Photodynamic therapy involving thiamine or a derivative thereof

Abstract

The present disclosure relates generally to methods of utilizing photosensitizers in the presence of thiamine or a derivative thereof.

Description

Attorney Docket No.: UCSF-813WO PHOTODYNAMIC THERAPY INVOLVING THIAMINE OR A DERIVATIVE THEREOF CROSS-REFERENCE TO RELATED APPLICATION

[0001] Pursuant to 35 U.S.C. § 119(e), this application claims priority to the filing date of United States Provisional Patent Application Serial No. 63 / 685,652 filed August 21, 2024, the disclosure of which is herein incorporated by reference in its entirety. BACKGROUND

[0002] Methods and compositions for enhancing photodynamic therapies (PDT) would represent an advancement in the art. Herein, a solution to this problem, among others, is presented. SUMMARY

[0003] Methods of producing singlet oxygen, stabilizing singlet oxygen, cross-linking animal tissue, and / or treating a condition in a subject, among others, by utilizing a photosensitizer in the presence of thiamine or a derivative thereof are described herein.

[0004] The foregoing summary is illustrative only and is not intended to be in any way limiting. In addition to the illustrative embodiments and features described herein, further aspects, embodiments, objects and features of the disclosure will become fully apparent from the drawings and the detailed description and the claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0005] The features of the present disclosure are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present disclosure will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the disclosure are utilized, and the accompanying drawings.

[0006] FIG 1 shows thiamine improves efficacy of photodynamic dynamic therapy ex vivo.

[0007] FIG 2 shows thiamine treatment in mice improves efficacy of photodynamic therapy.Attorney Docket No.: UCSF-813WO

[0008] FIG 3 shows human tissue treatment with thiamine during PDT improves resistance to elastase degradation post-PDT.

[0009] FIG 4 shows porcine tissue treatment with thiamine during PDT improves resistance to elastase degradation post-PDT.

[0010] FIG 5 shows high-dose thiamine treatment during PDT crosslinks veins and aortic tissue.

[0011] FIG 6 shows a dose-dependent increase in singlet oxygen production in photodynamic therapy Using ICG as Photosensitizer when Combined with Thiamine.

[0012] FIG 7 shows thiamine enhancing riboflavin based PDT in rabbit sclera.

[0013] FIG 8 shows thiamine potentiating the toxicity of photosensitizers in triple negative breast cancer.

[0014] FIG 9 shows porcine tissue treatment with photosensitizers in the presence of thiamine during PDT improves resistance to elastase-dependent degradation.

[0015] FIGs. 10A, 10B, 10C, 10D, and 10E show data demonstrating that photodynamic therapy (PDT) using methylene blue (MB) as a photosensitizer inhibits the induction of AAA. Abdominal aortas of mice were treated topically with methylene blue and exposed to light with indicated controls and were harvested on week 9 after induction of AAA using active elastase. Vehicle group are mice which have undergone AAA induction surgery with active elastase and treated with vehicle only and considered as positive controls. Sham group are mice undergoing AAA surgery that are treated with heat-inactivated elastase and considered healthy controls. FIG. 10A shows quantification of the weekly percent growth in maximal diameter of infrarenal abdominal aortas measured by micro-ultrasound imaging (MUI) using the B mode in indicated groups (n=8–15 per group). Measurements done weekly for 9 weeks following induction of aneurysms and treatment. Data were analyzed by one-way ANOVA followed by the Kruskal Wallis post hoc test. ***P<0.001. FIG. 10B shows quantification of the maximal diameter of infrarenal abdominal aortas measured by MUI and normalized to body-weight (BW) in indicated groups (n=8–15 per group). Data were analyzed by one-way ANOVA followed by the Kruskal Wallis post hoc test. ***P<0.001.FIG. 10C shows representative images of abdominal aortas visualized by MUI using the B mode in indicated groups. The aneurysm is highlighted by a white circle. FIG. 10D shows representative images of the macroscopic features of AAAAttorney Docket No.: UCSF-813WO formation in indicated groups. FIG. 10E shows quantification of the maximal diameter of infrarenal abdominal aortas measured by micrometry normalized to BW in indicated groups (n=6–15 per group). Data were analyzed by one-way ANOVA followed by the Kruskal Wallis post hoc test. AAA indicates abdominal aortic aneurysm; BW, body weight; MUI, micro- ultrasound imaging; Veh, vehicle (HBSS); MB, methylene blue; Veh+MB+Light, photodynamic therapy (PDT).

[0016] FIGs. 11A, 11B, and 11C show data demonstrating photodynamic therapy (PDT) using methylene blue (MB) as a photoactivator modulates immune cell infiltration and apoptosis in aneurysmal tissue from mice undergoing AAA induction and concurrently treated with PDT post-induction of AAA. Abdominal aortas of mice were topically treated with methylene blue and exposed to light with indicated controls and were harvested on week 9 after induction of AAA. FIG. 11A shows representative aortic aneurysm cross-sections from the indicated groups stained with Verhoeff-Van Gieson (VVG) stain for Elastin, Masson Trichrome for type I and type III collagen, CD3 antibody stain for T-cells and CD68 antibody stain for macrophages imaged by brightfield and polarized light. For illustrative purposes, a magnification of 20X was used for vehicle group, 50X was used for MB PDT group and 100X was used for sham group to account for the variable size of whole aortic cross-sections. FIG. 11B shows a semi-quantitative grade of elastin degradation in the aortic wall from aortic aneurysms sections of mice treated with vehicle, PDT, and healthy Sham mice. FIG. 11C shows IL1B, IL-6, and IL-10 expression from serum of mice assessed by ELISAs in indicated groups (n=7-12 per group). Data were analyzed by Student t test. **P<0.01; ***P<0.001. AAA indicates abdominal aortic aneurysm; VVG staining, Verhoeff-Van Gieson staining; Trichrome, Massons Trichrome staining, Veh, vehicle (HBSS); MB, methylene blue; Veh+MB+Light, photodynamic therapy.

