HK1097465A - Conjugates for photodynamic therapy - Google Patents

Description

Conjugates for photodynamic therapy

RELATED APPLICATIONS

According to 35U.S.C. § 119(e), the benefit of priority of U.S. provisional patent application No. 60/506,378 entitled "target-binding activated singlet oxygen photosensitizers that enhance selectivity of targeted PDT agents" filed by Pallenberg et al on 23/9/2003 is claimed. The above-referenced applications are incorporated by reference in their entirety into the present application.

Technical Field

Provided herein are compositions and methods of making compositions for enhancing the effects of fluorescence detection or photodynamic therapy for the purpose of detecting or destroying tumors, hyperproliferative tissue or other undesirable biological structures.

Background

Photodynamic therapy ("PDT") is a treatment that destroys tumors and hyperproliferative tissue. PDT is based on the accumulation of photosensitizers in malignant and hyperproliferative tissues after administration of the photosensitizers. Subsequent irradiation with light of an appropriate wavelength produces a photochemical reaction, a so-called photodynamic effect (e.g. a photochemical reaction producing singlet oxygen), which leads to tumor destruction.

Photodynamic therapy is effective in destroying abnormal tissues such as cancer cells. In this therapy, a photoreactive material having a characteristic light absorption band is first administered to the patient, usually orally or by injection. It is known that abnormal or hyperproliferative tissues in the body selectively take up certain photoreactive substances to a much greater extent than normal tissues, for example, tumors of the pancreas and colon can take up 2 to 3 times the amount of these substances compared to normal tissues.

Photosensitizers, such as porphyrins and related tetrapyrrole compounds, tend to concentrate in abnormal tissues, including malignant tumors and other hyperproliferative tissues, such as hyperproliferative blood vessels, at much higher concentrations than normal tissues, so they are useful as tools for the treatment of different types of cancer and other hyperproliferative tissues by photodynamic therapy (PDT) (t.j.dougherty, c.j.gomer, b.w.henderson, g.jori, d.kessel, m.korberik, j.moan, q.peng, j.natl.cancer inst.90: 889(1998), which is incorporated herein by reference). Most porphyrin-based photosensitizers that have been approved for the treatment of tumors and hyperproliferative tissues are slowly cleared from normal tissues, so in order to minimize the unwanted activity of the photosensitizer in non-target tissues, the patient must avoid prolonged exposure to sunlight after receiving treatment. Although photodynamic therapy is effective, there are also adverse side effects caused, for example, by the required dose and inadvertent activation in non-target tissues. Thus, there is a need to improve targeting and delivery of such therapies. Therefore, among the objects in the present application, it is an object to provide methods and compositions for targeted and delivered photodynamic therapy.

SUMMARY

Provided herein are methods and conjugates for photodynamic therapy targeting and delivery, and for imaging. The conjugates are targeted and designed such that they are inactive until they interact with a target, such as a target tissue or cell. The conjugates are used in methods of photodynamic therapy and imaging, and any method in which targeted delivery of a photo-generating agent is used. Also provided are methods in which the conjugates are used or administered, such as probes in microscopy, enzymology, clinical chemistry, molecular biology, and medicine, as well as other such applications. The conjugates are also useful as therapeutics (modalities) such as therapeutics in photodynamic therapy, and as diagnostic agents in imaging methods such as fluorescence immunoassays, in vivo fluorescence imaging, and magnetic resonance imaging.

The conjugates provided herein include a donor moiety (e.g., a fluorophore or a photosensitizer), an acceptor moiety (e.g., a quencher), and a targeting moiety. The conjugates comprise a donor, such as a fluorophore, a photosensitizer, and other such substances, linked to a targeting moiety and an acceptor moiety, such as a quencher, in such a manner that activation of the donor, such as the fluorophore or the photosensitizer, is quenched unless and until the targeting moiety binds to the target. When bound to the target, an acceptor moiety, such as a quencher, dissociates or moves away from the donor species, such as a photosensitizer, whereby the donor is activated or active. For example, for conjugates containing a photosensitizer, binding to the target results in activation of the photosensitizer when irradiated with light of a suitable wavelength.

Also provided are conjugates comprising a photosensitizer and a quencher, wherein the photosensitizer and quencher comprise a linking component linked to an amino or hydroxy fatty acid or sulfonic acid using an ester, amide, or sulfonamide linkage.

Also provided are conjugates comprising a photosensitizer and a quencher, wherein the photosensitizer and quencher comprise an oligonucleotide as a linking component, wherein the oligonucleotide comprises a specific sequence that binds to a desired target, and at least one pair of mutually complementary regions that cause it to adopt a conformation in which, in the absence of the target, the quencher is sufficiently close to the photosensitizer that the photosensitizer is inactive, and wherein binding of the target-specific sequence to the target disrupts said conformation, allowing the photosensitizer to become active when illuminated with light of an appropriate wavelength.

Also provided are conjugates comprising a photosensitizer and a quencher, wherein the photosensitizer comprises a porphyrin or porphyrin-derived tetrapyrrole and has a physiologically acceptable metal atom in its central coordination cavity, and one or more suitable functional groups are located on or near the quencher, which is effectively coordinated to the axial position of the metal coordinated inside the photosensitizer; and the targeting moiety is positioned in such a way that the presence of the target disrupts the association of the axial ligand with the metal, releasing the quencher and rendering the fluorophore or photosensitizer active.

Also provided are conjugates comprising a photosensitizer and a quencher linked to a targeting moiety, wherein the photosensitizer and the quencher are in an energy transfer conformation that allows interaction in such a manner that activation of the photosensitizer is quenched unless the targeting moiety and target are bound; and the targeting moiety is positioned such that when the targeting moiety binds to the target, the quencher is displaced by the photosensitizer from an energy transfer conformation that allows interaction, enabling activation of the photosensitizer upon illumination with light of a suitable wavelength.

Also provided are conjugates comprising a tetrapyrrole or tetrapyrrole derivative photosensitizer comprising a physiologically acceptable metal atom in its central coordination cavity; a quencher comprising one or more suitable functional groups that coordinate to an axial position of the metal coordinated within the photosensitizer and position the quencher in an energy transfer conformation with the photosensitizer such that activation of the photosensitizer is quenched; and a targeting moiety, wherein binding of the targeting moiety to the target disrupts the association of the axial ligand of the quencher with the metal, releasing the quencher and enabling activation of the photosensitizer upon illumination with light of a suitable wavelength.

Also provided are methods of detecting a target tissue or target component. Further provided herein are methods of photodynamic therapy using the conjugates provided herein. Also provided herein are methods of detecting hyperproliferative tissue using the conjugates provided herein.

Also provided is the use of the conjugates provided herein to treat target components or target tissues, including hyperproliferative tissues and neovascular tissues.

Also provided herein are methods of detecting the presence of hyperproliferative tissue in a subject. Also provided are methods of diagnosing a hyperproliferative disorder in a patient. Further provided are methods of diagnosing an infectious agent (infecting agent) in a patient.

Also provided herein are methods of generating an image of a target tissue in a subject. Further provided are kits for treating hyperproliferative disorders. Also provided are kits for labeling specific tissues for diagnostic analysis. Further provided is a combination comprising any of the conjugates provided herein and a light source.

Brief description of the drawings

FIG. 1 is a schematic representation of a targeted photosensitizer and target binding reaction.

Figure 2 illustrates a photosensitizer associated with a linking agent.

FIG. 3 illustrates binding-activated photosensitizers.

Detailed Description

A. Definition of

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. All patents, patent applications, published applications and publications, Genbank sequences, databases, web sites, and other published materials cited throughout the disclosure of this application are incorporated by reference in their entirety unless otherwise noted. If there are multiple definitions of a term in this application, that definition will prevail in this section. When referring to a URL or other such identifier or address, it should be understood that such identifiers may vary and that the particular information on the internet may vary, but equivalent information may be found by retrieving the internet. Reference thereto demonstrates that such information is available and publicly available.

As used herein, the term "photodynamic therapy" refers to a method by which light of a particular wavelength is directed at tissue undergoing treatment or investigation that has been made photosensitive by the administration of a photoreactive or photosensitizer. The purpose may be diagnosis, where the wavelength of light is chosen such that the photoreactive material fluoresces, thus producing information about the tissue without damaging the tissue, or treatment, where the wavelength of light delivered to the target tissue under treatment causes the photoreactive material to undergo a photochemical interaction with oxygen within the tissue under treatment, which interaction produces a high energy species, such as singlet oxygen, that causes local tissue lysis or destruction. The van Lier method (Photobiologiceal Techniques 216: 85-98(Valenzo et al, 1991)) can be used to demonstrate the ability of any given component to efficiently produce singlet oxygen, thus making it a good candidate for use in photodynamic therapy.

As used herein, the term "photosensitizer" or "photosensitizing agent" means a chemical compound that is activated when exposed to photoactivating light, converting the photosensitizer itself or some other species into a cytotoxic form, thereby killing target cells or reducing their proliferative potential. Thus, photosensitizers may exert their effects directly or indirectly through a variety of mechanisms. For example, some photosensitizers become directly toxic when activated by light, whereas others act to generate toxic species, e.g., oxidants such as singlet oxygen or oxygen-derived free radicals, which are very destructive to cellular material and biomolecules such as lipids, proteins and nucleic acids. Psoralens are typically direct acting photosensitizers; when exposed to light, they form adducts and cross-links between the two strands of a DNA molecule, thereby inhibiting DNA synthesis. Porphyrins are typical photosensitizers that act indirectly by generating toxic oxygen species. In fact, any chemical compound that is converted to or produces a cytotoxic form when exposed to photoactivating light can be used in the present invention. Generally, the chemical compound is non-toxic to the animal to which it is administered or can be formulated into a non-toxic composition, and the chemical compound in its photodegraded form is also non-toxic. A list of representative light-sensitive chemicals can be found in Kreimer-bibbaurn, sem. 157-73, 1989.

Photosensitive compounds include, but are not limited to, chlorins, bacteriochlorins, phthalocyanines, porphyrins, purpurinimides (purpurinimides), pheophorbides, pyropheophytin, merocyanines, psoralens, benzoporphyrin derivatives (BPD), talaporfin sodium and porfimer sodium, and prodrugs such as delta-aminolevulinic acid, which can be used to prepare pharmaceuticals such as protoporphyrins. Other compounds include indocyanine green, methylene blue, toluidine blue, tesafolin (texaphyrin); and any other substance that absorbs light in the range of 400nm to 1200 nm.

As used herein, the term "tetrapyrrole" denotes a macrocyclic compound comprising four pyrrole rings, which has the following general structure:

wherein the dashed lines indicate that the indicated bonds may be saturated or unsaturated, and wherein any atom on the ring may be substituted or unsubstituted.

As used herein, the term "porphyrin" refers to a cyclic structure, which is typically composed of four pyrrole rings, and refers to a porphyrin or porphyrin derivative. Such derivatives include porphyrins with an additional ring ortho-or ortho-perifused (periferated) to the porphyrin core, porphyrins in which one or more carbon atoms in the porphyrin ring are replaced by atoms of another element (framework substitution), derivatives in which the nitrogen atom in the porphyrin ring is replaced by atoms of another element (framework substitution of nitrogen atoms), derivatives having substituents other than hydrogen at the periphery (meta-, □ -) or at the position of the nuclear atom of the porphyrin, derivatives in which one or more bonds of the porphyrin are saturated (hydrogenated porphyrins, such as chlorins, bacteriochlorins, isobacteriochlorins, coprins, pyrrocorphins, etc.), derivatives obtained by coordination of one or more metals to one or more porphyrin atoms (metalloporphyrins), derivatives in which one or more atoms include a pyrrole unit and a pyrrolymethyl unit inserted into the porphyrin ring (expanded porphyrins), Derivatives that remove one or more groups from the porphyrin ring (contracted porphyrins, e.g., corrins, corroles), and combinations of the above derivatives (e.g., phthalocyanines, porphyrazines, naphthalocyanines, subphthalocyanines, and porphyrin isomers).

As used herein, "chlorin" refers to a class of porphyrin derivatives having a cyclic structure generally composed of four pyrrole rings with one partially saturated pyrrole ring, such as the basic chromophore of chlorophyll.

As used herein, "bacteriochlorin" refers to a class of porphyrin derivatives having a cyclic structure generally consisting of four pyrrole rings with two partially saturated non-adjacent (i.e., trans) pyrrole rings, and "isobacteriochlorin" includes those porphyrin derivatives having a cyclic structure generally consisting of four pyrrole rings with two partially saturated adjacent (i.e., cis) pyrrole rings.

As used herein, "molecule" refers to any molecular entity and includes, but is not limited to, small organic molecules, biopolymers, biomolecules, macromolecules or components or precursors thereof, such as peptides, proteins, organic compounds, oligonucleotides, or monomeric units of peptides, organics, nucleic acids and other macromolecules. The monomer unit is one of the components constituting the resulting compound. Thus, monomeric units include nucleotides, amino acids, and pharmacophores for the synthesis of small organic molecules.

As used herein, a "biomolecule" is any molecule that occurs in nature, or a derivative thereof. Biomolecules include biopolymers and macromolecules, and all molecules that can be isolated from living organisms and viruses, including but not limited to cells, tissues, prions, animals, plants, viruses, bacteria, prions, and other organisms. Biomolecules also include, but are not limited to, oligonucleotides, oligonucleosides, proteins, peptides, amino acids, lipids, steroids, Peptide Nucleic Acids (PNAs), oligosaccharides and monosaccharides, organic molecules such as enzyme cofactors, metal complexes such as heme, iron sulfur clusters, porphyrins and metal complexes thereof, metals such as copper, molybdenum, zinc, and others.

As used herein, "macromolecule" refers to any molecule having a molecular weight from hundreds to millions. Macromolecules include, but are not limited to, peptides, proteins, nucleotides, nucleic acids, carbohydrates, and other such molecules that are generally biosynthesized by biology, but that can be made synthetically or using recombinant molecular biology methods.

As used herein, "biopolymer" refers to a biomolecule, including macromolecules, that consist of two or more monomeric subunits, or derivatives thereof, that are linked by a bond or a macromolecule. The biopolymer may be, for example, a polynucleotide, polypeptide, carbohydrate, or lipid, or a derivative or combination thereof, such as a peptide nucleic acid portion or glycoprotein comprising a nucleic acid molecule.

As used herein, "donor molecule" refers to a chemical or biological compound that is capable of donating or transferring energy from itself to other molecules. The energy transferred includes, but is not limited to, electrons, photons, or fluorescence resonance energy.

As used herein, an "acceptor molecule" refers to a chemical or biological compound that is capable of accepting or accepting energy from other molecules. The energy transferred may include, but is not limited to, electrons, photons, or fluorescence resonance energy. The acceptor molecule accepts energy from the donor molecule through an energy transfer mechanism, resulting in a significant reduction in the energy of the donor molecule. Energy transfer from the donor molecule to the acceptor molecule can occur by a variety of mechanisms, including but not limited to resonance dipole induced dipole interaction, electron transfer, or charge transfer. Energy transfer occurs only over very short distances (typically less than 200nm) and so the donor and acceptor molecules need to be so very close together.

As used herein, "Fluorescence Resonance Energy Transfer (FRET)" refers to non-radiative energy transfer between donor and acceptor molecules. Fluorescence Resonance Energy Transfer (FRET) is a process of technical identification (art-recognited) in which one fluorophore (acceptor) receives energy from an electronically excited second fluorophore (donor) via quantum mechanical coupling, which can be promoted to an excited electronic state. For FRET to occur efficiently, the absorption and emission spectra between the donor and acceptor must generally overlap. The donor/acceptor pairs are characterized by their spectral overlap properties. The emission spectrum of the donor generally must overlap with the absorption spectrum of the acceptor. The degree of overlap determines the efficiency of the energy transfer. The degree of overlap also determines the optimal distance between the donor and acceptor molecules. When the overlap of the spectra is large, the transfer is efficient, so it can occur over long distances.

As used in this application, "fluorescence" refers to the emission of light caused by absorption of radiation of a certain wavelength (excitation) followed by almost immediate re-radiation (emission), usually at a different wavelength, which stops almost immediately when the incident radiation stops. At the molecular level, fluorescence occurs when certain compounds, called fluorophores, are transitioned by light energy from the ground state to a higher excited state; when the molecules return to their ground state, they emit light, usually at different wavelengths (Lakowicz, J.R., "Principles of Fluorescence Spectroscopy," (Plenum Press, NY, (1983)); Herman, B., "Resonance energy transfer Microcopy," in Fluorescence Microcopy of Living Cells in culture, Part B, Methods in Cell Biology, Vol.30, (tylor, D.L. & Wang, Y. -L., eds., (Academic Press, San Diego (1989), p.219-243).

As used herein, "chromophores" refer to those groups that have good absorption characteristics, i.e., they are capable of excitation when irradiated with any of a variety of photon sources. The chromophore may be fluorescent or non-fluorescent. Non-fluorescing chromophores generally do not emit energy in the form of photon energy. Non-fluorescing chromophores can be characterized as having a low quantum yield, which is the ratio of emitted photon energy to absorbed photon energy, typically less than 0.01.

As used herein, "fluorophore" refers to a compound that fluoresces, such as a fluorescing chromophore. Fluorescence is a physical process in which light is emitted from a compound after absorption of radiation. Generally, the emitted light has lower energy and longer wavelength than the absorbed light. A fluorophore is a molecule or moiety that fluoresces and/or is capable of generating a fluorescent signal. In particular, fluorophores are capable of absorbing energy, such as photons, and re-emitting the energy. Fluorophores typically emit photon energy at moderate to high quantum yields of 0.01 to 1. Sometimes, the energy of the fluorophore is re-emitted as radiation, which usually has a longer wavelength than the radiation absorbed by the fluorophore (i.e., fluorescence), and sometimes there is a time delay in re-emitting the energy of the fluorophore (i.e., fluorescence). Sometimes, the energy of the fluorophore can be transferred to other molecules by non-radiative processes (i.e., FRET). As used herein, "excited" refers to absorption of radiation by a molecule, resulting in an increase in the energy of the molecule and a transition to a higher energy state.

