WO2009038776A1 - Therapeutic nanoconjugates - Google Patents

Abstract

The construction and use of nanoconjugate molecules that incorporate a linker cleavable by an exogenous agent along with a therapeutic or diagnostic moiety are described. These novel nanoconjugates utilize a drug release mechanism which may incorporate photodynamic therapy drugs or similar compounds and a paired cleavable linker to target, image and/or deliver therapeutic concentrations of free drug to target cells such as cancer cells. Thus, this nanomedicine provides a highly specific and potent drug delivery system with highly improved therapeutic index over the free drug.

Description

THERAPUETIC NANOCONJUGATES

FIELD OF THE INVENTION

[0001] The present invention relates to therapeutic and imaging nanoconjugate molecules.

BACKGROUND OF THE INVENTION

[0002] The following discussion is provided solely to assist the understanding of the reader, and does not constitute an admission that any of the information discussed or references cited constitute prior art to the present invention.

[0003] As the number of cancer patients continues to rise, promising new therapies offer hope in improving the treatment of so many individuals. One of the most exciting of these new approaches involves the use of nanotechnology to create innovative drug delivery systems. These "nanomedicines" are designed to more precisely deliver drugs to tumor sites and maintain therapeutic concentrations of drugs for longer periods of time (Huang et al., 2001 ; Moses et al., 2003).

[0004] Currently approved nanomedicines rely on biodegradable polymers containing entrapped anticancer drugs, which are placed in the body but which offer only a modicum of specificity and controlled release. These medicines rely upon their increased duration of circulation and release relative to low molecular weight drugs alone. The slow release facilitates passive tumor targeting caused by the leakiness of angiogenic tumor blood vessels by the Enhanced Permeability and Retention effect (EPR effect), Examples of such medicines include treatments for prostate cancer (Tsukagoshi, 2002; Heyns et al., 2003) and brain cancer (glioblastoma multiforme) (Brem, 1995; Westphal, 2003).

[0005] Although these are examples of important advances, scientists are now looking to develop more sophisticated, covalently bound, biologically active, polymer-based drug delivery systems to replace those where drugs are non- covalently complexed or simply entrapped. These new medical nanotechnologies offer hope of targeting cancers more precisely. There have been recent developments in creating polymer-drug conjugates (Duncan, 2003) which are administered intravenously. Researchers have worked with linear polymers such as N-(2-hydroxypropyl)methacrylamide (HPMA) copolymers, polyglutamic acid (PGA), polyethyleneglycol (PEG), and polysaccharides (e.g., dextran); and they are now creating new polymeric architectures called dendrimers.

[0006] Several polymer-drug conjugates have progressed into clinical testing (e.g., Polyglutamate-paclitaxel with tradename Xyotax, and Dextran-doxorubicin), and the first product approval of a polymer-drug conjugate is expected to occur within the next two years. In addition, preclinical studies suggest opportunities for tumor-specific targeting of polymer conjugates using antibodies (Allen, 2002).

[0007] The aim of polymer-drug conjugation is to (a) improve drug targeting and therapeutic index, (b) reduce drug toxicity through limited access to sites of toxicity, and (c) overcome the mechanisms of drug resistance (Duncan, 2006). Whereas low molecular weight anticancer drugs distribute themselves randomly throughout the body shortly after administration and typically leave the circulation system within minutes, polymer-drug conjugates enhance drug targeting and limit toxicity by reducing their cellular uptake while in route to the tumor site. Furthermore, the ability to attach targeting ligands (i.e. antibodies, peptides, sugars) to the conjugate that are specific to the tumor allows true drug targeting by receptor mediated delivery (Kopecek et al., 2000).

[0008] Although researchers have succeeded in improving drug characteristics with polymers, the recently failed clinical trials with polymer-paclitaxel and camptothecin underline the importance of designing an improved nanomedicine (Meerum Terwogt et al., 2001 ; Schoemaker et al., 2002). Several obstacles remain for creating highly effective, targeted polymer conjugates. These include:

• use of unsuitable polymer-drug linkers, which are either too stable (preventing drug release), or too unstable (causing premature drug release);

• inadequate drug loading; and

• ineffective accumulation of the drug at the site of action. SUMMARY OF THE INVENTION

[0009] The present invention provides a method and compositions for controlled release of therapeutic agents (e.g., active therapeutic or prophylactic agents) and/or imaging or other diagnostic agents at a desired site, such as a desired site of action. A particular advantageous application of this method is for destroying and/or imaging abnormal tissue or cells, e.g., cancer tissue or cells, in a patient, typically at an internal treatment site. The method of the invention commonly involves using a therapeutic and/or diagnostic nanoconjugate, e.g., an anticancer nanoconjugate. (In describing the present invention, emphasis is placed on therapeutics and especially anticancer agents. Such description also applies to the other therapeutic nanoconjugates and applications, as well as imaging and other diagnostic applications unless the context dictates a narrower application.) Such nonoconjugates can provide targeted delivery and temporal control of the release of an active agent, which is often a therapeutic agent such as a small molecule drug, e.g., anticancer drug. Particularly useful examples utilize photocleavable linkers to release the active agent, with the cleavage accomplished by reaction with singlet oxygen produced by a photosensitizer in response to an appropriate light exposure. The nanoconjugate can be configured to include multiple releasable moieties (which may be the same or different), which may, for example, be linked on a polymer backbone or scaffold. Such an arrangement allows rapid release of large numbers of therapeutic agent molecules, which can provide locally high concentrations of the agent or agents. Thus, the invention also concerns such nanoconjugate molecules.

[0010] As indicated, in addition to therapeutic applications, the invention can be applied to assist in imaging or diagnosis, either in conjunction with delivery of therapeutic agents, or separately. That is, the nanoconjugate that includes a therapeutic agent moiety may also include an imaging agent moiety and/or a portion of the nanoconjugate (e.g., a portion exclusive of the therapeutic agent moiety) may function as an imaging moieity. Such an imaging moiety may, for example, be a contrast agent. Alternatively, a nanoconjugate with an imaging agent but without a therapeutic agent moiety may be used, either in conjunction with a separate nanoconjugate having a therapeutic moiety or alone. [0011] Thus, a first aspect of the invention concerns a method for destroying and/or imaging abnormal cells in a patient at an internal treatment site by administering to a subject having such abnormal cells a conjugate that includes at least one therapeutic or imaging agent moiety, a photosensitizer, a photocleavable linker, targeting agent, e.g., a specific binding agent targeting the abnormal cells, and a polymer or particle. The method further involves delivering light of a wavelength effective to cause generation of singlet oxygen by the photosensitizer to the internal treatment site. The conjugate is configured such that generation of the singlet oxygen causes release of the therapeutic or imaging agent moiety.

[0012] In certain embodiments, the conjugate is linked with a moiety or moieties which decrease immune system response (or enhance immune system tolerance) to the conjugate; such moiety is linked to the polymer or particle, and/or to the linker; such moiety is polyethyleneglycol (PEG) or a derivative thereof such as monomethoxypolyenthylene glycol, e.g., of about 2000-10000, 3000-8000, 3000- 5000, or about 3000 daltons average molecular weight.

[0013] In particular embodiments, the polymer is biocompatible and/or the polymer is a high molecular weight polymer with a highly flexible main chain; the polymer moiety bears a plurality (e.g., at least 2, 3, 4, 5, 7, 10, 15, 20, 30, 40, 50, 70, 100, 2-10, 10-20, 20-50, 50-100, 100-1000) of therapeutic agent moieties linked to the polymer through photocleavable linkers, where activation of the photosensitizer causes cleavage of a plurality of the photocleavable linkers; at least a portion of the polymer moiety is a polymer selected from the group consisting of N-(2-hydroxypropyl)methacrylamide (HPMA) copolymers, polyglutamic acid (PGA), polyethyleneglycol (PEG), polysaccharides such as polydextrans, dendrimers, liposomes, micelles, polymeric particles, linear cyclodextrins, polymerized Λ/-isopropylacrylamide, polyglutamic acid, polylysine, and polyaspartic acid; the polymer or nanoparticle includes a nanoparticle; the polymer or nanoparticle includes a nanoparticle which is 10-200, 10-100, 10-50, 20-100, 50-100, or 100-200 nm in diameter. [0014] Also in particular embodiments, the photosensitizer absorbs light within a defined waveband and generates singlet oxygen in response to absorbing that light; the photosensitizer absorbs light above 450 nm, above 550 nm, above 600 nm, above 700 nm, above 800 nm, above 900 nm, in the range of 450-1000 nm, in the range of 550-900 nm, in the range of 550-700 nm, in the range of 650-800 nm, or in the range of 700-900 nm, and generates singlet oxygen in response to absorbing that light; the photosentizer is selected from the group consisting of pthalocyanines, naphthalocyanines, 7,8-dihydro-5, 10, 15, 20- tetrakis (3- hydroxyphenyl)-21-23-[H]porphyrin (THPC), PEG-m-THPC, temoporfin, meta-tetra (hydroxyphenyl)chlorine, and photofrin; the photosensitizer further provides a contrast agent enabling imaging of the treatment site.

[0015] Also in certain embodiments, the photocleavable linker is hydrolyzed upon interaction with singlet oxygen; the photocleavable linker is selected from the group consisting of oxazoles, thiazoles, olefins, thioethers, selenoethers, and imidazoles.

[0016] In many embodiments, the targeting moiety includes a specific binding agent, e.g., a moiety which directly or indirectly specifically binds with one or more accessible components of the target cells; the targeting moiety includes a member of a specific binding pair, e.g., where the other member of the specific binding pair is on the target cells; the specific binding agent is a moiety that specifically binds with one or more molecules at the surface of the abnormal cells; the specific binding agent is a small molecule that specifically binds with a cell surface receptor, e.g., a folate receptor; the specific binding agent is an antibody (which may be a specific binding antibody fragment; the antibody preferentially binds to an antigen which is present substantially only at target cells, e.g., abnormal cells, in the patient; the antibody links with the abnormal cells at the treatment site, and the polymer increases an in vivo residence time of the conjugate proximal to the abnormal cells.

[0017] In advantageous embodiments, the singlet oxygen is generated in close proximity to the cleavable linker, resulting in hydrolysis of the cleavable linker and release of the therapeutic agent; the therapeutic agent is released quickly and in high concentrations at the treatment site; the therapeutic agent is an anticancer drug, an antiinfective agent, a hormone, a small molecule drug; the at least one therapeutic agent includes a plurality of different therapeutic agents, e.g., 2, 3, 4, 5, or even more; the therapeutic agent is highly toxic; the therapeutic agent is considered too toxic for non-targeted administration to a patient by the majority of medical practitioners. Similarly in particular embodiments, the imaging agent is a contrast agent, a fluorescent moiety, a chemiluminescent moiety.

[0018] In particular embodiments, the conjugate includes each discrete combination of polymer, therapeutic agent (or imaging or other diagnostic agent), photosensitizer, cleavable linker, and targeting moiety described for embodiments above; the conjugate includes each discrete combination of polymer, photosensitizer, cleavable linker, and targeting moiety described for embodiments above; the conjugate includes each discrete combination of polymer, therapeutic agent, photosensitizer, and cleavable linker, described for embodiments embodiments above; the conjugate includes each discrete combination of polymer, photosensitizer, and cleavable linker described for embodiments above.

[0019] A related aspect provides a method for treating a disease or condition in an animal, by administering to a subject suffering from or at risk of a disease of condition, a pharmacologically effective amount of a nanoconjugate molecule, where the nanoconjugate molecule includes at least one therapeutic moiety; at least one photocleavable linker; at least one photosensitizer moiety; and at least one targeting moiety that preferentially targets said molecule to a biological target (e.g., target cells). Photoactivation of the sensitizer causes cleavage of the photocleavable linker releasing at least one of the therapeutic moieties.