[0017] FIGs. 12A, 12B, 12C, 12D, and 12E show data demonstrating that MB PDT reduces the growth of AAA and expands the therapeutic window for treating an aneurysm. MB PDT was administered to mice with pre-established and progressive aneurysms 21 days after the initial aneurysm induction surgery with active elastase applied topically on the aorta. Vehicle group are mice which have undergone AAA induction surgery with active elastase applied topically on the aorta and vehicle only applied topically at the same region. This group is considered as positive controls. Sham group are mice undergoing AAA surgery with topically applied heat-inactivated elastase and considered healthy controls. FIG. 12A shows quantification of the weekly percentAttorney Docket No.: UCSF-813WO growth in maximal diameter of infrarenal abdominal aortas measured by micro-ultrasound imaging (MUI) using the B mode in indicated groups (n=10–15 per group). Measurements done weekly for 9 weeks following induction of aneurysms. Treatment done on Day 21 post-induction of aneurysms. Data were analyzed by one-way ANOVA followed by the Kruskal Wallis post hoc test. *P<0.001. FIG. 12B shows quantification of the maximal diameter of infrarenal abdominal aortas measured by MUI and normalized to body-weight (BW) in indicated groups (n=10–15 per group). Data were analyzed by one-way ANOVA followed by the Kruskal Wallis post hoc test. ***P<0.001. FIG. 12C shows representative images of abdominal aortas visualized by MUI using the B mode in indicated groups. The aneurysm is highlighted by a white circle. FIG. 12D shows representative images of the macroscopic features of AAA formation in indicated groups. FIG. 12E shows quantification of the maximal diameter of infrarenal abdominal aortas measured by micrometry normalized to body-weight (BW) in indicated groups (n=10–15 per group). Data were analyzed by one-way ANOVA followed by the Kruskal Wallis post hoc test. AAA indicates abdominal aortic aneurysm; BW, body weight; MUI, micro-ultrasound imaging; Veh, vehicle (HBSS); MB, methylene blue; Veh+MB+Light, photodynamic therapy.

[0018] FIGs. 13A and 13B show peptide mapping analysis of differential drug-to-antibody ratio (DAR) in anakinra methylene blue conjugates. FIG. 13A shows the sequence of Anakinra, where conjugated lysines are marked as *. FIG. 13B shows MS spectra for Ana-MB conjugate- site peptides for differential DAR levels. Ana-MB, anakinra conjugated to methylene blue; DAR, drug-to-antibody ratio.

[0019] FIGs. 14A, 14B, 14C, 14D, 14E, 14F, 14G, and 14H show data demonstrating that Targeted Delivery of Methylene Blue (anakinra attached to methylene blue, Ana-MB) to Inflammatory Aneurysmal Tissue Improves Efficacy of Photodynamic Therapy. Vehicle group are mice which have undergone AAA induction surgery with active elastase applied topically on the aorta and vehicle only applied topically at the same region. This group is considered as positive controls. Sham group are mice which have undergone AAA surgery treated heat- inactivated elastase and considered healthy controls. FIG. 14A shows representative images of IL-1R expression in cross-sections of aneurysms in vehicle and sham mice, with a higher density of IL-1R expression in vehicle mice. FIG. 14B presents quantification of the IL-1R positive area in cross-sections of aneurysms in vehicle and sham mice. FIG. 14C displays a bar graph representing localization of recombinant Anakinra to aneurysms of mice upon intravenousAttorney Docket No.: UCSF-813WO treatment with different concentrations (50µg and 400µg) of Ana-MB as assessed by ELISAs in indicated groups (n=4-5 per group). Healthy tissue was considered as negative control. Y-axis modified to account for differences in quantity. Data were analyzed by Student t test. FIG. 14D is a representative image of the increase in fluorescence in the aneurysms tissue, adjacent distal aorta and serum of the same mice depicting increase in localization of fluoresceine-labeled Anakinra conjugated methylene blue after tissues have been exposed to 600nm light for fluorescence. FIG. 14E depicts quantification of the weekly percent growth in the maximal diameter of infrarenal abdominal aortas measured by micro-ultrasound imaging (MUI) using the B mode in indicated groups (n=10–15 per group). Measurements were performed weekly for 9 weeks following the induction of aneurysms. Treatment was administered on Day 21 post- induction of aneurysms. Data were analyzed by one-way ANOVA followed by the Kruskal Wallis post hoc test. *P<0.001. FIG. 14F shows representative images of abdominal aortas (white circles) visualized by MUI using the B mode in the indicated group. FIG. 14G displays representative images of the macroscopic features of AAA formation in indicated groups. FIG. 14H demonstrates quantification of the maximal diameter of infrarenal abdominal aortas measured by micrometry normalized to BW in indicated groups (n=10–15 per group). Data were analyzed by one-way ANOVA followed by the Kruskal Wallis post hoc test. AAA indicates abdominal aortic aneurysm; Ana-MB indicates methylene conjugated Anakinra; Ana-50, 50µg of Anakinra-MB; Ana-400, 400µg of Anakinra-MB; MB, methylene blue; Veh+MB+Light, photodynamic therapy; Veh+Ana-MB+Light, targeted photodynamic therapy.

