Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K49/00—Preparations for testing in vivo
  • A61K49/06—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K49/00—Preparations for testing in vivo
  • A61K49/06—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations
  • A61K49/08—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by the carrier
  • A61K49/085—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by the carrier conjugated systems
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K39/385—Haptens or antigens, bound to carriers
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K41/00—Medicinal preparations obtained by treating materials with wave energy or particle radiation ; Therapies using these preparations
  • A61K41/0057—Photodynamic therapy with a photosensitizer, i.e. agent able to produce reactive oxygen species upon exposure to light or radiation, e.g. UV or visible light; photocleavage of nucleic acids with an agent
  • A61K41/0071—PDT with porphyrins having exactly 20 ring atoms, i.e. based on the non-expanded tetrapyrrolic ring system, e.g. bacteriochlorin, chlorin-e6, or phthalocyanines
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
  • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
  • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
  • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
  • A61K47/645—Polycationic or polyanionic oligopeptides, polypeptides or polyamino acids, e.g. polylysine, polyarginine, polyglutamic acid or peptide TAT
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K49/00—Preparations for testing in vivo
  • A61K49/06—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations
  • A61K49/08—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by the carrier
  • A61K49/10—Organic compounds
  • A61K49/14—Peptides, e.g. proteins
  • A61K49/146—Peptides, e.g. proteins the peptide being a polyamino acid, e.g. poly-lysine
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents

Definitions

• the invention relates to the field of biotechnology and cancer treatment, and more particularly to the use of magnetic resonance imaging-guided photodynamic treatment of tumors and other target tissues and/or sites.
• PDT i.e., photochemotherapy
• Photochemotherapy is an emerging cancer treatment based on the combined effects of visible light and a photosensitizing agent that is activated by exposure to light of a specific wavelength.
• Photochemotherapy is well known (Hsi R. A., Rosenthal D. I., Glatstein E., "Photodynamic therapy in the treatment of cancer: current state of the art," Drugs 1999, 57(5):725-734; Moore J. V., West C. M. L., Whitehurst C, "The biology of photodynamic therapy,” Phys. Med. Biol.
• a photosensitizing agent is injected into a subject systemically, which results in preferential uptake of the photosensitizing agent in tumor cells.
• the tumor site is then illuminated with visible light of a particular energy and wavelength that is absorbed by the photosensitizing agent. This illumination activates the photosensitizing agent, for instance, resulting in the generation of cytotoxic excited state oxygen molecules in those cells in which the agent has localized.
• These molecules are highly reactive with cellular components, and provide a treatment (a decrease in size, number, or mass) of tumor cells.
• Photodynamic therapy initially garnered clinical interest in the mid-20 ⁇ century when it was demonstrated that porphyrin compounds accumulated preferentially in tumors, resulting in photosensitization and, due to the fluorescence of these compounds, aided in tumor detection.
• Dougherty is credited with the creation of modern photodynamic therapy, recognizing the potential of photodynamic therapy for tumor treatment and demonstrating its use in treating metastatic tumors of the skin in the 1970's (Oleinick N. L., Evans H. H., "The photobiology of photodynamic therapy: cellular targets and mechanisms," Radial Res. 1998; 150:S146-56).
• photodynamic therapy generally involves the administration of compounds that are capable of absorbing light, typically in the visible range, (but also in the near ultraviolet), followed by irradiation of locations of the subject for which a modifying or inhibitory effect is desired.
• PDT was initially developed using hematoporphyrin and related compounds to treat tumors, as it appeared that these compounds would localize in rapidly dividing cells (such as in tumors). The tumor could then be irradiated with light. The light is absorbed by the hematoporphyrin and the tumor destroyed.
• PDT has since been shown to be useful for treating of atherosclerotic plaques, restenosis, infections in the blood stream, rheumatoid arthritis, psoriasis and in the treatment of ocular conditions not necessarily limited to tumors.