As used herein, "emission" refers to the emission of photon energy from a molecule, resulting in a reduction in the energy of the molecule and a transition to a lower energy state.

As used herein, "energy transfer" refers to the transfer of energy between molecules such that a molecule that emits energy transitions to a lower energy state while a second molecule that absorbs the energy emitted by the first molecule transitions to a higher energy state.

As used herein, "quenching group" or "quencher" refers to any fluorescence-modulating group of the present invention that can at least partially attenuate light emitted by a fluorescent group. As used herein, "quenching" refers to any process that causes a reduction in the quantum yield of a given fluorescence process. Thus, illumination of the fluorescent group in the presence of the quenching group produces an emission signal that is not as intense or even completely absent as desired. Quenching typically occurs by energy transfer between the fluorescent group and the quenching group. The quenching group has the capacity to accept the energy transfer of the donor molecule, but does not emit significantly. The quenching group includes an acceptor molecule that is shaped to draw an energy potential away from the excited acceptor such that the acceptor does not emit.

As used herein, "EDANS" refers to the fluorophore 5- ((2-aminoethyl) -amino) naphthalene-1-sulfonic acid.

As used herein, "DABCYL" refers to the acceptor chromophore 4- (4' -dimethylaminophenylazo) benzoic acid. As used herein, "DABSYL" refers to the acceptor chromophore 4- (4' -dimethylamino-phenylazo) sulfonic acid.

As used herein, "energy transfer" refers to the process by which the fluorescence emission of a fluorophore is altered by a fluorescence-modifying group. If the fluorescence-modifying group is a quencher, the fluorescence emission of the fluorescent group is attenuated or eliminated. The energy transfer may occur by fluorescence resonance energy transfer or by direct energy transfer. The precise energy transfer mechanism is different in these two cases. It should be understood that any reference to energy transfer in this application includes all such mechanistically different phenomena.

As used herein, an "energy transfer pair" refers to any two molecules involved in energy transfer. Typically, one molecule acts as a fluorophore and the other molecule acts as a fluorescence-modulating group. In one embodiment, the energy transfer pair comprises a fluorophore and a quencher group. In another embodiment, the energy transfer pair comprises a photosensitizer and a quenching group. In the present application, there is no limitation on the identity of the individual members of the energy delivery pair. All that is required is that the spectral properties of the energy transfer pair as a whole change in some measurable way if the distance between the individual members is changed by some critical amount.

As used herein, "fluorescence-modifying group" refers to a molecule that can alter the emission of fluorescence from a fluorescent group in any manner. The fluorescence-modifying group typically accomplishes this via an energy transfer mechanism. Depending on the nature of the fluorescence-modifying group, the fluorescent emission may undergo a number of changes, including but not limited to attenuation, complete quenching, enhancement, change in wavelength, change in polarity, and change in fluorescence lifetime. One example of a fluorescence-modifying group is a quenching group. If the fluorescence-modifying group is a quenching group, the quenching group will not generally emit a substantial portion of the absorbed light as light, but will generally consume it as heat.

As used herein, "coordination cavity" or "coordination pocket" refers to the spatial arrangement of chelated metal complexes formed by the interaction of metal-binding ligands. For example, in a porphyrin system, the coordination cavity is a "hole" in the macrocycle, the size of which is generally defined as the distance from the center to the midpoint of the four nitrogen atoms.

As used herein, "treatment" refers to any means of ameliorating or otherwise beneficially altering one or more symptoms of a disease or disorder. Treatment also includes any pharmaceutical use of the conjugates of the present application, such as the use to treat a hyperproliferative tissue or neovascularization mediated disease or disorder, or to treat a disease or disorder in which hyperproliferative tissue or neovascularization is implicated.

As used herein, "amelioration of symptoms" of a particular disorder by administration of a particular compound or pharmaceutical composition refers to any alleviation, whether permanent or temporary, permanent or transient, that may be attributed to or associated with the administration of the composition. As used herein, "antibodies and antibody fragments" generally refer to immunoglobulins or fragments thereof that specifically bind to an antigen to form an immune complex. The antibody may be a whole immunoglobulin of any class, e.g., IgG, IgM, IgA, IgD, IgE, a chimeric antibody or a hybrid antibody having two or more antigenic or epitope specificities. It may be a polyclonal antibody, such as an affinity purified antibody derived from a human or suitable animal, e.g., from a primate, goat, rabbit, mouse, etc. Monoclonal antibodies are also suitable for use in the present invention and are useful because of their high specificity. They are readily prepared by what is now considered conventional as follows: immunization of mammals with immunogenic antigen preparations, fusion of immune lymphocytes or splenocytes with an immortal myeloma cell line and isolation of specific hybridoma clones. More unconventional methods of making monoclonal antibodies, such as interspecies fusions and genetic engineering manipulations of hypervariable regions, are not excluded because it is primarily the antigen specificity of the antibodies that affects their utility. Newer techniques for making monoclonals may also be used, such as human monoclonal, interspecies monoclonal, chimeric (e.g., human/mouse) monoclonal, genetically engineered antibodies, and the like.

As used herein, "tumor" means a neoplasm, and includes both benign and malignant tumors. The term specifically includes malignancies, which may be solid (such as breast, liver or prostate cancer) or non-solid (such as leukemia). Tumors can be further divided into subtypes, such as adenocarcinomas (e.g., breast adenocarcinoma, prostate adenocarcinoma, or lung adenocarcinoma).

As used herein, "target" means an object that is intended to be detected, diagnosed, damaged or destroyed by the methods of the present application, including target cells, target tissues and target components. As used herein, "target tissue" and "target cells" are those tissues that are intended to be damaged or destroyed by the present treatment method. Binding of the photosensitizing compound to these target tissues or target cells; these tissues or cells are then damaged or destroyed when irradiation suitable for activating the photosensitizer is applied. The target cells are cells in a target tissue, including, but not limited to, vascular endothelial tissue, abnormal vessel walls of tumors, solid tumors such as, but not limited to, tumors of the head and neck, tumors of the eye, tumors of the gastrointestinal tract, tumors of the liver, tumors of the breast, tumors of the prostate, tumors of the lung, non-solid tumors, and malignant cells of hematopoietic and lymphoid tissues, neovascular tissue, other lesions in the vascular system, bone marrow, and tissues or cells associated with autoimmune diseases. Also included in the target cells are cells that undergo substantially more rapid division compared to non-target cells.

As used herein, a "non-target tissue" is all tissues of a subject that are not intended to be damaged or destroyed by the treatment method. These non-target tissues include, but are not limited to, healthy blood cells and other normal tissues not considered to be targeted.

As used herein, "Target compositions" are those compositions intended to be damaged or destroyed by the treatment methods of the present invention and may include one or more pathogenic agents including, but not limited to, bacteria, viruses, fungi, protozoa and toxins, and cells and tissues infected or infiltrated therewith. The term "target component" also includes, but is not limited to, infectious organic particles such as toxins, peptides, polymers, and other compounds that can be selectively and specifically identified as organic targets that are intended to be damaged or destroyed.

As used herein, "hyperproliferative tissue" refers to tissue that grows uncontrolled and includes neoplastic tissue, tumors, and unconstrained vascular growth, such as that found in age-related macular degeneration and which often occurs after glaucoma surgery.

As used herein, "hyperproliferative disorders" refer to those disorders sharing as an underlying pathology excessive cell proliferation caused by unregulated or abnormal cell growth, and include uncontrolled angiogenesis. Examples of such hyperproliferative disorders include, but are not limited to, cancer or carcinoma, tumors, acute and membranoproliferative glomerulonephritis, myeloma, psoriasis, atherosclerosis, psoriatic arthritis, rheumatoid arthritis, diabetic retinopathy, macular degeneration, corneal neovascularization, choroidal hemangioma, recurrence of pterygium, and scarring from excimer laser surgery and glaucoma filtration surgery.

As used herein, "amino acid" refers to a natural or unnatural amino acid. The amino acids include, but are not limited to, 4-aminobutyric acid, 6-amino-hexanoic acid, alanine, asparagine, aspartic acid, arginine, 3-cyclohexylalanine, citrulline, cysteine, 2, 4-diaminobutyric acid, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, naphthylalanine, norleucine, ornithine, phenylalanine, 4-halo-phenylalanine, phenylglycine, proline, 3- (2-pyridyl) -alanine, serine, thienylalanine, threonine, tryptophan, tyrosine, and valine.

As used herein, "therapeutically effective dose" and "therapeutically effective amount" are doses sufficient to arrest the progression of a disease or cause regression of a disease, or to alleviate symptoms caused by the disease.

"agent" or "drug" refers to a chemical compound or composition that, when properly administered to a subject, is capable of producing a desired therapeutic or prophylactic effect.

Where relevant, chemical compounds include any of the (+) and (-) enantiomers, as well as racemic mixtures.

As used herein, "irradiating" and "irradiation" includes exposing a subject to light of all wavelengths. The illumination wavelength is selected to include the wavelength of light that excites the photosensitizer. In some embodiments, the irradiation wavelength is selected to match the excitation wavelength of the photosensitizer and has low absorption by non-target tissues of the subject, including blood proteins.

The radiation of the present application is further defined by its coherence (laser) or non-coherence (non-laser) and by the intensity, duration and timing of administration of the photosensitizing compound. The intensity or flow rate must be sufficient to allow the light to reach the target tissue. The duration or total flux dose must be sufficient to photoactivate enough photosensitizing compound to act on the target tissue. Timing of administration with the photosensitizing compound is important because 1) the administered photosensitizing compound requires some time to be directed to the target tissue of interest and 2) the blood levels of many photosensitizing compounds decline over time. The radiant energy is provided by an energy source, such as a laser source or cold cathode light source, which is external to the subject, or which is implanted in the subject, or which is introduced into the subject, such as disclosed in U.S. Pat. No. 6,273,904(2001), for example, with a catheter, fiber optic, or by ingesting the light source in capsule or pellet form.

Although one embodiment of the present invention relates to the use of light energy for the administration of PDT to destroy tumors, one of ordinary skill in the art will appreciate that other forms of energy are also within the scope of the present invention. These forms of energy include, but are not limited to: thermal, acoustic, ultrasonic, chemical, electromagnetic radiation, mechanical, and electrical. For example, sonodynamic inducers or activators include, but are not limited to: gallium porphyrin complexes (see Yumita et al, Cancer Letters 112: 79-86(1997)), other porphyrin complexes, such as protoporphyrin and hematoporphyrin (see Umemura et al, Ultrasonics Sonochhemistry 3: S187-S191 (1996)); other cancer drugs such as daunorubicin and doxorubicin used in the presence of ultrasound therapy (see Yumita et al, Japan J. hyperthermic Oncology 3 (2): 175-182 (1987)).

As used herein, "destroy" or "destruction" refers to killing the desired target tissue or target component, including infectious agents. "injury" or "damage" refers to altering a target tissue or target component in such a way as to interfere with its function or reduce its growth. For example, it was observed by North et al that upon exposure of virus-infected T Cells treated with benzoporphyrin derivatives to light, pores form in the T cell membrane and increase in size until the membrane is completely broken down (Blood Cells 18: 12940 (1992)). Even if the target tissue or target composition is ultimately processed by macrophages, it is understood that the target tissue or target composition is damaged or destroyed.

The present invention provides a method of providing medical treatment to an animal, the term "animal" including but not limited to humans and other mammals.

The term "coupling agent" as used herein relates to a reagent capable of coupling a photosensitizer to a targeting agent.

The term "linking agent" or "linking component" as used herein relates to an agent that links a photosensitizer to a targeting agent. In some embodiments, a "linking component" may also serve as a targeting moiety.

As used herein, "targeting agent" or "targeting moiety" refers to a compound that targets or preferentially associates or binds to a particular tissue, receptor, infectious agent, or other area of the subject's body, such as a target tissue or target component. Examples of targeting agents include, but are not limited to, an oligonucleotide, an antigen, an antibody, a ligand, a receptor, one member of a specific binding pair, a polyamide comprising a peptide having affinity for a biological receptor, an oligosaccharide, a polysaccharide, a Low Density Lipoprotein (LDL) or APO-protein of LDL, a steroid or steroid derivative, a hormone such as estradiol or histamine, a hormone mimetic such as morphine, or other compound having binding specificity for a target.

As used herein, "specific binding pair" and "ligand-receptor binding pair" refer to two different molecules, wherein one molecule has a region on the surface or in the cavity that specifically attracts or binds to a particular spatial or polar tissue of the other molecule, such that the two molecules have an affinity for each other. The members of a specific binding pair are called ligand and receptor (anti-ligand). The terms ligand and receptor are intended to encompass the entire ligand or receptor or portions thereof sufficient for binding to occur between the ligand and receptor. Examples of ligand-receptor binding pairs include, but are not limited to, hormones and hormone receptors, such as epidermal growth factor and epidermal growth factor receptor, tumor necrosis factor- □ and tumor necrosis factor receptor, and interferon receptor; avidin and biotin or avidin; antibodies and antigen pairs; enzymes and substrates, drugs and drug receptors; cell surface antigens and lectins; two complementary nucleic acid strands; nucleic acid strands and complementary oligonucleotides; interleukins and interleukin receptors; as well as stimulating factors and their receptors such as granulocyte-macrophage colony stimulating factor (GMCSF) and GMCSF receptors, Macrophage Colony Stimulating Factor (MCSF) and MCSF receptors.

As used herein, "receptor" refers to a molecule having affinity for a given ligand. The receptor may be a naturally occurring or synthetic molecule. Receptors may also be referred to in the art as anti-ligands. As used herein, receptor and anti-ligand are interchangeable. The receptors may be used in their unaltered state or as aggregates with other species. Receptors may be linked to binding members, either covalently or non-covalently, or in physical contact, either directly or indirectly through specific binding substances or linkers. Examples of receptors include, but are not limited to, antibodies, cell membrane receptor surface and intrinsic receptors, monoclonal antibodies reactive with specific antigenic determinants, and antisera, such as viruses, cells or other substances, drugs, polynucleotides, nucleic acids, peptides, cofactors, lectins, sugars, polysaccharides, cells, cell membranes, and organelles.

As used herein, "specific binding" or "selective binding" refers to binding of a targeting agent to its target greater than to non-targets such as other receptors. The description that a particular compound is targeted to a target cell or target tissue means that its affinity for such cell or tissue in a host or in vitro or in vivo is greater than its affinity for other cells and tissues in a host or in an in vitro environment.

As used herein, "sample" refers to any substance containing a target for which a target assay is desired. The sample may be a biological sample, such as a biological fluid or a biological tissue. Examples of biological fluids include urine, blood, plasma, serum, saliva, semen, stool, sputum, cerebrospinal fluid, tears, mucus, sperm, amniotic fluid, and the like. Biological tissue is an aggregate of cells (usually of a particular kind) joined together with their intercellular substance that forms one of the structural substances of a human, animal, plant, bacterial, fungal or viral structure, including connective, epithelial, muscle and neural tissue. Examples of biological tissues also include organs, tumors, lymph nodes, arteries, and individual cells.

As used herein, "pharmaceutically acceptable derivatives" of a compound include salts, esters, enol ethers, enol esters, acetals, ketals, orthoesters, hemiacetals, hemiketals, acids, bases, solvates, hydrates, or prodrugs thereof. Such derivatives can be readily prepared by those skilled in the art using known methods of such derivatization. The conjugates can be administered to animals or humans without substantial toxic effects and are also pharmaceutically active or prodrugs.

Pharmaceutically acceptable salts include, but are not limited to, amine salts such as, but not limited to, N '-dibenzylethylenediamine, chloroprocaine, choline, ammonia, diethanolamine and other hydroxyalkylamines, ethylenediamine, N-methylglucamine, procaine, N-benzylphenethylamine, 1-p-chlorophenylmethyl-2-pyrrolidin-1' -ylmethyl-benzimidazole, diethylamine and other alkylamines, piperazine and tris (hydroxymethyl) aminomethane; alkali metal salts such as, but not limited to, lithium, potassium and sodium; alkaline earth metal salts such as, but not limited to, barium, calcium, and magnesium; transition metal salts such as, but not limited to, zinc; and other metal salts such as, but not limited to, sodium hydrogen phosphate and disodium phosphate; also included, but not limited to, salts of inorganic acids such as, but not limited to, hydrochlorides and sulfates; salts of organic acids such as, but not limited to, acetate, lactate, malate, tartrate, citrate, ascorbate, succinate, butyrate, valerate, and fumarate. Pharmaceutically acceptable esters include, but are not limited to, alkyl, alkenyl, alkynyl, aryl, heteroaryl, aralkyl, heteroaralkyl, cycloalkyl, and heterocycloalkyl of acidic groups including, but not limited to, carboxylic acid, phosphoric acid, phosphinic acid, sulfonic acid, sulfinic acid, and boronic acid. Pharmaceutically acceptable enol ethers include, but are not limited to, derivatives of the formula C ═ C (or), where R is hydrogen, alkyl, alkenyl, alkynyl, aryl, heteroaryl, aralkyl, heteroaralkyl, cycloalkyl, or heterocycloalkyl. Pharmaceutically acceptable enol esters include, but are not limited to, derivatives of the formula C ═ C (oc (o) R), where R is hydrogen, alkyl, alkenyl, alkynyl, aryl, heteroaryl, aralkyl, heteroaralkyl, cycloalkyl, or heterocycloalkyl.

As used herein, "treatment" refers to any manner in which one or more symptoms of a disease or disorder are ameliorated or otherwise beneficially altered. Treatment also includes any pharmaceutical use of the conjugates herein, such as for the treatment of a hyperproliferative tissue or neovascularization mediated disease or disorder, or a disease or disorder in which hyperproliferative tissue or neovascularization is implicated.