[0020] FIGs. 15A, 15B and 15C show data demonstrating that MB PDT protects extracellular matrix from elastase-induced degradation. FIG. 15A shows representative image of porcine aortic cross-sections from indicated groups before and after treatment with elastase stained for elastin with Verhoeff-Van Gieson (VVG). FIG. 15B shows a bar graph of Ne- (carboxymethyl)lysine (CML) levels, a known marker of elastin crosslinking, in recombinant tropoelastin elastin treated with PDT and indicated controls. FIG. 15C shows a bar graph of CML levels in porcine tissue treated with PDT and indicated control groups, normalized to total elastin within tissue. AAA indicates abdominal aortic aneurysm; VVG staining, Verhoeff-Van staining; CML, Ne-(carboxymethyl)lysine; Veh, Vehicle (HBSS), Trop elastin, tropoelastin, PGG, Pentagalloyl glucose.Attorney Docket No.: UCSF-813WO

[0021] FIGs. 16A, 16B, and 16C show data demonstrating that photodynamic therapy improves mechanical strength of aortic tissue. FIG. 16A shows a representative image of healthy mouse aortic tissue subjected to treatment with vehicle, methylene blue without light, and with light (30mW / cm2) at different timepoints (30 mins, 90 mins, and 120 mins). Glutaraldehyde treatment for 120 mins was used as a positive control. The relative change in stiffness was measured on a scale while holding the edge of the tissue with forceps. FIG. 16B shows a representative image of healthy porcine aortic tissue subjected to treatment with vehicle and with light for 120 mins at 60 mW / cm² and 120 mW / cm² intensity. Tissues were treated with recombinant elastase for 1 hour following light exposure to demonstrate resistance to elastin degradation. FIG. 16C shows the effect of MB PDT on stiffness of the aneurysm in aneurysms in vivo and the stiffness was assessed ex vivo (FIG 7C).

[0022] FIG. 17 shows a schematic overview of an animal model for aortic aneurysms (AAA).

[0023] FIG. 18 shows show schematic overview of the treatment procedure. DETAILED DESCRIPTION OF THE DISCLOSURE I. Definitions

[0024] Unless otherwise defined, all terms of art, notations, and other scientific terms or terminology used herein are intended to have the meanings commonly understood by those of skill in the art to which this application pertains. In some cases, terms with commonly understood meanings are defined herein for clarity and / or for ready reference, and the inclusion of such definitions herein should not necessarily be construed to represent a substantial difference over what is generally understood in the art. Many of the techniques and procedures described or referenced herein are well understood and commonly employed using conventional methodology by those skilled in the art.

[0025] The singular form “a”, “an”, and “the” include plural references unless the context clearly dictates otherwise. For example, the term “a cell” includes one or more cells, comprising mixtures thereof. “A and / or B” is used herein to include all of the following alternatives: “A”, “B”, “A or B”, and “A and B”.

[0026] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between theAttorney Docket No.: UCSF-813WO upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the disclosure. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the disclosure, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure.

[0027] Certain ranges are presented herein with numerical values being preceded by the term “about.” The term “about” is used herein to provide literal support for the exact number that it precedes, as well as a number that is near to or approximately the number that the term precedes. In determining whether a number is near to or approximately a specifically recited number, the near or approximating unrecited number may be a number which, in the context in which it is presented, provides the substantial equivalent of the specifically recited number. If the degree of approximation is not otherwise clear from the context, “about” means either within plus or minus 10% of the provided value, or rounded to the nearest significant figure, in all cases inclusive of the provided value. In some embodiments, the term “about” indicates the designated value ± up to 10%, up to ± 5%, or up to ± 1%.

[0028] Crosslinking, as used herein, refers to the process of chemically joining two or more molecules by a covalent bond.

[0029] MB, MetB, or Met B, as used herein, refers to methylene blue.

[0030] As used herein, percent (%) sequence identity or “sequence identity”, and terms related thereto, in the context of amino acid sequences or nucleic acid sequences, generally refer to and include the percentage of amino acid residues or nucleic acid residues in a candidate sequence that are identical with the amino acid residues or nucleic acid residues, respectively, in a selected sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity or percent nucleic acid identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as Clustal Omega, BLAST, BLAST-2, ALIGN, ALIGN-2 or Megalign (DNASTAR) software, with BLAST being the alignment algorithm of preference. Those skilled in the art can determine appropriateAttorney Docket No.: UCSF-813WO parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full-length of the sequences being compared, although for simplicity it maybe preferred to use default parameters.

[0031] The term “antibody” has used herein is used in the broadest sense and generally refers to and / or includes monoclonal antibodies, multi-valent antibodies, multi-specific, antigen-binding fragments of antibodies. Antigen-binding fragments of antibodies (antibody fragments) generally refer to and / or include antibody-derived proteins that comprise a functional set of CDRs (e.g., a CDR-H1-3 and CDR-L1-3) that bind a target protein and have a molecule weight less than a full length IgG antibody (e.g., a molecular weight less than ~150,000 Daltons). In certain embodiments, an antigen-binding antibody fragment includes: fragment antigen binding (Fab) fragments, F(ab’)2 fragments, Fab' fragments, Fv fragments, IgG (rIgG) fragments, and single chain antibody fragments, including single chain variable fragments (sFv or scFv). Antibodies and antigen-binding fragments of antibodies generally encompass genetically engineered, , and / or otherwise modified forms of immunoglobulins, such as intrabodies, peptibodies, chimeric antibodies, fully human antibodies, humanized antibodies, and heteroconjugate antibodies, multi- specific antibodies, multi-valent antibodies, diabodies, triabodies, and tetrabodies, tandem di- scFv, tandem tri-scFv. A full-length antibody or antibody fragment, intact antibody or antibody fragment, and / or whole antibody are interchangeable, and generally include and / or refer to an antibody having a structure substantially similar to a native antibody structure having heavy chains that contain an Fc region and / or include antibodies of any class or sub-class, including IgG and sub-classes thereof (e.g., IgG1 and IgG4), IgM, IgE, IgA, and IgD.