• Macromolecular Gd(III) complexes have been developed by conjugating these Gd(III) chelates to bio-medical polymers, including poly(amino acids)' 2 ' 3) , polysaccharides ⁇ ' 5) , dendrimers (6 8) , and proteins (9 ' 10) , to improve image contrast enhancement. These macromolecular agents have demonstrated superior contrast enhancement for blood pool imaging and cancer imaging in animal models. Unfortunately, the clinical application of macromolecular agents is limited by their slow excretion after MRI exams'"' 12) and potential unwanted side-effects of Gd(III) ions released by the metabolism of the agents (13"15) .
• MRI-guided therapies for cancer like LITT (Laser-Induced Interstitial Thermotherapy), or RFA (Radio Frequency Ablation), are invasive and have problematic disadvantages such as difficulty in treating non-uniform lesions and safety concerns due to skin burns.
• LITT Laser-Induced Interstitial Thermotherapy
• RFA Radio Frequency Ablation
• low molecular weight contrast agents have fast clearance, low relaxivity, and lower contrast enhancement, especially in tumors.
• high molecular weight contrast agents are now more commonly used to passively target tumors (due to the EPR effect). These higher molecular weight contrast agents show higher relaxivity and consequently better contrast enhancement in tumors.
• the present invention involves a new technology for cancer treatment with MRI-guided photodynamic cancer therapy.
• this technology includes administration of MRI contrast agent labeled polymer-photosensitizer conjugates, detection and localization of tumor or cancer tissues with contrast-enhanced MRI and illumination of target tissues, such as, but not limited to, tumor or cancer tissues, with a laser.
• target tissues such as, but not limited to, tumor or cancer tissues
• the delivered laser energy will activate the photosensitizer accumulated in the target tissue, resulting in target cell death and treatment.
• This method as disclosed herein, is more non-invasive as compared to other image-guided therapies, including image-guided ablation.
• the present invention includes use of MRI contrast-agent-labeled polymer-photosensitizer conjugates and a combination of contrast-enhanced MRI with photodynamic therapy.
• Contrast-enhanced MRI is an effective approach to non-invasive tumor detection.
• Photodynamic therapy is a clinically used therapy in the treatment of diseases, such as cancer.
• Embodiments of the invention combine contrast-enhanced MRI and PDT to provide MRI-guided photodynamic therapy.
• the drug delivery system contains a drug carrier, including polymers, proteins, liposomes, nanoparticles, MRI contrast agents, including Gd, Fe, Mn complexes or iron oxide particles; a photosensitizer or a tissue targeting agent, such as a tumor targeting agent.
• the delivery system carries the contrast agent and photosensitizer into the target tissue, which tissue is then localized in the subject by contrast-enhanced MRI.
• a laser beam is directed to the target tissue site. Laser energy activates the photosensitizer, which, in an embodiment, generates highly reactive species that kill or destroy the target cells and tissue.
• the method is used to treat cancer or tumor cells.
• the method of the present invention is not limited to MRI-targeted photodynamic cancer treatment of target tissues. It can also be used for positron emission tomography (PET) and single photon emission computed tomography (SPECT)-targeted photodynamic treatment.
• PET positron emission tomography
• SPECT single photon emission computed tomography
• one of skill in the art can substitute (for the MRI contrast agent) a PET and SPECT probe in the delivery system.
• the photosensitizer conjugates are used to detect and treat breast cancer cells.
• the method of the present invention is used to detect and treat breast cancer cells, pancreatic cancer cells, prostate cancer cells, skin cancer cells, and other malignancies able to be treated using the method of the present invention.
• MRI contrasting agents conjugated to photosensitizers useful in the treatment of target tissues in a subject.
• FIG. 1 Synthesis of Poly-L-Glutamic Acid.
• y-Benzyl glutamate is reacted with triphosgene in tetrahydrofuran ("THF") for three hours at 50 0 C, then cooled to -2O 0 C for 24 hours to produce the N-carboxy anhydride ("NCA") of y-benzyl glutamate.
• NCA N-carboxy anhydride
• This product is then reacted with tributylamine in dichloromethane and ethylacetoacetate (“EtOAc,” 1 :6, v:v) to produce poly-(benzyl glutamic acid).