[0020] In particular embodiments, the conjugate is as described above for the preceding aspect or otherwise described herein for conjugates that include a therapeutic moiety.

[0021] A further related aspect of the invention concerns a therapeutic nanoconjugate molecule which includes at least one therapeutic moiety; at least one photocleavable linker; at least one photosensitizer moiety; and at least one targeting moiety that preferentially targets the molecule to a biological target, e.g., target cells, where photoactivation of the sensitizer causes cleavage of the photocleavable linker releasing at least one of the therapeutic moieties.

[0022] Particular embodiments are as described for the conjugate in aspects above or otherwise described herein for this invention.

[0023] In particular embodiments, the conjugate includes each discrete combination of polymer, therapeutic agent, photosensitizer, cleavable linker, and targeting moiety described for aspects and embodiments above; the conjugate includes each discrete combination of polymer, photosensitizer, cleavable linker, and targeting moiety described for aspects and embodiments above; the conjugate includes each discrete combination of polymer, therapeutic agent, photosensitizer, and cleavable linker, described for aspects and embodiments embodiments above; the conjugate includes each discrete combination of polymer, photosensitizer, and cleavable linker described for aspects and embodiments above.

[0024] Similarly, another aspect concerns a therapeutic or diagnostic (e.g., imaging) nanoconjugate molecule which includes at least one therapeutic or diagnostic (e.g., imaging) moiety; at least one exogenously cleavable linker; and at least one targeting moiety that preferentially targets said molecule to a biological target (e.g., target cells), where introduction of an exogenous cleaving agent causes cleavage of the exogenously cleavable linker releasing at least one of the therapeutic moieties.

[0025] In certain embodiments, the nanoconjugate molecule also includes a polymer moiety which bears a plurality of therapeutic moieties each linked to said polymer through an exogenously cleavable linker, where activation of the photosensitizer causes cleavage of a plurality of the exogenously cleavable linkers; the therapeutic moieties include at least 2, 3, 4, 5, or more different therapeutic moieties; the exogenously cleavable linker is cleaved in vivo and/or in vitro by an exogeneously introduced agent selected from the group consisting of an enzyme, X-rays, and high energy radiation, the nanoconjugate is as described for the preceding aspect or otherwise described herein for therapeutic conjugates. [0026] The invention also provides, in another aspect, a therapeutic kit which includes a packaged, pre-measured quantity of a nanoconjugate that includes at least one therapeutic moiety; at least one photocleavable linker; at least one photosensitizer moiety; and at least one targeting moiety that preferentially or specifically targets the molecule to a biological target (usually target cells), where photoactivation of the sensitizer causes cleavage of the photocleavable linker releasing at least one of the therapeutic moieties. The kit also includes instructions for administering the nanoconjugate to a subject suffering from or at risk of a disease or condition treatable or at least potentially treatable by administration of the therapeutic agent.

[0027] In particular embodiments, the nanoconjugate is as described for an aspect above or otherwise described herein.

[0028] Another aspect of the invention concerns a method for preparing a therapeutic nanoconjugate molecule by covalently linking together a targeting moiety, e.g., a cell-targeting moiety, at least one photosensitizer, a polymer backbone, a plurality of photocleavable linkers, and a plurality of therapeutic agents, where illumination of the conjugate in physiological solution causes the photosensitizer to generate singlet oxygen which causes cleavage of the photocleavable linkers, releasing the therapeutic agents from the polymer backbone.

[0029] In particular embodiments, the nanoconjugate is as described for an aspect above or otherwise described herein.

[0030] Yet another aspect concerns a method for therapeutic use of a drug having unacceptably high toxicity when administered systemically, by administering to a subject suffering from or at risk of a disease or condition against which the drug has activity a conjugate as described above or otherwise described herein for therapeutic conjugates, where the conjugate includes that drug.

[0031] In certain embodiments, the drug is substantially non-toxic when attached in said conjugate; the method also includes selecting a drug having unacceptably high system toxicity; the conjugate includes a moiety allowing removal of circulating conjugate from the bood of subject, such as by affinity purification.

[0032] In yet another aspect, the invention concerns a method for temporally and/or spatially controlled (e.g., at a target site) delivery and release of a release moiety such, as a molecule, conjugate or complex, intended for delivery to a target, e.g., an active moiety such as a therapeutic agent, a prodrug, an imaging agent or other diagnostic agent, in a subject. The method involves delivering to a target environment, e.g., a cellular environment such as to a subject, in a tissue culture, or in a cell suspension, a conjugate, pair of conjugates or complex which includes a photosensitizer moiety, a photocleavable linker, an active moiety or other release moiety, and usually but not necessarily a target binding moiety. The photocleavable linker is located proximal (often adjacent) to the active moiety or other release moiety such that cleavage of the photocleavable linker releases the release moiety.

[0033] In one configuration, a single conjugate is used, which is configured such that upon exposure of the photosensitizer to light of an appropriate wavelength, singlet oxygen is produced by the photosensitizer causing cleavage of the photocleavable linker, and release of the active moiety from the conjugate.

[0034] In another configuration, a pair of conjugates are used, where one conjugate includes a photosensitizer, and the other includes a photocleavable linker and an active moiety. Either or both of the conjugates may include a target binding moiety. In most cases, one of the conjugates will include a target binding moiety, and each of the conjugates will include a member of a specific binding pair which binds to the other, e.g., one conjugate will include streptavidin and the other will include biotin. Thus, the conjugate which includes the target binding moiety will bind to the target, and the two conjugates will bind together, thus bringing the photosensitizer into close proximity to the photocleavable linker. Upon exposure of the photosensitizer to light of an appropriate wavelength, singlet oxygen will be generated causing cleavage of the photocleavable linker and release of the active moiety. [0035] Likewise, in certain embodiments, other conjugates and complexes may be used which include the photosensitizer and photocleavable linker; highly preferably the conjugate or complex is stable in the delivery environment. In certain embodiments, the complex includes a small number of non-covalently linked molecules, e.g., 2, 3, 4, 5, 2-5, or 5-10; the complex is or includes a particle, which may be or include a solid phase particle; the complex is or includes a liposome.

[0036] In certain embodiments, the conjugate is as described for an aspect above or otherwise described herein for the present invention; the photosensitizer is as described herein for the present invention; the photocleavable linker is as described herein for the present invention; the release moiety is as described herein for the present invention; a polymer bearing photocleavable linkers and release moieties is as described herein for the present invention.

[0037] A further related aspect concerns a conjugate, set of complementary conjugates, or complex as specified for the preceding aspect, but in which the target binding moiety is optional. Thus, the single conjugate, set of linked or linkable conjugates, or complex includes a photosensitizer and a release moiety linked in a molecule or conjugate through a photocleavable linker which is cleaved by reaction with singlet oxygen. In many embodiments, the conjugate, set of conjugates, or complex include at least one target binding moiety, e.g., an antibody (which may be an antibody fragment).

[0038] Another related aspect concerns a method for imaging a tumor or other tissue or selected group of cells. The method involves the targeted delivery of a conjugate, conjugates, or complex as described for an aspect above or otherwise described for the present invention, where the conjugate or complex includes at least one binding moiety targeted to the selected tumor, tissue, or group of cells, and also includes at least one contrast agent or other imaging agent (e.g., fluorescent or chemiluminescent agent). The conjugate or complex is administered to a subject (e.g., a human), tissue culture, or cell suspension. The imaging agent is released by exposing target-bound conjugate or complex to a suitable exteneral agent suitable to cause release of the imaging agent. In many embodiments, the conjugate or complex includes a photosensitizer and a photocleavable linker, and the conjugate or complex is exposed to light of a wavelength suitable to cause generation of singlet oxygen by the photosensitizer with cleavage of the photocleavable linker.

[0039] In addition to the therapeutic methods, conjugates, kits, and methods of preparation described above, the invention also provides similar conjugates that include releasable diagnostic moieties, e.g., imaging agents, probes, and the like. Thus, the invention also provides diagnostic methods utilizing such diagnostic moieties, diagnostic conjugates, diagnostic kits including such conjugates along with instructions for use, and methods for making diagnostic conjugates in the same manner as for therapeutic conjugates.

[0040] The term "abnormal cells" refers to cells within a subject that are different from normal cells, e.g., cells that are not properly growth-regulated by the subject (for example, neoplastic cells such as cancer cells), virus-infected cells, and cells of pathogenic organisms (e.g., pathogenic bacterial cells, pathogenic fungi, and the like).

[0041] As used in the context of antibody binding, the term "antigen" means a molecule bearing an epitope recognized by the particular antibody. It does not require that the "antigen" be the particular molecule against which the antibody was raised.

[0042] Indication that a conjugate or a polymer or particle is "biocompatible" means that the indicated entity does not cause or elicit significant adverse effects when administered in vivo to a selected subject, e.g., a human subject. Examples of possible adverse effects include excessive inflammation and/or an excessive or adverse immune response, as well as toxicity.

[0043] Indication that singlet oxygen is generated in "close proximity" to a cleavable linker means that the singlet oxygen is generated sufficiently close that the concentration of singlet oxygen is sufficient at the cleavable linker such that the cleavable linker will more likely than not react with a singlet oxygen within one minute. In most cases, "close proximity" will be within the diffusion distance for the lifetime of the singlet oxygen in the particular intended in vivo environment. In many cases, "close proximity" will be 100 nm or less, and often 50 nm or less, 40 nm or less, 30 nm or less, 20 nm or less, 10 nm or less, or 5 nm or less.

[0044] In the context of small particles, particularly nanoparticles, the term "diameter" refers to the mean linear dimension of the particle for lines passing through the center of mass of the particle. Acceptable approximation of the diameter of non-spherical particles is provided by taking the mean of the thickness of the particle along 3 orthogonal axes of a coordinate system, with one of the axes aligned with the longest dimension of the particle.

[0045] In reference to polymers, the term "highly flexible" means that the polymer has a short persistence length, generally less than 100 nm (preferably determined using single molecule measurements, though bulk property measurements can be used if single molecule measurements are not available), usually less than 50 nm, often less than 10, 1 nm, 500 angstroms, 100 angstroms, 50 angstroms, or even 10 angstroms.

[0046] As used in connection with a drug entity, the term "highly toxic" means that the drug entity exhibits toxicity for cells different from target cells in a subject at a level such that a reasonable medical practitioner would not use the drug in a non-targeted manner.

[0047] The term "internal treatment site" refers to a location in the body of a subject that is at least partially below the outer skin of the subject.

[0048] In reference to components of the present conjugates, the term "polymer" refers to a molecule (which may be linear, branched, cyclic, or even a network) which is formed of many covalently linked small molecules. The covalently linked small molecules may be of one type or may be a combination of two or more different types.

[0049] As used herein, the term "nanoparticle" refers to a particle that is not more than 1000 nm in at least one dimension, often no more than 100 nm, 70 nm, 50 nm, 40 nm, 30 nm, or 20 nm. In many cases, a nanoparticle will have a diameter of a just-specified value (as defined herein for particles).

[0050] For the present conjugates, the term "photosensitizer" refers to a molecule or moiety of the conjugate which generates singlet oxygen when exposed to light of a particular wavelength.

[0051] Also in reference to the present conjugate molecules, the term "photocleavable linker" refers to a moiety which reacts with singlet oxygen resulting in the cleavage of the moiety. For the present conjugates, in most cases a photocleavable linker links a therapeutic or diagnostic agent moiety with a carrier portion of the molecule, e.g., a polymer or particle.