[0032] “Complementarity determining regions” (CDRs) as used herein are synonymous with “hypervariable regions” (HVRs) generally refer to and / or include non-contiguous sequences of amino acids within antibody variable regions that confer antigen specificity and / or binding affinity. In general, there are three CDRs in each heavy chain variable region (CDR-H1, CDR- H2, CDR-H3) and three CDRs in each light chain variable region (CDR-L1, CDR-L2, CDR-L3). Framework regions (FRs) generally refer to and / or include non-CDR regions of the heavy and light chain variable regions. In general, there are four FRs in each full-length heavy chain variable region (FR-H1, FR-H2, FR-H3, and FR-H4), and four FRs in each full-length light chain variable region (FR-L1, FR-L2, FR-L3, and FR-L4). Variable regions (also referred to as variable domains) generally refer to and / or include the domain of an antibody heavy or lightAttorney Docket No.: UCSF-813WO chain that is involved in binding the antibody to antigen (e.g., a single variable domain comprises a CDR 1, CDR 2, and CDR 3). The variable domains of the heavy chain and light chain (VH and VL, respectively) of a native antibody generally have similar structures, with each domain comprising four conserved framework regions (FRs) and three CDRs In certain instances, a single VH or VL domain can be sufficient to confer antigen-binding. Fc region generally encompasses and / or refers to a C-terminal region of an immunoglobulin heavy chain that contains at least a portion of the constant region. The term includes native sequence Fc regions and variant Fc regions. In one embodiment, a human IgG heavy chain Fc region extends from Cys226, or from Pro230, to the carboxyl-terminus of the heavy chain. However, the C-terminal lysine (Lys447) of the Fc region may or may not be present. Unless otherwise specified herein, numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system, also called the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest , 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991. In certain embodiments, the Fc region include IgG and sub-classes thereof (e.g., IgG1 and IgG4), IgM, IgE, IgA, and / or IgD heavy chain constant regions and / or heavy chain constant regions derived from IgG and sub-classes thereof (e.g., IgG1 and IgG4), IgM, IgE, IgA, and IgD.

[0033] An “individual” as used herein is synonymous with “patient” and / or “subject” and includes and / or refers to a human and may be a human that has been diagnosed as needing to treat a disease or condition as disclosed herein. However, examples are not limited to humans and include, chimpanzees, marmosets, cows, horses, sheep, goats, pigs, rabbits, dogs, cats, rats, mice, guinea pigs, and the like. The individual is typically a human and may be a human that has been diagnosed as needing to treat a disease or condition as disclosed herein.

[0034] “Treating” or “treatment” as herein includes and / or refers to ameliorating the disease or disorder or symptoms thereof (e.g., slowing or arresting or reducing the development of the disease or at least one of the clinical symptoms thereof). In certain embodiments, treating or treatment also includes and / or refers to alleviating or ameliorating at least one physical and / or biological parameters including those which may not be discernible by the patient. In certain embodiments, treating or treatment includes and / or refers to modulating a disease, disorder, or biological process either physically, (e.g., stabilization of a discernible symptom), physiologically, (e.g., stabilization of a physical and / or biological parameter), or both. In certainAttorney Docket No.: UCSF-813WO embodiments, treating or treatment includes and / or refers to preventing or delaying the onset or development or progression of the disease or disorder. In certain embodiments, treating or treatment includes and / or refers to preventing or delaying or inhibiting the deterioration of (i) a healthy physiological state or (ii) a baseline physiological state (e.g., the progression of a disease or disorder).

[0035] As used herein, in any instance or embodiment described herein, “comprising” may be replaced with “consisting essentially of” and / or “consisting of,” unless context clearly connotes otherwise. Similarly, as used herein, in any instance or embodiment described herein, “comprises” may be replaced with “consists essentially of” and / or “consists of,” unless context clearly connotes otherwise.

[0036] As used herein, the term “about,” in the context of a given value or range, includes and / or refers to a value or range that is within 10% of the given value or range.

[0037] As used herein, the term “and / or” is to be taken as specific disclosure of each of the two specified features or components with or without the other. For example, “A and / or B” is to be taken as specific disclosure of each of (i) A, (ii) B and (iii) A and B, just as if each were set out individually herein.

[0038] It is understood that aspects and embodiments of the disclosure described herein include "comprising", "consisting", and "consisting essentially of" aspects and embodiments. As used herein, "comprising" is synonymous with "including", "containing", or "characterized by", and is inclusive or open-ended and does not exclude additional, unrecited elements or method steps. As used herein, "consisting of" excludes any elements, steps, or ingredients not specified in the claimed composition or method. As used herein, "consisting essentially of" does not exclude materials or steps that do not materially affect the basic and novel characteristics of the claimed composition or method. Any recitation herein of the term "comprising", particularly in a description of components of a composition or in a description of steps of a method, is understood to encompass those compositions and methods consisting essentially of and consisting of the recited components or steps.