• EtOAc tributylamine in dichloromethane and ethylacetoacetate
• Poly-(benzyl glutamic acid) is then incubated with hydrogen bromide (Br 2 + tetralin) for three hours and stirred for 72 hours to yield poly-(Z-glutamic acid).
• FIG. 2 Synthesis of Poly-L-Glutamic Acid Active Ester.
• Poly(L-glutamic acid) (“PLGA,” MW 67 kDa, 810 mg) is dissolved in 4 ml of TVyV-dimethylformamide (“DMF") with 2.166 g of N-hydroxy succinimide (“NHS,” MW 115, 3X moles based on glutamic acid monomer). A minimum amount of DMF is used.
• DMF TVyV-dimethylformamide
• NHS N-hydroxy succinimide
• EDC l-ethyl-3-(3-dimethyl aminopropyl)carbodiimide
• FIG. 3 Synthesis of PLGA-Mce 6 -DOTA-Gd Complexes.
• PLGA-NHS, mesochlorin e 6 ((25, 35)-18-carboxy-20-(carboxymethyl)-8, 13-diethyl-3, 7, 12, 17-tetramethyl- chlorin-2-propionic acid, "MCe 6 ") and 1 , 4, 7, 10-Tetraazacyclododecane-N,N',N",N"'-tetraacetate (“DOTA”)-hexane diamine are incubated in dimethylaminopyridine ("DMAP") and DMF for 24 hours.
• DMAP dimethylaminopyridine
• DMF dimethylaminopyridine
• the DMF is evaporated and the conjugate dissolved in water using 0.1 N NaOH and adjusted to pH 11.
• Mce 6 and other unreacted components are extracted and the product reacted with excess Gd(OAc) 3 .
• EDTA ethyl enediaminetetraacetic acid
• FIG. 4 Determination of Molecular Weight of Polymer-Gd Complexes. Plot of elution profile, milli-absorbane units ("mAU”) v. time in minutes, showing elution of polymer-Gd complexes. Sample was applied to a Sepharose 6 size-exclusion gel FPLC column (Pharmacia) at 0.5 ml/min in Tris buffer with 30% acetonitrile.
• mAU milli-absorbane units
• FIG. 5 Determination of Relaxivity of Polymer-Gd Complexes. Plot of TI v. [Gd] in mM allows determination of the relaxivity of the polymer-Gd complex.
• the Tl of Polymer-Gd complexes at various concentrations in aqueous solution were measured using Bl homogeneity corrected Look-Locker technique on a 1.5T GE NV/CVi scanner with the LX 8.4 operating system at room temperature* l8) .
• the coordination of bulky water molecules is crucial for the relaxivity of Gd(III) complexes.
• FIG. 6 MRI of tumors (athymic nu/nu mice) at pre-, 5, 30, 60, 120 minutes and 24 hours post-injection.
• the slices are through the heart and tumor separately, for mouse 1, and through the heart and tumor simultaneously, for mouse 2.
• the 24 hour images show contrast enhancement through the tumor as compared to pre-contrast while there is contrast enhancement in the heart at five minutes post-injection.
• FIG. 7. 2D coronal MR images for animals receiving polymer with Mce6 (Poll, upper panel) and control (Pol2, lower panel) through tumor cells.
• FIG. 8 2D coronal MR images for animals receiving polymer with Mce6 (Poll, upper panel) and control (Pol2, lower panel) through heart cells.
• FIG. 9. 3D MIP images for animals receiving polymer conjugates with Mce6 (Poll, upper panel) and control (Pol2, lower panel).
• FIG. 10 Spin echo images for Poll (upper panel) and Pol2 (lower panel) showing the accumulation of contrast agent in tumor tissues, post treatment.
• FIG. 11 An illustration of the efficacy of photodynamic therapy. DETAILED DESCRIPTION OF THE INVENTION
• Treatment of a tumor or cancer cells is defined as including the provision of a dose of the polymer-photosensitizer conjugate in conjunction with irradiation using laser energy enabling a decrease in the size, shape, mass, viability or number of viable cancer or tumor cells.
