Classifications

• • 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
  • A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
  • A61K51/02—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by the carrier, i.e. characterised by the agent or material covalently linked or complexing the radioactive nucleus
  • A61K51/04—Organic compounds
  • A61K51/041—Heterocyclic compounds
  • A61K51/044—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine, rifamycins
  • A61K51/0446—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine, rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
  • A61K51/0451—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine, rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil having four such rings, e.g. phorphine derivatives, bilirubin, biliverdine
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D487/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00
  • C07D487/22—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00 in which the condensed system contains four or more hetero rings

Definitions

• the three critical components of image-guided therapy are navigation, control and monitoring the therapy delivery. They all rely on the target identification - Precise navigation requires clear identification of the target, to monitor treatment delivery, the target lesions and all adjacent tissues must be identified accurately while controlling the intervention of procedure.
• PDT photodynamic therapy
• PS photosensitizers
• cytotoxic species mainly singlet oxygen
• Nanoscience is being developed in conjunction with advanced medical science for further precision in diagnosis and treatment.
• Multidisciplinary biomedical scientific teams including biologists, physicians, mathematicians, engineers and clinicians are working to gather information about the physical properties of intracellular structures upon which biology's molecular machines are built.
• a new emphasis is being given to moving medical science from laboratory to the bedside and the community.
• This platform development program brings together an outstanding laboratory that is pioneering biomedical applications of PAA nanovectors (Kopelman), together with an innovative porphyrin chemistry and a world-class PDT group at RPCI that is highly experienced in the high volume screening and in vitro/in vivo evaluation of novel compounds, and in developing new therapies from the test tube to FDA approval for clinical use.
• nanoplatforms and nanovectors i.e. a nanoplatform that delivers a therapeutic or imaging agent
• biomedical applications show enormous promise for cancer diagnosis and therapy.
• the approach has been the subject of several recent reviews2
• Therapeutic examples include NP containing PDT agents, folate receptor-targeted, boron containing dendrimers for neutron capture and NP-directed thermal therapy.
• NP containing PDT agents folate receptor-targeted
• boron containing dendrimers for neutron capture and NP-directed thermal therapy.
• PAA NP the post-loading efficiency showed enhanced in vitro/in vivo therapeutic and imaging potential.
• PAA NP have core matrixes that can readily incorporate molecular or small NP payloads, and can be prepared in 10-150 nm sizes, with good control of size distributions.
• the surfaces of NPs can be readily functionalized, to permit attachment of targeting ligands, and both are stable to singlet oxygen (102) produced during PDT.
• PAA-NP have the advantages of (1) A relatively large knowledge base on cancer imaging, PDT, chemical sensing, stability and biodegradation. (2) No known in-vivo toxicity. (3) Long plasma circulation time without surface modification (see Preliminary Data), but with biodegradation and bioelimination rates controllable via the type and amount of selective cross-linking (introduced during polymerization inside reverse micelles).
• PDT is a clinically effective and still evolving locally selective therapy for cancers.
• the utility of PDT has been demonstrated with various photosensitizers for multiple types of disease. It is FDA approved for early and late stage lung cancer, obstructive esophageal cancer, high-grade dysplasia associated with Barrett's esophagus, age-related macular degeneration and actinic keratoses.
• PDT employs tumor localizing PSs that produce reactive IO2 upon absorption of light which is responsible for the destruction of the tumor.
• oxidation-reduction reactions also can produce superoxide anions, hydrogen peroxide and hydroxyl radicals which contribute to tumor ablation4.
• Photosensitizers have been designed which localize relatively specifically to certain subcellular structures such as mitochondria, which are highly sensitive targets5.
• On the tumor tissue level direct photodynamic tumor cell kill, destruction of the tumor supporting vasculature and possibly activation of the innate and adaptive anti-tumor immune system interact to destroy the malignant tissue6.
• the preferential killing of the targeted cells (e.g. tumor), rather than adjacent normal tissues, is essential for PDT, and the preferential target damage achieved in clinical applications is a major driving force behind the use of the modality.
• RPCI Roswell Park Cancer Institute
• Photofrin® the first generation FDA approved hematoporphyrin-based compound.
• our group has investigated structure activity relationships for tumor selectivity and photosensitizing efficacy, and used the information to design new PSs with high selectivity and desirable pharmacokinetics.
• Photosensitizers have several very desirable properties as therapeutic agents deliverable by NP: (1) Only a very small fraction of administered targeted drug makes it to tumor sites and the remainder can cause systemic toxicity. However, PDT provides dual selectivity in that the PS is inactive in the absence of light and is innocuous without photoactivation. Thus the PS contained by the NP can be locally activated at the site of disease. (2) PDT effects are due to production of 102, which can readily diffuse from the pores of the NP (see Preliminary Data). Thus, in contrast to chemotherapeutic agents, release of encapsulated drug from the NP, is not necessary. Instead, stable NP with long plasma residence times can be used, which increases the amount of drug delivered to the tumors.
• NP platforms also provide significant advantages for PDT: (1) High levels of imaging agents can be combined with the PS in the NP permitting a "see and treat" approach, with fluorescence imageguided placement of optical fibers to direct the photoactivating light to large or subsurface tumors, or to early non clinically evident disease. (2) It is possible to add targeting moieties, such as cRGD or F3 peptide to the NP so as to increase the selective delivery of the PS. (3) The NP can carry large numbers of PS, and their surface can be modified to provide the desired hydrophilicity for optimal plasma pharmacokinetics. Thus, they can deliver high levels of PS to tumors, reducing the amount of light necessary for tumor cure.