As used herein, an "effective amount" of a compound for treating a particular disease is an amount sufficient to ameliorate or in some way reduce the symptoms associated with the disease. Such amounts may be administered as a single dose or may be administered according to a regimen with which it is effective. The amount may cure the disease, but is generally administered to ameliorate the symptoms of the disease. Achieving the desired improvement in symptoms may require repeated administrations.

As used in this application, "combination" refers to any combination of two or more items.

As used herein, a "kit" is a packaged combination, wherein the components of the combination are contained within the package, optionally including instructions and reagents.

As used herein, "composition" refers to any mixture. It may be a solution, suspension, liquid, powder, paste, aqueous, non-aqueous, or any combination thereof.

As used herein, "fluid" refers to any composition that can flow. Fluids thus include compositions in the form of semi-solids, pastes, solutions, aqueous mixtures, gels, lotions, creams and other such compositions.

The conjugates, kits, articles of manufacture, and methods discussed in the following sections are generally representative of the disclosed conjugates and methods in which such conjugates can be used. The following discussion is intended as an illustration of selected aspects and embodiments of the invention, and should not be construed as limiting the scope of the disclosure.

B. Conjugates

Conjugates that enhance the effect of fluorescence detection or photodynamic therapy for the purpose of detecting or destroying tumors, hyperproliferative tissue, or other undesirable biological structures are disclosed in the present application. When used for diagnostic purposes, in order to minimize unwanted activity of donor molecules such as fluorophores or targeted photosensitizers for PDT and to improve selectivity of the donor molecule, the donor molecule is made part of a macromolecule or conjugate into which at least two other moieties and the necessary linking components are incorporated. In one embodiment, the first of these components is a Targeting Moiety (TM), which may be an antibody or any other ligand or binding agent having the desired binding affinity and specificity for the target cell or structure. The second component incorporated is an acceptor molecule, such as a Quencher (QA) that is intercalated into the conjugate in such a way that when it is not bound to its intended target, the quencher is in a position from which it can effectively quench the excited state of the sensitizer (sensitizer) (or consume energy, typically in the form of thermal energy transferred to the medium).

1. Energy transfer pair

a. Donor molecule

i. Fluorophores

In one embodiment, the donor molecule is a fluorophore. Fluorophores are chromophores that fluoresce, or molecules that emit light at a specific wavelength when stimulated by absorption of light of a different wavelength. Any fluorophore known in the art is suitable for use in the disclosed conjugates. Typical compounds include, but are not limited to, cyanine, indocarbocyanine, tetramethylrhodamine, indodicarbocyanine, carbocyanine, calcein, FITC, rhodamine 110, 5-carboxyfluorescein, fluorescein succinimidyl ester, 2 ', 7' -difluorofluorescein, carboxyfluorescein succinimidyl ester, 6-carboxy-4 ', 5' -dichloro-2 ', 7' -dimethoxyfluorescein ester, 6-carboxy-2 ', 4, 7, 7' -tetrachlorofluorescein succinimidyl ester, 6-carboxy-2 ', 4, 4', 5 ', 7, 7' -hexachloro-fluorescein ester, rhodamine green, phycoerythrin, rhodamine phalloidin, rhodamine B, rhodamine red-X, X-rhodamine, sulforhodamine 101, rhodamine B, and the like, Pyronine Y, TAMRA, ROX, R-phycocyanin, C-phycocyanin, and thiodicarbocyanine. When the conjugate is used in vivo, the fluorophore of the composition is generally selected to absorb light in the near infrared spectrum (600-1000nm), minimizing absorption by physiologically abundant absorbers such as hemoglobin (< 550nm) or water (> 1200nm), maximizing tissue penetration. Many such fluorophores are known in the art, including, but not limited to, allophycocyanins, indodicarbocyanines, indotricarbocyanines, thiadicarbocyanines, fluorescein, sulforhodamine, ROX, sulforhodamine, Nile Red, R-phycocyanins, C-phycocyanins, and thiadicarbocyanines. Many other fluorophores are commercially available, for example, from Frontier Scientific (log, UT), the SIGMA Chemical Company (Saint Louis, Mo.), Molecular Probes (Eugene, Oreg.), R & D Systems (Minneapolis, min.), Pharmacia LKB Biotechnology (Piscataway, n.j.), CLONTECH laboratories, Inc. (Palo alf., Calif.), Aldrich Chemical Company milwauke, Wis.), GIBCO l Life Technologies, Inc. (Gaithersburg, Md.), Fluka Chemica-bioryka analysis (uk chemistry, swich, and Company), and many other sources are known to practitioners of commercial origin, AG.

A photosensitizing agent

In another embodiment, the donor molecule is a photosensitizing agent. Photosensitizers are chemical compounds that are activated upon exposure to photoactivating light, converting the photosensitizing agent into a cytotoxic form, thereby killing target cells or reducing their proliferative potential. The photosensitizing agent of the conjugates disclosed herein can be any of the variety of synthetic and naturally occurring photosensitizing agents known in the art, including pyrrole-based photosensitizing agents such as porphyrins and porphyrin derivatives, for example, chlorins, bacteriochlorins, isobacteriochlorins, phthalocyanines, naphthalocyanines, and other tetra-and poly-macrocyclic compounds, as well as related compounds (e.g., pyrophiles, cerines (sapphyrins), and tesoferins) and metal complexes (e.g., without limitation, complexes formed from tin, aluminum, zinc, lutetium). Tetrahydrochlorins, purpurins, porphycenes, and phenothiaziniums are also within the scope of the disclosure. Generally, any macrocyclic photosensitizing compound of a polypyrrole that is hydrophobic may be used.

Examples of these and other photosensitizers include, but are not limited to, angelicin, chalcogenogenarium dye, chlorin, chlorophyll, coumarin, cyanine, keratin daunorubicin, 5-iminodaunorubicin, doxycycline, furosemide, gilvacricin M, gilvacricin V, hydroxy-chloroquine sulfate, lumimazocycline (lumixycyline), mefloquine hydrochloride, mequitazine, merbromin (mercurochrome), primaquine diphosphate, quinacrine dihydrochloride, quinine sulfate, and tetracycline hydrochloride, certain flavins and related compounds such as alloxazines, flavin mononucleotides, 3-hydroxyflavonoids, chromium restrictors (limichromes), flavins (limiflavins), 6-methyl alloxazines, 7-methyl alloxazines, 8-methyl alloxazines, 9-methyl alloxazines, 1-methyl chromium restrictors, methyl-2-methoxy benzoate, 5-Nitrosalicylic acid, proflavins and riboflavin, metalloporphyrins, metallophthalocyanines, methylene blue derivatives, naphthalimides, naphthalocyanines, pheophorbides, pheophytins, photosensitizer dimers and conjugates, phthalocyanines, porphycene, porphyrins, psoralens, purpurins, quinones, retinoids, rhodamines, thiophenes, verdins, vitamins and xanthene dyes (Redmond and Gamlin, Photochem. Photobiol., 70 (4): 391-475 (1999)).

(a) Typical metalloporphyrins

Typical metalloporphyrins include meso-tetra- (4-N-methylpyridyl) -porphine cobalt, meso- (4-sulfonatophenyl-porphine cobalt (II), hematoporphyrin copper, meso-tetra- (4-N-methylpyridyl) -porphine copper, meso- (4-sulfonatophenyl) -porphine copper (II), dimethyltesaline dihydroxide (txampyrindihydrazide) europium (III), tetraphenylporphyrin gallium, meso-tetra (4-N-methylpyridyl) -porphine iron, tetra (N-methyl-3-pyridyl) -porphyrin chlorolutetium (III), meso-diphenyltetrabenzoporphyrin magnesium (II), tetraphenylporphyrin magnesium, meso- (4-sulfonatophenyl) -porphine magnesium (II), tesaflavolin hydroxide metalloporphyrin magnesium (II), Meso-tetra- (4-N-methylpyridyl) -porphine magnesium, meso-tetra (4-N-methylpyridyl) -porphine nickel, meso-tetra (4-sulfonatophenyl) -porphine nickel (II), meso-tetra- (4-N-methylpyridinyl) -porphine palladium, tetraphenylporphyrin palladium, meso- (4-sulfonatophenyl) -porphine palladium (II), meso- (4-sulfonatophenyl) -porphine platinum (II), dimethyltesaferin dihydroxide samarium (II), meso- (4-sulfonatophenyl) -porphine silver (II), Tin protoporphyrin (IV), meso-tetra- (4-N-methylpyridyl) -porphine tin, meso-tetra (4-sulfophenyl) -porphine tin, tetra (4-sulfophenyl) porphyrin chloride tin (IV), cadmium (II) chlorotesaerine nitrate, cadmium (II) meso-diphenyltetrabenzoporphyrin, cadmium (II) meso-tetra- (4-N-methylpyridyl) -porphine, cadmium (II) tesaerine nitrate, 15-aza-3, 7, 12, 18-tetramethyl-porphryno (porphyrinato) -13, 17-diyl-dipropionate-dimethylester zinc (II), zinc (II) chlorotesaerine chloride, zinc coproporphyrin III, 2, 11, 20, 30-tetra- (1, 1-dimethyl-ethyl) tetranaphtho (2, 3-b: 2 ', 3' -g: 2 "3" -1: 2 * 3 * -q) porphyrazine zinc (II), 2- (3-pyridyloxy) benzo [ b ] -10, 19, 28-tris (1, 1-dimethylethyl) trinaphtho [2 ', 3' -g: 2 "3" 1: : 2 *,3 * -q ] porphyrazine zinc (II), 2, 18-bis- (3-pyridyloxy) dibenzo [ b, 1] -10, 26-bis (1, 1-dimethyl-ethyl) dinaphtho [2 ', 3' -g: 2 *,3 * -q ] porphyrazine zinc (II), 2, 9-bis- (3-pyridyloxy) dibenzo [ b, g ] -17, 26-bis (1, 1-dimethyl-ethyl) dinaphtho [2 ", 3" -1: 2 *,3 * -q ] porphyrazine zinc (II), 2, 9, 16-tris- (3-pyridyloxy) tribenzo [ b, g, 1] -24 ═ (1, 1-dimethyl-ethyl) naphtho [2 *,3 * -q ] porphyrazine zinc (II), 2, 3-bis- (3-pyridyloxy) benzo [ b ] -10, 19, 28-tris (1, 1-dimethyl-ethyl) trinaphtho [2 ', 3' -g: 2 ", 3" 1: 2 *,3 * -q ] porphyrazine zinc (II), 2,3, 18, 19-tetrakis- (3-pyridyloxy) dibenzo [ b, 1] -10, 26-bis (1, 1-dimethyl-ethyl) trinaphtho [2 ', 3' -g: 2 *,3 * -q ] porphyrazine zinc (II), 2,3, 9, 10-tetrakis- (3-pyridyloxy) dibenzo [ b, g ] -17, 26-bis (1, 1-dimethyl-ethyl) dinaphtho [2 ", 3" -1: 2 *,3 * -q ] porphyrazine zinc (II), 2,3, 9,10, 16, 17-hexa- (3-pyridyloxy) tribenzo [ b, g, 1] -24- (1, 1-dimethyl-ethyl) naphtho [2 *,3 * -q ] porphyrazine zinc (II), 2- (3-N-methyl) pyridyloxy) benzo [ b ] -10, 19, 28-tris (l, l-dimethyl-ethyl) trinaphtho [2 ', 3' -g: 2 ", 3" 1: 2 *,3 * -q ] porphyrazine zinc (II) monoiodide, 2, 18-bis- (3- (N-methyl) pyridyloxy) dibenzo [ b, 1] -10, 26-bis (l, l-dimethylethyl) dinaphtho [2 ', 3' -g: 2 *,3 * -q ] porphyrazine zinc diiodide (II), 2, 9-bis- (3- (N-methyl) pyridyloxy) dibenzo [ b, g ] -17, 26-bis (1, 1-dimethylethyl) dinaphtho [2 ", 3" -1: 2 *,3 * -q ] porphyrazine diiodozinc (II), 2, 9, 16-tris- (3- (N-methyl-pyridyloxy) tribenzo [ b, g, 1] -24- (1, 1-dimethylethyl) naphtho [2 *,3 * -q ] porphyrazine triiodide zinc (II), 2, 3-bis- (3- (N-methyl) pyridyloxy) benzo [ b ] -10, 19, 28-tris (1, 1-dimethylethyl) trinaphtho [2 ', 3' -g: 2 ', 3' -1: 2 *,3 * -q ] porphyrazine diiodozinc (II), 2,3, 18, 19-tetrakis- (3- (N-methyl) pyridyloxy) dibenzo [ b, 1] -10, 26-bis (1, 1-dimethyl) dinaphtho [2 ', 3' -g: 2 *,3 * -q ] porphyrazine zinc tetraiodide (II), 2,3, 9, 10-tetrakis- (3- (N-methyl) pyridyloxy) dibenzo [ g, g ] -17, 26-bis (1, 1-dimethylethyl) dinaphtho [2 ", 3" -1: 2 *,3 * -q ] porphyrazine zinc tetraiodide (II), 2,3, 9,10, 16, 17-hexa- (3- (N-methyl) pyridyloxy) tribenzo [ b, g, 1] -24- (1, 1-dimethylethyl) naphtho [2 *,3 * -q ] porphyrazine zinc hexaiodide (II), meso-diphenyltetraphenylporphyrin, meso-triphenyltetrabenzoporphyrin zinc (II), meso-tetra (2, 6-dichloro-3-sulfonatophenyl) porphyrin zinc (II), meso-tetra- (4-N-methylpyridinyl) -porphin zinc (II), 5, 10, 15, 20-meso-tetra (4-octyl-phenylpropynyl) -porphin zinc (II), porphyrin c zinc, protoporphyrin zinc (II), zinc (I) and zinc (I) porphin, Zinc protoporphyrin IX, zinc meso-triphenyl-tetraphenylporphyrin (II), zinc tetraphenylporphyrin (II), zinc tetranaphthoporphyrin, zinc tetraphenylporphyrin, zinc 5, 10, 15, 20-zinc tetraphenylporphyrin (II), meso- (4-sulfonatophenyl) -porphine and zinc (II) tesaferin zinc chloride (II).

(b) Typical pheophorbide

Typical pheophorbides include pheophorbide a, methyl 13-1-deoxy-20-formyl-7, 8-mono-dihydro-bacteria-m-pheophorbide a, methyl-2- (1-dodecyloxyethyl) -2-devinyl-pyropheol a, methyl-2- (1-heptyl-oxyethyl) -2-devinyl-pyropheol a, methyl-2- (1-hexyl-oxyethyl) -2-devinyl-pyropheol a, methyl-2- (1-methoxy-ethyl) -2-devinyl-pyropheol a, methyl-2- (l-phenyl-oxyethyl) -2-devinyl-pyropheol a, methyl-2-ethyl-2-devinyl, Magnesium methylobacterium pheophorbide d, methyl-bacteriopheophorbide d and pheophorbide.

(c) Typical porphyrins

Typical porphyrins include dimethyl 5-azaprotoporphyrin, bis-porphyrin, coproporphyrin III, tetrapotalmethyl coproporphyrin III, deuteroporphyrin IX dimethyl ester, diformyldeuteroporphyrin IX dimethyl ester, dodecaphenylporphyrin, hematoporphyrin IX, hematoporphyrin monomer, hematoporphyrin dimer, hematoporphyrin derivative A, hematoporphyrin IX dihydrochloride, hematoporphyrin IX dimethyl ester, mesoporphyrin IX dimethyl ester, mono-formyl-vinyl-deuteroporphyrin IX dimethyl ester, monoethyl vinyl deuteroporphyrin, 5, 10, 15, 20-tetrakis (o-hydroxyphenyl) porphyrin, 5, 10, 15, 20-tetrakis (m-hydroxyphenyl) porphyrin, 5, 10, 15, 20-tetrakis (p-hydroxyphenyl) porphyrin, 5, 10, 15, 20-tetrakis (3-methoxyphenyl) porphyrin, 5, 10, 15, 20-tetrakis (3, 4-dimethoxyphenyl) porphyrin, 5, 10, 15, 20-tetrakis (3, 5-dimethoxyphenyl) porphyrin, 5, 10, 15, 20-tetrakis (3, 4, 5-trimethoxyphenyl) porphyrin, 2,3, 7, 8, 12, 13, 17, 18-octaethyl-5, 10, 15, 20-tetraphenylporphyrin, Photofrin^®Photofrin II, porphyrin c, protoporphyrin IX, dimethyl protoporphyrin IX, protoporphyrin propyl aminoethyl formamide iodide, protoporphyrin N, N-dimethylaminopropyl-formamide, protoporphyrin propylaminopropyl formamide iodide, protoporphyrin butyl formamide, protoporphyrin N, N-dimethylamino-formamide, protoporphyrin formamide, cyhaloline 13, 12, 13, 22-tetraethyl-2, 7, 18, 23 tetramethylcyhaloline-8, 17-dipropanol, cyhaloline 23, 12, 13, 22-tetraethyl-2, 7, 18, 23 tetramethylcyhaloline-8-monoglycoside, cyhaloline 3, m-tetrakis- (4-N-carboxyphenyl) -porphin, tetra- (4-N-carboxyphenyl) -porphin3-methoxyphenyl) -porphine, tetrakis- (3-methoxy-2, 4-difluorophenyl) -porphine, 5, 10, 15, 20-tetrakis (4-N-methylpyridinyl) -porphine, meta-tetrakis- (4-N-methylpyridinyl) -porphine tetrachloride, meta-tetrakis (4-N-methylpyridinyl) -porphine, meta-tetrakis- (3-N-methylpyridinyl) -porphine, meta-tetrakis- (2-N-methylpyridinyl) -porphine, tetra (4-N, N, N-trimethylphenylammonium) porphine, meta-tetrakis- (4-N, N, N "-trimethylaminophenyl) -porphine tetrachloride, tetranaphthyrin, porphyrin, 5, 10, 15, 20-tetraphenylporphyrin, m-tetra- (4-N-sulfonatophenyl) -porphin, tetraphenylporphyrin tetrasulfonate, m-tetra (4-sulfonatophenyl) -porphin, tetra (4-sulfonatophenyl) porphin, tetraphenylporphyrin sulfonate, m-tetra (4-sulfonatophenyl) porphin, tetra (4-sulfonatophenyl) porphyrin, meta-tetrakis (4-sulfonatophenyl) porphine, meta-tetrakis (4-sulfonatophenyl) porphine, tetrakis (4-sulfonatophenyl) porphyrin, meta-tetrakis (4-N-trimethylphenylammonium) -porphine, uroporphyrin I, uroporphyrin IX and uroporphyrin I.

