphors into the cavity to be activated for PDT. The area can then be radiated with infra red light or x-rays to activate the PDT.

Emphysema:

[0049] There are millions of patients with emphysema. The pathophysiology of emphysema is that there are "baggy" portions of the lung that trap air and take up space, but do not have enough blood flow to contribute to lung function. In fact, these areas of the lung compress the portions of the lung that do have blood flow and prevent them from getting aerated and, hence, further decrease lung function. The disease is presently treated by surgical removal of the "baggiest" portions of the lung to allow the better portions to re-expand - lung volume reduction surgery. In an exemplary embodiment, PDT can be delivered as an aerosolized photosensitizer and trapped in the baggy portions of the lung. Once the sensitizer was delivered, phosphors that produce visible light when irradiated with infra red light or x-rays can then also be aerosolozied in a similar manner. One or both components could also be delivered bronchoscopically. Subsequently, localized radiation can be applied to effect PDT in that part of the lung. The technique will cause scarring and contracture of the most emphysematous lung and effect "lung volume reduction surgery without surgery. This technique would be doubly selective in that one could select the part of the lung to perform PDT - no light, no PDT - and that the "lungs would select" the most diseased portion because that is where the air is normally trapped and that is the area where the aerosolized sensitizer and/or phosphors would be retained.

Imaging Applications

[0050] X-ray excitable luminescent compounds may also be used for medical diagnostic imaging. In one embodiment, luminescent compounds are employed that tend to collect in certain types of tissue can be used to seek out that tissue If these compounds are made to emit infra-red radiation when excited with x-rays, the patient can be irradiated with x-rays and the compound detected with infra red detection equipment. In further embodiments, x-ray excitable luminescent compounds can be coupled to probes that bind specifically to biological markers. U.S. Patent No. 5,698,397 discloses such probes in conjunction with down up-converting luminescent compounds that are typically excited with infra-red light. U.S. Patent No. 5,698,397 is incorporated by reference herein.

[0051] In general, preparation of inorganic phosphor particles and linkage to binding reagents is performed essentially as described Tanke U.S. Pat. No. 5,043,265. Alternatively, a water- insoluble polyfunctional polymer which exhibits glass and melt transition temperatures well above room temperature can be used to coat the up-converting phosphors in a nonaqueous medium. For example, such polymer functionalities include: carboxylic acids (e.g., 5% acrylic acid/95% methyl acrylate copolymer), amine (e.g., 5% aminoethyl acrylate/95% methyl acrylate copolymer) reducible sulfonates (e.g., 5% sulfonated polystyrene), and aldehydes (e.g., polysaccharide copolymers).

[0052] The phosphor particles are coated with water-insoluble polyfunctional polymers by coacervative encapsulation in non-aqueous media, washed, and transferred to a suitable aqueous buffer solution to conduct the heterobifunctional crosslinking to a protein (e.g., antibody) or polynucleotide probe molecule. An advantage of using water-insoluble polymers is that the polymer microcapsule will not migrate from the surface of the phosphor upon aging the encapsulated phosphors in an aqueous solution (i.e., improved reagent stability). Another advantage in using copolymers in which the encapsulating polymer is only partially functional- ized is that one can control the degree of functionalization, and thus the number of biological probe molecules which can be attached to a phosphor particle, on average. Since the solubility and coacervative encapsulation process will depend on the dominant nonfunctionalized component of the copolymer, the functionalized copolymer ratio can be varied over a wide range to generate a range of potential crosslinking sites per phosphor, without having to substantially change the encapsulation process.

[0053] A preferred functionalization method employs heterobifunctional crosslinkers that can be made to link the biological macromolecule probe to the insoluble phosphor particle in three steps: (1) bind the crosslinker to the polymer coating on the phosphor, (2) separate the unbound crosslinker from the coated phosphors, and (3) bind the biological macromolecule to the washed, linked polymer-coated phosphor. This method prevents undesirable crosslinking interactions between biological macromolecules and so reduces irreversible aggregation as described by Tanke et al. Examples of suitable heterobifunctional crosslinkers, polymer coating functionalities, and linkable biological macromolecules include, but are not limited to:

Coating Heterobifunctional Biological

Functionality Crosslinker Macromolecule

carboxylate N-hydroxysuccimide Proteins l-ethyl-3-(3-dimethyl- (e.g., Ab, avidin) aminopropyl)carbodiimide

(EDC) primary amine

N-5-azido-2-nitrobenzovl All having 1° ami] oxysuccimide (ANB-NOS) N-succinimidyl (4-iodoacetyl) aminobenzoate (SIAB)

thiol(reduced N-succinimidyl (4-iodoacetyl) Proteins sulfonate) aminobenzoate (SIAB) [0054] Detection and quantization of inorganic phosphor(s) is generally accomplished by: (1) illuminating a sample suspected of containing phosphors with an x-ray, and (2) detecting infrared or visible light at one or more emission wavelength band(s).

[0055] Detection and quantization of luminescence from excited phosphors can be accomplished by a variety of means. Various means of detecting emission(s) can be employed, including but not limited to: avalanche photodiodes, charge-coupled devices (CCD), CID devices, photographic film emulsions, photochemical reactions yielding detectable products, and visual observation (e.g., fluorescent light microscopy). Detection can employ time-gated and/or frequency- gated light collection for rejection of residual background noise.

[0056] Time-gated detection is generally desirable, as it provides a method for recording long- lived emission(s) after termination of illumination; thus, signal(s) attributable to phosphorescence or delayed fluorescence of a phosphor is recorded, while short-lived autofluoresence and scattered illumination light, if any, is rejected. Time-gated detection can be produced either by specified periodic mechanical blocking by a rotating blade (i.e., mechanical chopper) or through electronic means wherein prompt signals (i.e., occurring within about 0.1 to 0.3 microseconds of termination of illumination) are rejected (e.g., an electronic-controlled, solid-state optical shutter such as Pockel's or Kerr cells).

[0057] When the tissue to be imaged is a tumor, the phosphors of the invention are attached to one or more probe(s) that bind specifically to tumors tissue. The phosphors serve as contrast agents for tumor detection.

[0058] Imaging compositions may be prepared in which the phosphors of the invention with one or more probe(s) attached that bind specifically to biological markers in tissues are suspended in a tissue-compatible carrier. The composition may be administered systemically or locally to a patient for tissue-imaging purposes by means of a syringe or catheter. Other imaging or contrast agents may also be present. The tissue may be imaged in situ or a biopsy may be performed for external analysis. The composition may also be applied ex-vivo to a biopsy sample for imaging purposes.

[0059] When the tissue is tumor tissue, the composition may also be used to identify tissue to be removed during cancer surgery and confirm that the tumor was completely removed. That is, any tumor tissue remaining will have phosphors present from the composition that was first administered to image the tumor. The surgical site can be illuminated with infra red light or x- rays and any tumor tissue remaining will emit visible light from the phosphors present.

[0060] With reference to Figure 3, an exemplary method is shown for imaging and optionally treating tissue with a probe-bound luminescent compound. At step 300 a luminescent compound that is bound to a tissue- specific probe is introduced into a living being. At step 310, the compound is given time to collect at the tissue site for which the probe is intended. At step 320, x-rays are then externally applied to excite the luminescent compound, causing it to emit infra-red or visible light. At step 330, the tissue (or the sites within the tissue that have attracted the probe-bound luminescent compound) is imaged with an imaging device for detecting infra- red or visible light emitted by the compound. Optionally, if the tissue is to be treated with PDT, a photosensitizing compound can be introduced to the tissue site at step 340 and x-rays again applied at step 350, this time to excite the luminescent compound in order to activate the photosensitizing compound.

EXAMPLES

Example 1 - Ex vivo PDT Treatment of Lung Cancer Cells (H460) Using Photofrin

[0061] 30 Nm particles of erbium and ytterbium doped gadolinium oxysulfide (Sunstones) doped with erbium and ytterbium were suspended in cell growth media with Photofrin photosensitizer and illuminated with 810 nm infrared wavelength (5 mW cm² power). FIG. 4 depicts that IR wavelengths alone or in combination with erbium and ytterbium doped gadolinium oxysulfide or photosensitizer dye does not produce the dramatic reduction in metabolic activity as does the combination of IR wavelengths, erbium and ytterbium doped gadolinium oxysulfide and photsensitizer dye. This confirms that the excitation of the photosensitizer dye by the visible wavelengths emitted by the erbium and ytterbium doped gadolinium oxysulfide upon exposure to IR wavelengths effectively kills lung tumor cells.