• F3 peptide is a 31 -amino acid synthetic peptide derived from a fragment of the nuclear protein, high mobility group protein 2 (HMGN2)15.
• HMGN2 is a highly conserved nucleosomal protein thought to be involved in unfolding higher-order chromatin structure and facilitating the transcriptional activation of mammalian genes 62 when injected i.v., F3 peptide internalizes and accumulates in the nuclei of HL-60 cells and human MDA-MB- 35 breast cancer cells.
• Tissue and cellular localization of F3 peptide indicated that it homes selectively to tumor blood vessels and tumor cells and has the remarkable property of being able to carry a payload into the cytoplasm and nucleus of the target cells.
• NPs with surface attached F3 behave similarly, attaching selectively to nucleolin expressing cells, and then channeled towards the cell nucleus.
• F3 peptide binds to cell surface-expressed nucleolin on the target cells.
• nucleolin is expressed on the surface of MDA-MB- 35 cells and shuttles between the cytoplasm and the nucleus and between the cell surface and the nucleus. Nucleolin is also overexpressed in 9L glioma cells.
• the mechanism of F3 targeting is recognition by nucleolin at the surface of actively growing cells (tumor cells and neovascular endothelial cells), which then binds and internalizes it, and transports it into the nucleus. While nucleolin can carry F3- targeted molecules from the cell surface into the nucleu, F3-labelled PAA nanoparticles containing Photofrin accumulated in the cytoplasm, which is useful because mitochondria are the primary target of PDT-produced 102. F3 targeting has been used recently to deliver nano-sized particles composed of lipids or quantum dots to tumor vasculature.
• Integrins are a major group of cell membrane receptors with both adhesive and signaling functions. They influence behavior of neoplastic cells by their interaction with the surrounding extracellular matrix, participating in tumor development 16. Integrin ⁇ 3 in tumor cells binds to matrix metalloprotease-2 in a proteolytically active form and facilitates cell-mediated collagen degradation and invasion. It over-expresses in U87 and 9L glioma tumors. An increase in its expression is correlated with increased malignancy in melanomas. ⁇ 3 plays a critical role in angiogenesis and is up-regulated in vascular cells within human tumors. Significant overexpression of ⁇ 3 is reported in colon, lung, pancreas, brain and breast carcinomas, which was significantly higher in metastatic tumors.
• Optical imaging includes measurement of absorption of endogenous molecules (e. g. hemoglobin) or administered dyes, detection of bioluminescence in preclinical models, and detection of fluorescence from endogenous fluorophores or from targeted exogenous molecules. Fluorescence, the mission of absorbed light at a longer wavelength, can be highly sensitive: a typical cyanine dye with a lifetime of 0.6 nsec can emit up to 1032 photons/second/mole. A sensitive optical detector can image ⁇ 103 photons/second. Thus even with low excitation power, low levels of fluorescent molecular beacons can be detected.
• a challenge is to deliver the dyes selectively and in high enough concentration to detect small tumors.
• Use of ICG alone to image hypervascular or "leaky” angiogenic vessels around tumors has been disappointing, due to its limited intrinsic tumor selectivity.
• Multiple approaches have been employed to improve optical probelocalization, including administering it in a quenched form that is activated within tumors, or coupling it to antibodies or small molecules such as receptor ligands.
• Recent studies have focused on developing dye conjugates of small bioactive molecules, to improve rapid diffusion to target tissue and use combinatorial and high throughput strategies to identify, optimize, and enhance in vivo stability of the new probes.
• Some peptide analogs of ICG derivatives have moderate tumor specificity and are entering pre-clinical studies. However, none of these compounds are designed for both tumor detection and therapy. It is important to develop targeting strategies that cope with the heterogeneity of tumors in vivo, where there are inconsistent and varying expressions of targetable sites.
• PS Photosensitizers
• PS generally fluoresce and their fluorescence properties in vivo has been exploited for the detection of early-stage cancers in the lung, bladder and other sites 17
• the fluorescence can be used to guide the activating light.
• PS are not optimal fluorophores for tumor detection for several reasons: (i) They have low fluorescence quantum yields (especially the long wavelength photosensitizers related to bacteriochlorins). Efficient PS tend to have lower fluorescence efficiency (quantum yield) than compounds designed to be fluorophores, such as cyanine dyes because the excited singlet state energy emitted as fluorescence is instead transferred to the triplet state and then to molecular oxygen .
• Porphyrin-based PS have small Stokes shifts. Porphyrin-based PS have a relatively small difference between the long wavelength absorption band and the fluorescence wavelength (Stokes shift), which makes it technically difficult to separate the fluorescence from the excitation wavelength, (iii) Most PS have relatively short fluorescent wavelengths, ⁇ 800 nm, which are not optimal for detection deep in tissues.
• PET Positron emission tomography
• PET is a technique that permits non-invasive use of radioisotope labeled molecular imaging probes to image and assay biochemical processes at the level of cellular function in living subjects20.
• PET predominately has been used as a metabolic marker, without specific targeting to malignancies.
• radiolabeled peptide ligands to target malignancies.
• PET is important in clinical care and is a critical component in biomedical research, supporting a wide range of applications, including studies of gene expression, perfusion, metabolism and substrate utilization, neurotransmitters, neural activation and plasticity, receptors and antibodies, stem cell trafficking, tumor hypoxia, apoptosis and angiogenesis21.