Photosensitizing agents for use in the conjugates disclosed herein include porphyrin derivatives obtained by reacting a porphyrin core with an alkyne in a Diels-Alder type reaction to give a monohydrobenzoporphyrin, such as those described in detail by Levy et al in U.S. Pat. No. 5,171,749, which is incorporated herein by reference. The absorption spectrum of the photosensitizing agent is typically between 400nm and 1200nm, and in some embodiments between 500-900nm or 600-900 nm.

(d) Typical psoralens

Typical psoralens include psoralen, 5-methoxypsoralen, 8-methoxy-psoralen, 5, 8-dimethoxypsoralen, 3-ethoxyformylpsoralen, 3-ethoxyformyl-pseudopsoralen, 8-hydroxypsoralen, pseudopsoralen, 4,5 ', 8-trimethyl-psoralen, isopsoralen, 3-acetyl-isopsoralen, 4, 7-dimethyl-isopsoralen, 4, 7, 4 ' -trimethyl-isopsoralen, 4, 7, 5 ' -trimethyl-isopsoralen, 3-acetyl-isopsoralen, 4,5 ' -dimethyl-isopsoralen, 5 ', 7-dimethyl-isopsoralen, 5, 8-dimethoxy-psoralen, 3-ethoxyformyl-isopsoralen, 3,7, Pseudoisopsoralen, 3-acetyl-pseudoisopsoralen, 3/4 ', 5' -trimethyl-aza-psoralen, 4 ', 8-trimethyl-5' -amino-methylpsoralen, 4 ', 8-trimethyl-phthalylamino (phthaloyl) -psoralen, 4, 5', 8-trimethyl-4 '-aminomethyl psoralen, 4, 5', 8-trimethyl-bromo-psoralen, 5-nitro-8-methoxy-psoralen, 5 ' -acetyl-4, 8-dimethyl-psoralen, 5 ' -acetyl-8-methyl-psoralen and 5 ' -acetyl-4, 8-dimethyl-psoralen. Typical purpurins include octaethyl purpurin, octaethyl purpurin zinc, oxidized octaethyl purpurin, reduced octaethyl purpurin tin, purpurin 18, purpurin-18-methyl ester, purpurin, stannyl purpurin I, Zn (II), benoxanthin ethyl ester, and zinzileucin.

(e) Typical quinones

Typical quinones include 1-amino-4, 5-dimethoxyanthraquinone, 1, 5-diamino-4, 8-dimethoxyanthraquinone, 1, 8-diamino-4, 5-dimethoxyanthraquinone, 2, 5-diamino-1, 8-dihydroxyanthraquinone, 2, 7-diamino-1, 8-dihydroxyanthraquinone, 4, 5-diamino-1, 8-dihydroxyanthraquinone, mono-methylated 4, 5-or 2, 7-diamino-1, 8-dihydroxyanthraquinone, anthralin (in the form of a ketone), anthralin, an anthralin anion, 1, 8-dihydroxyanthraquinone (chrysazine), 1, 2-dihydroxyanthraquinone (alizarin); 1, 4-dihydroxyanthraquinone (quinizarine), 2, 6-dihydroxyanthraquinone (Anthraflavin), 1-hydroxyanthraquinone (erythroxy-anthraquinone), 2-hydroxy-anthraquinone, 1, 2,5, 8-tetra-hydroxyanthraquinone (quinizarine), 3-methyl-1, 6, 8-trihydroxy anthraquinone (emodin), anthraquinone-2-sulfonic acid, benzoquinone, tetramethylbenzoquinone, hydroquinone, chlorohydroquinone, resorcinol and 4-chlororesorcinol.

(f) Typical retinoids

Typical retinoids include all-trans retinal, C₁₇Aldehyde, C₂₂Aldehyde, 11-cis retinal, 13-cis retinal, retinal and retinal palmitate.

(g) Typical rhodamine classes

Typical rhodamines include 4, 5-dibromo-rhodamine methyl ester, 4, 5-dibromo-rhodamine n-butyl ester, rhodamine 101 methyl ester, rhodamine 123, rhodamine 6G hexyl ester, tetrabromo-rhodamine 123, and tetramethyl-rhodamine ethyl ester.

(h) Examples of other photosensitizers

Other non-limiting examples of photosensitizing agents that may be suitable for use in the conjugates are bacteriochlorophyll-a derivatives described in U.S. patent nos. 5,171,741 and 5,173,504; photosensitized Diels Alder (Diels-Alder) porphyrin derivatives described in U.S. Pat. No. 5,308,608; porphyrin-like compounds described in U.S. Pat. nos. 5,405,957, 5,512,675, and 5,726,304; porphyrin derivatives and imines of porphyrins described in U.S. patent nos. 5,424,305 and 5,744,598; alkyl ether analogs of benzoporphyrin derivatives described in U.S. patent No. 5,498,710; rhodopsin-18, bacteriorhodopsin-18 and related compounds described in U.S. patent No. 5,591,847; meta-substituted chlorins, isopenicillin, and bacteriochlorin described in U.S. Pat. No. 5,648,485; meta-substituted tetracyclines described in U.S. Pat. No. 5,703,230; carbodiimide analogs of chlorins and bacteriochlorins described in U.S. patent No. 5,770,730; meta-substituted chlorins, isopenicillin, and bacteriochlorin described in U.S. Pat. No. 5,831,088; meta-substituted tri-pyrans of the macrocyclic forms of polypyrroles described in U.S. Pat. nos. 5,703,230, 5,883,246 and 5,919,923; the isoimides of chlorins and bacteriochlorins described in U.S. Pat. No. 5,864,035; alkyl ether analogs of chlorins with N-substituted imide rings described in U.S. Pat. No. 5,952,366; ethylene glycol esters described in U.S. patent 5,929,105; carotene analogs of porphyrins, chlorins, and bacteriochlorins described in U.S. patent No. 6,103,751; fatty acid ester derivatives of porphyrins, chlorins or bacteriochlorins described in U.S. Pat. No. 6,245,811; indium photosensitizers described in U.S. patent No. 6,444,194; porphyrins, chlorins, bacteriochlorins and their related tetrapyrrole compounds described in U.S. Pat. No. 6,534, 04; 1, 3-propane diol and ether derivatives of porphyrins, chlorins, and bacteriochlorins described in U.S. Pat. No. 6,555,700; trans beta substituted chlorins described in U.S. patent No. 6,559,374; palladium substituted bacteriochlorophyll derivatives described in U.S. patent No. 6,569,846; and photosensitizer entities described in Wilson et al (curr. micro.25: 77-81, 1992) and Okamoto et al (Lasers in Surg. Med.12: 450-. Generally any hydrophobic or hydrophilic photosensitizing agent that absorbs in the ultraviolet, visible, and infrared spectral ranges will be suitable for use in the disclosed conjugates.

b. Acceptor molecules

The acceptor molecule of the disclosed conjugates is a chemical or biological compound that is capable of receiving or accepting energy from another molecule. In one embodiment, the conjugates disclosed herein comprise a quencher that is an acceptor molecule. Any fluorescence-modifying group that can at least partially attenuate light emitted by a fluorophore or prevent activation of a photosensitizing agent can be used as a quencher in the disclosed conjugates. Such attenuation typically occurs by energy transfer between a donor molecule, such as a fluorophore or photosensitizing agent, and an acceptor molecule, such as a quencher.

Fluorescence quenching typically occurs by a number of mechanisms, including direct and indirect energy transfer. In all cases, when the donor molecule of the disclosed conjugates, including a fluorophore or a photosensitizing agent, is excited by an input of energy, typically by irradiation with light of a particular wavelength, energy is transferred from the donor molecule, e.g., the fluorophore or the sensitizer, to an acceptor molecule, e.g., the quencher, rather than being consumed by the fluorescence or the conversion of the photosensitizing agent to an active state. A quencher that is an acceptor molecule has the capacity to accept the energy transferred, for example by dipole coupling but does not have significant emission.

Thus, a quencher is any chemical that can transfer or consume the energy of the excited state of a donor molecule, such as a fluorophore or photosensitizer of a conjugate, when the conjugate does not bind to its intended target. Quenchers include, but are not limited to, acceptor chromophores which do not exhibit significant emission, and aromatic compounds capable of accepting the energy delivered, such as nitrosated aromatic compounds, including nitrobenzene, nitrobenzyloxycarbonyl, nitrobenzoyl.

Typical quenching agents

Typical quenchers include 4- (4' -dimethylamino-phenylazo) benzoic acid (DABCYL); dabcyl succinimidyl ester; 4- (4' -dimethylamino-phenylazo) sulfonic acid (DABSYL); dabsyl succinimidyl ester, tetramethyl-rhodamine (TAMRA), 4- [ (4-nitrophenyl) diazinyl ] -aniline and 4- [ 4-nitrophenyl ] diazinyl ] -naphthylamine; dabcyl nitro-thiazole; 6- (N- [ 7-nitrophenyl-2-oxa-1, 3-oxadiazol-4-yl ] amino) hexanoic acid; 6-carboxy-X-Rhodamine (ROX); QSY-7; 2- [4- (4-nitrophenylazo) -N-ethylphenyl-amino ] ethanol (disperse red 1); 2- [4- (2-chloro-4-nitrophenyl-azo) -N-ethylphenylamino ] -ethanol (disperse red 13); tetrarhodamine isothiocyanate (TRITC); allophycocyanin; beta-carotene, diarylrhodamine derivatives such as QSY 7; QSY 9 and QSY 21 dyes; QSY 35 acetic acid succinimidyl ester; QSY 35 iodoacetamide and aliphatic methylamine; naphthalenedicarboxylate (napthalate); reactive red 4 and malachite green.

There is a great deal of practical guidance available in the literature for selecting the appropriate donor-acceptor pair for use in the disclosed conjugates. See, for example, Pesce et al, "Fluorescence Spectroscopy" (Marcel Dekker, New York, 1971), White et al, "Fluorescence Analysis: a Practical Approach "(Marcel Dekker, New York, 1970). The literature also includes references providing an exhaustive list of fluorescing and chromophoric Molecules and their associated optical properties for selection of acceptor-quencher (donor-acceptor) pairs (see, e.g., Berlman, "Handbook of Fluorescence Spectra of aromatic Molecules," second edition (Academic Press, New York, 1971), "Griffiths," Color and consistency of Organic Molecules, "(Academic Press, New York, 1976)," Bishop, "Indicators" (Pergamon Press, Oxford, 1972) and Haughland, "Handbook of Fluorescence Probe Chemicals," (Molecular Probes, Eugene, 1992).

c. Selection of energy transfer pairs

The ability of a donor molecule to transfer energy to an acceptor molecule depends on a number of factors. These factors include, but are not limited to, energy transfer efficiency, spectral overlap between acceptor and donor molecules, dipoles, fluorescence quantum yield of the donor, extinction coefficient of the acceptor, and fluorescence emission intensity of the donor. Because these factors are dependent on the environment, the actual values observed in a particular experimental situation are somewhat variable.

i. Fluorescence Resonance Energy Transfer (FRET)

FRET refers to non-radiative energy transfer between chemical and/or biological luminescent molecules (Curr. biol. 6: 178-182(1996) of Heim et al; Gene 173: 13-17(1996) of Mitra et al; meth. enzymol. 246: 300-345(1995) of Selvin et al; Matyus, J. Photochem. Photobiol. B: biol. 12: 323-337 (1992); anal. biochem. 218: 1-13(1994)) of Wu et al). The efficiency of FRET depends on the minus 6 th power of the intermolecular spacing, which makes it useful within distances comparable to the size of biological macromolecules (Stryer and Haughand, Proc Natl Acad Sci USA 58: 719-726 (1967)). Thus, the sensitivity of FRET to molecular proximity is described (dos Remedios et al, J Structure Biol 115: 175-185 (1995); Selvin Methods Enzymol 246: 300-334 (1995); Boyde et al, Scanning 17: 72-85 (1995); Wu et al, AnalBiochem 218: 1-13 (1994); Van der Meer et al, "resource EnergyTransfer therapy and Data," page 133-. When FRET is used as a control mechanism, co-localization of proteins and other molecules can be imaged with spatial resolution beyond the limits of traditional optical microscopy. (Kenworthy, Methods 24: 289-296 (2001); Gordon et al, Biophys J74: 2702-2713 (1998)).

The efficiency of energy transfer depends on many factors, including the transfer efficiency and the distance (r) between the donor and acceptor. For example, the basic F ö rster energy transfer process involves the absorption of one wavelength of photon energy (hv) by a donor group₁) And through a non-radiative process, transmits its ability to an acceptor group, which in turn emits photon energy of longer wavelength (hv)₂) Or to dissipate energy non-radiatively. When energy transfer is through nonradiative ground or F ö rster energy transfer, equations describing the relationship between the efficiency of energy transfer and the efficiency of energy transfer are known (see, e.g., Youvan et al, U.S. patent No. 6,456,734 and Heller, U.S. patent No. 6,416,953).

i) F ö rster distance

The rate of energy transfer between the acceptor molecule and the donor molecule in FRET is inversely proportional to the 6 th power of the distance between the donor and the acceptor, and therefore, the energy transfer efficiency is very sensitive to changes in distance. It is said that energy transfer occurs with a detectable efficiency in the distance range of 1-10 nm. The distance at which 50% of the energy transfer is effective (i.e., 50% of the excited donor is inactivated by FRET) is defined as the F ö rster radius (R)_o)。R_oThe order of magnitude of (a) depends on the spectral properties of the donor and acceptor molecules and can be calculated from the spectral overlap integral using the following equation:

R_o＝[8.8×10²³·κ²·n^-4QY_D·J(λ)]^1/6tea tree (Angel)

Wherein κ²Dipole orientation factor (range 0 to 4, k)²2/3 for randomly oriented donors and acceptors)

QY_DFluorescence quantum yield of donor without acceptor

n-refractive index

J (λ) ═ spectral overlap integral (see below)

＝∫ε_A(λ)·F_D(λ)·λ⁴dλcm³M^-1

Wherein epsilon_AExtinction coefficient of acceptor

F_DIntensity of fluorescence emission of the donor as a fraction of the total overall intensity

The F ö rster distance must be considered in selecting the donor and acceptor molecules of the energy transfer pair of the conjugate. The F ö rster distance should also be considered in selecting the placement of the linking component or energy transfer pair such that interaction of the targeting moiety with its target causes a change in the distance between the donor molecule and the acceptor molecule. These distances may be determined empirically or may be calculated. As a non-limiting example, the donor and acceptor molecules may be placed within about 1 to about 10nm (10 angstroms to about 100 angstroms) to observe energy transfer. Measurement of energy transfer includes monitoring quenching of the signal from the excited energy donor, which decreases as the energy transfer compounds come into proximity with each other.

iii) selection criteria

The fluorophore and/or quencher used as the energy transfer pair in the disclosed conjugates can be selected based on factors such as, but not limited to, cost, availability, size, absorption wavelength, and emission wavelength. For example, because the conjugate activates when the targeting moiety interacts with its target, the use of certain fluorophore molecules or quencher molecules may be excluded due to size or electrostatic inhibition. In addition, the selection of the photosensitizer and/or fluorophore and/or quencher for use in the disclosed conjugates must also meet various criteria to facilitate the energy transfer process. These criteria include, but are not limited to, acceptor-donor distance, overlap of donor emission and acceptor absorption, discrimination of donor and acceptor peaks, quantum yield, and orientation of the fluorophore

a) Distance between two adjacent plates

As an energy transfer reaction, e.g., FRET, involves energy transfer from one luminescent molecule (i.e., a donor molecule) to another luminescent molecule (i.e., an acceptor molecule) in a distance-dependent manner, the donor and acceptor molecules must be in close proximity to facilitate energy transfer. By way of non-limiting example, the donor and acceptor molecules may be placed within about 1 to about 10nm to observe energy transfer. One skilled in the art can vary the placement of the donor molecule and the acceptor molecule such that they are within the desired proximity for energy transfer, thus quenching the donor molecule. In particular, one skilled in the art can select the location and placement of the donor and acceptor molecules in the conjugate, perform sample FRET experiments to measure energy transfer and adjust the location of the donor and acceptor molecules until the donor molecule is quenched. Alternatively, one skilled in the art can use literature, manuals, guidelines, the internet, experimental results, and other sources well known in the art to determine the placement distance of the donor molecule and the acceptor molecule, thereby achieving quenching of the donor molecule by the acceptor molecule.

When all other parameters are optimal, the efficiency of energy transfer decreases with increasing distance between the donor and acceptor molecules, e.g., 1/r⁶. For example, a donor-to-acceptor distance of less than 3.5nm (35 angstroms) can produce 50% efficient FRET energy transfer (see Heller, 6,416,953). The conjugates disclosed herein are designed in such a way that the donor molecule and the acceptor molecule are at a donor-acceptor transfer distance when the conjugate is not interacting with the target. In one embodiment, the donor molecule and the acceptor molecule are in close proximity such that quenching of the donor molecule is from about 25% to 100% effective.