[0039] All genes, gene names, and gene products disclosed herein are intended to correspond to homologs from any species for which the compositions and methods disclosed herein are applicable. Thus, the terms include, but are not limited to genes and gene products from humansAttorney Docket No.: UCSF-813WO and mice. It is understood that when a gene or gene product from a particular species is disclosed, this disclosure is intended to be exemplary only, and is not to be interpreted as a limitation unless the context in which it appears clearly indicates. Thus, for example, for the genes or gene products disclosed herein, which in some embodiments relate to mammalian nucleic acid and amino acid sequences, are intended to encompass homologous and / or orthologous genes and gene products from other animals including, but not limited to other mammals, fish, amphibians, reptiles, and birds. In some embodiments, the genes, nucleic acid sequences, amino acid sequences, peptides, polypeptides and proteins are human. The term “gene” is also intended to include variants thereof.

[0040] It is appreciated that certain features of the disclosure, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the disclosure, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable sub- combination. All combinations of the embodiments pertaining to the disclosure are specifically embraced by the present disclosure and are disclosed herein just as if each and every combination was individually and explicitly disclosed. In addition, all sub-combinations of the various embodiments and elements thereof are also specifically embraced by the present disclosure and are disclosed herein just as if each and every such sub-combination was individually and explicitly disclosed herein. II. General Thiamine / Thiamine Derivative Embodiments

[0041] It has been found that thiamine or a derivative thereof enhances the reactions of photosensitizers. In one aspect, the invention provides a method of producing singlet oxygen, the method comprising: subjecting a photosensitizer in the presence of thiamine or a derivative thereof to photoactivating conditions; thereby producing singlet oxygen. In one aspect, the invention provides a method of increasing the production of singlet oxygen from a photosensitizer, the method comprising: subjecting a photosensitizer in the presence of thiamine or a derivative thereof to photoactivating conditions; thereby increasing the production of singlet oxygen from the photosensitizer. In one aspect, the invention provides a method of stabilizing singlet oxygen, the method comprising: subjecting a photosensitizer in the presence of thiamine or a derivative thereof to photoactivating conditions, thereby stabilizing singlet oxygen. In oneAttorney Docket No.: UCSF-813WO aspect, the invention provides a method of increasing the stability of singlet oxygen from a photosensitizer, the method comprising: subjecting a photosensitizer in the presence of thiamine or a derivative thereof to photoactivating conditions, thereby increasing the stability of singlet oxygen from the photosensitizer. In an exemplary embodiment, for any of the methods described herein, the photosensitizer is part of a photoactivatable drug conjugate described herein.

[0042] In another aspect, the invention provides a method of cross-linking animal tissue, the method comprising: (a) administering to the animal tissue a photosensitizer and thiamine or a derivative thereof; and (b) subjecting the photosensitizer and the thiamine or a derivative thereof to photoactivating conditions at the site of or near the animal tissue, thereby cross-linking the animal tissue, wherein the animal tissue comprises collagen and / or elastin. In another aspect, the invention provides a method of strengthening animal tissue, the method comprising: (a) administering to the animal tissue a photosensitizer and thiamine or a derivative thereof; and (b) subjecting the photosensitizer and the thiamine or a derivative thereof to photoactivating conditions at the site of or near the animal tissue, thereby strengthening the animal tissue, wherein the animal tissue comprises collagen and / or elastin. In another aspect, the invention provides a method of stiffening animal tissue, the method comprising: (a) administering to the animal tissue a photosensitizer and thiamine or a derivative thereof; and (b) subjecting the photosensitizer and the thiamine or a derivative thereof to photoactivating conditions at the site of or near the animal tissue, thereby stiffening the animal tissue, wherein the animal tissue comprises collagen and / or elastin. In another aspect, the invention provides a method of producing singlet oxygen within or at the site of animal tissue, the method comprising: (a) administering to the animal tissue a photosensitizer and thiamine or a derivative thereof; and (b) subjecting the photosensitizer and the thiamine or a derivative thereof to photoactivating conditions at the site of or near the animal tissue, thereby producing singlet oxygen within or at the site of animal tissue, wherein the animal tissue comprises collagen and / or elastin. In another aspect, the invention provides a method of increasing the production of singlet oxygen within or at the site of animal tissue, the method comprising: (a) administering to the animal tissue a photosensitizer and thiamine or a derivative thereof; and (b) subjecting the photosensitizer and the thiamine or a derivative thereof to photoactivating conditions at the site of or near the animal tissue, thereby increasing the production of singlet oxygen within or at the site of animal tissue,Attorney Docket No.: UCSF-813WO wherein the animal tissue comprises collagen and / or elastin, and wherein the increase is compared with the photosensitizer in the absence of thiamine or a derivative thereof. In another aspect, the invention provides a method of stabilizing singlet oxygen within or at the site of animal tissue, the method comprising: (a) administering to the animal tissue a photosensitizer and thiamine or a derivative thereof; and (b) subjecting the photosensitizer and the thiamine or a derivative thereof to photoactivating conditions at the site of or near the animal tissue, thereby stabilizing singlet oxygen within or at the site of animal tissue, wherein the animal tissue comprises collagen and / or elastin. In another aspect, the invention provides a method of increasing the stability of singlet oxygen within or at the site of animal tissue, the method comprising: (a) administering to the animal tissue a photosensitizer and thiamine or a derivative thereof; and (b) subjecting the photosensitizer and the thiamine or a derivative thereof to photoactivating conditions at the site of or near the animal tissue, thereby increasing the stability of singlet oxygen within or at the site of animal tissue, wherein the animal tissue comprises collagen and / or elastin, and wherein the increase is compared with the photosensitizer in the absence of thiamine or a derivative thereof. In an exemplary embodiment, for any of the methods described herein, the animal tissue is contacted with the photosensitizer, and then contacted with the thiamine or a derivative thereof. In an exemplary embodiment, for any of the methods described herein, the animal tissue is contacted with the thiamine or a derivative thereof, and then contacted with the photosensitizer. In an exemplary embodiment, for any of the methods described herein, the animal tissue is contacted with the photosensitizer and the thiamine or a derivative thereof contemporaneously (e.g. at about the same time, such as within 10 seconds). In an exemplary embodiment, for any of the methods described herein, the photosensitizer is part of a photoactivatable drug conjugate described herein.