• a long circulation time may be desirable, as it can increase delivery of the agent into tumors.
• HPPH and the iodobenzyl pheophorbide-a have plasma half lives ⁇ 25 h.
• the long radiological half life of 1241 is well matched to the pheophorbides; it permits sequential imaging with time for clearance from normal tissue. Labeling techniques with radioiodine are well defined with good yield and radiochemical purity22.
• NPs can optimize tumor detection and treatment of brain tumors.
• a photosensitizer (PS) with increased selectivity and longer wavelength could be a more suitable candidate for brain and deeply seated tumors (especially breast, brain and lung).
• PDT photodynamic therapy
• Chang et al reported an effective radius of tumor cell kill in 22 glioma patients of 8 mm compared with the 1.5 cm depth of necrosis noted by Pierria with the intracavitary illumination method. It is believed that tumor resection is important so that the numbers of tumor cells remaining to treat are minimized. With stereotactic implantation of fibers for interstitial PDT there is no cavity to accommodate swelling and a considerable volume of necrotic tumor which causes cerebral edema. However, cerebral edema can be readily controlled with steroid therapy. Compared to chemotherapy and radiotherapy, patients with brain tumors treated with PDT have definitely shown long-term survival, whereas glioma patients treated with adjuvant chemotherapy or radiotherapy do not show additional benefits as reported by Kostron et al. and Kaye et al. On the basis of our preliminary data, the ⁇ 3 targeted NPs may improve tumor-selectivity and PDT outcome.
• the present invention relates to PAA (polyacrylic acid and its derivatives at the carboxy groups, e.g. polyacrylamide) nanoparticles containing a photosensitizer and an imaging enhancing agent.
• the imaging agent is preferably a PET imaging agent and more preferably an 124 I labeled compound.
• the photosensitizer is preferably selected from chlorins, bacteriochlorins, pyropheophorbides, and mixtures thereof.
• the nanoparticles preferably contain at least one photosensitizer comprises a moiety containing 124 I and also acts as an imaging agent.
• the photosensitizer and imaging agent are preferably post loaded onto the nanoparticle after nanoparticle formation.
• the photosensitizer is preferably a tetrapyrollic photosensitizer having the structural formula:
• X is an aryl or heteroaryl group
• n is an integer of 0 to 6;
• R 2 o is methyl, butyl, heptyl, docecyl or 3,5-bis(trifluoromethyl)-benzyl
• R 2 i is 3,5,-bis(trifluoromethyl)benzyl
• Ri a and R 2a are each independently hydrogen or substituted or unsubstituted alkyl, or together form a covalent bond;
• R 3 and R4 are each independently hydrogen or substituted or unsubstituted alkyl;
• R 3a and R4 a are each independently hydrogen or substituted or unsubstituted alkyl, or together form a covalent bond;
• R5 is hydrogen or substituted or unsubstituted alkyl
• R 9 and Rio are each independently hydrogen, or substituted or unsubstituted alkyl and R9 may be -CH 2 CH 2 COOR 2 where R 2 is an alkyl group that may optionally substituted with one or more fluorine atoms;
• the photosensitizer is preferably a chlorophyll-based photosensitizer post- loaded to biodegradable and biocompatible polyacrylamide (PAA) nanoparticles.
• PAA polyacrylamide
• the photosensitizer may be conjugated with an image enhancing agent prior to incorporation into the nanoparticle, after incorporation into the nanoparticle or the photosensitizer and/or image enhancing agent may chemically bound to the nano particle and/or one or more of the photosensitizer and image enhancing agent may be physically bound to the nanoparticle.
• Imaging enhancing agents may be for essentially any imaging process, e.g.
• imaging enhancing agents are discussed in the background of the invention previously discussed and in the list of references incorporated by reference herein as background art.
• Figure 1A shows the structural formula of HPPH-CD (cyanine dye) conjugate used as a photosensitizer and imaging agent.
• Figure IB is a graph showing in vivo photosensitizing efficacy of HPPH-CD conjugate 1 in C3H mice bearing RIF tumors (10 mice/ group) at variable drug doses. The tumors were exposed to light (135J/cm2/75mW/cm2) at 24h post-injection.
• Figure 1 C shows a scanned image showing localization of the conjugate 1 in a live mouse 24 h after injection (drug dose 0.3 (Without PAA NP0).
• Figure 2 shows whole body images of BALB/c mice bearing Colon26 tumors with
• PAA NPs formulations HPPH and cyanine dye (CD) were post-loaded in 2 to 1 ratio).
• the CD concentration was kept constant (0.3 ⁇ /kg) at the images were obtained at variable time points.
• Figure 3 is a graph showing in vivo PDT efficacy of HPPH and CD post loaded in a ratio of 2: 1 and 4: 1 in PAA and ORMOSIL NPs. Note: HPPH dose: 0.47 ⁇ /kg in PAA NPs and 0.78 ⁇ /kg in ORMOSIL NPs.
• Figure 5 A is a diagram showing structure of PAA nanoparticles (PAA NP's).
• Figure 5B shows comparative in vivo imaging at variable time points of BALB/c mice bearing Colon26 tumors with HPPH-CD conjugate 1 and CD-conjugated with PAA NPs/post;-loaded with HPPH. The NPs were more tumor specific. (Mouse 1)
• FIG. 6 shows a series of scans wherein Panel 1 (4T1 tumors): Primary (PT) and metastasized tumors (MT) dissected and Panel 2 (4T1 tumors): PET imaging of the dissected primary and metastasized tumors.