The optimal placement or spacing of the donor molecule and acceptor molecule to form the donor-acceptor transfer distance can be determined empirically. Generally, the conditions required for energy transfer, such as FRET, are (i) the donor molecule and the acceptor molecule are in close proximity to each other (typically 1 or 10 to 100 or 200 angstroms) and (ii) the absorption spectrum of the acceptor overlaps the fluorescence emission spectrum of the donor.

b) Overlap of donor emission and acceptor absorption

A second criterion for determining a donor-acceptor energy transfer pair for use in the conjugates of the present application is that the emission spectrum of the donor molecule should at least partially overlap the energy absorption spectrum of the acceptor molecule such that energy transfer from the donor to the acceptor can occur. Typically, the energy transfer donor compound has an emission peak wavelength (Dλ)_em) At the excitation peak wavelength (A lambda) of the acceptor molecule_ex) Within a few nm. D lambda_emAnd A λ_exThe difference in (a) is typically about 70nm to about 20nm, or less. D lambda_emAnd A λ_exThe difference in (b) may be about 60nm, about 50nm, about 30nm, about 20nm, about 15nm, about 10nm, about 5nm, about 4nm, about 3nm, about 2nm, or about 1 nm. In some examples, if Dλ_emPeak sum A lambda_exPeaks partially overlap and Dλ if the donor and acceptor are within proximity for detectable energy transfer to occur_emAnd A λ_exThe difference in (a) may be greater than 70nm (i.e., light having an excitation peak wavelength that is distal from the donor and/or an emission peak wavelength of the acceptor may be used). An integral table of spectral overlap is readily available to those skilled in the art (see, e.g., Berlman, i.b., "energy transfer parameters for aromatics" Academic press, new york and london (1973)).

c) Limited environmental sensitivity

Another criterion that can be used to select donor and acceptor molecules of an energy transfer pair is their sensitivity to assay or physiological conditions. As non-limiting examples, quenchers that are not affected by changes in pH, ionic concentration, temperature, and solvent media may be selected for the conjugates disclosed herein.

d) Quantum yield

Energy transfer is most efficient when a donor fluorophore with a high fluorescence quantum yield (e.g., close to 100%) is paired with an acceptor that has a large extinction coefficient at a wavelength that coincides with the emission wavelength of the donor. The dependence of Fluorescence energy transfer on the above parameters has been reported (Lakowicz, J.R., "Principles of Fluorescence Spectroscopy," New York: Plenum (1983); and Herman, B., "Resonance energy transfer Microscopy," in: Fluorescence Microscopy of living Cells in Culture, Part B, Methods in Cell Biology, Vol.30 (Taylor, D.L. & Wang, Y. -L., eds.), San Diego, Academic Press (1989), p.219-243).

e) Effective attachment site

Another factor to consider when selecting a donor molecule, such as a photosensitizer or fluorophore, and an acceptor molecule, such as a quencher, is the effective attachment site. Most attachment is conveniently achieved by thiol or amine interactions. Synthetic and commercially available alternatives are available, depending on the photosensitizing agent, fluorophore or quencher, molecule or linking component used in the selected conjugate, the environment in which it will be present. As noted above, the distance is selected such that interaction of the targeting moiety results in a change in the position of the quencher from the fluorescence-quenching interaction-permissive position. If the donor molecule and acceptor molecule are too close, the interaction of the targeting agent may not terminate quenching of the donor molecule, as energy transfer will continue to occur. If the distance between the donor molecule and the acceptor molecule is too large, energy transfer may not occur at all. The distance may be determined by any method, such as by calculation or empirically.

Techniques of synthetic chemistry provide methods for attachment of donor molecules using a linking component that provides a donor-acceptor transfer distance (see, e.g., Heller et al, U.S. Pat. No. 4,996,143). For example, synthetic linkage techniques are known which allow for the incorporation of donor and acceptor molecules within the same oligonucleotide (see Heller et al, U.S. Pat. No. 4,996,143). Using a specific linker arm, the most efficient energy transfer (in terms of re-emission of the acceptor) was found to occur when the donor and acceptor were separated by 5 intervening nucleotide units or by approximately 2 nm. Heller et al, U.S. Pat. No. 4,996,143, also show that as the nucleotide spacing is increased from 6 to 12 units (about 2nm to about 4nm), the energy transfer efficiency is also found to be reduced, consistent with F ö rster theory. There is extensive guidance in the literature for derivatizing reporter and quencher molecules covalently attached via readily available reactive groups that can be added to the molecule. The diversity and utility of chemistries available for conjugating fluorophores to other molecules and surfaces is exemplified by the extensive literature on preparing nucleic acids derivatized with fluorophores. See, for example, Ullman et al, U.S. Pat. No. 3,996,345 and Khanna et al, U.S. Pat. No. 4,351,760.

f) Regulation of energy transfer

The components of the energy transfer pairs used in the disclosed conjugates are generally selected so that the absorption band of the acceptor molecule overlaps the fluorescence emission band of the donor molecule. Another factor to consider in selecting a donor/acceptor energy transfer pair is the efficiency of energy transfer between them. The efficiency of energy transfer can be easily tested empirically using methods known in the art. The efficiency of energy transfer between a donor-acceptor pair can be modulated by varying the ability of the donor and acceptor to associate tightly.

For example, by adjusting the length of the linking component between the fluorophore or photosensitizing agent and the quencher, an increase or decrease in association can be facilitated. The ability of the donor-acceptor pair to associate can also be increased or decreased by modulating the hydrophobic or ionic interaction or steric repulsion between the two moieties in the disclosed conjugates. Thus, the intramolecular interactions that cause the association of the donor-acceptor pair can be enhanced or attenuated. Thus, for example, the association between a donor-acceptor pair can be increased, e.g., by utilizing a generally negatively charged donor and a generally positively charged acceptor.

2. Targeting moieties

The conjugates disclosed herein include targeting moieties that preferentially associate or bind to a particular cell, tissue, receptor, infectious agent, or body part of the subject to be treated, such as a target cell, target tissue, or target component. The targeting moiety may be a polypeptide (which may be linear, branched or cyclic). The targeting moiety may comprise a polypeptide having affinity for a polysaccharide target, such as a lectin (e.g., seed, bean, root, bark, seaweed, fungal, bacterial or invertebrate lectin). Particularly useful lectins include concanavalin A obtained from Canavalia gladiata and lectins obtained from Lens culiaris. The targeting moiety can be a molecule or macromolecular structure (e.g., a liposome, micelle, lipid vesicle, etc.) that preferentially associates or binds to a particular tissue, receptor, infectious agent, or other part of the body of the subject to be treated.

a. Typical targeting moieties

Examples of targeting moieties include, but are not limited to, oligonucleotides, carbohydrates, carbohydrate polymers (such as dextran sulfate or heparin), receptors, ligands, and one member of a ligand-receptor binding pair. Ligands suitable for use as targeting moieties include those ligands that are receptor specific and immunoglobulins and fragments thereof. For example, immunoglobulins suitable for use as targeting moieties generally include antibodies and monoclonal antibodies and immunologically active fragments thereof.

For example, the following receptors may be used to target macrophages: complement receptors (Rieu et al, J.cell biol.127: 2081-2091, 1994), scavenger receptors (Brasseur et al, Photochem.Photobiol.69: 345-352, 1999), transferrin receptors (Dreier et al, bioconjug.chem.9: 482-489, 1998; Hamblin et al, J.Photochem.Photobiol.26: 4556, 1994), Fc receptors (Rojanasakul et al, pharm.Res.11: 1731-1733, 1994), mannose receptors (Frankeyl et al, Carbodr.Res.300: 258, 1997; Chakrabarty et al, J.Protozool.37: 358-364, 1990). Targeting moieties which can be conjugated to photosensitizers, such as target-bound macrophages, include low density lipoproteins (Mankertz et al, biochem. Biophys. Res. Commun.240: 112. Bufonis 115, 1997; von Baeyer et al, int. J. Clin. Pharmacol. Ther. Toxicol.31: 382. sup. 386, 1993), very low density lipoproteins (Tabas et al, J. cell biol.115: 1547. sup. 1560, 1991), mannose residues and other carbohydrate moieties (Pittt et al, Nucl. Med. biol. 22: 355. sup. 365, 1995), polycationic molecules such as poly-L-lysine (Hamblin et al, J. Photochem. Photobiol.26: 45-56, 1994), liposomes (Bakker-Woudeng et al, J. drug. 2: 717. 371; Bermeuk. J. Pharma. J. photonic. multidrug. 92. 1994, Immun. J. 10. J. 92. Mahonex. 1994, and E. Menkel. 92. Menke. 92. kappa. 92, 1994).

Many targeting moieties and methods for targeting compounds are well known to those skilled in the art. All such targeting methods are contemplated for use in the present conjugates. For example, see, e.g., U.S. patent nos. 6,316,652, 6,274,552, 6,271,359, 6,253,872, 6,139,865, 6,131,570, 6,120,751, 6,071,495, 6,060,082, 6,048,736, 6,039,975, 6,004,534, 5,985,307, 5,972,366, 5,900,252, 5,840,674, 5,759,542, and 5,709,874 for non-limiting examples of targeting methods.

b. Placing

The donor and acceptor molecules of the conjugates disclosed herein are positioned at a donor-acceptor transfer distance such that when the conjugate does not interact with the target, it is in a non-reactive state. When the conjugate interacts with a target cell or target tissue through the targeting moiety, the donor molecule and the acceptor molecule are separated such that energy transfer between them no longer occurs. Thus, only after the targeting moiety interacts with its target does spatial rearrangement of the donor and acceptor molecules occur in the conjugate. Thus, the targeting moiety in the conjugate is selected and positioned so that when the targeting moiety interacts with its target, the spatial arrangement of the conjugate is altered so that the donor molecule and the acceptor molecule are no longer at the donor-acceptor transfer distance.

For example, in one embodiment, when the conjugate binds its target, the three-dimensional structure of the conjugate is altered in such a way that the quencher is no longer in sufficient proximity to quench the excited state of the photosensitizer-thus allowing the photosensitizer to function as a fluorescence-based diagnostic method or by generating singlet oxygen: (¹O₂) The function required for PDT. In the latter case, the singlet oxygen can then be used to breakAnd (4) damaging the target. When this embodiment is used for diagnostic purposes, the photosensitizer need only function as a fluorophore. When the quencher of the present invention does not bind to the target, it then serves to prevent the generation of false positive signals from the fluorophore.

In another embodiment, the donor molecule is a porphyrin or porphyrin derivative tetrapyrrole that includes a metal atom in its central coordination cavity, and the acceptor molecule is a quencher that has one or more suitable functional groups coordinated to the axial position of the metal coordinated within the photosensitizing agent. The targeting moiety is positioned in the conjugate in such a way that interaction of the targeting moiety and the target disrupts the association of the axial ligand and the metal, releasing the quencher and allowing the porphyrin or porphyrin derivative tetrapyrrole to activate when irradiated.

C. Preparation of conjugates

The conjugates provided herein can be prepared according to methods known to those skilled in the art, for example, according to the methods provided below and exemplified herein (see, e.g., examples 1-3).

1. Coupling agent

Techniques for constructing conjugates of ligands and photosensitizers are well known to those of ordinary skill in the art. For example, Rakestraw et al teach the use of a modified dextran carrier to conjugate tin (IV) chlorins to monoclonal antibodies via covalent bonds (Rakestraw, S.L., Tompkins, R.D., and Yarmush, M.L., Proc. Nat. Acad. Sci. USA 87: 4217-. The conjugates disclosed in the present application may be conjugated to a ligand, such as an antibody, using a coupling agent. Any linkage capable of linking the components such that they are stable under physiological conditions for the time required for administration and treatment is suitable, but covalent linkages are preferred. The linkage between the two components may be direct, e.g., the photosensitizer is directly linked to the targeting agent; or indirectly, e.g., a photosensitizer is linked to the linking component and the linking component is linked to the targeting agent.

The coupling agent should function under conditions of temperature, pH, salt, solvent system and other reactants that substantially maintain the chemical stability of the donor molecule, acceptor molecule and targeting moiety. The coupling agent should be stably attached to the component moiety, but such that there is minimal or no denaturation or inactivation of the donor molecule, acceptor molecule, or targeting moiety. Many coupling agents react with amines and carboxylates to form amides, or with alcohols and carboxylates to form esters. Coupling agents are known in the art (see, e.g., M.Bodansky, "Principles of Peptide Synthesis", second edition with T.Greene and P.Wuts, "Protective Groups in organic Synthesis", second edition, 1991, John Wiley, NY). Representative combinations of such groups are amino and carboxyl groups forming amide linkages, or carboxyl and hydroxyl groups forming ester linkages, or amino and alkyl halide groups forming alkylamine linkages, or thiol and thiol forming disulfides, or thiol and maleimide or alkyl halide groups forming thioethers. Obviously, hydroxyl, carboxyl, amino and other functionalities, when absent, may be introduced using techniques known in the art.

The conjugates provided herein can be prepared, for example, by coupling a photosensitizer to a targeting moiety such as an antibody, by cleaving an effective ester moiety on the photosensitizer and coupling the compound to the antibody through an N-terminus with a peptide bond, or by other methods known in the art. A number of coupling agents, including cross-linking agents, may be used for covalent conjugation. Examples of crosslinking agents include N, N' -Dicyclohexylcarbodiimide (DCC), N-succinimidyl-S-acetyl-thioacetate (SATA), N-succinimidyl-3- (2-pyridyldithio) propionate (SPDP), o-phenylene-dimaleimide (o-PDM), and thiosuccinimidyl 4- (N-maleimido-methyl) cyclohexane-1-carboxylate (thio-SMCC). See, e.g., Karpovsky et al j.exp.med.160: 1686 (1984); and Liu, MA et al, proc.natl.acad.sci.usa 82: 8648(1985). Other methods include Brennan et al Science 229: 81-83(1985) and Glennie et al, J.Immunol.139: 2367 those described in 2375 (1987). Coupling agents for many polypeptides and proteins, as well as buffers, solvents and methods of use are described in Pierce Chemical co, catalog, pages O-90 to O-110(1995, Pierce Chemical co., 3747n. meridian Rd., Rockford il., 61105, u.s.a., which is incorporated herein by reference.

For example, DCC is a useful coupling agent that can be used to facilitate the coupling of alcohol NHS and chlorin e6 in DMSO to form an activated ester that can be crosslinked to polylysine. DCC is a carboxyl-reactive cross-linking agent, commonly used as a coupling agent in peptide synthesis, with a molecular weight of 206.32. Another useful crosslinker is SPDP, a heterobifunctional crosslinker used with primary amines and mercapto groups. The molecular weight of SPDP is 312.4, the spacer arm length is 6.8 angstroms, is reactive with NHS-ester and pyridyldithio groups, and produces cleavable crosslinks such that upon further reaction, the crosslinker is eliminated so that the photosensitizer can be directly linked to the linking component or targeting agent. Other useful conjugating agents are SATA, for introducing block SH groups for two-step crosslinking, which is deblocked with hydroxylamine-HCl, and thio-SMCC, which is reactive towards amines and sulfhydryls. Other cross-linking and coupling agents are also available from Pierce chemical company. The remaining compounds and methods for conjugating proteins to other proteins or to other compositions (e.g., to reporter groups or to chelators for metal ion labeling of proteins), particularly those involving schiff bases as intermediates, are disclosed in EPO243,929 a2 (disclosed in 1987.11.4).

2. Reactive group

The acceptor molecule or targeting moiety may be conjugated to the donor molecule directly or through a linking component using a reactive group on the donor molecule or on the acceptor molecule or on the targeting moiety. For example, a carboxyl-containing molecule may be bound to the □ -amino group of a lysine in a target polypeptide by a preformed active ester (such as an N-hydroxysuccinimide ester) or an ester conjugated in situ by a carbodiimide-mediated reaction. Using the same method for molecules containing sulfonic acid groups, the sulfonic acid groups can be converted to sulfonyl chlorides which react with amino groups. Molecules having carboxyl groups can be attached to, for example, amino groups on polypeptides by in situ carbodiimide methods. The molecule may also be attached to a hydroxyl group of a serine or threonine residue, or to a sulfhydryl group of a cysteine residue.

Methods of linking the conjugate components, such as linking a photosensitizer with a polyamino acid chain to an antimicrobial polypeptide, can use heterobifunctional crosslinking reagents. These agents bind one functional group on one strand to a different functional group on a second strand. These functional groups are typically amino, carboxyl, mercapto and aldehyde. There are many substitutions of suitable moieties which will react with these groups and have different chemical structures to conjugate them together. See Pierce catalog and Merrifield, r.b. et al, Ciba Found symp.186: 5-20(1994).

The photosensitizer component of the conjugate may optionally be functionalized to include a linking component that allows the photosensitizer component to be linked to a targeting moiety such as an analyte, antigen, antibody or other molecule. For example, the linking component may include, but is not limited to, an oligonucleotide, polynucleotide, nucleic acid, oligosaccharide, polysaccharide, or □, □ -diaminoalkane linking species such as 1, 3-diaminopropane. Various linking components suitable for this purpose have been described in the literature. See, e.g., Kricka, J.J., "Ligand-Binder Assays; labels and Analytical Strategies ", pages 15-51 (Marcel Dekker, Inc., New York, N.Y. (1985)). The photosensitizer component is linked to a linking component and the linking component is linked to an analyte, antigen, antibody or other molecule using conventional techniques.

a. Typical reactive groups and reactions

Reactive groups and reactive species suitable for use in preparing the disclosed conjugates are generally those well known in the art of bioconjugate chemistry. The kind of reaction includes those carried out under relatively mild conditions. These include, but are not limited to, nucleophilic substitutions (e.g., amine and alcohol reactions with acyl halides, active esters), electrophilic substitutions (e.g., enamine reactions), and addition of carbon-carbon and carbon-heteroatom multiple bonds (e.g., Michael reactions). These and other useful reactions are discussed, for example, in Morrison et al, "organic chemistry," 4 th edition, Allyn and Bacon, Inc., 1983, and Hermanson, "Bioconjugate Techniques," Academic Press, San Diego, 1996.