[0043] In another aspect, the invention provides a method of treating a condition in a subject, the method comprising: (a) administering to the subject in need of treatment thereof with a photosensitizer and thiamine or a derivative thereof; and (b) subjecting the photosensitizer in the presence of the thiamine or a derivative thereof to photoactivating conditions, thereby treating the condition in the subject. In another aspect, the invention provides a method of treating a condition in a subject, the method comprising: (a) administering to the subject in need of treatment thereof with a photoactivatable drug conjugate and thiamine or a derivative thereof; and (b) subjecting the photosensitizer in the presence of the thiamine or a derivative thereof toAttorney Docket No.: UCSF-813WO photoactivating conditions, thereby treating the condition in the subject. In an exemplary embodiment, for any of the methods described herein, the photosensitizer is administered to the subject, and then the thiamine or a derivative thereof is administered to the subject. In an exemplary embodiment, for any of the methods described herein, the thiamine or a derivative thereof is administered to the subject, and then the photosensitizer is administered to the subject. In an exemplary embodiment, for any of the methods described herein, the photosensitizer and the thiamine or a derivative thereof are administered to the subject contemporaneously (e.g. at about the same time, such as within 10 seconds). In an exemplary embodiment, for any of the methods described herein, the photosensitizer is part of a photoactivatable drug conjugate described herein. Photosensitizers

[0044] The photosensitizers herein can be activated by any source of light energy. Photosensitizers provided and described herein are generally advantageous in that they are activated when exposed to light at a wavelength. In certain embodiments, activation induces cross-linking of animal tissue. In certain embodiments, activation induces the killing of a cancer cell.

[0045] In some embodiments, the photosensitizer is a thiazine, porfimer sodium, a porphyrin, a chlorin, a pheophorbide, a phthalocyanine, an anthraquinone, a phenothiazine, a xanthene, or a cyanine, that generates singlet oxygen when exposed to light within its excitation wavelength window. In some embodiments, the photosensitizer is selected from Table 1. Table 1 Photosensitizer PubChem CID Excitation Wavelength WindowAttorney Docket No.: UCSF-813WO indocyanine green 5282412 ~600-900 nm IR-780 118987185 ~740-800 nm [00g bacteriochlorins, pheophorbides, pyropheophorbide-a, photochloride, chlorins, chlorin e6, mono- L-aspartyl 10 chlorin e6, di-L-aspartyl chlorin e6, tin (IV) chlorin e6, palladium bacteriochlorophylls, palladium bacteriopheophorbides, synthetic chlorins, synthetic bacteriochlorins, metatetrahydroxyphenyl chlorin, a bacteriopheophorbide, bacteriochlorin, benzoporphyrin, monobenzoporphyrins, verteporfin, sulfonated aluminum phthalocyanines (disulfonated and tetrasulfonated), sulfonated aluminum naphthalocyanines, tin and zinc octaethylpurpurine, tin ethiopurpurine, vegetables porficenos, synthetic porphyrins, chlorins synthetic, synthetic bacteriochlorins, mesotrietinylporphyrins without metals, metallized mesotrietinylporphyrins, modified core porphyrins, expanded porphyrins (texaphyrins), tocopherol, Lutetium motexaphine, gadolinium motexafin, merocyanine 540, acridine dyes, hypericin, halogenated squarin dyes, halogenated xanthene dyes, eosin, talaporfin sodium, 2-(1- Hexyloxyethyl)-2-Devinyl Pyropheophorbide-a (HPPH-Photochlor), benzo-porphyrin derivative monoacid ring A, redaporfin (LUZ11), chlorin E6 / P6 / purpurin, Ru(II) polypyridyl complex (TLD-1433), padeliporfin di-potassium, or 5-aminolevulinic acid hydrochloride.

[0047] In certain embodiments, the photosensitizer is riboflavin, which can be excited by light of a wavelength of from about 440 nm to about 460 nm. In certain embodiments, the photosensitizer is methylene blue, which can be excited by light of a wavelength of from about 640 nm to about 680 nm. In certain embodiments, the photosensitizer is indocyanine green, also known as ICG, which can be excited by light of a wavelength of from about 840 nm to about 860 nm. In certain embodiments, the photosensitizer is a cyanine dye which is cardiogreen, which can be excited by light of a wavelength of from about 600 nm to about 900 nm. In certain embodiments, the photosensitizer is a chlorin which is temoporfin, which can be excited by lightAttorney Docket No.: UCSF-813WO of a wavelength of from about 630 nm to about 670 nm. In certain embodiments, the photosensitizer is aminolevulinic acid.