• Panel 3 (BALB/C mouse bearing 4T1 tumor): Whole body PET imaging. The tumor metastasis in lung was clearly observed.
• Panel 4 The position of the lung is shown by the transmission scan using 57Co source in mice with no lung metastasis.
• Panel 5 (BALB/C mouse bearing Colo-26 (non-metastatic tumor): Whole body imaging by PET. A high accumulation of the 1241- photosensitizer in tumor is clearly observed without any significant accumulation in lungs (injected dose: 100 ⁇ ).
• T Tumor
• PT Primary tumor
• MT Metastatic tumor.
• Figure 8 A shows in vivo comparative in vivo PET imaging (72 h post injection) and biodistribution (24h, 48h and 72h postinjection) of 1241-labeled photosensitizer 2 without PAA nanoparticles in BALB/c mice bearing Colon26 tumors (see the text). (Biodistribution of PET imaging agent 2 : No PAA, with PAA).
• Figure 8B shows in vivo comparative in vivo PET imaging (72 h post injection) and biodistribution (24h, 48h and 72h postinjection) of 1241-labeled photosensitizer 2 with PAA nanoparticles in BALB/c mice bearing Colon26 tumors (see the text). (Biodistribution of PET imaging agent 2: No PAA, with PAA ).
• Figure 8C shows biodistribution of PET imaging agent 2, no PAA and with PAA.
• Figure 9 Fluorescence intensity of cells targeted by F3- targeted (A series), F3-Cys targeted (B series) and nontargeted NPs (F series) in nucleolin rich MDA-MB-435 cell lines.
• FIG. 10 Fluorescence (left) & Live/dead cell assay (right) of HPPH conjugated PAA NPs + or - F3-Cys peptide incubated for 15 min with MDA-MB- 435 cells.
• Figure 1 Confocal images showing the target-specificity of F3-Cys peptide in 9L
• Glioma tumor cells Left: F3-Cys PEG Rhodamine-PAA NPs (9L cells). Right: PEG Rhodamine-PAA NPs (9L Cells).
• FIG. 12 In vivo biodistribution of 14 C-labeled HPPH, and 14 C-labeled HPPH post- loaded into PAA NPs in BALB/c mice bearing Colon26 tumors.
• 14 C-labeled PS (3.8 ⁇ /0.2 mL) were administered to 12 mice/group. At 24, 48, 72h after injection, three mice/time- point were sacrificed. The organs of interest were removed and the radioactivity was measured. The raw data were converted to counts/ gram of tissue.
• Figure 13A shows In vivo biodistribution of iodinated photosensitizer at 24, 48 and 72h post injection.
• Figure 13B shows In vivo biodistribution of iodinated photosensitizer using variable sizes of PAA NPs at 24, 48 and 72h post injection 531-ME Post-Loaded into 30 nm PAA Nanoparticles.
• Figure 13C shows In vivo biodistribution of iodinated photosensitizer using variable sizes of PAA NPs at 24, 48 and 72h post injection 531-ME Post-Loaded into 150 nm PAA Nanoparticles.
• Figure 14 shows the structural formula of HPPH.
• Figure 15 is a diagram of Multifunctional PAA Nanoparticles.
• Figure 16 shows flow diagrams for preparation of postloaded nanoparticles.
• FIG 17A shows the structure of photosensitizer 1(PS1).
• Figure 17B shows the corresponding 124 I- labeled analog 2 of PS 1.
• Figure 17C shows the structure of 18 F-fluoro- deoxyglucose (FDG).
• FDG F-fluoro- deoxyglucose
• Figure 17D shows a schematic representation of photosensitizer (1 or 2) post-loaded in polyacrylamide (PAA) nanoparticles (NPl).
• PAA polyacrylamide
• Figure 18A is a curve showing electronic absorbance spectra for NPl at various times during the post-loading procedure.
• Figure 18B shows fluorescence spectra of NPl at various times during the post- loading procedure.
• Figure 18C shows electronic absorbance spectra for PSl and NPl in drug solution form (aqueous tween-80 solution) and in 17% Bovine Calf Serum in PBS (BCS-PBS).
• Figure 18D shows the fluorescence spectra for PSl, NPl, PSl in 17% BCS-PBS and
• NP l in 17% BCS-PBS The concentration for photosensitizer 1 in all samples is three ⁇ and the concentration of Tween-80 is less than 1%.
• Figure 19A shows whole body PET Images of BALB/c mice bearing subcutaneous Colon26 rumors on the light shoulder with 124 I-PS2 at 24, 48, and 72 h post-injection (i.v.).
• Figure 19B shows whole body PET Images of BALB/c mice bearing subcutaneous
• Figure 19C is a bar graph showing relative uptake values (RUV) of PS2 and NP2.
• Figure 20A is a bar graph showing comparative in-vivo biodistribution of (A): NP2, PS2 and FDG at 90 minutes post injection in BALB/c mice (3 mice/group) bearing subcutaneous Colon26 tumors on the right shoulder
• Figure 20B is a bar graph showing comparative in vivo biodistribution of NP2 at 24, 48 anf 72 hours post injection in BALB/c mice (3 mice/group) bearing subcutaneous Colon 26 tumors on the right shoulder.
• Figure 20C is a table showing the ratio of tumor to various organs/tissues/fluids for PS2, NP2 at 24 h, and for 18 F-FDG at 90 min post- injection in BALB/c mice (3 mice/group) bearing subcutaneous Colon26 tumors on the right shoulder.