For example, useful reactive functional groups include:

(a) carboxyl and various derivatives thereof including, but not limited to, N-hydroxysuccinimide ester, N-hydroxybenzotriazole ester, acid halides, acylimidazoles, thioesters, p-nitrophenyl esters, alkyl, alkenyl, alkynyl, and aromatic esters;

(b) hydroxyl groups, which can be converted to esters, ethers, aldehydes, and the like;

(c) haloalkyl groups, for example wherein the halide can later be replaced with a nucleophilic group such as an amine, carboxylate anion, thiol anion, carbanion, or alkoxide ion, resulting in covalent attachment of a new group at the halogen atom site;

(d) dienophile groups, which can, for example, participate in Diels-Alder reactions, such as maleimido groups;

(e) carbonyl groups, such that subsequent derivatization is possible by formation of carbonyl derivatives such as imines, hydrazones, semicarbazones or oximes or by mechanisms such as Grignard addition or alkyllithium addition;

(f) sulfonyl groups that are subsequently reacted with amines, e.g., to form sulfonamides;

(g) a sulfhydryl group which can be converted to a disulfide or reacted with an acyl halide;

(h) amines or thiols, which may, for example, be acylated, alkylated or oxidised;

(i) olefins, for example, which may undergo cycloaddition, acylation, Michael addition, and the like;

(j) epoxides, for example, which can react with amines and hydroxyl compounds; and

(k) phosphoramidates (phosphoramides) and other standard functional groups suitable for nucleic acid synthesis.

There is extensive guidance in the literature for derivatizing photosensitizer and quencher molecules for covalent attachment via readily available reactive groups that can be added to the molecule. The diversity and utility of chemistries suitable for conjugating fluorophores, including photosensitizers, to other molecules and surfaces is exemplified by the extensive literature on preparing nucleic acids derivatized with fluorophores. See, for example, Ullman et al, U.S. Pat. No. 3,996,345 and Khanna et al, U.S. Pat. No. 4,351,760.

D. Pharmaceutical composition

1. Formulation of pharmaceutical compositions

The pharmaceutical compositions provided herein comprise a therapeutically effective amount of one or more of the conjugates provided herein in a pharmaceutically acceptable carrier, which conjugates are useful for preventing, treating or ameliorating one or more symptoms of a disease or disorder associated with hyperproliferative tissue or neovascularization, or in which hyperproliferative tissue or neovascularization is implicated. Diseases or disorders associated with hyperproliferative tissue or neovascularization include, but are not limited to, cancer or carcinoma, tumors, acute glomerulonephritis, membranoproliferative glomerulonephritis, myeloma, psoriasis, atherosclerosis, psoriatic arthritis, rheumatoid arthritis, diabetic retinopathy, macular degeneration, neovascularization of the cornea, choroidal hemangioma. Pharmaceutical carriers suitable for administration of the conjugates provided herein include any such carrier known to those skilled in the art to be suitable for a particular mode of administration.

In addition, the compositions may be formulated in the composition as the sole pharmaceutically active ingredient, or may be combined with other active ingredients.

Pharmaceutical formulations include one or more of the conjugates provided herein. In one embodiment, the composition is formulated into suitable pharmaceutical formulations such as solutions, suspensions, tablets, dispersible tablets, pills, capsules, powders, sustained release formulations or elixirs for oral administration, or sterile solutions or suspensions for parenteral administration, as well as transdermal patch preparations and dry powder inhalers. In one embodiment, the conjugates described above are formulated into Pharmaceutical compositions using techniques and procedures well known in the art (see, e.g., Ansel, "Introduction to Pharmaceutical dosage forms," 4 th edition, 1985, page 126).

An effective concentration of one or more conjugates or pharmaceutically acceptable derivatives thereof is combined with a suitable pharmaceutical carrier. As mentioned above, the conjugates may be derivatized to the corresponding salts, esters, enol ethers or esters, acetals, ketals, orthoesters, hemiacetals, hemiketals, acids, bases, solvates, hydrates or prodrugs prior to formulation. The concentration of the compound in the composition is effective for delivering an amount that, upon administration, treats, prevents or ameliorates one or more symptoms of a disease or disorder associated with, or in which, hyperproliferative tissue or neovascularization is implicated.

In one embodiment, the conjugates disclosed herein are formulated for single dose administration. To formulate the composition, parts by weight (weight fraction) of the compound are dissolved, suspended, dispersed, or otherwise mixed in a carrier of choice at concentrations effective to alleviate, prevent, or ameliorate one or more symptoms of the condition being treated.

The components are included in a pharmaceutically acceptable carrier in an amount sufficient to produce a therapeutically beneficial effect in the treated patient without adverse side effects. Therapeutically effective concentrations can be determined empirically by testing compounds in vitro and in vivo systems known in the art, such as described in U.S. Pat. No. 5,952,366 to Pandey et al (1999), from which to extrapolate dosages for human use.

The concentration of the pharmaceutical composition will be determined by the rate of absorption, inactivation and excretion of the active ingredient, the physicochemical properties of the components, the dosing regimen and amount of administration, and other factors known to those skilled in the art. For example, the amount delivered is sufficient to ameliorate one or more symptoms of a disease or disorder associated with hyperproliferative tissue or neovascularization, or in which hyperproliferative tissue or neovascularization is implicated.

In one embodiment, a therapeutically effective dose should result in a serum concentration of the active ingredient of about 0.1ng/ml to about 50-100 μ g/ml. In another embodiment, the pharmaceutical composition should provide a dose of the compound of about 0.001mg to about 2000mg per kilogram body weight per day. Pharmaceutical dosage unit forms are prepared to provide from about 0.01mg, 0.1mg, or 1mg to about 500mg, 1000mg, or 2000mg, and in one embodiment from about 10mg to about 500mg of the active ingredient or combination of essential ingredients per dosage unit form.

The active ingredient may be administered at one time, or may be divided into a number of smaller doses to be administered at intervals. It will be understood that the precise dose and duration of treatment is a function of the disease being treated and may be determined empirically using known assay protocols or by extrapolation of in vivo or in vitro assay data. It should be noted that concentrations and dose values may vary with the severity of the disorder to be alleviated. It will be further understood that for any particular subject treated, the particular dosage regimen will be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the compositions, and that the concentration ranges set forth herein are exemplary only and are not intended to limit the practice or scope of the conjugates provided herein.

In the case where the component exhibits insufficient solubility, a method of dissolving the component may be used. Such methods are known to those skilled in the art and include, but are not limited to, the use of co-solvents such as dimethyl sulfoxide (DMSO), the use of surfactants such as TWEEN ®, or dissolution in aqueous sodium bicarbonate.

When the components are mixed or blended, the resulting mixture may be a solution, suspension, emulsion, or the like. The form of the resulting mixture will depend on a number of factors, including the intended mode of administration and the solubility of the components in the selected carrier or vehicle. The effective concentration is sufficient to ameliorate symptoms of the disease, disorder, condition being treated and is empirically determined.

The pharmaceutical compositions are provided for administration to humans and animals in unit dosage forms, such as tablets, capsules, pills, powders, granules, sterile parenteral solutions or suspensions, oral solutions or suspensions, and oil-water emulsions, which contain a suitable amount of the conjugate or a pharmaceutically acceptable derivative thereof. In one embodiment, the pharmaceutically therapeutically active composition is formulated or administered in a unit-dose form or a multi-dose form. Unit-dosage forms, as used herein, refer to physically discrete units suitable for human and animal subjects and packaged individually as is known in the art. Each unit-dose comprises a predetermined amount of the therapeutically active component sufficient to produce the desired therapeutic effect in combination with the required pharmaceutical carrier, vehicle or diluent. Examples of unit-dose forms include ampoules and syringes, and individually packaged tablets or capsules. The unit-dose form may be divided into several or multiple administrations. A multi-dose form is a plurality of identical unit-dose forms packaged in a single container for administration as separate unit dose forms. Examples of multi-dose forms include vials, bottles of tablets or capsules, or bottles of pints or gallons. Thus, a multi-dose form is a plurality of unit doses that are not divided on a package.

For example, liquid pharmaceutically administrable compositions may be prepared by dissolving, dispersing or otherwise mixing the active ingredient as defined above and optional pharmaceutical adjuvants in a carrier, such as water, saline, aqueous dextrose, glycerol, ethylene glycol, ethanol, and the like, to thereby form a solution or suspension. If desired, the pharmaceutical compositions for administration may also contain minor amounts of non-toxic auxiliary substances such as wetting agents, emulsifying agents, solubilizing agents, pH buffering agents and the like, for example, acetate, sodium citrate, cyclodextrin derivatives, sorbitan monolaurate, triethanolamine sodium acetate, triethanolamine oleate and other such agents.

The actual method of preparing such dosage forms is known or will be apparent to those skilled in the art; see, for example, "Remington's Pharmaceutical Sciences" (Mack Publishing Company, Easton, Pa., 15 th Ed., 1975).

Dosage forms or compositions containing the active ingredient in the range of 0.005% to 100% (the remainder consisting of non-toxic carriers) can be prepared. Methods for preparing these compositions are known to those skilled in the art. Contemplated compositions may contain from 0.001% to 100% of the active ingredient, for example from 0.1 to 95% in one embodiment, and from 75 to 85% in another embodiment.

2. Orally administrable compositions

Oral pharmaceutical dosage forms are solid, gel or liquid. The solid dosage forms are tablets, capsules, granules and powder (bulk) preparations. Types of oral tablets include compressed tablets, chewable lozenges and tablets which may be enteric-coated, sugar-coated or film-coated. Capsules may be hard or soft gelatin capsules, while granules and powders may be provided in non-effervescent or effervescent form in combination with other ingredients well known to those skilled in the art.

a. Solid composition for oral administration

In certain embodiments, the formulation is a solid dosage form; in one embodiment, it is a capsule or tablet. Tablets, pills, capsules, lozenges, and the like, may comprise one or more of the following ingredients or compounds of similar properties: binders, lubricants, diluents, glidants, disintegrants, colorants, sweeteners, flavoring agents, wetting agents, enteric coatings and film coatings. Examples of binders include microcrystalline cellulose, tragacanth gum, xanthan gum, dextrose solution, acacia syrup, gelatin solution, molasses, polyvinyl pyrrolidine, povidone, crospovidone, sucrose and starch paste. Lubricants include talc, starch, magnesium or calcium stearate, lycopodium and stearic acid. Diluents include, for example, lactose, sucrose, starch, kaolin, salt, mannitol, and dicalcium phosphate. Glidants include, but are not limited to, colloidal silicon dioxide. Disintegrating agents include croscarmellose sodium, sodium starch glycolate, alginic acid, corn starch, potato starch, bentonite, methylcellulose, agar and carboxymethylcellulose. Colorants include, for example, any approved standard-compliant water-soluble FD and C dyes, mixtures thereof; water insoluble FD and C dyes suspended on hydrated alumina. Sweetening agents include sucrose, lactose, mannitol, and artificial sweetening agents such as saccharin, and any number of spray dried flavors. Flavoring agents include natural flavors extracted from plants such as fruits and synthetic blends of compounds that produce a pleasant sensation such as, but not limited to, mint and methyl salicylate. Wetting agents include propylene glycol monostearate, sorbitan monooleate, diethylene glycol monolaurate and polyoxyethylene lauryl ether. Enteric coatings include fatty acids, fats, waxes, shellac, ammoniated shellac, and cellulose acetate phthalate. Film coatings include hydroxyethyl cellulose, gellan gum, sodium carboxymethyl cellulose, polyethylene glycol 4000, and cellulose acetate phthalate.

The conjugate or pharmaceutically acceptable derivative thereof may be provided in the form of a composition that protects it from the acidic environment of the stomach. For example, the composition may be formulated with an enteric coating that maintains its integrity in the stomach while releasing the active ingredient in the intestine. The components may also be formulated in combination with antacids or other such ingredients.

When the dosage unit form is a capsule, it may contain, in addition to materials of the above type, a liquid carrier such as a fatty oil. Moreover, the dosage unit form may contain a variety of other materials which modify the physical form of the dosage unit, such as sugar coatings or coatings of enteric agents. The compositions may also be administered as components of elixirs, suspensions, syrups, wafers, sprays (sprinkle), chewing gums and the like. Syrups may contain, in addition to the active ingredient, sucrose as a sweetening agent and certain preservatives, dyes and colorings and flavors.

The active substance may also be mixed with other active substances which do not impair the desired effect, or with substances which supplement the desired effect, such as antacids, H2 blockers and diuretics. The active ingredient is a conjugate as described above or a pharmaceutically acceptable derivative thereof. Higher concentrations, up to about 98% by weight of the active ingredient may be included.

In all embodiments, the tablet and capsule formulations may be coated in a manner known to those skilled in the art to modify or maintain the dissolution of the active ingredient. Thus, for example, they may be coated with conventional enteric coatings such as phenyl salicylate, waxes and cellulose acetate phthalate.

b. Liquid composition for oral administration

Liquid oral dosage forms include aqueous solutions, emulsions, suspensions, solutions and/or suspensions reconstituted from non-effervescent granules, and effervescent formulations reconstituted from effervescent granules. Aqueous solutions include, for example, elixirs and syrups. The emulsion is of the oil-in-water type or the water-in-oil type.

Elixirs are clear, sweetened, hydroalcoholic preparations. Pharmaceutically acceptable carriers used in elixirs include solvents. Syrups are concentrated aqueous solutions of sugars, such as sucrose, and may contain preservatives. An emulsion is a two-phase system in which one liquid is dispersed in another in the form of small spheres. Pharmaceutically acceptable carriers used in emulsions are non-aqueous liquids, emulsifiers and preservatives. Suspensions employ pharmaceutically acceptable suspending agents and preservatives. Pharmaceutically acceptable materials used in non-effervescent granules that are reconstituted into liquid oral dosage forms include diluents, sweeteners and wetting agents. Pharmaceutically acceptable materials used in effervescent granules that are reconstituted into liquid oral dosage forms include organic acids and sources of carbon dioxide. Coloring and flavoring agents are used in all of the dosage forms described above.

Solvents include glycerol, sorbitol, ethanol and syrup. Examples of preservatives include glycerol, methyl and propyl parabens, benzoic acid, sodium benzoate and alcohol. Examples of non-aqueous liquids utilized in emulsions include mineral oil and cottonseed oil. Examples of emulsifying agents include gelatin, gum acacia, gum tragacanth, bentonite, and surfactants such as polyethylene glycol sorbitan monooleate. Suspending agents include sodium carboxymethylcellulose, pectin, tragacanth, xanthan gum, magnesium aluminum silicate clay and acacia. Sweetening agents include sucrose, syrup, glycerin and artificial sweeteners such as saccharin. Wetting agents include propylene glycol monostearate, sorbitan monooleate, diethylene glycol monolaurate and polyoxyethylene lauryl ether. Organic acids include citric acid and tartaric acid. Sources of carbon dioxide include sodium bicarbonate and sodium carbonate. Colorants include any approved standard-compliant water-soluble FD and C dyes and derivatives thereof. Flavoring agents include natural flavors extracted from plants such as fruits and synthetic blends of compounds that produce a pleasant taste sensation.

For solid dosage forms, solutions or suspensions of, for example, propylene carbonate, vegetable oils or triglycerides, in one embodiment are encapsulated in gelatin capsules. Such solutions and their formulation and encapsulation are described in U.S. patent nos. 4,328,245, 4,409,239 and 4,410,545. For liquid dosage forms, for example, a solution of polyethylene glycol may be diluted with a sufficient amount of a pharmaceutically acceptable carrier, such as water, to allow easy metering.

Alternatively, liquid or semi-solid oral formulations can be prepared by dissolving or dispersing the active ingredient or salt in vegetable oils, glycols, triglycerides, propylene glycol esters (e.g., propylene carbonate) and other such carriers, and then encapsulating these solutions or suspensions in hard or soft gelatin capsule shells. Other useful formulations include those set forth in U.S. Pat. nos. RE28,819 and 4,358,603. Briefly, such formulations include, but are not limited to, those comprising the conjugates provided herein, dialkylated mono-or poly-alkylene glycols including, but not limited to, 1, 2-dimethoxymethane, diethylene glycol dimethyl ether, triethylene glycol dimethyl ether, tetraethylene glycol dimethyl ether, polyethylene glycol-350-dimethyl ether, polyethylene glycol-550-dimethyl ether, polyethylene glycol-750-dimethyl ether where 350, 550, 750 refer to the approximate average molecular weight of the polyethylene glycol, and one or more antioxidants such as Butylated Hydroxytoluene (BHT), Butylated Hydroxyanisole (BHA), propyl gallate, vitamin E, hydroquinone, ethanolamine, hydroxycoumarin, lecithin, cephalin, ascorbic acid, malic acid, sorbitol, phosphoric acid, thiodipropionic acid and its esters, and dithiocarbamates.

Other formulations include, but are not limited to, aqueous alcoholic solutions containing pharmaceutically acceptable acetals. The alcohol used in these formulations is any pharmaceutically acceptable water-miscible solvent having one or more hydroxyl groups, including but not limited to propylene glycol and ethanol. Acetals include, but are not limited to di (lower alkyl) acetals of lower alkyl aldehydes, such as acetaldehyde diethyl acetal.

3. Injections, solutions and emulsions

In one embodiment, parenteral administration characterized by injection subcutaneously, intramuscularly or intravenously is also contemplated by the present application. Injectables can be prepared in conventional forms, such as liquid solutions or suspensions, solid forms suitable for liquid solutions or suspensions prior to injection, or as emulsions. Injections, solutions and emulsions may also comprise one or more excipients. Suitable excipients are, for example, water, saline, glucose, glycerol or ethanol. In addition, if desired, the pharmaceutical compositions for administration may also contain minor amounts of non-toxic auxiliary substances such as wetting or emulsifying agents, pH buffering agents, stabilizers, solubilizing agents, and other such agents, for example, sodium acetate, sorbitan monolaurate, triethanolamine oleate and cyclodextrins.