[0048] In certain embodiments, the photosensitizer is administered in an amount of from about 1 µg / mL to about 500 µg / mL. In certain embodiments, the photosensitizer is administered in an amount of from about 80 µg / mL to about 120 µg / mL, from about 95 µg / mL to about 105 µg / mL, or about 100 µg / mL. In certain embodiments, the photosensitizer is administered in a concentration of from about 0.1 µM to about 3 µM. In certain embodiments, the photosensitizer is administered in a concentration of from about 0.4 µM to about 0.6 µM, or about 0.5 µM. In certain embodiments, the photosensitizer is administered in a concentration of from about 0.8 µM to about 1.2 µM, from about 0.95 µM to about 1.05 µM, or about 1.0 µM. In certain embodiments, the photosensitizer is administered in a concentration of from about 1.8 µM to about 2.2 µM, from about 1.95 µM to about 2.05 µM, or about 2.0 µM.

[0049] In some embodiments, the photosensitizer is one that is activatable with light that can penetrate through blood or blood cells. In certain embodiments, the photosensitizer is one that generates singlet oxygen when exposed to light within the tissue window. In some instances, such range is 600 nm to about 1,000 nm. Other ranges near or overlapping with tissue window can be used, for example, 350 nm to 1,200 nm. Thiamine or a derivative thereof

[0050] In an exemplary embodiment, the thiamine or a derivative thereof is thiamine. In an exemplary embodiment, the thiamine or a derivative thereof is a thiamine derivative. In an exemplary embodiment, the thiamine derivative is benfotiamine.

[0051] In certain embodiments, the thiamine or a derivative thereof is administered in an amount of from about 0.1 mg / mL to about 20 mg / mL. In certain embodiments, the thiamine or a derivative thereof is administered in an amount of from about 0.1 mg / mL to about 1.5 mg / mL, from about 0.5 mg / mL to about 1.5 mg / mL, from about 0.8 mg / mL to about 1.2 mg / mL, from about 0.95 mg / mL to about 1.05 mg / mL, or about 1 mg / mL. In certain embodiments, the thiamine or a derivative thereof is administered in an amount of from about 1.8 mg / mL to about 2.2 mg / mL, from about 1.95 mg / mL to about 2.05 mg / mL, or about 2 mg / mL. In certain embodiments, the thiamine or a derivative thereof is administered in an amount of from about 8 mg / mL to about 12 mg / mL, from about 9.5 mg / mL to about 10.5 mg / mL, or about 10 mg / mL.Attorney Docket No.: UCSF-813WO

[0052] In certain embodiments, the thiamine or a derivative thereof is administered in an amount of from about 0.01 µg / mL to about 300 µg / mL, from about 90 ng / mL to about 120 ng / mL, or about 100 ng / mL. In certain embodiments, the thiamine or a derivative thereof is administered in an amount of from about 0.1 µg / mL to about 5 µg / mL, from about 0.5 µg / mL to about 2 µg / mL, from about 0.90 µg / mL to about 1.1 µg / mL, or about 1 µg / mL. In certain embodiments, the thiamine or a derivative thereof is administered in an amount of from about 1 µg / mL to about 30 µg / mL, from about 5 µg / mL to about 20 µg / mL, from about 8 µg / mL to about 12 µg / mL, or about 10 µg / mL. In certain embodiments, the thiamine or a derivative thereof is administered in an amount of from about 25 µg / mL to about 300 µg / mL, from about 50 µg / mL to about 200 µg / mL, from about 80 µg / mL to about 120 µg / mL, from about 95 µg / mL to about 110 µg / mL, or about 100 µg / mL. Animal Tissue

[0053] In an exemplary embodiment, the animal tissue comprises elastin. In an exemplary embodiment, the animal tissue comprises collagen. In an exemplary embodiment, the animal tissue comprises elastin and collagen. In an exemplary embodiment, the animal tissue is mammalian. In an exemplary embodiment, the animal tissue is human, porcine, or murine. In an exemplary embodiment, the animal tissue is vascular, ocular, breast, muscle, ligament, bone, skin (such as dermal or subdermal). In an exemplary embodiment, the animal tissue is heart. In an exemplary embodiment, the animal tissue is aortic. In an exemplary embodiment, the animal tissue comprises a cancer cell. In an exemplary embodiment, at least 0.01%, at least 0.1%, at least 0.5%, at least 1%, or at least 2% of the protein by weight in the animal tissue is collagen. In an exemplary embodiment, at least 0.01%, at least 0.1%, at least 0.5%, at least 1%, or at least 2% of the protein by weight in the animal tissue is elastin. Photoactivating Conditions

[0054] In an exemplary embodiment, the photoactivating conditions involve a light source that provides light which excites a photosensitizer described herein.

[0055] In an exemplary embodiment, the light source has an irradiance sufficient to crosslink animal tissue without significantly damaging the underlying animal tissue. In an exemplary embodiment, the light source has an irradiance sufficient to kill cancer cells without significantly damaging non-cancer cells. In an exemplary embodiment, the light source has an irradianceAttorney Docket No.: UCSF-813WO sufficient to kill precancerous cells without significantly damaging non-cancer cells. In an exemplary embodiment, the light source has an irradiance of from about 10mW / cm2and about 200mW / cm2. In an exemplary embodiment, the light source has an irradiance of from about 30mW / cm2and about 170mW / cm2. In an exemplary embodiment, the light source has an irradiance of from about 45mW / cm2and about 150mW / cm2. In an exemplary embodiment, the light source has an irradiance of from about 90mW / cm2and about 150mW / cm2. In an exemplary embodiment, the light source has an irradiance of from about 45mW / cm2and about 90mW / cm2.