• Figure 21 shows whole-Body fluorescence reflectance images of BALB/c mice bearing subcutaneous Colon26 tumors.
• A-C PS1
• D-F NP1 at 24, 48, and 72 h post- injection (i.v.).
• the ⁇ 665 nm and the 700 nm.
• Figure 22A shows in vivo PDT data (% mice cured) by Kaplan-Meier survival curve show a significant difference in PDT efficacy of PS1 with and without NPs at a dose of 1.0
• the tumors were exposed to light (135 J/cm 2 and 75 mW/cm 2 ) 24 h post- injection.
• the P value for the for the two survival curves is ⁇ 0.0001 as determined by the Mantel-Cox test.
• Figure 22B is a curve showing the weights of BALB/c mice (3 mice/group) injected with 100 mg/kg or 400 mg/kg of blank PAA NPs, recorded daily for 29 days.
• Figure 23 shows formalin-fixed, paraffin embedded hematoxylin-eosin (H.E.) stained tissue sections (representative sample for 400 mg/kg): (a) Liver, (b) spleen, (c) heart, (d) kidney, and (e) lung [Magnification: 200x].
• H.E. formalin-fixed, paraffin embedded hematoxylin-eosin
• Figure 24A is a distribution curve characterizing of the size of the blank PAA nanoparticle formulation used for Photodynamic Therapy/fluorescence reflectance imaging and toxicology studies.
• the mean diameter is 30 nm.
• Figure 24B is a distribution curve characterizing of the size of NP 1 in Tween-80 / PBS (concentration of Tween-80 is ⁇ 1%). The mean diameter is 35.1 nm.
• Figure 25 shows biodistribution of PS2: 24, 48, and 72 H post- injection.
• Figure 26 shows the release profile of PS1 from NP 1.
• HPPH a tumor-avid photosensitizer for developing bifunctional agents for fluorescence imaging/ PDT and its limitations:
• tumor-avid PS(s) e. g., HPPH
• R absorbing fluorophore(s) non-tumor specific cyanine dyes
• HPPH was used as a vehicle to deliver the imaging agent to tumor.
• the limitation of this approach was that the conjugate exhibited significantly different dose requirements for the two modalities.
• the imaging dose was approximately 10-fold lower than the phototherapeutic dose (Fig.
• HPPH and the cyanine dye (fluorophore) were post-loaded in variable ratios (HPPH to CD: 1 : 1; 2: 1 ; 3 : 1 and 4: 1 molar concentrations).
• HPPH was postloaded to PAA NPs first. Free HPPH was removed by spin filtration and then cyanine dye was postloaded. It was spin-filtered again, washed several times with 1% bovine calf serum and the concentration was measured.
• the 2: 1 formulations produce the best tumor imaging and long-term tumor cure in BALB/c mice bearing Colon26 tumors.
• This formulation contained in a single dose the therapeutic dose of HPPH (0.47 ⁇ /kg) and the imaging dose of Cyanine dye (0.27 _mol/kg), which were similar to the components used alone for tumor imaging and therapy, but with much more tumor selectivity (skin to tumor ratio of HPPH was 4: 1 instead of 2: 1 without NPs). Under similar treatment parameters the ORMOSIL NPs showed a significantly reduced response (imaging and PDT, not shown).
• the stability of the drugs in PAA NP was established by repeated washing with aqueous bovine calf serum through AMICON centrifugal filter units with a lOOKDa or larger cut off membrane and drug in the filtrate was measured spectrophotometrically.
• Figs. 2-4 The comparative in vivo PDT efficacy of the ORMOSIL and PAA formulations, their tumor imaging potential and stability (in vitro release kinetics) is shown in Figs. 2-4, which clearly illustrate the advantages of PAA NPs in reducing the therapeutic dose by almost 8- fold without diminishing the tumor-imaging potential and also avoiding the Tween-80 formulation required for the HPPH-CD conjugate 1.
• the HPPH CD conjugate 1 was post-loaded to PAA NPs, which certainly enhanced the tumorimaging, but the therapeutic dose was still 10-fold higher (similar to the HPPH CD conjugate, Fig. 5B).
• the cyanine dye was conjugated peripherally to the PAA NPs first and then HPPH was post loaded. Again, compared to HPPH-CD conjugate 1, the PAA formulation showed enhanced tumor- specificity (imaging) (Fig.5B).
• PET imaging and PDT PAA NPs decreased the liver uptake of the 1241- photosensitizer (PET imaging agent) and enhanced the tumor-specificity.
• PET imaging agent 1241- photosensitizer
• Our initial investigation with an 1241-labeled PS 2 indicates its in vivo PDT efficacy and capability of detecting tumorsl04-106 (RTF, Colon26, U87, GL261, pancreatic tumor xenograft) and tumor metastases (BALB/c mice bearing orthotopic 4T1 (breast) tumors) (Fig 6).
• RDF tumor metastases
• BALB/c mice bearing orthotopic 4T1 (breast) tumors Fig 6
• 18F FDG PS 2 showed enhanced contrast in most of the tumors including those where 18F FDG-PET provides limited imaging potential (e.g., brain, lung and pancreatic tumors). See Fig.
• radioactive PS such as the 1241- labeled analog 2 (superior to 18F-FDG in PET-imaging of lung, brain, breast and pancreas tumors) with a T1 ⁇ 2 of 4.2 days could cause radiation damage to normal organs.