• an effective amount means an amount of the polymer-photosensitizer conjugate and laser energy administered to the subject that is effective to improve, prevent, or treat the disease condition in the subject.
• the pharmaceutical carriers acceptable for the purposes of this invention include carriers that do not adversely affect the drug, the host, or the material comprising the drug delivery device.
• Suitable pharmaceutical carriers include sterile water, saline, dextrose, dextrose in water or saline condensation products of castor oil and ethylene oxide (combining about 30 to 35 moles of ethylene oxide per mole of castor oil), liquid acid, lower alkanols, oils such as corn oil, peanut oil, sesame oil and the like, with emulsifiers such as mono- or diglyceride of a fatty acid; or a phosphatide, e.g., lecithin, and the like; glycols, polyalkylene glycols, aqueous media in the presence of a suspending agent, for example, sodium carboxymethyl cellulose, sodium alginate, poly(vinylpyrrolidone), and the like, alone, or with suitable dispensing agents such as lecithin, polyoxyethylene stearate, and the like
• the various embodiments of the present invention relate to a novel polymer-photosensitizer drug conjugate for photodynamic therapy. Further embodiments of the invention generally relate to a high molecular weight polymer-photosensitizer drug conjugates.
• the macro-molecule, or high molecular weight polymer is a paramagnetically labeled polymer conjugate for photodynamic therapy.
• the polymer paramagnetically labeled conjugate is paramagnetically labeled poly-(L-glutamic acid)-Mce 6 .
• the conjugated polymer comprises PLGA-Mce 6 -[(Gd-DOTA)-hexane diamine].
• those of ordinary skill in the art would readily identify other polymer-photosensitizer drug conjugates that would function in various embodiments of the present invention.
• PDT photodynamic therapy
• various methods of PDT can be used with embodiments of the present invention, such as those disclosed in US 5,829,448, US 5,736,563, US 5,630,996, US 5,482,698, and US 6,889,723, the contents of all of which are hereby incorporated by reference as if they were set forth herein their entirety.
• the PDT of the invention is a MRI guided therapy and the embodiment of the polymer-photosensitizer drug conjugates is contrast enhanced.
• Further embodiments comprise positron emission tomography (PET) and single photon emission computed tomography (SPECT)-targeted photodynamic treatment.
• PET and SPECT-guided therapy one of skill in the art can substitute for the MRI contrast agent a PET and SPECT probe in the delivery system.
• the present invention includes a delivery system containing a contrast agent and a photosensitizer (such as a MRI contrast agent); precise location of target tissue in the patient with contrast-enhanced MRI or other imaging modalities; and image-guided irradiation of target tissue with laser energy. This allows for precise location of tumor tissue in a subject and non-invasive treatment of target tissues with PDT.
• a drug delivery system of the present invention comprises a drug carrier, including polymers, proteins, liposomes, nanoparticles, MRI contrast agents, such as, but not limited to Gd, Fe, Mn complexes or iron oxide particles; a photosensitizer or other tissue targeting agent, such as a tumor targeting agent.
• a drug carrier including polymers, proteins, liposomes, nanoparticles, MRI contrast agents, such as, but not limited to Gd, Fe, Mn complexes or iron oxide particles; a photosensitizer or other tissue targeting agent, such as a tumor targeting agent.
• a delivery system carries or conveys the contrast agent and photosensitizer into or adjacent the target site.
• the delivery system is conveyed to a target site that is a target tissue and/or a target cell.
• FIG. 1 For purposes and/or method common in the art.
• the target site is localized by contrast-enhanced MRI or other form of MRI.
• Other embodiments direct the delivery system to the target site.
• An energy source such as a laser, other energy pulse, beam, and/or photo means, is then directed to/applied to/illuminates the target site.
• the energy pulse activates the photosensitizer, thereby killing and/or destroying the target site.
• the energy pulse initiates or causes the photosensitizer to generate a reactive specie(s) that kills and/or immobilizes the target site.