[0052] In the context of the present invention, the term "small molecule" refers to a molecule or a moiety of a conjugate that has a MW of 1000 daltons or less, usually 600, 500, 400, 300, 200, 100 daltons or less.

[0053] The term "specific binding agent" is used to refer to a molecule or moiety which binds to a particular other molecule or complex with a significant level of specificity. That is, the specific binding agent binds to the particular other molecule or complex to a substantially greater degree than to other-molecules ro complexes that are normally present in the particular environment. In many case the specific binding agent binds with another particular molecule, and the two binding molecules constitute a specific binding pair. Examples include antibody/antigen pairs (including specific binding antibody fragments), ligand/receptor pairs, and enzyme/substrate pairs (including substrate analogs).

[0054] Indication that a molecule (or molecule/complex or complex/complex) "specifically binds" with another means that the molecule binds with the other to a substantially greater extent than to other similar molecules in the environment. Highly preferably, the molecule does not bind to a significant extent to any other molecules in the relevant environment.

[0055] As used herein in the context of administration of the present conjugates, the term "subject" means an individual complex organism, e.g., a person or a non-human animal. The term "patient" refers to a human subject, i.e., a person.

[0056] The term "targeting moiety", in the context of the present invention, refers to a portion of a conjugate which causes the conjugate to preferentially locate to a particular target area or tissue of a subject, or to associate with particular types of cells. For example, a target moiety can cause a conjugate to preferentially associate with tumor cells. In many cases the association is mediated by the binding of specific binding pairs. ^'

[0057] In the context of the present invention, the terms "therapeutic agent", "therapeutic agent moiety", and similar terms referring to therapeutic function mean that the referenced molecule or conjugate moiety can beneficially affect the initiation, course, and/or one or more symptoms of a disease or condition in a subject.

[0058] Additional embodiments will be apparent from the Detailed Description and from the claims.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS A. Introduction

[0059] The present invention concerns the preparation and use of advantageous therapeutic and/or diagnostic nanoconjugate molecules (e.g., imaging nanoconjugates) designed to release therapeutic or diagnostic moieties (or other molecular entities for which temporal and/or spatial control is desired), e.g., small molecule drugs, dyes, and the like, with selected timing and/or location. Particularly advantageous nanoconjugates include a polymer-drug linker that is stable during its transit to the tumor or other site, specific in their binding, and possessing a novel mechanism for abrupt and rapid drug release at the tumor site.

[0060] The nanoconjugate molecules include a cleavable linker, where the cleavage is accomplished through the administration of an exogenous agent that is deliverable to the desired site. A particularly advantageous approach to accomplish this, though not the only one, is the use of photosensitizers and photocleavable linkers together in a nanoconjugate molecule, where cleavage of the photocleavable linker results in release and/or activation (e.g., conversion of a prodrug) of a therapeutic or diagnostic entity. Throughout this description, primary reference will be made to the combination of photosensitizers and photocleavable linkers, but it should be understood that other types of cleavable linkers, the cleavage of which can be controlled by an exogenously administered agent, e.g., an electromagnetic radiation, particle radiation, or small molecule, can similarly be used in the present invention with corresponding modification for providing the active cleaving entity to the cleavable linker at the desired location.

[0061] This approach of using cleavable linkers can be advantageously extended by using a polymeric or particulate backbone or carrier to simultaneously target a plurality of therapeutic or diagnostic entities to the target site. In this way, a high dose and/or an extended dose of the active entity (therapeutic or diagnostic entity) can be achieved with good localization. This can greatly assist in providing therapies with high therapeutic indices. [0062] As indicated above, a particularly advantageous application for the present invention is in cancer treatment, or more generally, the destruction of abnormal tissue. Providing a suitable therapeutic conjugate and using it to destroy the abnormal tissue can be illustrated by the following:

[0063] (i) Manufacturing -The conjugate can be manufactured by creating a conjugate comprised of (B)_x-P-(S-L-D)_y (1) a hydrophilic, nontoxic polymer (P), responsible for carrying all active ingredients. (2) A binding agent (B) specific for the cancer (or other target) such as an antibody or a specific molecule that binds to the receptor on the surface of the cancer cell, that is attached (most often covalently) to the polymer and is responsible for directing the conjugate to the tumor. (3) An anticancer drug (or other release moiety) (D) such as paclitaxel, (or other active agent) responsible for destroying abnormal tissue such as cancer. (4) A photosensitizer (S) (also referred to as PS) such as Phthalocyanine that generates singlet oxygen. (5) A cleavable linker (L) such as an oxazole. In such embodiments, the bond between the anticancer drug and the polymer has a cleavage-inducing moiety with an effective proximity to the singlet oxygen generating agent. (6) The number (y) of the complex molecules S-L-D which determine the number of drug molecules per conjugate. (7) The number of binding molecules per conjugate (x) that are used to direct the conjugate to the abnormal tissue.

[0064] (ii) Administering the nanoconjugate - the nanoconjugate is commonly administered either intravenously or i.m., with the binding reagent directing the nanoconjugate to the abnormal tissue. The cleavable linker bond between the polymer and the anticancer drug is highly stable enabling the conjugate to reach the cancer fully intact.

[0065] (iii) Activation and Drug Release - The PS may be activated by irradiation of appropriate wavelength (i.e. phthalocyanine = 680 nm) light. The irradiation may be provided externally by a lamp or a laser, or internally by an endoscopic laser or the like for deep seated tumors. Application of light activates the S and produces a high level of singlet oxygen which reacts very quickly with the cleavable linker and separates the anticancer drug (or other release moiety) from the polymer. The anticancer drug is then released in close proximity to the tumor and enters inside the cancer cell to produce anticancer activity.

[0066] (iv) Mechanism of cell uptake -The drug is usually either hydrophobic to allow passive diffusion inside the tumor cell and long action at the tumor site, or delivered inside the cell through active transport. In some cases,

[0067] Components of the present invention are additionally described below.

B. Photosensitizer Moieties

[0068] As indicated, in certain particularly advantageous embodiments, the present invention utilizes at least one photosensitizer moiety, often at least one photosensitizer moiety per cleavable linker.

[0069] Photosensitizers are compounds (attached as moieties in the present conjugates or complexes) that photochemically generate a reactive form of oxygen called singlet oxygen (Haugland et al., 2002). A number of these photosensitizers, such as phthalocyanine, have found utility as anticancer drugs when used as singlet oxygen generators in Photodynamic Therapy (PDT). Those photosensitizers identified for PDT can be used in the present invention also with corresponding suitable cleavable compounds, although the present invention is not limited to those entities.

[0070] In cancer PDT, the photosensitizer is commonly administered intravenously and is allowed to selectively localize and concentrate in the tumor while largely clearing from normal tissue. The drug is then activated by excitation with laser energy delivered to the diseased site, e.g., through a fiber optic device, generating large amounts of highly reactive singlet oxygen. The singlet oxygen destroys the diseased tissue in which the photosensitizer has concentrated with minimal damage to healthy tissue. A single molecule of the photosensitizer can generate half a million molecules of singlet oxygen per second (Youngjae et al., 2003).

[0071] The higher wavelengths (650 nm and above) of activating laser energy are preferred because they can penetrate up to three centimeters (over 1 inch) deep into human tissues. This means that targeted tissue beneath the skin's surface can be irradiated by light that is simply shined on the skin, avoiding any need for invasive surgical procedures. Alternatively, if deeper tissue illumination is needed, light can be delivered via fiber optic devices through blood vessels, the Gl tract, etc. (Zheng et al., 2001 ).

[0072] Because of the limited diffusion distance of singlet oxygen in biologic environments, the drug must be activated in close proximity to important cellular organelles (i.e. cell nucleus, mitochondria) to effectively damage the cell (Otsu et al., 2005). This limitation in singlet oxygen's diffusion distance therefore limits PDT's effectiveness. However, that limitation is utilized to great advantage in the present conjugates and methods.

[0073] A non-limiting list of examples of useful photosensitizers include the pthalocyanines, naphthalocyanines, 7,8-dihydro-5, 10, 15, 20- tetrakis (3- hydroxyphenyl)-21-23-[H]porphyrin (THPC), PEG-m-THPC, temoporfin, meta-tetra (hydroxyphenyl)chlorine, photofrin. Other photosensitiers are described in US Patents 5990149, 5880145, 5283255, 5171749, 5095030, 4920143, and 4883790, each of which is incorporated herein by reference in its entirety, including for purposes of their descriptions of photosensitizers and PDT. Other photosensitizers are also known and can be used in this invention.

C. Cleavable Linkers

[0074] A variety of cleavable linkers are known that may serve as linker L. In many cases such cleavable linkers include photocleavable linkers that react with singlet oxygen in solution, resulting in cleavage of the linker.

[0075] Such a photocleavable linkage includes an oxidation-labile linkage that is cleaved by singlet oxygen. Examples of such linkers are heterocyclic compounds, such as diheterocyclopentadienes, as exemplified by substituted imidazoles, thiazoles, oxazoles, etc., where the rings will usually be substituted with at least one aromatic which contains carbon-carbon double bonds. Upon reaction with singlet oxygen, these compounds form an oxo group which then hydrolyzes into two separate molecules (Ando et al., 1973). For example, see the example of the mechanism of oxazole cleavage below.

[0076] Thus, in many embodiments, the cleavable linkage is an oxidation-labile linkage, and preferably it is a linkage cleavable by reaction with singlet oxygen. The linker may, for example, be a thioether or its selenium analog; or an olefin, which contains carbon-carbon double bonds. Cleavage of a double bond to an oxo group releases the active moiety, e.g., an anticancer drug. Additional examples of olefins which may be used include vinyl sulfides, vinyl ethers, enamines, imines substituted at the carbon atoms with an a-methine (CH, a carbon atom having at least one hydrogen atom), where the vinyl group may be in a ring, the heteroatom may be in a ring, or substituted on the cyclicolefinic carbon atom, and there will be at least one and up to four heteroatoms bonded to the olefinic carbon atoms. The resulting dioxetane may decompose spontaneously or, highly preferably, by reaction with singlet oxygen from a photosensitizer. Such reactions are described in the following exemplary references: Adam and Liu, J. Amer. Chem. Soc. 94, 1206-1209,1972, Ando, etal., J. C. S. Chem. Comm. 1972,477-8, Ando, etal., J. Amer. Chem. Soc. 96,6766-8, 1974, Wasserman and Terao, Tetra. Lett. 21 ,1735-38,1975, and U. S. Patent No. 5,756,726 (which is incorporated herein by reference in its entirety).

[0077] The dioxetane occurs upon reaction of singlet oxygen with an activated olefin substituted with a drug moiety or other active moiety at one carbon atom and the second binding agent at the other carbon atom of the olefin. See, for example, U. S. Patent No. 5,807,675 (incorporated herein by reference in its entirety).

[0078] Exemplary cleavable linkages include S-3-thiolacrylic acid, -N, N-methyl 4-amino-4butenoicacid,-0, 3-hydroxyacrolein, N- (4-carboxyphenyl) 2-imidazole, oxazole, and thiazole. Other useful cleavable linkers include N-alkyl acridinyl derivatives, substituted at the 9 position with a divalent group of the formula: - (CO) X' (A) wherein: X'is a heteroatom selected from the group consisting of O, S, N, and Se, usually one of the first three; and A is a chain of at least 2 carbon atoms and usually not more than 6 carbon atoms substituted with anticancer drug, where preferably the other valences of A are satisfied by hydrogen, although the chain may be substituted with other groups, such as alkyl, aryl, heterocyclic groups, etc., A generally being not more than 10 carbon atoms.