• radioactive PS such as the 1241- labeled analog 2 (superior to 18F-FDG in PET-imaging of lung, brain, breast and pancreas tumors) with a T1 ⁇ 2 of 4.2 days could cause radiation damage to normal organs.
• 1241- imaging agent Based on the observation of high uptake of PAA NPs in liver and spleen (below) we postulated that saturating the organs with the non-toxic PAA NPs before injecting the PET agent might reduce uptake and radiation damage by 1241- imaging agent.
• For proof-of principle blank PAA NPs were first injected (i.v.) into mice bearing Colon26 tumors followed 24 h later by i.v. 1241-analog (100- 50
• PAA NPs The presence of PAA NPs made a remarkable difference in tumor contrast with brain, lung and pancreatic tumors). See Fig. 7 for comparative biodistribution.
• PAA NPs can be targeted to nucleolin with F3-Cys.
• F3-targeted NPs were prepared using two kinds of F3 peptides: F3 peptide conjugated to NP via one of the 8 lysines available in its sequence and F3-Cys peptide conjugated to NP via cysteine. Cysteine capped NPs served as non-targeted control.
• Three 25 mg batches of each type of NP contained: 2.6, 5.1 and 7.7 mg F3, (A3-A5) respectively; 2.7, 5.3 and 8 mg F3-Cys (B3-B5) respectively, and 0.29, 0.58 and 0.87 mg Cys (C3-C5) respectively.
• the fluorescence intensity from PAA NP incubated in vitro with nucleolin positive MDA-MB-435 cells is shown in Fig. 9.
• the F3-Cys conjugated NPs show considerably higher binding efficiency than non-targeted NPs, while F3 conjugated NPs do not. Conjugation via a cysteine link preserves the specificity of F3 peptide for nucleolin. In addition excess cysteine on the NPs helps to minimize the non-specific binding. Additional experiments (not shown) suggested that the amount of F3-Cys peptide (5.3 mg/25mg NP) used for B4 NPs was optimal.
• NPs post-loaded with both HPPH and cyanine dye clearly shows characteristic signatures for both the PS and dye, without aggregation-induced broadening, while the fluorescence spectrum shows strong signals from both components.
• HPPH conjugated PAA NPs with F3-Cys peptide at the outer surface show targeted specificity. F3 -mediated specificity is retained in the presence of conjugated HPPH. F3 targeted NPs did targeted NPs did not, indicating that F3-mediated specificity is retained in the presence of conjugated HPPH. F3 targeted NPs did not accumulate in the nucleus. On activation of cells with light at 660 nm only F3-targeted NP caused cell kill (Fig 1 1). Cell internalization of F3- targeted NPs was confirmed by fluorescence confocal microscopy.
• HPPH conjugated PAA NPs with F3-Cyspeptide at the outer surface show targeted specificity.
• the specificity of targeted NPs was tested by fluorescent imaging (Fig. 10).
• F3 targeted NPs did not accumulate in the nucleus.
• On activation of cells with light at 660 nm only F3 -targeted NP caused cell kill (Fig 1 1).
• Cell internalization of F3-targeted NPs was confirmed by fluorescence confocal microscopy.
• F3-Cys shows target-specificity in 9L glioma cells. Similar to F3-cys, a pegylated form of F3-Cys PEG on PAA NPs also showed remarkable target-specificity in 9L rat glioma cells which also expresses nucleolin, Fig 11. (Note: HPPH is replaced with a Rhodamine moiety). [0035] Biodistribution studies: PAA NP Enhances tumor uptake of HPPH.
• PET/fluorescence imaging photosensitizer derived from chlorophyll-a has significant unexpected advantages. Compared to a free photosensitizer (PS), the corresponding polyacrylamide-based nanoformulation shows a remarkable in vivo enhancement in tumor- imaging and photodynamic therapy.
• the non-toxic nanoparticles (30-35 nm) formulation drastically change the pharmacokinetic profile of the imaging/therapeutic agent (formulated in 1% Tween 80 and 5%/D5W) with remarkable enhancement in tumor uptake (10% of the injected dose) and reduced uptake in spleen and liver.
• the labeled ( 124 I-) and non-labeled PS in combination show great potential for tumor imaging (PET/fluorescence) and photodynamic therapy in BALB/c mice bearing Colon26 tumors and provides an opportunity for "See and Treat" approach.
• This invention shows the utility of porphyrin-based compounds in a "BIFUNCTIONAL AGENT" for imaging breast tumor and tumor metastasis. Similar to most NPs, PAA NP accumulate in liver and spleen. Their clearance rate from most organs is significantly faster than Ormosil NP and they do not show long-term organ toxicity. Even tumor-avid porphyrin based PS exhibit high uptake in liver and spleen, but are non-toxic until exposed to light. The PS clear from the system quickly (days) without organ toxicity.
• PET has widened its appeal for research at the drug development stage, as it allows studying the drug distribution in vivo.
• Most of the porphyrin- based compounds show significantly higher accumulation in the tumor at 24 to 48 h post injection. Therefore for developing multifunctional agents for PET/PDT, we introduced the iodobenzyloxyethyl group at position-3 of the pyropheophorbide-a, which showed tumor- avidity with significant PDT/optical imaging (excitation: 665 nm, emission: 715 nm) efficacy 24 h post- injection.
• the corresponding 124 I-analog half-life 4.2 days
• mice models U87, Colon26, RTF, 4T1, Panc-1
• FIG. 19C shows that the RUV for both PS2 and NP2, which increased over time, i.e., the visibility of the tumor compared to the background signal increases.