• various embodiments of methods of the present invention generally comprise a method for killing or destroying a target site in a subject or in vitro, said method comprising: administering to the subject or in vitro a pharmaceutically effective amount of a delivery system, said delivery system comprising a contrast agent and a photosensitizer; conveying the delivery system to the target tissue; localizing the target tissue with an imaging modality; and illuminating the target tissue and killing or destroying the target site.
• Delivery systems of the present invention may be administered by any method and/or applicator known in the art.
• a delivery system of the present invention is administered by injection, such as by a syringe, needleless injector, and/or the like.
• a delivery system of the present invention is administered by application, such as pouring, wiping, smearing, and/or the like.
• a delivery system of the present invention is administered orally.
• the step of administering to the subject a pharmaceutically effective amount of a delivery system comprises administering a pharmaceutically effective amount of a magnetic resonance imaging contrast agent labeled polymer-photosensitizing conjugate.
• the a pharmaceutically effective amount of a magnetic resonance imaging contrast agent labeled polymer-photosensitizing conjugate is poly-(Z-glutamic acid)- (25, 35)-18-carboxy-20-(carboxymethyl)-8, 13-diethyl-3, 7, 12, 17-tetramethyl- chlorin-2-propionate-[(Gd- 1 , 4, 7,10-tetraazacyclododecane-N,N',N",N'"-tetraacetate)-hexane diamine].
• Various embodiments comprise methods and/or systems used to treat or provide treatment to a desired tissue or cell, such as, but not by way of limitation, malignant cells, such as cancer and/or tumor cells, moles, warts, growths, and/or the like.
• a desired tissue or cell such as, but not by way of limitation, malignant cells, such as cancer and/or tumor cells, moles, warts, growths, and/or the like.
• an embodiment of the present invention comprises the use of a pharmaceutically acceptable composition for the treatment of a tumor or cancerous tissue or cancerous cells.
• a method of synthesis of an exemplary conjugate comprises the steps of: A method of synthesizing poly-(Z-glutamic acid)-(25', 35)-18-carboxy-20-(carboxymethyl)-8, 13-diethyl-3, 7, 12, 17-tetramethyl- chlorin-2-propionate-[(Gd-l, 4,
• the high molecular weight poly-glutamic acid polymers show higher relaxivity and higher accumulation in tumor tissue after 24 hours as compared to two hours which is indicated by an increase in contrast enhancement.
• the amount of polymer in or about the tumor can aid in determining the exact tumor volume to be irradiated, such as with the use of an imaging modality.
• the time of treatment may vary according to the size of the malignancy, the strength of the illumination, energy beam, and/or pulse, the depth of the malignancy in the tissue, the composition of the malignancy in the tissue, and/or the like.
• the malignancy is treated with laser at 650 run for about 1 to about 25 minutes.
• any size and/or strength laser may be used and/or modified to be used with various embodiments provided the laser does not cause excessive damage to surrounding tissues when used within correct parameters, such as, but not limited to beam width and/or time.
• illumination of the target site is delayed until time has been given for the administered delivery system to localize the target site.
• a delay from between 0.1 hours to 36 hours occurs.
• a delay from about 1 hour to 24 hour occurs.
• a delay of about 18 hours occurs.
• a polymer-photosensitizer conjugate of the present invention is administered in multiple doses.
• a target site can be illuminated multiple times or through multiple procedures.
• images of the target site are taken.
• an image is taken prior to administration of a delivery vehicle.
• an image is taken upon administration of a delivery vehicle.
• multiple images are taken at varying time points.
• an image is used to provide data, to provide direction, to monitor progress, and/or the like.
• the polymer used is poly-(Z-glutamic acid) with DOTA-Gd as the contrast agent and Mse 6 as the photosensitizer drug.
• the present invention also discloses a method of preparing a pharmaceutical composition useful in, among other things, treating cancer, hi an embodiment, the composition comprises a PLGA-Mce 6 -[(Gd-DOTA)]. In an embodiment, the composition comprises the compound PLGA-Mce 6 -[-l,6-hexanediamine-(Gd-DOTA)].