[0079] Other cleavable linkers are heterocyclic compounds, such as diheterocyclopentadienes, as exemplified by substituted imidazoles, thiazoles, oxazoles, etc., where the rings will, in some cases, be substituted with at least one aromatic group and in some instances hydrolysis will be necessary to release the drug. The oxazole cleavable linkage,"-CH2-oxazole- (CH2) n-C (=O)-NH-.

[0080] Still other cleavable linkers are tellurium (Te) derivatives, where the Te is bonded to an ethylene group having a hydrogen atom beta to the Te atom. The ethylene group is part of an alicyclic or heterocyclic ring that may have an oxo group, preferably fused to an aromatic ring and the other valence of the Te is bonded to the drug. The rings may be, for example, coumarin, benzoxazine, tetralin, etc.

[0081] Cleavage of the olefin linkage results in freeing the drug moiety (or other active agent moiety). Thus, the drug is generally in an active form"R - D" where "R" may be a substituent which does not destroy the desired activity of the core drug entity. Some such substitution, R, are described in, e. g Ullman et al, U. S. patent 6,251 ,581 ; Smith and March, March's Advanced Organic Chemistry: Reactions, Mechanisms, and Structure, 5th Edition (Wiley-lnterscience, New York, 2001 ). In certain cases, R is a group having from 1-8 carbon atoms and from 0 to 4 heteroatoms selected from the group consisting of O, S, and N which enhances cell uptake.

[0082] Photocleavable linkers suitable for use in this invention which are currently regarded as preferable include, for example, oxazoles, thiazoles, olefins, thioethers, selenoethers, and imidazoles.

[0083] As indicated above, other types of cleavable linkers and corresponding cleaving agents can also be used. Thus, for example, enzymes that cleave a corresponding substrate can be used (with the substrate serving as a cleavable linker). Such enzymes include, for example, alkaline phosphatases, penicillin amidases, arylsulfatases, cytosine deaminases, proteases, D-alanyl carboxypeptidases, carbohydrate-cleaving enzymes, beta-lactamases, DNA nucleases, and RNA nucleases.

[0084] An exemplary cleavage reaction for oxazole linker cleaved by reaction with singlet oxygen is shown below.

D. Combining These Approaches

[0085] As indicated above, the present invention provides a new generation of drug delivery systems (as well as diagnostic agent delivery systems) made of a synthetic nanovector to achieve high specificity to a target site (e.g., a tumor), which utilizes a cleavable release mechanism, e.g., using photocleavable linkers and photosensitizer agents. In these systems, cleavage of the linker(s) releases the therapeutic or diagnostic agent. This system can, for example, readily deliver therapeutic concentrations of free anti-cancer drugs to a tumor.

[0086] In a present delivery system using photosentizers and photocleavable linkers, a photosensitizer is introduced in close proximity (typically less than 1 nm) to a photocleavable linker to release an active entity, e.g., a long acting lipophilic anticancer drug such as paclitaxel, thereby avoiding the limitations of singlet oxygen's extremely short diffusion distance. The components of the delivery system can advantageously be loaded onto a polymer backbone (as described below) functionalized with a specific binding agent (e.g., an antibody) to achieve the desired concentration of drug at the tumor site.

[0087] As outlined in the background sections on nanomedicine, photosensitizers, and photocleavable linkers, the raw materials and fundamental research relating to the components of such a nanovector are readily available. From bio-compatible polymer backbones, potent anti-cancer drugs, highly specific antibodies, photocleavable linkers and their activation with photosensitizers, each component has demonstrated the characteristics utilized for the function of the nanovector. The exemplary embodiments of the present invention combines each of these component therapies into a single, highly effective therapeutic entity.

[0088] The resulting nanomedicine can overcome the limitations of current nanomedicine and PDT approaches, and be of great use in the treatment of common solid tumors such as breast, prostate, lung and gastrointestinal cancers.

[0089] Thus, the present invention provides advantageous treatment for a varieity of conditions. Particulary advantageous is the use of the invention against solid tumors In the case of tumors that are closer to the surface, light can penetrate from outside to the tumor site, thereby effecting cleavage of the linker and release of the active agent. For tumors that are to far away from the surface for effective cleavage and release from external light, light can be provided by inserting an endoscopic laser to the tumor site, operated using remote control from outside.

E. Carrier Polymers

[0090] In advantageous embodiments, the present conjugates include a nontoxic polymer to which multiple delivery/targeting moieties and/or therapeutic moieties are attached. Thus, the polymer itself, as well as degradation products, if any, should be suitable for in vivo applications. In addition, the polymer should have or be able to be modified to have suitable functional groups for attaching cleavable linker and active agent combinations. Selection of appropriate functional groups and the corresponding reaction conditions and any protective or blocking groups useful for the reactions are well within the skill of synthetic chemists and can be utilized in preparation of the present conjugates.

[0091] The polymer can advantageously have the ability to deliver a wide range of therapeutic payloads, ranging from small molecules to proteins and peptides. Thus, the polymer and resulting conjugate should be useful in a variety of diseases such as cancer, infectious diseases, and the like.

[0092] Polymers such as N-(2-hydroxypropyl)methacrylamide (HPMA) copolymers, polyglutamic acid (PGA)₁ polyethyleneglycol (PEG), and polysaccharides such as polydextrans, and dendrimers have been used for polymer-drug conjugates and can be utilized in the present invention. Of course, other biocompatible polymers can also be utilized, including but not limited to liposomes, micelles, polymeric particles, linear cyclodextrins, polymerized N- isopropylacrylamide, polyglutamic acid, polylysine, and polyaspartic acid.

[0093] Selection of appropriate polymer backbone candidates may be guided by criteria such as water solubility, size and/or size distribution, density of chemical functional groups (usually a high density is desirable), and capability of delivering a wide range of therapeutic payloads, e.g., ranging from small molecules to proteins and peptides.

[0094] Dendrimers in particular are a useful class of polymeric nanoparticles that have found use in biological applications ranging from drug delivery to tissue repair. Dendrimers are polymers that are branched (as opposed to conventional linear polymers) where the branches radiate in a symmetric fashion from a central core. (Lee et al., 2005) While they can be made from many different polymeric materials, dendrimers found in biological applications are usually based on polyamidoamines, polyamides, carbohydrates or polypeptides. (Lee et al., 2005) Dendrimers are commercially available in a variety of sizes and chemistries.

[0095] Another type of useful polymer materials are nanogels. For example, the DeSimone group utilized inverse microemulsion polymerization techniques to synthesize stable, biocompatible polymeric nanogels less than 200 nm in size, for antisense and gene delivery to HeLa cells via the exploitation of charge. (McAllister et al., 2002)

[0096] Additionally, monodisperse, shape-specific 200 nm trapezoidal particles from poly(pyrrole) (Ppy) have been generated. Ppy has been used in a variety of applications, ranging from electronic devices and sensors to cell-scaffolds. (Curran, 1991) Ppy can also be used in the present invention.

F. Delivery and Targeting Moieties

[0097] A number of different delivery and/or targeting moieties have been utilized in therapeutic applications. Perhaps the most common is the use of site or target specific antibodies. Other targeting moieties include, for example, targeting peptides, nucleic acid molecules, ligand analogs, oligosaccharides, and the like. Such targeting moieties have been used, for example, to target molecules to tumors.

[0098] The use of antibodies (including antibody fragments) has been well documented, and serves as an exemplary targeting method herein. Usually, the antibody is a monoclonal antibody or a fragment thereof, which can be produced by conventional methods.

[0099] For anti-tumor therapy, for example, the antibody can be selected to recognize and bind to a tumor marker. In many cases, the marker will be a protein accessible on the outside of tumor cells, e.g., a tumor-specific receptor or the like.

[00100] Similarly, other binding pairs may be used, such as receptors with their specific binding partner, e.g., folate and folate receptor. Folate receptors are known to be overexpressed in many different types of tumor cells.

G. Therapeutic Moiety or Other Active Agent

[00101] The present invention is adapted to delivery of a variety of different active agents, especially therapeutic agents, including many different small molecule drugs. WhIe the present conjugates are advantageous for therapeutic agents, they are also useful for localized delivery of certain diagnostic agents also. The therapeutic moiety can be selected to have desirable properties for the particular application.

[00102] Non-limiting examples of therapeutic moieties or agents include paclitaxel, doxorubicin, daunorubicin, epirubicin, idarubicin, mitoxandrone, chloroambucil, melphalan, 5-fluorouracyl, 5'-desoxy-5-fluorouridine, thioguanine, methotrexate, docetaxel, topotecan, 9-aminocamptothecin, mitopodoside, vinblastine, vincristine, vindesine, vinorelbine, etoposide, teniposide, daunomycin, carminomycin, aminopterin, dactinomycin, mitomycins, cis-platinum and cis- platinum analogues, bleomycins, esperamicins, melphalan and other nitrogen mustards.

[00103] Thus, for example, a therapeutic agent may be selected that facilitate its entry into target cells, e.g., tumor cells, once the agent is freed from the conjugate. For example, the agent may be recognized and internalized through particular receptors, or may have such properties as to enable it to pass through the cellular membranes passively. Desirably, the therapeutic agent and the other components of the conjugate are selected so that they have little or no effect while not associated with the target cells. For example, the linkage of the therapeutic agent can be selected so that the agent is essentially inactive until it is released by cleavage of the cleavable linker.

[00104] Other properties may be selected as appropriate for the particular application.

[00105] As described above, in addition to therapeutic applications, the present conjugates are also useful for imaging and other diagnostic applications in which it is advantageous to spatially and/or temporally control release of a particular agent. Such agents may include, for example, contrast agents and the like. H. In vivo Function and Applications of Therapeutic Conjugate Molecules

[00106] The present conjugates are designed such that the attached therapeutic moiety is preferentially delivered to a desired site or sites before being released from the conjugate. In general, as discussed above, the release is accomplished using a cleavable linker that can be temporally controlled using an exogenous agent, e.g., light, penetrating radiation, or other type of electromagnetic radiation that is sufficiently energetic to directly or indirectly cause cleavage of a selected cleavable linker, exogenously administered chemical moiety, and the like. Thus, the present therapeutic conjugate molecules are adaptable to a variety of different therapeutic, prophylactic, and/or diagnostic applications.

[00107] A particularly appropriate and important application is in treatment of cancers or other localized cells or groups of cells that may be targeted with physical targeting (e.g., by localized injection) or preferably using a targeting moiety that specifically or preferentially causes the conjugate molecule to locate to or in the desired cells.

I. Administration of Therapeutic Conjugates

[00108] The present therapeutic conjugates can be administered by methods suitable for administering other molecules of similar size.

[00109] The present cleavable conjugates may be administered by any route appropriate to the condition to be treated. The conjugates will usually be administered parenterally, e.g., by injection or infusion, such as subcutaneous, intramuscular, intravenous, intradermal, intrathecal and epidural. Thus, pharmaceutical formulations of the present conjugates are typically prepared for parenteral administration with a pharmaceutically acceptable parenteral vehicle and in a unit dosage injectable form. A conjugate having the desired degree of purity can also be optionally mixed with pharmaceutically acceptable diluents, carriers, excipients or stabilizers (Remington's Pharmaceutical Sciences (1980) 16th edition, Osol, A. Ed.). The pharmaceutical formulation can be prepared, for example, as a lyophilized formulation or an aqueous solution. [00110] Pharmaceutically acceptable diluents, carriers, excipients, and stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, and other organic acids; antioxidants such as ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions such as sodium; metal complexes (e.g. Zn-protein complexes); and/or non-ionic surfactants such as TWEEN™, PLURONICS™ or polyethylene glycol (PEG).

[00111] The active pharmaceutical ingredients may also be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin-microcapsules and poly-(methylmethacylate) microcapsules, respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano- particles and nanocapsules) or in macroemulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).