• the RUV value was consistently higher starting at 3.17 (24 h post- injection) and optimizing at 8.7 (72 h post- injection), whereas for the PS2 the RUV increases with time from 2 to 8.2 72 h post- injection.
• This reflects a 223% increase in PS2 (NP formulation) present in the tumor as compared to free PS2 (without NP formulation) and interestingly an increase of 239% if compared against 18 F- FDG.
• the NP2 formulation showed a remarkable decrease in accumulation in the spleen and liver and significantly less in the heart and muscle if compared to 18 F-FDG alone.
• the %ID/gram of PS2 present in the spleen and liver at 24 h post- injection was 15.51 and 9.32%, respectively. With nanoparticle formulation NP2, the amount decreased to 2.02 and 3.99%, respectively.
• the tumors were exposed to light at 665 nm (dose: 135 J/cm 2 , 75 mW/cm 2 ) at 24 h post injection and the tumor response was recorded daily following the animal protocol approved by the institutional IACUC committee.
• the percentages of tumor cure are shown in Figure 22 A.
• the Kaplan-Meier survival graph highlights the remarkable enhancement of long-term tumor cure with NP formulation, from 20% (2/10 mice were tumor-free with the PS alone) to 80% (8/10 mice were tumor free with NPs-PS formulation).
• the resulting solution was stirred vigorously overnight.
• hexane was removed by rotary evaporation and the particles were precipitated by addition of ethanol (50 mL).
• the surfactant and residual monomers were washed away from the particles with ethanol (150 mL, Pharmaco-Aaper, USA) followed by washing with water (100 mL) five times each in an Amicon ultra-filtration cell equipped with a Biomax 300 kDa cutoff membrane (Millipore, USA).
• the concentrated nanoparticles were lyophilized for two days, and stored in the freezer.
• the nanoparticles were reconstituted by suspending in PBS. Once in liquid form, the nanoparticles are stored at 4 °C.
• AFPAA Amin Functionalized Polyacrylamide Nanoparticles
• AOT Dioctyl Sulfosuccinate Sodium Salt
• APMA 3-(aminopropyl) methacrylamide
• AHM 3- (acryloyloxy)-2-hydroxypropyl methacrylate
• PBS Phosphate Buffered Saline
• AFPAA NPs are dissolved in 1% Tween-80 / PBS (pH 7.4, 10 mM) to a final concentration of 10 mg / 1 mL.
• the NPs are sized by DLS prior to the post-loading of PSl to ensure that they are of the appropriate size.
• PS l is dissolved in DMSO to a final concentration of 20 mM.
• 20 of PSl in DMSO is added to 2 ml of NP solution and is magnetically stirred at a constant rpm for a minimum of 2 hours.
• the NP solution is transferred to an Amicon Ultra-4 30 kDa centrifuge filter and centrifuged at 4,000 rpm for 40 minutes to remove excess DMSO, Tween-80, and PSl that did not post-load.
• the filtrate is spectrophotometrically measured and if signal for PSl is detected, the retentate is reconstituted to the original volume with PBS and recentrifuged. This is continued until no signal is detectable in the filtrate spectrophotometrically.
• the nanoparticle solution is syringed filtered and then the concentration of PS1 is measured in ethanol using the Beer's-Lambert Law (molar exctinction coefficient: 47,500 L m "1 cm "1 ).
• the nanoparticles may cause scattering in the absorbance spectra. If this occurs, the nanoparticle solution can be centrifuge filtered in a microfuge membrane-filter ( ANOSEP 100K OMEGA, Pall Corporation) at 14,000 RPM for 10 minutes. The filtrate is used to calculated the concentration of PS1 that was post- loaded to the PAA NPs. The nanoparticles are syringe filtered with a 0.2 ⁇ syringe filter and stored at 4°C for further use.
• ANOSEP 100K OMEGA Pall Corporation
• Post-Loading of the PS2 to Blank AFPAA Nanoparticles The lyophilized AFPAA NPs are dissolved in 1% Tween-80 / PBS (pH 7.4, 10 mM) to a final concentration of 10 mg / 1 mL. The NPs are sized by DLS prior to the post-loading of 124 I-labeled PS2 to ensure that they are of the appropriate size. 2.1 mL of the NP solution is added to the vial containing PS2 dissolved in a 100 of DMSO. The solution is magnetically stirred at a constant rpm for a minimum of 2 hours.
• the NP solution is transferred to an Amicon Ultra-4 30 kDa centrifuge filter to remove excess DMSO, Tween-80, and PS2 that did not post-load. 1.3 mL of additional PBS is used in the transfer process to ensure that the entire radioactivity is transferred from the vial to the centrifuge filter.
• the NP solution was transferred to the centrifuge filter and was centrifuged at 4,000 rpm for 40 min. Post-centrifuge filtration, the amount of radioactivity released from the NP is measured. If the activity in the filtrate is greater than 5%, then the retentate is reconstituted to the original volume and recentrifuged. This process is repeated until less than 5% of the total radioactivity is found in the filtrate.
• the rententate is reconstituted to 1.5 mL with PBS to ensure that each 100 of NP solution will contain ⁇ 60 ⁇ of activity.
• the nanoparticles post-loaded PS1 were mixed with 1% aqueous Human Serum Albumin, HSA, solution (w/v). The absorbance is measured of the solution and is marked as the stock absorbance. The solution is then centrifuged in an Amicon Ultracel-4, 100 kDa centrifuge filter at 4,000 RPM for 30 minutes. The filtrate is marked as filtrate #1 and is measured spectrophotometrically. The retentate is reconstituted to the original volume with 1% HSA, thoroughly mixed with a pippet, and re- centrifuge filtered. The filtrate is marked filtrate #2 and is measured spectrophotometrically.