• Various PLGA-Mce 6 -(Gd-DOTA) conjugates contain a disulfide linker between the contrast agent and the polymeric carrier, allowing rapid clearing of the remaining Gd complex after treatment from the subject. (See reference 17, Lu, et al.
• biodegradable linkers similar to this are known in the art and it is understood that one of skill in the art is able to substitute other such linkers to provide a biodegradable character to the conjugate.
• Such biodegradable polymeric -conjugates are covered by the present invention and constitute a further exemplary embodiment of the present invention.
• various embodiments of the present invention comprise a pharmaceutical composition useful in the treatment of a target tissue in a subject, said pharmaceutical composition comprising: a pharmaceutically acceptable amount of the compound poly-(Z-glutamic acid)-(25, 3,S)-18-carboxy-20-(carboxymethyl)-8, 13-diethyl-3, 7, 12, 17-tetramethyl- chlorin-2-propionate-[(Gd-l, 4,
• Further embodiments comprise the use of a pharmaceutically acceptable composition
• a pharmaceutically acceptable composition comprising poly-(Z-glutamic acid)-(25', 35)-18-carboxy-20-(carboxymethyl)-8, 13-diethyl-3, 7, 12, 17-tetramethyl- chlorin-2-propionate-[l,6-hexanediamine-(Gd-l, 4, 7,10-tetraazacyclododecane-N,N',N",N"'-tetraacetate)] in the manufacture of a medicament for the treatment of a target site.
• Further embodiments comprise the use of a pharmaceutically effective amount of a magnetic resonance imaging contrast agent labeled polymer-photosensitizing conjugate to localize or locate a target site.
• the agent is a poly-(Z-glutamic acid)-(25, 35)-18-carboxy-20-(carboxymethyl)-8, 13-diethyl-3, 7, 12, 17-tetramethyl- chlorin-2-propionate- [ 1 ,6-hexane-diamine-(Gd- 1, 4, 7,10-tetraazacyclododecane-N,N',N",N'"-tetraacetate)] .
• a system for localizing a target tissue comprising: a magnetic resonance imaging contrast agent, a photosensitizer, and an imaging modality.
• a system for treating a target site is disclosed, the system comprising: a magnetic resonance imaging contrast agent; a photosensitizer, an imaging modality; and a laser.
• Further embodiments of systems of the present invention may comprise an applicator for applying the delivery vehicle.
• ⁇ -benzyl-glutamate was purchased for VWR (West Chester, PA). Extra dry solvents; Tetrahydrofuran (THF), Ethyl acetate, and Methylene chloride were purchased from Arcos Organics, NJ. Meso chlorin e 6 and Cyclen were purchased from Macrocyclic Inc. Gd (OAc) 3 and di-tert-butyl dicarbonate (t-boc), tert-butyl bromoacetate, N-ethyl diisopropylamine, and mono-boc-l,6-diamino hexane were purchased from Alfa Aesar (Ward Hill, MA).
• EDC [1 -ethyl - 3-(3-Dimethylaminopropyl)carbodiimide HCl]
• EDC was purchased from TCI America (Portland, OR).
• Silica gel, mesh size 230-400 was purchased from Natland International Corp., NC. All solvents were purchased from Fisher Scientific and used without further purification unless otherwise stated.
• PD-IO desalting columns were purchased from Amersham Bioscience (Uppsala, Sweden).
• Cell line (MDA-MB-231), LH- 15 media containing 2 ⁇ M glutamine and Trypsin were purchased from American Type Culture Collection (ATCC, Manassas VA).
• Female nu/nu athymic mice were purchased from NCI (Frederick, MD). Animals were maintained according to IACUC, University of Utah guidelines.