[00112] Sustained-release preparations may be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the conjugates, which matrices are in the form of shaped articles, e.g. films, or microcapsules. Examples of sustained-release matrices include polyesters, hydrogels (for example, poly(2-hydroxyethyl- methacrylate), or poly(vinylalcohol)), polylactides (U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and gamma-ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as the LUPRON DEPOT™ (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), and poly-D-(-)-3-hydroxybutyric acid.

[00113] The formulations to be used for in vivo administration should be sterile. Sterilization can be accomplished by any of a variety of methods compatible with the components present, e.g., by filtration through sterile filtration membranes.

[00114] The formulations may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy. Techniques and formulations generally are found in Remington's Pharmaceutical Sciences (Mack Publishing Co., Easton, Pa.). Such methods include the step of bringing into association the active ingredient with the carrier which constitutes one or more accessory ingredients. In general the formulations are prepared by uniformly and intimately bringing into association the active ingredient with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product.

[00115] Aqueous suspensions of the invention contain the active materials in admixture with excipients suitable for the manufacture of aqueous suspensions. Such excipients may include a suspending agent, such as sodium carboxymethylcellulose, croscarmellose, povidone, methylcellulose, hydroxypropyl methylcelluose, sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia, and dispersing or wetting agents such as a naturally occurring phosphatide (e.g., lecithin), a condensation product of an alkylene oxide with a fatty acid (e.g., polyoxyethylene stearate), a condensation product of ethylene oxide with a long chain aliphatic alcohol (e.g., heptadecaethyleneoxycetanol), a condensation product of ethylene oxide with a partial ester derived from a fatty acid and a hexitol anhydride (e.g., polyoxyethylene sorbitan monooleate). The aqueous suspension may also contain one or more preservatives such as ethyl or n-propyl p-hydroxy-benzoate, one or more coloring agents, one or more flavoring agents and one or more sweetening agents, such as sucrose or saccharin.

[00116] The pharmaceutical compositions may be in the form of a sterile injectable preparation, such as a sterile injectable aqueous or oleaginous suspension. This suspension may be formulated according to the known art using those suitable dispersing or wetting agents and suspending agents, e.g., as mentioned above. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent, such as a solution in 1 ,3-butane-diol or prepared as a lyophilized powder. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution and isotonic sodium chloride solution. In addition, sterile fixed oils may conventionally be employed as a solvent or suspending medium. For this purpose any bland fixed oil may be employed including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid may likewise be used in the preparation of injectables.

[00117] The amount of active ingredient that may be combined with the carrier material to produce a single dosage form will vary depending upon the host treated and the particular mode of administration. For example, an aqueous solution intended for intravenous infusion may contain from about 3 to 500 microgram of the active ingredient per milliliter of solution in order that infusion of a suitable volume at a rate of about 30 mL/hr can occur.

[00118] Formulations suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents.

[00119] Although oral administration of high molecule weight polymer therapeutics are less common due to potential hydrolysis and/or low uptake, formulations of the present conjugates suitable for oral administration may be prepared as discrete units such as capsules, cachets or tablets each containing a predetermined amount of the conjugate.

[00120] The formulations may be packaged in unit-dose or multi-dose containers, for example sealed ampoules and vials. The formulations may be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example water, for injection immediately prior to use. Extemporaneous injection solutions and suspensions are prepared from sterile powders, granules and tablets of the kind previously described. Exemplary unit dosage formulations include a daily dose or unit daily sub-dose, as herein above recited, or an appropriate fraction thereof, of the active ingredient.

[00121] The invention further provides veterinary compositions comprising at least one conjugate together with a veterinary carrier therefore. Veterinary carriers are materials useful for the purpose of administering the composition and may be solid, liquid or gaseous materials which are otherwise inert or acceptable in the veterinary art and are compatible with the active ingredient. These veterinary compositions may be administered parenterally, orally or by any other desired route.

J. Cleavable Conjugate Treatments

[00122] The present conjugates may be used to treat various diseases or conditions, e.g., diseases or conditions characterized by the overexpression of a tumor antigen. Examples of such diseases or conditions include benign or malignant tumors; leukemia and lymphoid malignancies; as well as other disorders such as neuronal, glial, astrocytal, hypothalamic, glandular, macrophagal, epithelial, stromal, blastocoelic, inflammatory, angiogenic and immunologic disorders.

[00123] In many cases, the disease or condition to be treated is cancer. Examples of cancer to be treated herein include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia or lymphoid malignancies. More particular examples of such cancers include squamous cell cancer (e.g. epithelial squamous cell cancer), lung cancer including small-cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung and squamous carcinoma of the lung, cancer of the peritoneum, hepatocellular cancer, gastric or stomach cancer including gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney or renal cancer, prostate cancer, vulval cancer, thyroid cancer, hepatic carcinoma, anal carcinoma, penile carcinoma, as well as head and neck cancer.

[00124] The cancer may include HER2-expressing cells, such that the conjugates are able to bind to the cancer cells. To determine ErbB2 expression in the cancer, various diagnostic/prognostic assays are available. In one embodiment, ErbB2 overexpression may be analyzed by IHC, e.g. using the HERCEPTEST (Dako). Parrafin embedded tissue sections from a tumor biopsy may be subjected to the IHC assay and accorded a ErbB2 protein staining intensity criteria as follows: Score 0, no staining is observed or membrane staining is observed in less than 10% of tumor cells; Score 1+, a faint/barely perceptible membrane staining is detected in more than 10% of the tumor cells, the cells are only stained in part of their membrane; Score 2+, a weak to moderate complete membrane staining is observed in more than 10% of the tumor cells; Score 3+, a moderate to strong complete membrane staining is observed in more than 10% of the tumor cells. Those tumors with 0 or 1+ scores for ErbB2 overexpression assessment may be characterized as not overexpressing ErbB2, whereas those tumors with 2+ or 3+ scores may be characterized as overexpressing ErbB2.

[00125] Alternatively, or additionally, FISH assays such as the INFORM™ (Ventana Co., Ariz.) or PATHVISION™ (Vysis, III.) may be carried out on formalin- fixed, paraffin-embedded tumor tissue to determine the extent (if any) of ErbB2 overexpression in the tumor.

[00126] The cancer to be treated herein may be one characterized by excessive activation of an ErbB receptor, e.g. HER2. Such excessive activation may be attributable to overexpression or increased production of the ErbB receptor or an ErbB ligand. In one embodiment of the invention, a diagnostic or prognostic assay will be performed to determine whether the patient's cancer is characterized by excessive activation of an ErbB receptor. For example, ErbB gene amplification and/or overexpression of an ErbB receptor in the cancer may be determined. Various assays for determining such amplification/overexpression are available in the art and include the IHC, FISH and shed antigen assays described above. Alternatively, or additionally, levels of an ErbB ligand, such as TGF-alpha., in or associated with the tumor may be determined according to known procedures. Such assays may detect protein and/or nucleic acid encoding it in the sample to be tested. In one embodiment, ErbB ligand levels in the tumor may be determined using immunohistochemistry (IHC); see, for example, Scher et al. (1995) Clin. Cancer Research 1 :545-550. Alternatively, or additionally, one may evaluate levels of ErbB ligand-encoding nucleic acid in the sample to be tested; e.g. via FISH, southern blotting, or PCR techniques.

[00127] Moreover, ErbB receptor or ErbB ligand overexpression or amplification may be evaluated using an in vivo diagnostic assay, e.g. by administering a molecule (such as an antibody) which binds the molecule to be detected and is tagged with a detectable label (e.g. a radioactive isotope) and externally scanning the patient for localization of the label.

[00128] For the prevention or treatment of disease, the appropriate dosage of a conjugate will depend, for example, on the type of disease to be treated, the severity and course of the disease, whether the conjugate is administered for preventive or therapeutic purposes, previous therapy, the patient's clinical history and response to the antibody, and the discretion of the attending physician. The conjugate is suitably administered to the patient at one time or over a series of treatments. Depending on the type and severity of the disease, about 1 μg/kg to 15 mg/kg (e.g. 0.1-20 mg/kg) of conjugate is an initial candidate dosage for administration to the patient, whether, for example, by one or more separate administrations, or by continuous infusion. A typical daily dosage might range from about 1 μg/kg to 100 mg/kg or more, depending on the factors mentioned above. An exemplary dosage of conjugate to be administered to a patient is in the range of about 0.1 to about 10 mg/kg of patient weight.

[00129] For repeated administrations over several days or longer, depending on . the condition, the treatment is sustained until a desired suppression of disease symptoms occurs. The progress of this therapy is easily monitored by conventional techniques and assays.

Combination Therapy [00130] The present conjugates may be combined in a pharmaceutical combination formulation, or dosing regimen as combination therapy, with a second active compound, e.g., a compound also having anti-cancer properties. The second compound of the pharmaceutical combination formulation or dosing regimen may have complementary activities to the present conjugates such that they do not adversely affect each other.

[00131] The second compound may, for example, be a chemotherapeutic agent, cytotoxic agent, cytokine, growth inhibitory agent, anti-hormonal agent, and/or cardioprotectant. Such molecules are suitably present in combination in amounts that are effective for the purpose intended. A pharmaceutical composition containing the present conjugates may also have a therapeutically effective amount of a chemotherapeutic agent such as a tubulin-forming inhibitor, a topoisomerase inhibitor, or a DNA binder.

[00132] Other therapeutic regimens may be combined with the administration of an anticancer conjugate as described herein. The combination therapy may be administered as a simultaneous or sequential regimen. When administered sequentially, the combination may be administered in two or more administrations. The combined administration includes coadministration, using separate formulations or a single pharmaceutical formulation, and consecutive administration in either order, wherein there is a time period while both (or all) active agents simultaneously exert their biological activities.

[00133] In one embodiment, treatment with the present conjugates involves the combined administration of an anticancer agent, e.g., as identified herein, and one or more chemotherapeutic agents or growth inhibitory agents, including coadministration of cocktails of different chemotherapeutic agents. Chemotherapeutic agents include, for example, taxanes (such as paclitaxel and doxetaxel) and/or anthracycline antibiotics. Preparation and dosing schedules for such chemotherapeutic agents may be used according to manufacturers¹ instructions or as determined empirically by the skilled practitioner. Preparation and dosing schedules for such chemotherapy are also described in Chemotherapy Service Ed., M. C. Perry, Williams & Wilkins, Baltimore, Md. (1992).

[00134] The anticancer agent may be combined with an anti-hormonal compound; e.g., an anti-estrogen compound such as tamoxifen; an anti- progesterone such as onapristone (EP 616812); or an anti-androgen such as flutamide, in dosages known for such molecules. Where the cancer to be treated is hormone independent cancer, the patient may previously have been subjected to anti-hormonal therapy and, after the cancer becomes hormone independent, the anti-ErbB2 antibody (and optionally other agents as described herein) may be administered to the patient. It may be beneficial to also coadminister a cardioprotectant (to prevent or reduce myocardial dysfunction associated with the therapy) or one or more cytokines to the patient. In addition to the above therapeutic regimes, the patient may be subjected to surgical removal of cancer cells and/or radiation therapy.

[00135] Suitable dosages for any of the above coadministered agents are those presently used and may be lowered due to the combined action (synergy) of the newly identified agent and other chemotherapeutic agents or treatments.