Example 2 - In vivo Treatment of Mesothelioma Tumors in Mice Animals:

[0062] Six to twelve week old, female BALB/cJ (ABl tumor host) mice, obtained pathogen- free from Taconic Farms, were used for all of the experiments. All animal studies were performed following a protocol approved by the University of Pennsylvania Institutional Animal Care and Use Committee. Animals were kept in conventional conditions in micro-isolator cages in laminar flow unit under ambient light with full access to food and water during experiments.

Cell Line:

[0063] AB 12, murine malignant pleural mesothelioma (MPM) was utilized for tumor inoculation. Cells were maintained in complete DMEM/F12 culture media in a 37⁰C humidified 5% CO₂ incubator. AB 12 cell line was donated by Dr. Steven Albelda, from the Hospital of the University of Pennsylvania.

Treatment Protocol: [0064] Seventy balb/C mice were inoculated with 5x 10⁵ AB 12, malignant mesothelioma cells via subcutaneous flank injection. The subsequent tumors were measured using digital calipers three times per week until the tumors reached ~200-300mm³ at which time the mice were given their first treatment. Mice were divided into five groups. The groups were as follows: i. Control, it Infrared-Nanophosphor-Photosensitizer (IR-NP-PS), Hi. IR-NP, iv. IR-PS, v. IR alone, vi. NP alone, and vii. PS alone. Groups H, Hi, and vi were administered intra- tumoral injections with lOOul of 25mg/ml nanophosphor suspension in saline at 72 and 48h prior to only the first treatment. Groups H, iv, and vii were administered weekly intra-peritoneal (i.p.) injections of 10mg/kg Photofrin™, twenty-four hours before first treatment of each week.

[0065] For this procedure mice were anesthetized with i.p. ketamine and xylazine, 80mg/kg and 12mg/kg, respectively. A maximum dose of LOW of 980nm light (BW Tek) at the treatment surface was measured using an IR thermopile detector (Coherent, Inc) for a total dose of 28.3 J/cm². Pulsed light delivery method of 1 second ON, 2 seconds OFF for 1.5 hours was utilized to minimize damage of healthy tissue due to heat from the infrared light by allowing the tissue to dissipate the heat between delivery cycles. Treatments were administered bi-weekly until tumors completely regressed or showed no regression and reached tumor endpoint. Over the course of the entire treatment program tumors were measured three times weekly and mice were monitored for signs of morbidity for 60 days or until tumors reached endpoint of 500 mm³.

Results:

[0066] Significant external PDT-mediated tumor regression and eradication of a stably growing murine mesothelioma was obtained. The IR-NP-PS group exhibited an initial decrease in tumor size after the second treatment, day 4, while the control groups experienced a steady rate of tumor growth with very little or no delay. All control groups receiving IR light exhibited a minimal delay in tumor growth with no significant difference between the groups. By day 18 (Six treatments) no palpable tumor was present in 8 of 10 animals in the IR-NP-PS group. These results can be seen in Fig. 6. The two mice from the IR-NP-PS group that did not exhibit effective PDT induced tumor regression eventually grew to the mandatory 500mm³ endpoint and were euthanized. All remaining animals in each group were monitored for ninety days and tumor site pathologically examined for any signs of residual disease.

[0067] Upon completion of the ninety day observation period 80% of the IR-NP-PS treated animals survived, 8/10 of the animals exhibited complete tumor regression from an initial average tumor size of 239.21 mm³ (Fig. 7). The remaining two animals from the IR-NP-PS treated group exhibited very little PDT-mediated cytotoxicity and tumors continued to grow to endpoint. The total number of surviving animals from each group is as follows: Control - 1/10;

IR-NP-PS - 8/10; IR-NP - 0/9; IR-PS - 0/10; IR alone - 0/9; NP alone - 1/10; and PS alone - 1/10. Deaths related to anesthesia occurred in the IR-NP and IR alone treated groups, 1/10 mice for both groups.

[0068] The results demonstrate that up-converting nanophosphors can efficiently excite photosensitizers and provide an effective method of PDT delivery using infrared light.