It is also contemplated herein to implant a sustained release or sustained release system in order to maintain a constant level of dosage (see, e.g., U.S. Pat. No. 3,710,795). Briefly, the conjugates provided herein are dispersed in a solid internal matrix, such as polymethylmethacrylate, polybutylmethacrylate, plasticized or unplasticized polyvinyl chloride, plasticized nylon, plasticized polyethylene terephthalate, natural rubber, polyisoprene, polyisobutylene, polybutadiene, polyethylene, ethylene-vinyl acetate copolymers, silicone rubber, polydimethylsiloxane, silicone-carbonate copolymers, hydrophilic polymers such as hydrogels of acrylic and methacrylic acid esters, collagen, crosslinked polyvinyl alcohol, and partially crosslinked hydrolyzed polyvinyl acetate, which is encapsulated with an external polymeric film, such as polyethylene, polypropylene, ethylene/propylene copolymers, ethylene/ethyl acrylate copolymers, ethylene/vinyl acetate copolymers, silicone rubber, polydimethylsiloxane (rubber), Neoprene, chlorinated polyethylene, polyvinyl chloride, copolymers of vinyl chloride and vinyl acetate, vinylidene chloride, ethylene and propylene, polyethylene terephthalate ionomers, butyl rubber epichlorohydrin rubber, ethylene/vinyl alcohol copolymers, ethylene/vinyl acetate/vinyl alcohol terpolymers, and ethylene/vinyloxyethanol copolymers, which are insoluble in body fluids. In the release rate controlling step, the components diffuse through the outer polymeric membrane. The percentage of active ingredient contained in such parenteral compositions is highly dependent upon its particular nature, as well as the activity of the ingredient and the needs of the subject.

Parenteral administration of the compositions includes intravenous, subcutaneous and intramuscular administration. Formulations for parenteral administration include sterile solutions ready for injection, sterile dry soluble products such as lyophilized powders ready for combination with a solvent immediately prior to use, including subcutaneous tablets, sterile suspensions ready for injection, sterile dry insoluble products ready for combination with a vehicle immediately prior to use, and sterile emulsions. The solution may be aqueous or non-aqueous.

If administered intravenously, suitable carriers include physiological saline or Phosphate Buffered Saline (PBS), and solutions containing thickening and solubilizing agents such as glucose, polyethylene glycol, polypropylene glycol, and mixtures thereof.

Pharmaceutically acceptable carriers for use in parenteral formulations include aqueous vehicles, non-aqueous vehicles, antimicrobial agents, isotonic agents, buffers, antioxidants, local anesthetics, suspending and dispersing agents, emulsifying agents, sequestering or chelating agents, and other pharmaceutically acceptable materials.

Examples of aqueous vehicles include sodium chloride injection, ringer's injection, isotonic glucose injection, sterile water injection, dextrose and lactated ringer's injection. Non-aqueous parenteral vehicles include fixed oils of vegetable origin, cottonseed oil, corn oil, sesame oil and peanut oil. Antimicrobial agents at bacteriostatic or fungistatic concentrations must be added to parenteral preparations packaged in multi-dose containers, which include phenols or cresols, mercurials, benzeneMethanol, chlorobutanol, methyl and propyl parabens, thimerosal, benzalkonium chloride and benzethonium chloride. Isotonic agents include sodium chloride and glucose. Buffers include phosphates and citrates. The antioxidant comprises sodium bisulfate. Local anesthetics include procaine hydrochloride. Suspending and dispersing agents include sodium carboxymethylcellulose, xanthan gum, hydroxypropyl methylcellulose and polyvinylpyrrolidone. The emulsifier comprises polysorbate 80 (TWEEN)^®80). Sequestering or chelating agents for metal ions include EDTA. The pharmaceutical carrier also includes ethanol, polyethylene glycol and propylene glycol for water miscible vehicles; sodium hydroxide, hydrochloric acid, citric acid or lactic acid for adjusting the pH.

The concentration of the pharmaceutically active ingredient is adjusted so that the injection provides an effective amount to produce the desired pharmacological or therapeutic effect. The exact dosage will depend on the age, weight and condition of the patient or animal, as is known in the art.

Unit-dose parenteral formulations are packaged in ampoules, vials or syringes with needles. All parenteral formulations must be sterile, as is known and practiced in the art.

Illustratively, intravenous or intra-arterial infusion of a sterile aqueous solution containing the active ingredient is an effective mode of administration. Another embodiment is a sterile aqueous or oily solution or suspension containing □ the active substance necessary for injection to produce the desired pharmacological effect.

Injections are designed for local and systemic administration. In one embodiment, a therapeutically effective dose is formulated to contain a concentration of at least about O.1% w/w up to about 90% w/w or more, and in some embodiments, greater than l% w/w of the active ingredient to the tissue being treated.

The components may be suspended in micronized or other suitable form, or may be derivatized to produce a more soluble active product or to produce a prodrug. The form of the resulting mixture will depend on a number of factors, including the intended mode of administration and the solubility of the components in the selected carrier or vehicle. The effective concentration is sufficient to ameliorate the symptoms of the condition and can be determined empirically.

4. Freeze-dried powder

Of interest herein are also lyophilized powders, which can be reconstituted as a solution, emulsion, or other mixture for administration. They may also be reconstituted and formulated as solids or gels.

Sterile lyophilized powders are prepared by dissolving the conjugates provided herein, or pharmaceutically acceptable derivatives thereof, in a suitable solvent. The solvent may also contain excipients to improve stability, or other pharmacological components of the powder or solutions reconstituted from the powder. Excipients that may be used include, but are not limited to, glucose, sorbitol, fructose, corn syrup, xylitol, glycerol, glucose, sucrose, or other suitable agents. In one embodiment, the solvent may also comprise a buffer such as citrate, sodium phosphate or potassium phosphate, or other such buffers known to those skilled in the art, at about a neutral pH. The solution is then sterile filtered and subsequently lyophilized under standard conditions known to those skilled in the art to give the desired formulation. In one embodiment, the resulting solution is dispensed into vials for lyophilization. Each vial contains a single dose or multiple doses of the components. The lyophilized powder can be stored under suitable conditions, such as from about 4 ℃ to room temperature.

The lyophilized powder is reconstituted with water for injection to give a formulation for parenteral administration. For reconstitution, the lyophilized powder is added to sterile water or other suitable carrier. The exact amount depends on the composition selected. Such amounts may be determined empirically.

5. Topical administration of drugs

Topical mixtures were prepared as described for local and systemic administration. The resulting mixture may be in the form of a solution, suspension, emulsion, etc., and formulated as a cream, gel, ointment, emulsion, solution, elixir, lotion, suspension, tincture, paste, foam (foams), aerosol, rinse, spray, suppository, bandage, skin patch, or any other formulation suitable for topical administration.

The conjugate or pharmaceutically acceptable derivative thereof may be formulated as an aerosol for topical application, such as by inhalation (see, e.g., U.S. Pat. nos. 4,044,126, 4,414,209, and 4,364,923, which describe aerosols for delivery of steroids suitable for use in the treatment of inflammatory diseases, particularly asthma). These formulations for administration to the respiratory tract may be in the form of an aerosol or solution for a nebulizer, or a fine powder for inhalation, alone or in combination with an inert carrier such as lactose. In this case, the particles of the formulation will have a diameter of less than 50 microns in one embodiment; in another embodiment, has a diameter of less than 10 microns.

The compositions are formulated for topical application, such as to the skin and mucous membranes, such as for the eye, in the form of gels, creams and lotions, and for the eye or for intracisternal or intraspinal application. Topical administration is contemplated for transdermal administration and administration to the eye or mucosa or for inhalation therapy. Nasal solutions of the active ingredient alone or in combination with other pharmaceutically acceptable excipients may be administered. These solutions, particularly those intended for ophthalmic use, can be formulated as 0.01% to 10% isotonic solutions, pH about 5 to 7, containing appropriate salts.

6. Compositions for other routes of administration

Other routes of administration, such as transdermal patches including iontophoretic and electrophoretic devices, and rectal administration, are contemplated herein.

Transdermal patches including iontophoresis devices and electrophoresis devices are well known to those skilled in the art. Such patches are disclosed, for example, in U.S. patent nos. 6,267,983, 6,261,595, 6,256,533, 6,167,301, 6,024,975, 6,010,715, 5,985,317, 5,983,134, 5,948,433 and 5,860,957.

For example, pharmaceutical dosage forms for rectal administration are rectal suppositories, capsules and tablets which produce a systemic effect. Rectal suppositories are used herein refer to solid objects inserted into the rectum which melt or soften at body temperature to release one or more pharmacologically or therapeutically active ingredients. The pharmaceutically acceptable substances used in rectal suppositories are bases or vehicles and agents that raise the melting point. Examples of bases include cocoa butter (theobroma oil), glycerol-gelatin, carbowaxes (polyethylene glycols) and suitable mixtures of mono-, di-and triglycerides of fatty acids. Combinations of different matrices may be used. Agents that raise the melting point of suppositories include spermaceti and wax. Rectal suppositories may be prepared by compression methods or by molding methods. In one embodiment, the rectal suppository weighs about 2-3 grams.

Tablets and capsules for rectal administration are prepared using the same pharmaceutically acceptable materials and by the same methods as for oral administration of the formulations.

E. Article of manufacture

Thus, the composition or pharmaceutically acceptable derivative thereof can be packaged as an article of manufacture comprising a packaging material, a conjugate provided herein or a pharmaceutically acceptable derivative thereof within the packaging material effective to modulate the activity of hyperproliferative tissue or neovascularization, or to treat, prevent or ameliorate a hyperproliferative tissue or neovascularization-mediated disease or disorder or one or more symptoms of a disease or disorder in which hyperproliferative tissue or neovascularization activity is implicated, and a label identifying the use of the conjugate or pharmaceutically acceptable derivative thereof for modulating the activity of hyperproliferative tissue or neovascularization or treating, preventing or ameliorating a hyperproliferative tissue or neovascularization-mediated disease or disorder or one or more symptoms of a disease or disorder in which hyperproliferative tissue or neovascularization is implicated.

Articles of manufacture provided herein comprise packaging materials. Packaging materials for packaging pharmaceutical products are well known to those skilled in the art. See, for example, U.S. patent nos. 5,323,907, 5,052,558, and 5,033,252. Examples of pharmaceutical packaging materials include, but are not limited to, blister packs, bottles, tubes, inhalers, pumps, bags, vials, containers, syringes, bottles, and any packaging material suitable for the formulation selected and the intended mode of administration and treatment. Numerous formulations of conjugates and compositions provided herein are contemplated, as are various treatments for diseases or disorders in which hyperproliferating tissue or neovascularization is implicated as a mediator or contributor or cause of the symptoms.

F. Reagent kit

Any of the conjugates disclosed herein or a pharmaceutically acceptable derivative thereof can be provided in a kit with instructions for use in guiding any of the methods disclosed herein. The instructions may be in any tangible form such as printed paper, a computer diskette that instructs a person how to perform the method, a video tape or digital video device containing instructions that instruct a person how to perform the method, or a computer memory that receives data from a remote location and that instructs or otherwise provides instructions to the person (e.g., via the internet).

Further provided are kits for detecting a target tissue or target component, e.g., in a sample, or for diagnosing an infectious agent, comprising any of the conjugates described herein comprising a targeting moiety that targets a target tissue or target component and instructions, e.g., for performing an assay or for interpreting a result or for aiding in determining whether a target tissue is present in a subject, or whether a subject is infected with an infectious agent. The kit also optionally contains one or more containers (microtiter plates, eppendorf tubes, etc.) for holding the conjugate or performing the assay. The kit also includes standards for calibrating any detection reaction or assay using the conjugate.

G. Methods of using conjugates

PDT, methods of diagnostic and therapeutic application

Briefly, the composition is typically administered to the subject prior to the target tissue, target composition, or subject receiving light. The compositions are administered in the manner described elsewhere herein.

The dosage of the conjugates disclosed herein to achieve optimal therapeutic levels can be determined clinically. A certain length of time is allowed to elapse for the circulating or locally delivered conjugate to be absorbed by the target tissue. During this waiting period, unbound conjugate is cleared from the circulation, or additional time may optionally be provided for clearing unbound conjugate from non-target tissue. The duration of waiting will be determined clinically and may vary depending on the composition of the composition.

When the waiting period is over, the bound conjugate is activated using a light source. The light source may provide incoherent light (non-laser light) or coherent light (laser light). For example, incoherent light sources include, but are not limited to, mercury or xenon arc lamps with filters, tungsten lamps, cold cathode fluorescent lamps, halogen lamps, Light Emitting Diodes (LEDs), LED arrays, incandescent sources, and other electroluminescent devices. The light source is used when it is not necessary to precisely define the illumination area or when a large area is to be illuminated. Focused incoherent light can be used to illuminate small areas, such as by using lenses to focus the light or filters to direct or transmit the light. Laser sources are often used to illuminate small, well-defined areas because of their higher specific illumination and more controllable beam properties. Coherent light sources include, but are not limited to, dye lasers, argon ion lasers, laser diodes, tunable lasers, titanium-sapphire lasers, ruby lasers, alexandrite lasers, helium-neon lasers, GaAlAs and InGaAs diode lasers, Nd-YLF lasers, Nd-glass lasers, Nd-YAG lasers, and fiber lasers. For example, lasers are commonly used as excitation sources in confocal devices and produce very high fluxes. Laser sources are limited in that they emit a limited, often discontinuous set of wavelengths as compared to lamps, which typically produce a continuous spectrum that can be filtered to provide any desired spectral band within a certain range.

The area of illumination is determined by the size and location of the pathological site to be detected, diagnosed or treated. The duration of the illumination period will depend on whether detection or treatment is to be performed and can be determined empirically. A total or cumulative time period of approximately 1 minute to 72 hours may be used. In one embodiment, the illumination period is between about 4 minutes and 48 hours. In another embodiment, the illumination period is between about 30 minutes and 24 hours.

The total fluence or energy of light used for illumination is between about 10 joules and about 25,000 joules; in some embodiments, the total fluence is between about 100 joules and about 20,000 joules or between about 500 joules and about 10,000 joules. Whether for fluorescence detection or therapeutic treatment to destroy or damage target tissue or target components, light of a fluence and wavelength sufficient to produce the desired effect is selected. Light having a wavelength at least partially corresponding to the characteristic light absorption wavelength of the photosensitizing agent is used to illuminate the target tissue.

The energy delivered by the light used is measured in watts, where 1 watt equals 1 joule/second. Intensity is the energy per area. Thus, the intensity may be measured in watts per square centimeter. Therefore, the intensity of light for irradiation in the present invention may be about 5mW/cm²To about 500mW/cm². Since the total fluence or energy of light in joules is divided by the duration of the total exposure time in seconds, the longer the target is exposed to irradiation without increasing the amount of light intensity used, the greater the amount of fluence or total energy used. The total fluence of irradiation used in the present invention is high enough to activate the conjugates disclosed herein.

In one embodiment of photodynamic therapy using the conjugates disclosed herein, the conjugate is injected into a mammal, such as a human, to be diagnosed or treated. The level of injection is generally between about 0.1 to about 0.5. mu. mol/kg body weight. In the case of treatment, the site to be treated is exposed to light of the desired wavelength and energy, e.g., about 10-200J/cm². In the case of detection, fluorescence is measured when exposed to light of a wavelength sufficient to cause the conjugate to fluoresce, which is different from the wavelength of light used to illuminate the conjugate. The energy used in the detection is sufficient to cause fluorescence and is generally significantly less than the energy required for treatment.

2. Detecting target tissue or target composition

In addition to PDT, the compositions provided herein can be used to detect target cells, target tissues, or target components of a subject. When a conjugate provided herein is used to detect a target tissue or target component, the conjugate is introduced into a subject and allowed sufficient time for the conjugate to accumulate in the target tissue or associate with the target component. The treated area is then irradiated, typically with light of an energy sufficient to cause the conjugate to fluoresce, and the energy used is typically significantly lower than that required for photodynamic therapy treatment. Fluorescence is measured when exposed to light of a desired wavelength, and the amount of fluorescence can be qualitatively or quantitatively correlated with the presence of the conjugate, according to methods known in the art.

3. Diagnosis of infectious agents

The conjugates provided herein can be used to diagnose the presence of an infectious agent or the identity of an infectious agent in a subject. In this embodiment, the targeting moiety of the conjugates provided herein is selected to be specific for an infectious agent. For example, the targeting moiety of choice may be an antibody or antibody fragment that selectively associates with the infectious agent, and after allowing sufficient time for the disclosed conjugate to associate with the infectious agent and clear from non-target tissues, the conjugate is visualized, such as by exposure to light of an energy sufficient to cause the conjugate to fluoresce. As an example, any one of the conjugates provided herein can include an antibody as a targeting moiety that targets a suitable Helicobacter pylori (Helicobacter pylori) antigen. The conjugate is formulated as a pharmaceutical preparation which, when introduced into a patient, releases the conjugate to the gastric mucus/epithelial layer where the bacteria are found. After a sufficient time for the conjugate to selectively associate with the target infectious agent and for the unbound conjugate to clear from non-target tissue, the subject may be examined to determine whether any H.pylori is present. For example, the protocol may be carried out by illuminating the suspected target area with light of an energy sufficient to cause fluorescence of the conjugate, for example using an optical fibre and detecting any fluorescence of the conjugate.

4. Fluorescence immunoassay

One problem that plagues fluorescent immunoassays is the discrimination of the fluorescent signal of the target from background illumination. The signal intensity from background illumination may be up to 10,000 times greater than the fluorescence signal intensity of the target. The problem of background detection is particularly significant in the measurement of biological samples. For example, in the analysis of plasma, the presence of a naturally occurring fluorescent substance, biliverdin, causes substantial background illumination. Such compounds are highly fluorescent and contribute significant background signals that interfere with the signal of the label, thus reducing the sensitivity of assays using fluorescein labels.