[00136] The combination therapy may provide "synergy" and prove "synergistic", i.e. the effect achieved when the active ingredients used together is greater than the sum of the effects that results from using the compounds separately. A synergistic effect may be attained when the active ingredients are: (1 ) co- formulated and administered or delivered simultaneously in a combined, unit dosage formulation; (2) delivered by alternation or in parallel as separate formulations; or (3) by some other regimen. When delivered in alternation therapy, a synergistic effect may be attained when the compounds are administered or delivered sequentially, e.g. by different injections in separate syringes. In general, during alternation therapy, an effective dosage of each active ingredient is administered sequentially, i.e. serially, whereas in combination therapy, effective dosages of two or more active ingredients are administered together.

[00137] Combination therapy may be accomplished in a variety of ways. For example, the present conjugates may be provided containing two or more different therapeutic agent moieties, which may be releasable under the same or different conditions. As an example, a polymer may be used to which at least two different types of therapeutic agents are linked through photocleavable linkers. Activation of attached photosensitizers will essentially simultaneously release the different therapeutic agents. Similar results can be obtained by using separate, concurrently administered conjugates, where the different conjugates include different therapeutic agents linked through photocleavable linkers.

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EXAMPLES

[00138] The following examples illustrate the capability to provide controlled cleavage to release a desired active agent using photosensitizers and photocleavable linkers, and further explains an approach for identifying and optimizing the present conjugates.

EXAMPLE 1 : Demonstration of release of selected moiety using a photosensitizer and a photocleavable linkage.

[00139] This example serves as a basic demonstration of the nanovector's technical approach using a photosensitizer nanoconjugate, a cleavable linker, and a drug surrogate (a fluorophore in order to follow the release of the surrogate, Molecular Weight = 1079 g/mol).

Materials/Methods

[00140] A photosensitizer (Phthalocyanine) was incorporated into white, carboxy modified, polystyrene nanoparticles. These particles were then aminated by coating them with amino-dextran polymer in the presence of EDC. After extensive washing, the amine-modified nanoconjugate was resuspended, characterized for the presence of amines and monodispersity, and stored in double-deionized water for later use (see Construct 1).

Construct 1 - Amine-modified nanoconjugate. [00141] The photocleavabe linker 2-Amino-4-thiazoleacetic acid (Alpha-Aesar) was modified with amine reactive, N-hydroxysuccinimide activated Alexa-546 dye (NHS-Alexa-546, Invitrogen) as a drug surrogate to create the following subcomponent (see Construct 2). The use of this drug surrogate enables tracking its presence throughout the experiment.

Surrogate

Construct 2 - Photocleavable Linker with conjugated Drug Surrogate.

[00142] This subcomponent was then reacted with an excess of amine-modified, photosensitizer nanoconjugate from above in the presence of EDC. After extensive washing, the final nanoconjugate (see Construct 3) was resuspended and stored in double-deionized water for later use.

Surrogate

Construct 3 - Final nanoconjugate.

Experimental

[00143] Ten microliters of the final nanoconjugate were activated for ten seconds using a 680 nm, 300 mW laser diode (Perkin-Elmer). The resulting nanoconjugate suspension and its fluorescence characteristics were compared to the non-Alexa-dyed amine-modified nanoconjugate and the non-activated, final nanoconjugate using a fluorescence microscope equipped with a scientific grade, CCD camera (Leica). Briefly, each nanoconjugate suspension was diluted 1 :10 in double-deionized water and applied to a glass slide for analysis. Each suspension was then centrifuged at 13,000 x g for five minutes and the resulting supernatants applied to glass slides for analysis. Fluorometric measurements from the phthalocyanine photosensitizer dye (Excitation 680 nm / Emission 700 nm) and the Alexa-546, drug surrogate dye (Excitation 556 nm / Emission 570 nm) were performed. The nanoconjugate was then resuspended and washed again by centrifugation. The resulting nanoconjugate suspensions were applied to glass slides and measured fluorometrically as well.

Results

[00144] Each of the nanoconjugate suspension samples were analyzed microscopically to confirm the presence and release of the Alexa-546 fluorophore (drug surrogate). The analysis included imaging of the photosensitizer nanoconjugate using the excitation/emission wavelengths of the phthalocyanine dye, as well as imaging of the location of signal from the drug surrogate using excitation/emission wavelengths for Alexa-546 (Excitation at 556 nm / Emission at 570 nm). Comparison of the images indicated that (1) amine-modified photosensitizer (pthalocyanine-containing) nanoconjugate without Alexa-546 attached, and fluoresces only in the phthalocyanine channel; (2) the final nanoconjugate pre-laser activation includes both phthalocyanine and Alexa-546 dye present, and fluoresces in both channels; and (3) the final nanoconjugate post-laser activation and two cycles of washing retains fluorescence in the pthalocyanine channel but has little Alexa-546 fluorescence remaining associated with the nanoconjugate. These results indicate that upon activation of the photosensitizer the bond between the polymer and drug surrogate is cleaved, releasing the drug surrogate as a free compound.

[00145] To confirm the release of Alexa-546 fluorophore following phthalocyanine activation, fluorescence images and intensities (in Relative Fluorescence Units) from the Alexa-546 fluorescence channel for each of the nanoconjugate suspensions' supernatant were analyzed and intensities are shown in the following table.

[00146] Fluorescence images from the nanoconjugate supernatants illustrated that the amine-modified nanoconjugate in (1) and the final nanoconjugate pre- laser activation in (2) show no fluorescence in their supernatants in the Alexa-546 channel. The final nanoconjugate post-laser activation in (3) shows high levels of Alexa-546 fluorophore in its supernatant, confirming the release of the fluorophore from the nanoconjugate surface into solution following activation of the phthalocyanine.

[00147] Thus, the fluorescence intensity measured in the final nanoconjugate post-laser activation's supernatant exhibits the successful cleavage of the photocleavable linker holding the Alexa-546 fluorophore to the nanoconjugate. It is clear from the lack of fluorescence measured in the pre-laser activated and amino-modified nanoconjugate supernatants that it is the activation of the photosensitizer dye that has caused this cleavage.

Conclusion

[00148] The preparation of a photosensitizer-cleavable linker-drug complex conjugated to a nanopolymer has been achieved. Activation of the photosensitizer with a long wavelength laser cleaves the linker between the drug and polymer and initiates the rapid release of the drug surrogate from the final nanoconjugate.

EXAMPLE 2 - Preparation of Exemplary Nanovector Conjugate. [00149] An example of a group of related nanovectors containing a drug moiety, a photocleavable linker, a photosensitizer moiety, and a targeting moiety is shown below.

Phthalocyanine ) _m- Polymer - (Anti-HER2 -Ab ) ,

[00150] This resulting compound is a novel conjugate with a stable, covalent attachment of its drug to a polymer backbone. As a result of such a stable linkage, the drug is not released from the polymer during delivery of the drug to the tumor. The conjugate binds specifically to tumor cells, such as breast cancer cell line MDA-MB-453, by way of its tumor specific antibody - Anti-HER2. The drug is released by activating the phthalocyanine with 680 nm light, causing this photosensitizer to rapidly produce singlet oxygen. The singlet oxygen reacts with the cleavable linker that is situated in close proximity, breaking the bond between the polymer and Paclitaxel to release the Paclitaxel at the tumor site. Since free Paclitaxel is very hydrophobic, it will diffuse inside the tumor cell at high concentration for efficient cell killing.

[00151] This compound improves in the following areas relative to current therapeutic approaches:

• Highly potent drug (Paclitaxel) chosen for its anti-cancer activity

• Improved stability of the linker (an oxazole) between the anti-cancer drug and the polymer to minimize toxicity during transport to the tumor

• Highly efficient photosensitizer compound (Phthalocyanine) generates large amounts of singlet oxygen upon irradiation with 680 nm light

• Relatively long wavelength for photosensitzer excitation (680 nm) allows penetration of the exciting light through tissue

• Photosensitizer compound is in extremely close proximity to the cleavable linker (an oxazole) ensuring highly efficient cleavage by the generated singlet oxygen (diminished half-life of singlet oxygen in biologic systems no longer a limiting factor) • Highly loaded conjugate ensures delivery of high concentrations of drug to the tumor site (dendrimers, polysaccharides, and other polymers can have thousands of sites for drug loading)

• Improved release of the drug at the tumor site at high concentrations to achieve therapeutic effect

• A hydrophobic drug molecule that diffuses passively inside the cell and has a relatively long half life around the tumor could be used

• Improved targeting of the drug conjugate through use of an affinity binding molecule (an anti-HER2 antibody) that is specific for tumor cells

Details of Exemplary Conjugation Method

[00152] The lmmunopolymer part of the conjugate can be constructed utilizing many different antibodies, e.g., different monoclonal antibody (MAb) fragments. For example, a demonstration conjugate can include rhuMAb HER2-Fab' (Genentech, Inc.). The antibody can be modified for immunopolymer conjugation by addition of a cysteine residue near the COOH terminus of the recombinant sequence and reacting this with a maleimide activated polymer (Sigma-Aldrich, Inc. or Molecular Probes, Inc.). A negative control consisting of inactivated rhuMAb HER2-Fab', in which the antibody is exposed to a specific inactivating agent resulting in disruption of the antigen-binding domain essential for binding activity can also be prepared.

[00153] The Phthalocyanine-cleavable linker-Paclitaxel (PCT) portion of the conjugate can be prepared by conjugation of amine-modified Paclitaxel (Sigma- Aldrich, Inc.) to a COOH group on the cleavable linker (Alfa-Aesar, Inc) in the presence of EDC. The remaining amino group on the cleavable linker can then be complexed with Phthalocyanine (Sigma-Aldrich, Inc).

[00154] The final conjugate can then be prepared by combining the two subcomponents (immunopolymer with remaining amine groups and PCT conjugate). The immunopolymer can be conjugated to the PCT at a ratio of 1000 PCT's per 1 immunopolymer. EXAMPLE 3 - Nanovector Optimization

[00155] In many cases, after construction of an initial nanovector or set of nanovectors, it will be useful to select a suitable nanovector, or a candidate nanovector, or to optimize from a particular nanovector or set of nanovectors. Such optimization can utilize assays such as the following.

Binding Assay

[00156] Assays to measure binding specificity can be used to demonstrate specific conjugate binding to tumor cells. As an example, anti-HER2 antibody conjugated to the nanovector will provide specific binding to MDA-MB-453, a cell line expressing this known tumor antigen. Direct measurement of the conjugate's fluorescence (Excitation 680 nm / Emission 700 nm) on a fluorescence microscope will allow its level of specific binding to a cancer cells to be assessed. Briefly, MDA-MB-453 cells will be incubated with the nanovector at various concentrations and centrifuged to separate cells from unbound nanovector. The cells will then be applied to glass microscope slides and measured fluorometrically to determine the level of nanovector binding.

Details of Drug Release Assay

[00157] ^'A separate assay measuring the degree of drug release from the conjugate can also advantageously be used. For example, after activation of the photosensitizer and separation of free drug from the conjugate, the levels of free drug relative to conjugated drug can be quantified by homogeneous Taxol immunoassay (ALPHAscreen, Perkin-Elmer). Briefly, a sample of nanovector can be activated with a 680 nm laser. This released drug and conjugate can then be separated via dialysis, and the level of drug remaining on the conjugate can be measured and compared to the amount on the conjugate initially.

EXAMPLE 4 - Nanovector Evaluation

[00158] Once a candidate nanovector or set of such nanovectors has been identified for a particular application, it will generally be desirable to evaluate the nanovector, e.g., using cell-based and/or in vivo evaluations. Tumor Cell Line

[00159] As an example, cell line MDA-MB-453 can be obtained from American Type Culture Collection. This cell line has been extensively characterized for HER2 expression by flow cytometry, homogeneous immunoassay, ELISA, and immunohistochemistry (Lewis et a!.. 1993). lmmunohistochemistry (IHC) scoring of HER2 expression on a semiquantitative scale (0-3+) was first developed for trastuzumab clinical trials and is now routinely used clinically (Park. 2000; Mass et al., 2001) . Recent studies have suggested that the presence of HER2 gene amplification as detected by fluorescence in situ hybridization may be more predictive of response to trastuzumab than IHC assays of HER2 (Vogel et al., 2001). Therefore, direct measurement of the conjugate's fluorescence (Excitation 680 nm / Emission 700 nm) on a fluorescence microscope will allow its level of specific binding to a HER2 cell to be assessed.