[0069] While the invention has been described in detail and with reference to specific examples thereof, it will be apparent to one skilled in the art that various changes and modifications can be made therein without departing from the spirit and scope thereof.

Claims

CLAIMS WHAT IS CLAIMED IS:

1. A system for treating a living being comprising: at least one photosensitizing agent for application to diseased tissue that is responsive to visible or infra-red light stimulation to generate species toxic to said diseased tissue; a luminescent compound that is excitable by tissue-penetrating radiation to produce visible or infra-red light to stimulate said photosensitizing agent to generate said toxic species; and a non-invasive or minimally invasive source of said tissue-penetrating radiation to excite said luminescent compound.

2. The system of claim 1, wherein said photosensitizing agent generates singlet oxygen from molecular oxygen in said diseased tissue;

3. The system of claim 1 wherein said at least one photosensitizing agent is selected from the group consisting of : Porfimer sodium, 5 -Aminolevulinic acid (ALA); Benzoporphyrin derivative monoacid ring A (BPD-MA); Tin ethyl etiopurpurin (SnET2); 5, 10, 15,20-tetrakis(m- hydroxyphenyl) chlorin (Foscan®); 2-devinyl-2-(l'-hexyloxy)ethyl-pyropheophorbidea (HPPH); Lutetium Texaphyrin (Lutex) and Benzoporphyrin Derivative (BPD).

4. The system of claim 1 wherein said luminescent compound is selected from the group consisting of: Calcium Tungstate (CaWO₄); Gadolinium Oxysulphide (Gd₂O₂S); Yttrium

Oxide:Terbium (Yt₂θ₃:Tb); Lanthanam Oxysulphide: Terbium (La₂O₂S:Tb); Gadolinium Oxysulphide, Europium (Gd₂O₂S:Eu); Lanthanam Oxysulphide: Europium (La₂O₂S: Eu); and Gadolinium Oxysulphide: Promethium, Cerium, Fluorine (Gd₂O₂S :Pr,Ce,F).

5. The system of claim 1 wherein said luminescent compound had a particle size less than 100 nanometers.

6. The system of claim 1 wherein said luminescent compound and said photosensitizing agent are introduced in a single mixture.

7. The system of claim 1 wherein said luminescent compound and said photosensitizing agents are introduced into the lungs in an aerosolized form.

8. The system of claim 1 wherein said luminescent compound contains Gadolinium and is adapted to accumulate at the site of a brain tumor.

9. The system of claim 1 wherein said photosensitizing compound is adapted to destroy cancer cells.

10. The system of claim 1 wherein said photosensitizing compound is adapted to kill a pathogenic fungus.

11. The system of claim 1 wherein said photosensitizing compound is adapted to destroy emphysematic lung tissue.

12. The system of claim 1 wherein said luminescent compound is cathodo- luminescent and said tissue penetrating radiation is x-ray radiation.

13. The system of claim 1 wherein said tissue penetrating radiation source is a noninvasive source of infra red radiation.

14. The system of claim 13 wherein said infra red radiation is transmitted from said non-invasive source to said luminescent compound by a non-invasive delivery means.

15. The system of claim 14 wherein said non-invasive delivery means is a fiber optic diffuser.

16. A diagnostic system for imaging an internal portion of the body of a living being comprising: a luminescent compound arranged to be introduced into the body of the living being for residence at a desired location therein, said luminescent compound being excitable by x-rays from outside the body of the living being to produce visible or infra-red light; a source of x-rays for transmitting said x-rays into the body of the living being to excite said luminescent compound; and an imaging device arranged to locate the portion of the body of the living being where said luminescent compound is resident by the infra-red or visible light produced by said luminescent compound when excited by the received x-rays.

17. The system of claim 16 wherein said luminescent compound is selected from the group consisting of: Calcium Tungstate (CaWO₄), Gadolinium Oxysulphide (Gd₂O₂S), Yttrium Oxide: Terbium (Yt₂O₃ITb); Lanthanam Oxysulphide: Terbium (La₂O₂SiTb); Gadolinium Oxysulphide, Europium (Gd₂O₂SiEu); Lanthanam Oxysulphide: Europium (La₂O₂SiEu); and Gadolinium Oxysulphide: Promethium, Cerium, Fluorine (Gd₂O₂S :Pr,Ce,F).