• Gd content was determined using Inductively Coupled argon Plasma- Optical Emission Spectrometer (ICP-OES) (Perkin Elmer, Norwalk, CT, Optima 3100XL). Tl relaxivity measurements for the final conjugates were acquired on a Siemens Trio 3T MRI scanner using standard inversion recovery sequence. Photodynamic therapy was carried out via Diode Odyssey laser (650nm) (CAO Group, UT) A. Synthesis of paramagnetically labeled drug- polymer complexes 1. Synthesis of poly-(L-glutamic acid) active ester (PLGA-OSu).
• PLGA poly-L-glutamic acid
• High molecular weight poly- (L-glutamic acid) was synthesized by methods previously described. Briefly, the first step in the synthesis involved the formation of N-carboxy anhydride by the reaction of ⁇ - benzyl glutamate (15g, O.O ⁇ mol) with triphosgene (9.37g, 0.03mol) in THF (15ml). The reaction was carried out at 5O 0 C for 3 hours under N 2 and the reaction mixture was poured into n-hexane (150ml) to precipitate the NCA.
• PBLG high molecular weight poly-(benzyl- ⁇ -glutamate)
• PBLG was precipitated in methanol: ether (2:1) mixture, filtered, washed with ether and dried in vacuum.
• the final step involved removal of the benzyl group on PBLG, dissolved in dichloromethane (l.Ogm in 300ml), by bubbling HBr through the solution until poly-L-glutamic acid precipitated out.
• HBr gas was produced by the reaction of Tetralin and Bromine.
• PLGA was further purified using acetone. Molecular weight determination was carried out using SEC on Superdex 200 column equipped with UV and refractive index (RI) detectors. Absence of UV peak, corresponding to PJ peak confirmed near complete deprotection of PLGA.
• DO 3 A was synthesized via cyclen in 2 steps according to methods previously described but with few changes to obtain DO 3 A of high purity.
• ter/-butyl bromoacetate (leq, lOmmol, 1.96gm) dissolved in 20ml was added dropwise to the reaction mixture.
• the reaction was stirred for another 18 hours at R.T.
• the solution was concentrated in vacuum, solid dissolved in minimum amount of Methylene chloride and applied to Silica gel column for further purification. Gradient elution using ethyl acetate: methanol (100%, 50:1, 20: 1 and 10:1) as eluent system was carried out.
• TB- cyclen (t ⁇ rt-butyl protected cyclen) was obtained in 20: 1 fraction.
• TB cyclen was deprotected using minimum amount of cold trifluoroacetic acid (25°C, overnight) to form DO 3 A.
• the product was purified using diethyl ether.
• Tl relaxivity of the polymer complexes, Poll and Pol2 was determined on a Siemens Trio 3T scanner using a standard inversion recovery sequence with varying TI of 22- 1600 ms.
• Gd (III) of Pol, and PoI 2 content was determined by Inductively Coupled Plasma- Optical Emission Spectrometry (ICP-OES) using Gd standards to construct standard curve.
• ICP-OES Inductively Coupled Plasma- Optical Emission Spectrometry
• Mce ⁇ content Meso chlorin e 6 content for PoU was determined by UV spectrophotometry. Mce 6 standard the polymer conjugate solutions were prepared in methanol and UV absorbance measured at 650nm.
• MDA-MB-231 human breast carcinoma cell line was grown in LH- 15 culture media with 2mM glutamine and 10% FBS, in 5% CO 2 . The cells were harvested at confluence and counted after staining with trypan blue. 2x10 6 cells were injected per animal to generate tumor models.
• mice Female athymic nu/nu mice were purchased from NCI (Frederick, MD) at the age of 6 weeks. To generate tumor models, each mouse was injected subcutaneously with 2x10 6 MB-231 cells in lOO ⁇ l of culture media mixed with lOO ⁇ l of BD MATRIGEL, a solubulized basement membrane preparation extracted from EHS mouse sarcoma, in the flank region. Magnetic resonance imaging and photodynamic therapy studies were carried out at an average tumor size of 25- 40mm 3 .
• mice were injected with MDA-MB231 human breast carcinoma cells (ATCC). About 1 to 3x10 6 cells were mixed with MATRIGEL (BD Biosciences) and were injected per mouse. The mice were observed for tumor growth. Tumor volume was measured using digital calipers. The mice were selected for imaging and therapy when the tumor volume was approximately 20 mm 3 .