Pharmacokinetic Studies

[00160] It can also be beneficial to conduct pharmacokinetic studies using a selected therapeutic nanoconjugate. For example, healthy, adult, Sprague Dawley rats can be used which will receive single or multiple intravenous injections of the complete nanovector conjugate, polymer-phthalocyanine, or free drug via indwelling jugular venous catheters. After the injection, blood will be serially sampled via catheter up to 48 h post injection, and plasma will be assayed for nanovector and Paclitaxel. Paclitaxel will be measured by homogeneous immunoassay method while nanovector or polymer-phthalocyanine concentration will be determined by flourometric analysis after extraction from plasma.

[00161] For two-component PK studies, plasma samples will be divided and analyzed for both Paclitaxel and HER2-Fab' concentrations. The Ab concentration will be determined by ELISA and homogeneous immunoassay using recombinant HER2 extracellular domain for capture and flourescence detection. Paclitaxel concentration will be determined by homogenous immunoassay method. [00162] For multiple-dose PK studies, rats will be given intravenous injections of nanovector conjugate every week for 3 weeks as in the therapy studies (see below).

Details of Therapeutic Studies

[00163] It can also be beneficial to conduct therapeutic studies initially in animal model systems.

[00164] For example, in one exemplary type of study, tumor cells can be implanted subcutaneously with or without Matrigel in the dorsum of athymic nude mice or SCID mice. When tumor xenografts become fully established and tumors are 200 mm³ on average, mice will be randomly assigned to different treatment groups (5-15 mice/group per condition). All intravenous treatments will be performed via tail vein injection.

[00165] Anti-HER2 nanovector will be administered intravenously at 20 mg Paclitaxel/kg/dose every week for 3 weeks, for a total dose of 60 mg/kg. Additional treatment groups will be included as in the pharmacokinetic studies. Saline (PBS) will be administered intravenously at the same injection volume and schedule as the nanovector. Free Paclitaxel will also be administered intravenously at its MTD of 25 mg/kg on the same schedule as the nanovector.

[00166] Negative control nanovector will be prepared identically as described above with the omission of the MAb conjugation step or the photosensitizer. All preparations of the negative control conjugate will be administered intravenously at the same dose and schedule as the complete nanovector.

[00167] All mice will receive illumination of the tumor site with a 6.2 mm diameter, 670 nm, 300 mW PDT laser (Edmund Optics) following standard PDT administration energies (i.e. 25 J/cm²) and techniques (Kolarova et al., 1999).

[00168] Tumors will be measured twice a week by caliper, and tumor volumes will be calculated using the equation: length x width x depth x 0.5. Mice with complete tumor regressions by measurement will be necropsied at the end of the study and classified as "cured" if no residual tumor cells are detected on histopathological examination of the tumor injection site.

[00169] Although these xenograft models do not typically support metastatic tumor growth, necropsy will also include gross examination of viscera to confirm absence of metastases. Studies will be terminated at ~2 months after treatment initiation in these models because of their requirement for estrogen supplementation; estrogen pellets implanted at the time of tumor injection potentially lose potency over extended periods. Mice will also be weighed and examined for toxicity twice a week. In most of the studies, the experimenter measuring tumor volumes will be blinded to treatment condition.

Statistical Methods and Definitions

[00170] All assay measurements will be performed in triplicate (at minimum) with resulting mean and standard deviation from the mean calculated.

[00171] Noise in an assay is defined as the standard deviation from the mean of a zero input (negative control). Assay sensitivity is defined as the input resulting in a signal-to-noise ratio of three.

[00172] Precision information from all assays will be calculated by the percent coefficient of variation (%CV) defined as the standard deviation from mean / mean x 100%.

[00173] Assay Linearity will be calculated by the R² metric using the linear regression line through a plot of signal vs. input.

[00174] Assay Dynamic Range will be calculated by dividing the highest input yielding a change in signal by the sensitivity of the assay.

Vertebrate Animals

[00175] All research involving vertebrate animals should be conducted in accordance with accepted practices and guidelines.

1. For pharmacokinetic studies, 5 to 15 adult, Sprague Dawley rats of either sex will receive injections of the nanovector and plasma will be assayed for nanovector and Paclitaxel. For therapeutic studies, 5 to 15 athymic nude mice or SCID mice of either sex, approximately 6 weeks of age,. will have tumor cells implanted subcutaneously and treated with the proposed nanovector and therapy.

2. These species and their numbers are typical per NIH guidelines. Sprague Dawley rats are historically acceptable models for pharmacokinetic studies and the mice species are immune compromised strains allowing tumor implantation and study.

3. All animals should be under veterinary care.

4. Any discomfort, pain, etc. will be inspected by the attending vet. If necessary, medication will be administered. Animals with pain or distress that cannot be alleviated by medication will be euthanized humanely according to AVMA guidelines.

5. Euthanasia will be performed according to AVMA guidelines. Mice will be euthanized by anesthetic overdose (Pentobarbital > 150 mg/kg).

[00176] All patents and other references cited in the specification are indicative of the level of skill of those skilled in the art to which the invention pertains, and are incorporated by reference in their entireties, including any tables and figures, to the same extent as if each reference had been incorporated by reference in its entirety individually.

[00177] One skilled in the art would readily appreciate that the present invention is well adapted to obtain the ends and advantages mentioned, as well as those inherent therein. The methods, variances, and compositions described herein as presently representative of preferred embodiments are exemplary and are not intended as limitations on the scope of the invention. Changes therein and other uses will occur to those skilled in the art, which are encompassed within the spirit of the invention, are defined by the scope of the claims.

[00178] It will be readily apparent to one skilled in the art that varying substitutions and modifications may be made to the invention disclosed herein without departing from the scope and spirit of the invention. For example, variations can be made to therapeutic agents, targeting agents, carrier polymers, cleavable moieties, and cleaving agents utilized. Thus, such additional embodiments are within the scope of the present invention and the following claims.

[00179] The invention illustratively described herein suitably may be practiced in the absence of any element or elements, limitation or limitations which is not specifically disclosed herein. Thus, for example, in each instance herein any of the terms "comprising", "consisting essentially of and "consisting of may be replaced with either of the other two terms. The terms and expressions which have been employed are used as terms of description and not of limitation, and there is no intention that in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed. Thus, it should be understood that although the present invention has been specifically disclosed by preferred embodiments and optional features, modification and variation of the concepts herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention as defined by the appended claims.

[00180] In addition, where features or aspects of the invention are described in terms of Markush groups or other grouping of alternatives, those skilled in the art will recognize that the invention is also thereby described in terms of any individual member or subgroup of members of the Markush group or other group.

[00181] Also, unless indicated to the contrary, where various numerical values or value range endpoints are provided for embodiments, additional embodiments are described by taking any 2 different values as the endpoints of a range or by taking two different range endpoints from specified ranges as the endpoints of an additional range. Such ranges are also within the scope of the described invention. Further, specification of a numerical range including values greater than one includes specific description of each integer value within that range. [00182] Thus, additional embodiments are within the scope of the invention and within the following claims.

Claims

WHAT IS CLAIMED IS:

1. A method for destroying or imaging abnormal cells in a patient at an internal treatment site, comprising administering to a subject having said abnormal cells a conjugate comprising at least one therapeutic or imaging agent moiety, a photosensitizer, a photocleavable linker, a targeting moiety targeting said cells, and a polymer or nanoparticle; and delivering to said internal treatment site light of a wavelength effective to cause generation of singlet oxygen by said photosensitizer, wherein said conjugate is configured such that generation of said singlet oxygen causes release of said therapeutic agent moiety or imaging agent moiety.

2. The method of claim 1 , wherein said conjugate further comprises at least one immunotolerant moiety that decreases immune system activation by said conjugate.

3. The method of claim 2, wherein said immunotolerant moiety comprises polyethyleneglycol or a derivative thereof.

4. The method of claim 1 , wherein said polymer is biocompatible.

5. The method of any of claims 1-4, wherein said polymer is a high molecular weight polymer with a highly flexible main chain.

6. The method of any of claims 1-5, wherein said polymer moiety bears a plurality of said therapeutic agent moieties linked to said polymer through a said photocleavable linker, wherein activation of said photosensitizer causes cleavage of a plurality of said photocleavable linkers.

7. The method of any of claims 1-6, wherein at least a portion of said polymer moiety comprises a polymer selected from the group consisting of N- (2-hydroxypropyl)methacrylamide (HPMA) copolymers, polyglutamic acid (PGA), polyethyleneglycol (PEG), polysaccharides such as polydextrans, dendrimers, liposomes, micelles, polymeric particles, linear cyclodextrins, polymerized /V-isopropylacrylamide, polyglutamic acid, polylysine, and polyaspartic acid.

8. The method of any of claims 1-3, wherein said polymer or nanoparticle comprises a nanoparticle.

9. The method of claim 8, wherein said nanoparticle is 10-200 nm in diameter.

10. The method of claim 8, wherein said nanoparticle is 20-100 nm in diameter.

11. The method of any of claims 1-10, wherein said photosensitizer absorbs light within a defined waveband and generates singlet oxygen in response to absorbing said light.

12. The method of claim 11 , wherein said photosensitizer absorbs light above 450 nm and generates singlet oxygen in response to absorbing said light.

13. The method of claim 11 , wherein said photosensitizer absorbs light above 550 nm and generates singlet oxygen in response to absorbing said light.

14. The method of claim 11 , wherein said photosensitizer absorbs light between 550 and 900 nm and generates singlet oxygen in response to absorbing said light

15. The method of any of claims 1-14, wherein said photosentizer is selected from the group consisting of pthalocyanines, naphthalocyanines, 7,8-dihydro- 5, 10, 15, 20- tetrakis (3-hydroxyphenyl)-21-23-[H]porphyrin (THPC), PEG-m- THPC, temoporfin, meta-tetra (hydroxyphenyl)chlorine, and photofrin.

16. The method of any of claims 1-15, wherin said photocleavable linker is hydrolyzed upon interaction with singlet oxygen.

17. The method of any of claims 1-16, wherein said photocleavable linker is selected from the group consisting of oxazoles, thiazoles, olefins, thioethers, selenoethers, and imidazoles.

18. The method of any of claims 1 -17, wherein said photosensitizer further provides a contrast agent enabling imaging of said treatment site.

19. The method of any of claims 1-18, wherein said targeting moiety comprises a specific binding agent.

20. The method of claim 19, wherein said specific binding agent is a moiety that specifically binds with one or more molecules at the surface of said abnormal cells.

21. The method of claim 19, wherein said specific binding agent is a small molecule that specifically binds with a cell surface receptor.

22. The method of claim 21 , wherein said cell surface receptor is a folate receptor.

23. The method of claim 19, wherein said specific binding agent is an antibody.

24. The method of claim 23, wherein said antibody preferentially binds to an antigen which is present substantially only at said abnormal cells in said patient.

25. The method of claim 24, wherein said antibody links with the abnormal cells at the treatment site, and said polymer increases an in vivo residence time of the conjugate proximal to said abnormal cells.

26. The method of any of claims 1-25, wherein said singlet oxygen is generated in close proximity to the cleavable linker, resulting in hydrolysis of said cleavable linker and release of said therapeutic agent.