When any of the disclosed conjugates is used for diagnostic purposes, the photosensitizer component need only function as a fluorophore. The quencher of this embodiment serves to prevent the generation of false positive signals that would otherwise be produced by a fluorophore that is not bound to the target. The photosensitizing agent can function as a fluorophore only when the targeting moiety of the disclosed conjugate interacts with the target cell, target tissue, or target component, the quenching agent leaves the fluorescence-quenching interaction-permissive location where the photosensitizing agent is present.

Fluorescence immunoassays are well known to those skilled in the art. For example, in one embodiment, the sample can be analyzed for the presence of an infectious agent or target component. The sample may be immobilized on a solid support or the assay may be performed in solution. The disclosed conjugate is added and incubated with the sample under bioassay conditions. If the test sample is immobilized on a solid support, excess unbound conjugate can optionally be removed, for example, by washing the solid support with buffer, saline, or distilled water. Because of the nature of the conjugates disclosed herein, only conjugates that bind to the target through the targeting moiety will fluoresce when illuminated. The detection and measurement of the conjugate bound to the sample to be analyzed results in a value that can be compared to a comparative value for qualitative or quantitative determination.

The following examples are included for illustrative purposes only and do not limit the scope of the present invention.

Examples

Example 1

A photosensitizer, such as talaporfin sodium, is covalently linked to one end of the single stranded oligonucleotide. The oligonucleotide starts and ends with mutually complementary sequences, the remaining sequence of which consists of a binding sequence known to have a suitable degree of binding affinity for the target tissue or structure. The opposite end of the oligonucleotide is linked by a covalent bond to a non-fluorescing quencher.

A therapeutically beneficial amount of the conjugate is administered to a subject. After a sufficient time for the agent to bind to the intended target and clear from normal tissue, a therapeutically beneficial amount of light is delivered to a site including a lesion or region of hyperproliferative tissue using a light source of appropriate wavelength.

Example 2

In another embodiment, the photosensitizer talaporfin sodium is derivatized with a water soluble carbodiimide reagent and a commercially available alpha, omega-diaminoalkane linker such as 1, 3-diaminopropane to give the monoamino compound shown in figure 2. The linker is then attached by a targeting moiety, such as a thiol-reactive linking moiety on an antibody or polymer, using techniques known in the art, which demonstrates selective targeting in biological systems such as oligonucleotides or oligopeptides. These generally involve treatment with an electrophile (such as a haloacetyl or maleimido group) which chemically reacts with the thiol function. The mono-amino structured species depicted in fig. 2 allows for the preparation of regiochemically defined species in which the quencher is covalently linked to the remainder of the composition. One of ordinary skill in the art can use this method to link the quencher to an oligonucleotide, which is obtained in a commercially available form in which the thiol-terminated alkyl group is located on the 5' phosphate.

Example 3

Photosensitizers such as talaporfin sodium are attached to one end of the polymer via an amide bond, which polymer is known to exhibit selective binding to a target. The opposite end of the polymer is conjugated to a quencher such as a dabcyl (4- (4' -dimethylaminobenzazo) benzoyl) group by reaction with a commercially available reagent such as dabcyl chloride. This agent may be further modified by the addition of suitable metal ions to the aqueous solution of the composition. The metal binds to the coordination pocket of the porphyrin ring-system and also coordinates the amine or azo group of the quenching group, ensuring that the quenching agent is still sufficiently close to the photosensitizer to allow energy transfer and thereby quench the generation of singlet oxygen. However, binding of the targeting polymer to its target disrupts this coordination binding environment, releasing the quencher from the metal and allowing the quencher to leave the photosensitizer and restore its activity.

A therapeutically beneficial amount of this exemplary conjugate is administered to a subject. After a sufficient time for the conjugate to bind to the intended target and clear from normal tissue, a therapeutically beneficial amount of light is delivered to a site including a lesion or region of hyperproliferative tissue using a light source of appropriate wavelength.

Since modifications will be apparent to those skilled in the art, it is intended that the invention be limited only by the scope of the appended claims. All patents, published patent applications, and non-patent documents referred to in this application are incorporated herein by reference.

Claims (64)

1. A conjugate, comprising:

fluorophores or photosensitizers

A quenching agent; and

a targeting moiety, wherein:

the fluorophore or photosensitizer is attached to the quencher and the targeting moiety in such a way that activation of the fluorophore or photosensitizer is quenched until the targeting moiety binds to the target whereupon the quencher is moved away from the photosensitizer, enabling activation of the photosensitizer upon illumination with light of a suitable wavelength.

2. The conjugate of claim 1, wherein the fluorophore is 5- ((2-aminoethyl) -amino) naphthalene-1-sulfonic acid (EDANS).

3. The conjugate of claim 1, wherein the photosensitizer is a porphyrin.

4. The conjugate of claim 1, wherein the photosensitizer is a chlorin

5. The conjugate of claim 1, wherein the photosensitizer is bacteriochlorin.

6. The conjugate of claim 1, wherein the quencher is □ -carotene or a derivative thereof.

7. The conjugate of claim 1, wherein the targeting moiety is an antibody.

8. The conjugate of claim 1, wherein the targeting moiety is a fragment or other derivative of an antibody.

9. The conjugate of claim 1, wherein the targeting moiety is selected from the group consisting of an antigen, a ligand, a receptor, one member of a specific binding pair, a polyamide, a peptide, an oligosaccharide, a polysaccharide, a Low Density Lipoprotein (LDL) or an apoprotein of LDL, a steroid derivative, a hormone, and a hormone-mimetic.

10. The conjugate of claim 1, wherein the photosensitizer and quencher comprise a linking component linked to an amino or hydroxy fatty acid or sulfonic acid of 1 to 20 carbon atoms using an ester, amide or sulfonamide linkage.

11. The conjugate of claim 10, wherein the linking component is a 20-60 residue oligonucleotide.

12. The conjugate of claim 11, wherein the oligonucleotide comprises a specific sequence that binds to a desired target, and at least one pair of mutually complementary regions that cause it to adopt a conformation in which, in the absence of the target, the quencher is sufficiently proximal to the photosensitizer to render the photosensitizer inactive; wherein binding of the target-specific sequence to the target disrupts the conformation, allowing the photosensitizer to become active upon illumination with light of a suitable wavelength.

13. The conjugate of claim 1, wherein the quencher is 4- (4 '-dimethylaminophenylazo) benzoic acid (DABCYL) or 4- (4' -dimethylaminophenylazo) sulfonic acid (DABSYL).

14. The conjugate of claim 1, wherein the photosensitizer and quencher are linked by a polymer that exhibits binding specificity for a desired target, wherein in the absence of target, the linking system adopts a conformation in which the quencher is sufficiently close to the photosensitizer to render it photochemically inert, and wherein in the presence of target, the conformation is disrupted allowing the photochemical process to proceed.

15. The conjugate of claim 1, wherein the photosensitizer comprises a porphyrin or porphyrin derivative tetrapyrrole and has a physiologically acceptable metal atom in its central coordination cavity, and one or more suitable functional groups are located on or near the quencher that is effectively coordinated to the axial position of the metal coordinated inside the photosensitizer; and the targeting moiety is positioned in such a way that the presence of the target disrupts the relatively weak association of the axial ligand with the metal, releases the quencher and renders the fluorescer or PDT agent active.

16. The conjugate of claim 1, wherein the fluorophore emits light of a suitable wavelength for exciting more than one type of second fluorophore or photosensitizer, and the presence of said more than one second fluorophore or photosensitizer causes a different reaction of the component, which is set appropriate for the presence of each target that can react with said component.

17. The conjugate of claim 1, wherein the targeting moiety is a polymer having at least one sulfate or sulfonate functional group.

18. The conjugate of claim 17, wherein the targeting moiety is dextran sulfate.

19. The conjugate of claim 18, wherein the dextran sulfate has an average molecular weight of about 5,000.

20. The conjugate of claim 1, wherein the photosensitizer is talaporfin sodium.

21. A pharmaceutical composition comprising the conjugate of claim 1 or a pharmaceutically acceptable derivative thereof in a pharmaceutically acceptable carrier.

22. An article of manufacture, comprising:

a packaging material; and

the conjugate of claim 1, or a pharmaceutically acceptable derivative thereof, contained within a packaging material, wherein:

the conjugate or a pharmaceutically acceptable derivative thereof is effective in photodynamic therapy treatment for ameliorating the symptoms of a hyperproliferative disorder; and is

The packaging material includes a label indicating use of the component or salt thereof in photodynamic therapy treatment to ameliorate symptoms of a hyperproliferative disorder.

23. A method of administering photodynamic therapy to a target comprising:

(i) administering to a subject the conjugate of claim 1, or a pharmaceutically acceptable derivative thereof, that preferentially associates with a target; and

(ii) the subject is irradiated with light of a wavelength and total energy flow sufficient to produce a therapeutic effect.

24. The method of claim 23, wherein the target is selected from the group consisting of vascular endothelial tissue, neovascular structural tissue present in the eye, abnormal vessel walls of tumors, solid tumors, tumors of the head, tumors of the neck, tumors of the eye, tumors of the gastrointestinal tract, tumors of the liver, tumors of the breast, tumors of the prostate, tumors of the lung, non-solid tumors, malignant cells of one of the hematopoietic and lymphoid tissues, lesions in the vascular system, diseased bone marrow, and diseased cells, wherein the disease is one of an autoimmune disease and an inflammatory disease.

25. The method of claim 24, wherein the target component is selected from the group consisting of bacteria, viruses, fungi, protozoa, and toxins.

26. The method of claim 24, further comprising the step of allowing sufficient time for any conjugates that do not preferentially associate with the target to clear from non-target tissue of the subject prior to the irradiating step.

27. A method of photodynamic therapy for treating hyperproliferative tissue in a subject, comprising:

(i) administering to a subject the conjugate of claim 1, or a pharmaceutically acceptable derivative thereof, that preferentially associates with hyperproliferative tissue, and

(ii) the subject is irradiated with light having a wavelength and fluence sufficient to activate the conjugate, thereby destroying or damaging the hyperproliferative tissue.

28. A method of detecting the presence of a target tissue in a subject, comprising:

(i) administering to a subject a sufficient amount of the conjugate of claim 1, or a pharmaceutically acceptable derivative thereof, that preferentially associates with a target tissue; and

(ii) the conjugate is visualized in the subject.

29. The method of claim 28, wherein the step of visualizing is accomplished by exposing the conjugate with light of sufficient energy to cause the component to fluoresce.

30. A method of detecting a target in a biological sample, comprising:

(i) adding to a biological sample a conjugate of claim 1 or a pharmaceutically acceptable derivative thereof that binds to a target; and

(ii) and (4) detecting the components.

31. The method of claim 32, wherein the biological sample is selected from the group consisting of blood, urine, saliva, tears, synovial fluid, sweat, interstitial fluid, semen, cerebrospinal fluid, ascites fluid, and/or tumor tissue biopsy and circulating tumor cells.

32. A method of diagnosing an infectious agent in a patient, comprising:

(i) administering to a patient the conjugate of claim 1 or a pharmaceutically acceptable derivative thereof having a targeting moiety that binds an infectious agent; and

(ii) the conjugate is visualized in the patient.

33. The method of claim 32, wherein the step of visualizing is accomplished by exposing the conjugate with light of sufficient energy to cause the conjugate to fluoresce.

34. A method of imaging a target tissue or target component in a subject, comprising:

(i) administering to a subject the conjugate of claim 1 or a pharmaceutically acceptable derivative thereof; and

(ii) imaging at least a portion of the subject, the portion and the conjugate preferentially associate.

35. A kit for treating a hyperproliferative disorder comprising the conjugate of claim 1, or a pharmaceutically acceptable derivative thereof, and instructions teaching a method of photodynamic therapy.

36. A kit for specifically labeling a cell or tissue, comprising:

the conjugate of claim 1, or a pharmaceutically acceptable derivative thereof, comprising a targeting moiety that targets a particular cell or tissue; and

instructions for a fluorescence imaging method are taught.

37. A combination, comprising:

the conjugate of claim 1 or a pharmaceutically acceptable derivative thereof; and

a light source.

38. A conjugate, comprising:

a tetrapyrrole or tetrapyrrole derivative photosensitizer comprising a physiologically acceptable metal atom in its central coordination cavity;

a quencher comprising one or more suitable functional groups that coordinate to an axial position of the metal coordinated within the photosensitizer and position the quencher in an energy transfer conformation with the photosensitizer such that activation of the photosensitizer is quenched; and

a targeting moiety, wherein binding of the targeting moiety to the target disrupts the association of the axial ligand of the quencher and the metal, releasing the quencher and allowing the photosensitizer to activate.

39. The conjugate of claim 38, wherein the photosensitizer is a porphyrin.

40. The conjugate of claim 38, wherein the photosensitizer is a chlorin.

41. The conjugate of claim 38, wherein the photosensitizer is bacteriochlorin.

42. The conjugate of claim 38, wherein the photosensitizer is talaporfin sodium.

43. The conjugate of claim 38, wherein the quencher is □ -carotene or a derivative thereof.

44. The conjugate of claim 38, wherein the targeting moiety is an antibody and a fragment of an antibody or other derivative of an antibody.

45. The conjugate of claim 38, wherein the targeting moiety is selected from the group consisting of an antigen, a ligand, a receptor, one member of a specific binding pair, a polyamide, a peptide, an oligosaccharide, a polysaccharide, a Low Density Lipoprotein (LDL) or an apoprotein of LDL, a steroid derivative, a hormone, and a hormone mimetic.

46. The conjugate of claim 38, wherein the targeting moiety is dextran sulfate.

47. The conjugate of claim 38, wherein the quenching agent is 4- (4 '-dimethylaminophenylazo) benzoic acid (DABCYL) or 4- (4' -dimethylaminophenylazo) sulfonic acid (DABSYL).

48. A pharmaceutical composition comprising the conjugate of claim 38 or a pharmaceutically acceptable derivative thereof in a pharmaceutically acceptable carrier.

49. An article of manufacture, comprising:

a packaging material; and

the conjugate of claim 38, or a pharmaceutically acceptable derivative thereof, contained within a packaging material, wherein:

the conjugate or a pharmaceutically acceptable derivative thereof is effective in photodynamic therapy treatment to ameliorate the symptoms of a hyperproliferative disorder; and

the packaging material includes a label indicating use of the component or salt thereof in photodynamic therapy treatment to ameliorate symptoms of a hyperproliferative disorder.

50. A method of administering photodynamic therapy to a target comprising:

(i) administering to a subject the conjugate of claim 38, or a pharmaceutically acceptable derivative thereof, that preferentially associates with a target; and

(ii) the subject is irradiated with light of a wavelength and total energy sufficient to produce a therapeutic effect.

51. The method of claim 50, wherein the target is selected from the group consisting of vascular endothelial tissue, neovascular structural tissue present in the eye, abnormal vascular walls of tumors, solid tumors, tumors of the head, tumors of the neck, tumors of the eye, tumors of the gastrointestinal tract, tumors of the liver, tumors of the breast, tumors of the prostate, tumors of the lung, non-solid tumors, malignant cells of one of the hematopoietic and lymphoid tissues, lesions in the vascular system, diseased bone marrow, and diseased cells, wherein the disease is one of an autoimmune disease and an inflammatory disease.

52. The method of claim 50, wherein the target component is selected from the group consisting of bacteria, viruses, fungi, protozoa, and toxins.

53. The method of claim 50, further comprising the step of allowing sufficient time for any conjugates that do not preferentially associate with the target to clear from non-target tissue of the subject prior to the irradiating step.

54. A method of photodynamic therapy for treating hyperproliferative tissue in a subject, comprising:

(i) administering to a subject the conjugate of claim 38, or a pharmaceutically acceptable derivative thereof, that preferentially associates with hyperproliferative tissue, and

(ii) the subject is irradiated with light having a wavelength and fluence sufficient to activate the conjugate, thereby destroying or damaging the hyperproliferative tissue.

55. A method of detecting the presence of a target tissue in a subject, comprising:

(i) administering to a subject a sufficient amount of the conjugate of claim 38, or a pharmaceutically acceptable derivative thereof, that preferentially associates with a target tissue; and

(ii) the conjugate is visualized in the subject.

56. The method of claim 55, wherein the step of visualizing is accomplished by exposing the conjugate with light of sufficient energy to cause the component to fluoresce.

57. A method of detecting a target in a biological sample, comprising:

(i) adding to a biological sample a conjugate of claim 38 or a pharmaceutically acceptable derivative thereof that binds to a target; and are

(ii) The component is detected.

58. The method of claim 57, wherein the biological sample is selected from the group consisting of blood, urine, saliva, tears, synovial fluid, sweat, interstitial fluid, semen, cerebrospinal fluid, ascites fluid, and/or tumor tissue biopsy and circulating tumor cells.

59. A method of diagnosing an infectious agent in a patient, comprising:

(i) administering to a patient the conjugate of claim 38 or a pharmaceutically acceptable derivative thereof having a targeting moiety that binds an infectious agent; and

(ii) the conjugate is visualized in the patient.

60. The method of claim 59, wherein the step of visualizing is accomplished by exposing the conjugate with light of sufficient energy to cause the conjugate to fluoresce.

61. A method of imaging a target tissue or target component of a subject, comprising:

(i) administering to a subject the conjugate of claim 38 or a pharmaceutically acceptable derivative thereof; and

(ii) imaging at least a portion of the subject, the portion and the conjugate preferentially associate.

62. A kit for treating a hyperproliferative disorder comprising the conjugate of claim 38, or a pharmaceutically acceptable derivative thereof, and instructions teaching a method of photodynamic therapy.

63. A kit for specifically labeling a cell or tissue, comprising:

the conjugate of claim 38, or a pharmaceutically acceptable derivative thereof, comprising a targeting moiety that targets a particular cell or tissue; and

instructions for use of the fluorescence imaging method are taught.

64. A combination, comprising:

the conjugate of claim 38 or a pharmaceutically acceptable derivative thereof; and

a light source.