18. The system of claim 16 wherein said luminescent compound is coupled to probes that bind specifically to biological markers on said internal portion of the body.

19. A method for high resolution tissue imaging comprising labeling a tissue to be imaged with a luminescent compound being excitable by x-rays to produce visible or infra-red light, wherein said luminescent compound is coupled to probes that bind specifically to biological markers on said tissue; exciting said luminescent compound with x-rays so that said luminescent compound emits infra-red or visible light; and converting the infra-red or visible light to a visible image.

20. A method for treating a living being comprising: introducing at least one photosensitizing agent into the body of the living being in proximity to diseased tissue, wherein said photosensitizing agent generates species toxic to said diseased tissue in response to stimulation by visible or infra-red light; introducing a luminescent compound into the body of the living being, said luminescent compound being excitable by tissue penetrating radiation to produce visible or infra-red light, and said luminescent compound being in sufficient proximity to said photosensitizing agent to stimulate the production of said toxic species; and transmitting tissue-penetrating radiation for exciting said luminescent compound to said luminescent compound from a source that is within minimally invasive or non-invasive excitation proximity of said luminescent compound.

21. The method of claim 20 wherein said photosensitizing agent generates singlet oxygen from molecular oxygen in said diseased tissue.

22. The method of claim 20 wherein said at least one photosensitizing agent is selected from the group consisting of: Porfimer sodium, 5-Aminolevulinic acid (ALA); Benzoporphyrin derivative monoacid ring A (BPD-MA); Tin ethyl etiopurpurin (SnET2); 5,10,15,20-tetrakis(m-hydroxyphenyl) chlorin (Foscan®); 2-devinyl-2-(l'-hexyloxy)ethyl- pyropheophorbidea (HPPH); Lutetium Texaphyrin (Lutex) and Benzoporphyrin Derivative (BPD).

23. The method of claim 20 wherein said at least one photosensitizing agent is selected from the group consisting of: Porfimer sodium, 5-Aminolevulinic acid (ALA); Benzoporphyrin derivative monoacid ring A (BPD-MA); Tin ethyl etiopurpurin (SnET2); 5,10,15,20-tetrakis(m-hydroxyphenyl) chlorin (Foscan®); 2-devinyl-2-(l'-hexyloxy)ethyl- pyropheophorbidea (HPPH); Lutetium Texaphyrin (Lutex) and Benzoporphyrin Derivative (BPD).

24. The method of claim 20 wherein said luminescent compound had a particle size less than 100 nanometers.

25. The method of claim 20 wherein said luminescent compound and said photosensitizing agent are introduced in a single mixture.

26. The method of claim 20 wherein said luminescent compound and said photosensitizing agents are introduced into the lungs in an aerosolized form.

27. The method of claim 20 wherein said luminescent compound contains Gadolinium and is adapted to accumulate at the site of a brain tumor.

28. The method of claim 20 wherein said photosensitizing compound is adapted to destroy cancer cells.

29. The method of claim 20 wherein said photosensitizing compound is adapted to kill a pathogenic fungus.

30. The method of claim 20 wherein said photosensitizing compound is adapted to destroy emphysematic lung tissue.

31. The method of claim 20 wherein said luminescent compound is cathodo- luminescent and said tissue penetrating radiation is x-ray radiation.

32. The method of claim 20 wherein said tissue penetrating radiation source is a noninvasive source of infra red radiation.

33. The method of claim 32 wherein said infra red radiation is transmitted from said non-invasive source to said luminescent compound by a non-invasive delivery means.

34. The method of claim 33 wherein said non-invasive delivery means is a fiber optic diffuser.

35. A method for imaging an internal portion of the body of a living being comprising: introducing a luminescent compound into the body of the living being for residence at a desired location therein, said luminescent compound being excitable by x-rays from outside the body of the living being to produce visible or infra-red light; transmitting x-rays into the body of the living being to excite said luminescent compound; and directing at said internal portion of the body an imaging device arranged to locate the portion of the body of the living being where said luminescent compound is resident by the infra-red or visible light produced by said luminescent compound when excited by the received x-rays.