• the NPs are reconstituted with 1% HSA, thoroughly mixed with a pippet and is measured spectrophotometrically.
• the hydrodynamic diameter of the blank nanoparticle and NP 1 were measured using the Nicomp 370 Submicron Particle Analyzer (Nicomp, Santa Barbara, CA).
• the NPs were diluted in a borosilicate glass tube with PBS (10 mM, pH 7.4) to achieve an intensity count of 300 kHz.
• the samples were measured in triplicate with each run lasting five minutes. The volume intensity weighting was used when determining the mean hydrodynamic diameter.
• NP 1 is diluted in ethanol and measured spectrophotometrically using a Varian (Cary-50 Bio) with an extinction coefficient of 47,500 remove the scattering in the absorbance spectra, the nanoparticles were
• NP 1 The absorbance spectra was collected from 350 - 900 nm with the concentration of PS 1 and NP1 in either PBS or 17% BCS-PBS was three micromolar. The fluorescence measurements were recorded using a Cary Eclipse fluoremeter (Varian Inc, USA). The excitation wavelength for PS1 diluted in PBS, PS1 diluted in 17% BCS, NP 1, and NP1 diluted in 17% BCS was excited 413, 416, 413, 413 nm, respectively. The fluorescence emission was collected from 600 - 800 nm. For both formulations the excitation and emission slit was set to 5 nm and the PMT voltage was set to medium.
• the absorbance and fluorescence spectra was measured at various times throughout the post-loading procedure. The times points included, before magnetic stirring (0 min), after magnetic stirring: 30 min, 60 min, 90 min, and 120 min, after centrifuge filtration, and after syringe filtering NP 1 with a 0.2 ⁇ cellulose acetate syringe filter.
• the absorbance spectra was collected from 300 - 800 nm with the concentration of each sample equaling three micromolar. From time 0 to time 120 minutes post magnetic stirring, the excitation wavelength was 425 nm and the excitation wavelength for the nanoparticle sample that was centrifuge filtered, and syringe filtered was 414, and 413 nm, respectively.
• FOCUS 120 a dedicated 3D small-animal PET scanner (Concorde Microsystems Incorporated) at the State University of New York at Buffalo under the Institutional Animal Care and Use Committee (IACUC) guidelines.
• IACUC Institutional Animal Care and Use Committee
• Radioiodine uptake by the thyroid or stomach was not blocked. All mice that were imaged were marked with a cross-line on their back to provide a reference landmark for consistently positioning them in a similar position each day they were imaged. The acquired data were rebinned with FORE algorithm20 and reconstructed using the 2D OSEM algorithm. The dead-time and singles-based random coincidence corrections were applied to all the PET studies. The RUV results were calculated from PET images with attenuation and scatter corrections.
• mice were injected i.v. with 30-90 ⁇ of PS2 or NP2.
• Three mice were sacrificed at each of the following time points, 24, 48, and 72 h post-injection and the blood and body organs, tumor, heart, liver, spleen, kidney, lung, muscle, gut, and stomach were removed.
• the amount of radioactivity in each sample was measured by a ⁇ well counter.
• the radioactivity present in each sample was calculated as a percentage of the injected dose per gram of the tissue (% ID/g).
• a statistical analysis standard deviation and the unpaired, two-tailed student t-test) was performed using Microsoft Excel to assess if the difference in radioactive uptake of PS2 and NP2 was significantly different (P value ⁇ 0.05).
• mice were injected intravenously with 100, 200, 300, and 400 mg/kg of polyacrylamide nanoparticles with mice receiving 100 mg/kg per day. Over a thirty-day period, the weight and behavioral changes were monitored. Day 30, the mice were sacrificed, placed in 10% formalin, and the following organs were analyzed by conventional H.E. staining: trachea, esophagus, urinary bladder, diaphragm, colon, jejunum, duodenum, pancreas, lung (right and left), liver, spleen, thymus, heart, ovary (right and left), uterus, kidney, skin, brain, and bone marrow of the sternum.
• Figure 23 shows the representative stained images for the control and 400 mg/kg injected group for the liver, spleen, heart, kidney, and lung at 200x magnification.
• Dr. Karoly Toth performed the histopathological analysis at Roswell Park Cancer Institute.
• the argon-pumped dye laser was set to 665 nm with a monochromator, and the fluence and fluence rate used was 135 J/cm 2 and 75 mW/cm 2 .
• the mice were observed for, necrotic scabbing, weight loss, and tumor regrowth. Tumor regrowth is calibrated by two orthogonal measurements, length and width and the tumor volume is calculated according to f L * W 2 ⁇
• mice were euthanized according the institute policy.
• Figure 24A shows the DLS for Blank PAA NPs used for the toxicological studies and Figure 24B shows the DLS for NP1 in Tween-80 / PBS (concentration of Tween- 80 is ⁇ 1%).
• the mean diameter is 30 nm, and 35.1 nm for Figures 24A and 24B, respectively.
• Figure 25 shows in vivo biodistribution of PS2 24, 48, and 72 hours post tail vein injection in BALB/c (3 mice/group) mice bearing subcutaneous Colon26 tumors on the right shoulders.
• Figure 26 shows Release/Retention Profiles of PS1 from NP1 in a 1% Human
• HSA Serum Albumin

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