• MATRIGEL BD Biosciences
• MR imaging was performed on a Siemens Trio 3T MRI scanner.
• the animals were anesthetized by intra-peritoneal or intra-muscular injection of the anesthetic (mixture of ketamirie - 80 mg/kg body weight and xylazine - 12 mg/kg body weight).
• the animals were placed in the human wrist coil and pre-contrast images (prior to the injection of the polymer chelate system containing the drug and the contrast agent) were obtained using a 3D FLASH (Fast Low Angle Shot) sequence.
• 3D FLASH Flust Low Angle Shot
• the macromolecular complexes were injected at a dose of 0.09 mmol Gd/kg body weight and 6 mg/kg of Mc ⁇ 6 by tail vein injection.
• Image acquisition was set at 5, 30, 60, 120 minutes and 24 hours post injection.
• Animal imaging was performed using the multi -phase Tl weighted sequence. The repetition time was about 6 milliseconds to 2 milliseconds, respectively; consistent with the 4 to 8 cm FIV (field of view) and 0.5 mm slice thickness chosen for each animal.
• the imaging volume was acquired before injection and in rapid succession after injection. Each acquisition required about 30 seconds.
• dosage amounts are expected to be effective in other subjects, such as humans, hi humans, dosage amounts may include, for instance, 0.01 to 0.03 mmol Gd/kg body weight. Similarly, dosage amounts are expected to be effective in other subjects, such as humans wherein the dosage amount may include, for example, 1 to 10 mg/kg body weight of Mce 6 . However, the dosage may adjusted as determined by routine experimentation.
• the tumor was irradiated using laser at a wavelength of 650 run corresponding to the excitation wavelength of MCe 6 .
• Results are depicted in the magnetic resonance images of FIG. 6, showing images of tumors in athymic nu/nu mice at pre-, 5, 30, 60, 120 minutes and 24 hours post- injection.
• the slices are through the heart and tumor separately, for mouse 1, and through the heart and tumor simultaneously, for mouse 2.
• the 24 hour images show contrast enhancement through the tumor, compared to pre-contrast, while there is contrast enhancement also in the heart at five minutes post-injection.
• 6 mice were examined using the above methodology, all yielding similar results. These results indicate that the conjugates are capable of localizing in the target tissues within 24 hours.
• Example IV Example IV.
• Gd-DOTA a clinically approved contrast agent
• FIG. 7 and Figure 8 show 2D coronal MR images through tumor and heart respectively.
• the upper panel of Figure 7 represents mice with Pol / and the lower panel upper represents mice with Poh.
• the upper panel of Figure 8 represents mice with Poll and the lower panel represents mice with PoI 2 .
• the higher signal in heart may be due to high molecular weight fractions of the control polymer.
• Figure 11 illustrates that a reduction in tumor volume or slowed tumor growth was observed for mice receiving Pol l while the mice injected with P0I2 (control group) showed rapid increase in tumors. 4 out of 6 the mice in the drug group survived for the time period of the experiment (90 days). Out of the 6 mice in the control group, 5 had tumors larger than 10% of the body weight and were sacrificed.
• MR imaging post treatment a high molecular weight contrast agent, (Gd-DTP A)-cystine copolymer (GDCP) was used to determine the difference in tumor uptake for treated and untreated mice. This agent has been shown in previous work to produce relatively strong contrast enhancement in angiogenic blood vessels in similar tumor models. The molecular weight and relaxivity of the polymer were similar to that of Pol,, 34kDa and 5.33 mM ' W.
• Figure 9 illustrates 3D MIP echo MR images of mice receiving GDCC at 2, 5, 10, 15, 30 minutes post injection.
• Figure 10 illustrates 2D Spin echo MR images of mice receiving GDCC at 2, 5, 10, 15, 30 minutes post injection.
• the tumors showed a rim enhancement for mice in both groups.
• the mice in control group however showed higher contrast enhancement, indicative of absence of treatment.

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