27. The method of any of claims 1-26, wherein said therapeutic agent is released quickly and in high concentrations at said treatment site.

28. The method of any of claims 1-27, wherein said therapeutic agent is an anticancer drug.

29. The method of any of claims 1-28, wherein said at least one therapeutic agent comprises a plurality of different therapeutic agents.

30. The method of any of claims 1-29, wherein said therapeutic agent is highly toxic.

31. A method for treating a disease or condition in an animal, comprising administering to a subject suffering from or at risk of a disease of condition, a pharmacologically effective amount of a nanoconjugate molecule, wherein said nanoconjugate molecule comprises at least one therapeutic moiety; at least one photocleavable linker; at least one photosensitizer moiety; and at least one targeting moiety that preferentially targets said molecule to target cells, wherein photoactivation of said sensitizer causes cleavage of said photocleavable linker releasing at least one of said therapeutic moieties and said targeting moiety preferentially targets said nanoconjugate molecule to cells in said subject associated with said disease or condition.

32. The method of claim 31 , further comprising a polymer or nanoparticle, wherein said therapeutic moiety is linked to said polymer or nanoparticle through said photocleavable linker.

33. The method of claim 32, wherein said polymer is biocompatible.

34. The method of claim 32, wherein said polymer is a high molecular weight polymer with a highly flexible main chain.

35. The method of claim 32, wherein said polymer moiety bears a plurality of said therapeutic agent moieties linked to said polymer through a said photocleavable linker, wherein activation of said photosensitizer causes cleavage of a plurality of said photocleavable linkers.

36. The method of claim 32, wherein at least a portion of said polymer moiety comprises a polymer selected from the group consisting of N-(2- hydroxypropyl)methacrylamide (HPMA) copolymers, polyglutamic acid (PGA), polyethyleneglycol (PEG), polysaccharides such as polydextrans, dendrimers, liposomes, micelles, polymeric particles, linear cyclodextrins, polymerized Λ/-isopropylacrylamide, polyglutamic acid, polylysine, and polyaspartic acid.

37. The method of claim 32, wherein said polymer or nanoparticle comprises a nanoparticle.

38. The method of claim 37, wherein said nanoparticle is 10-200 nm in diameter.

39. The method of claim 37, wherein said nanoparticle is 20-100 nm in diameter.

40. The method of claim 31 , wherein said photosensitizer absorbs light within a defined waveband and generates singlet oxygen in response to absorbing said light.

41. The method of claim 31 , wherein said photosensitizer absorbs light above 450 nm and generates singlet oxygen in response to absorbing said light.

42. The method of claim 31 , wherein said photosensitizer absorbs light above 550 nm and generates singlet oxygen in response to absorbing said light.

43. The method of claim 31 , wherein said photosensitizer absorbs light between 550 and 900 nm and generates singlet oxygen in response to absorbing said light

44. The method of claim 31 , wherein said photosentizer is selected from the group consisting of pthalocyanines, naphthalocyanines, 7,8-dihydro-5, 10, 15, 20- tetrakis (3-hydroxyphenyl)-21-23-[H]porphyrin (THPC), PEG-m-THPC, temoporfin, meta-tetra (hydroxyphenyl)chlorine, and photofrin.

45. The method of claim 31 , wherin said photocleavable linker is hydrolyzed upon interaction with singlet oxygen.

46. The method of claim 31 , wherein said photocleavable linker is selected from the group consisting of oxazoles, thiazoles, olefins, thioethers, selenoethers, and imidazoles.

47. The method of claim 31 , wherein said photosensitizer further provides a contrast agent enabling imaging of said treatment site.

48. The method of claim 31 , wherein said targeting moiety comprises a specific binding agent.

49. The method of claim 48, wherein said specific binding agent is a moiety that specifically binds with one or more molecules at the surface of said abnormal cells.

50. The method of claim 48, wherein said specific binding agent is a small molecule that specifically binds with a cell surface receptor.

51. The method of claim 50, wherein said cell surface receptor is a folate receptor.

52. The method of claim 48, wherein said specific binding agent is an antibody.

53. The method of claim 52, wherein said antibody preferentially binds to an

. antigen which is present substantially only at said target cells in said patient.

54. The method of claim 53, wherein said antibody links with the target cells at the treatment site, and said polymer increases an in vivo residence time of the conjugate proximal to said target cells.

55. The method of claim 31 , wherein said singlet oxygen is generated in close proximity to the cleavable linker, resulting in hydrolysis of said cleavable linker and release of said therapeutic agent.

56. The method of claim 55, wherein said therapeutic agent is released quickly and in high concentrations at said treatment site.

57. The method of claim 31 , wherein said therapeutic agent is an anticancer drug.

58. The method of claim 31 , wherein said at least one therapeutic agent comprises a plurality of different therapeutic agents.

59. The method of claim 31 , wherein said therapeutic agent is highly toxic.

60. The method of claim 31 , wherein said therapeutic agent is an antiinfective agent.

61. A therapeutic nanoconjugate molecule, comprising at least one therapeutic moiety; at least one photocleavable linker; at least one photosensitizer moiety; and at least one targeting moiety that preferentially targets said molecule to a target cells, wherein photoactivation of said sensitizer causes cleavage of said photocleavable linker releasing at least one of said therapeutic moieties.

62. The nanoconjugate molecule of claim 61 , further comprising a polymer or nanoparticle, wherein said therapeutic moiety is linked to said polymer or nanoparticle through said photocleavable linker.

63. The nanoconjugate molecule of claim 62, wherein said polymer is biocompatible.

64. The nanoconjugate molecule of claim 62, wherein said polymer is a high molecular weight polymer with a highly flexible main chain.

65. The nanoconjugate molecule of claim 61 , further comprising a polymer or nanoparticle moiety which bears a plurality of said therapeutic moieties linked to said polymer through a said photocleavable linker, wherein activation of said photosensitizer causes cleavage of a plurality of said photocleavable linkers.

66. The nanoconjugate molecule of claim 62, wherein at least a portion of said polymer moiety comprises a polymer selected from the group consisting of N-(2-hydroxypropyl)methacrylamide (HPMA) copolymers, polyglutamic acid (PGA), polyethyleneglycol (PEG), polysaccharides such as polydextrans, dendrimers, liposomes, micelles, polymeric particles, linear cyclodextrins, polymerized Λ/-isopropylacrylamide, polyglutamic acid, polylysine, and polyaspartic acid.

67. The nanoconjugate molecule of claim 62, wherein said polymer or nanoparticle comprises a nanoparticle.

68. The nanoconjugate molecule of claim 67, wherein said nanoparticle is 10- 200 nm in diameter.

69. The nanoconjugate molecule of claim 67, wherein said polymer is 20-100 nm in diameter.

70. The nanoconjugate molecule of claim 61 , wherein said photosensitizer absorbs light within a defined waveband and generates singlet oxygen in response to absorbing said light.

71. The nanoconjugate molecule of claim 61 , wherein said photosensitizer absorbs light above 450 nm and generates singlet oxygen in response to absorbing said light.

72. The nanoconjugate molecule of claim 61 , wherein said photosensitizer absorbs light above 550 nm and generates singlet oxygen in response to absorbing said light.

73. The nanoconjugate molecule of claim 61 , wherein said photosensitizer absorbs light between 550 and 900 nm and generates singlet oxygen in response to absorbing said light

74. The nanoconjugate molecule of claim 61 , wherein said photosentizer moiety is selected from the group consisting of pthalocyanines, naphthalocyanines, 7,8-dihydro-5, 10, 15, 20- tetrakis (3-hydroxyphenyl)-21- 23-[H]porphyrin (THPC), PEG-m-THPC, temoporfin, meta-tetra (hydroxyphenyl)chlorine, and photofrin.

75. The nanoconjugate molecule of claim 61 , wherin said photocleavable linker is hydrolyzed upon interaction with singlet oxygen.

76. The nanoconjugate molecule of claim 61 , wherein said photocleavable linker is selected from the group consisting of oxazoles, thiazoles, olefins, thioethers, selenoethers, and imidazoles.

77. The nanoconjugate molecule of claim 61 , wherein said targeting moiety comprises a specific binding agent.

78. The nanoconjugate molecule of claim 77, wherein said specific binding agent is a moiety that specifically binds with one or more molecules at the surface of said target cells.

79. The nanoconjugate molecule of claim 77, wherein said specific binding agent is a small molecule that specifically binds with a cell surface receptor.

80. The nanoconjugate molecule of claim 79, wherein said cell surface receptor is a folate receptor.

81. The nanoconjugate molecule of claim 77, wherein said targeting moiety comprises an antibody.

82. The nanoconjugate molecule of claim 77, wherein said targeting moiety comprises a targeting peptide.

83. The nanoconjugate molecule of claim 77, wherein said targeting moiety comprises a targeting polysaccharide.

84. The nanoconjugate molecule of claim 61 , wherein said therapeutic moiety comprises an anticancer therapeutic agent.

85. The nanoconjugate molecule of claim 61 , wherein said therapeutic moiety comprises a plurality of different therapeutic moieties.

86. The nanoconjugate molecule of claim 61 , wherein said therapeutic moiety is an antiinfective agent.

87. The nanoconjugate molecule of claim 61 , wherein said therapeutic moiety is highly toxic.

88. A therapeutic nanoconjugate molecule, comprising at least one therapeutic moiety; at least one exogenously cleavable linker; and at least one targeting moiety that preferentially targets said molecule to a biological target, wherein contacting said exogenously cleavable linker with an exogenous cleaving agent causes cleavage of said exogenously cleavable linker releasing at least one of said therapeutic moieties.

89. The nanoconjugate of claim 88, further comprising a polymer moiety which bears a plurality of said therapeutic moieties each linked to said polymer through a said exogenously cleavable linker, wherein activation of said photosensitizer causes cleavage of a plurality of said exogenously cleavable linkers.

90. The nanoconjugate of claim 89, wherein said therapeutic moiety comprises at least two different therapeutic moieties.

91. The nanoconjugate of claim 88, wherein said exogenously cleavable linker is cleaved in vivo by an exogeneously introduced agent selected from the group consisting of an enzyme, X-rays, and high energy radiation.

92. A therapeutic kit, comprising a packaged, pre-measured quantify of a nanoconjugate that comprises at least one therapeutic moiety; at least one photocleavable linker; at least one photosensitizer moiety; and at least one targeting moiety that preferentially targets said molecule to a biological target, wherein photoactivation of said sensitizer causes cleavage of said photocleavable linker releasing at least one of said therapeutic moieties; and instructions for administering said nanoconjugate to a subject suffering from or at risk of a disease or condition potentially treatable by administration of said therapeutic agent.

93. The kit of claim 92, wherein said nanoconjugate is a nanoconjugate of any of claims 61-91.

94. A method for preparing a therapeutic nanoconjugate molecule, comprising covalently linking together a cell-targeting moiety, at least one photosensitizer, a polymer backbone, a plurality of photocleavable linkers, and a plurality of therapeutic agents, wherein illumination of said conjugate in physiological solution causes said photosensitizer to generate singlet oxygen which causes cleavage of said photocleavable linkers, releasing said therapeutic agents from said polymer backbone.

95. The method of claim 94, wherein said therapeutic nanoconjugate molecule is a nanoconjugate molecule of any of claims 61-91.

96. A method for therapeutic use of a drug having unacceptably high toxicity when administered systemically, comprising administering to a subject suffering from or at risk of a disease or condition a conjugate of any of claims 61-91 , wherein said conjugate comprises a highly toxic drug. The method of claim 96, wherein said highly toxic drug is substantially non-toxic when attached in said conjugate.