[0056] In an exemplary embodiment, the photoactivating conditions occur for a period of time sufficient to crosslink animal tissue without significantly damaging the underlying animal tissue. In an exemplary embodiment, the photoactivating conditions occur for a period of time sufficient to kill cancer cells without significantly damaging non-cancer cells. In an exemplary embodiment, the photoactivating conditions occur for a period of time sufficient to kill precancerous cells without significantly damaging non-cancer cells. In an exemplary embodiment, the time of the photoactivating conditions is from about 1 second to about 2 hours. In an exemplary embodiment, the time of the photoactivating conditions is from about 30 seconds to about 1 hour. In an exemplary embodiment, the time of the photoactivating conditions is from about 30 seconds to about 30 minutes. In an exemplary embodiment, the time of the photoactivating conditions is from about 1 minute to about 30 minutes. In an exemplary embodiment, the time of the photoactivating conditions is from about 5 minutes to about 30 minutes. In an exemplary embodiment, the time of the photoactivating conditions is from about 4 minutes to about 6 minutes, from about 8 minutes to about 12 minutes, and from about 18 minutes to about 22 minutes. Targeting Moieties

[0057] Photosensitizer(s) can be attached (coupled / conjugated) to a targeting moiety in order to target the photosensitizer(s) to a site near and / or within a desired activity site, concentrate the photosensitizer(s) at the site of near and / or within a desired activity site, and / or reduce the overall exposure of the subject to the photosensitizer(s). A targeting moiety is preferably one that targets one or more photosensitizers to an animal tissue described herein (e.g., by binding cell surface receptor associated with the animal tissue). In some embodiments, the targeting moietyAttorney Docket No.: UCSF-813WO does not selectively bind to a cancer cell. Alternatively, the targeting moiety can be one that selectively binds non-malignant cells. Photoactivatable Drug Conjugates

[0058] Any of the photosensitizers herein can be coupled to a targeting moiety using known conjugation technologies to form a photoactivatable drug conjugate (PDC). Conjugation can optionally include the addition of one or more spacers or linkers into the PDC. Techniques for conjugation of an activatable agent such as a photosensitizer to a targeting moiety contemplated herein include, for example: ThermoFisher. Bioconjugation and crosslinking technical handbook: Reagents for bioconjugation, crosslinking, biotinylation, and modification of proteins and peptides. 2022.; Bioconjugation: Methods and Protocols. 22 July 2019. ISBN: 978-1-4939- 9654-4; and Khongorzul P, Ling CJ, Khan FU, Ihsan AU, Zhang J. Antibody-Drug Conjugates: A Comprehensive Review. Mol Cancer Res. 2020 Jan;18(1):3-19. Epub 2019 Oct 28. PMID: 31659006; Su Z, Xiao D, Xie F, Liu L, Wang Y, Fan S, Zhou X, Li S. Antibody-drug conjugates: Recent advances in linker chemistry. Acta Pharm Sin B. 2021 Dec;11(12):3889- 3907. Epub 2021 Apr 6. PMID: 35024314; PMCID: PMC8727783, and Tong JTW, Harris PWR, Brimble MA, Kavianinia I. An Insight into FDA Approved Antibody-Drug Conjugates for Cancer Therapy. Molecules. 2021 Sep 27;26(19):5847. PMID: 34641391. Pharmaceutical Compositions

[0059] In some embodiments, provided herein are pharmaceutical compositions and / or formulations comprising the photosensitizer and the thiamine or a derivative thereof, or the photoactivatable drug conjugate and the thiamine or a derivative thereof. Pharmaceutical compositions include and / or refers a preparation that is in such form as to permit the biological activity of the photosensitizer alone or in the PDC to be effective, and that contains no additional components which are unacceptably toxic to a subject to which the formulation would be administered. In certain embodiments, the pharmaceutical composition comprises a pharmaceutically acceptable carrier (e.g., an ingredient in a pharmaceutical formulation, other than an active ingredient, which is nontoxic to a subject). A pharmaceutically acceptable carrier includes, but is not limited to, a buffer, excipient, stabilizer, or preservative.Attorney Docket No.: UCSF-813WO Systems

[0060] Provided and described herein are systems useful for treating a condition described herein. In some embodiments, a system comprises: a photoactivatable drug conjugate, thiamine or a derivative thereof, and an energy source to activate the photosensitizer (e.g. a light delivery unit such as an optical applicator). Such a system can also comprise a light generating unit comprising a light source. In certain embodiments, the optical applicator comprises a catheter configured to deliver light at the range required to activate the photosensitizer incorporated in the system.

[0061] In some instances, the catheter is a laser catheter and / or light-emitting diode catheter. In certain embodiments, the catheter contains optical fibers to deliver the light. In certain embodiments, the light delivery unit is attached to the light generator and facilitates the transmission of light (e.g., a laser beam). In certain embodiments, the light generating unit and / or the light delivery unit is configured to administer light at an irradiance of about 1 mW / cm2to about 600 mW / cm2. In certain embodiments, the light generating unit and / or the light delivery unit is configured to administer light at an irradiance of about 25 mW / cm2to about 100 mW / cm2.

[0062] In some embodiments, also provided and described herein are kits comprising one or more components of the systems described herein. For example, a kit can include all components of the photodynamic therapy systems, or only the photoactivatable drug conjugate (e.g., any one of the photoactivatable drug conjugates described herein), thiamine or a derivative thereof, and a light delivery unit. Specific Embodiments

[0063] In an exemplary embodiment, the invention is a method of treating cancer, the method comprising: (a) administering to a subject in need of treatment thereof a photosensitizer and thiamine or a derivative thereof; and (b) subjecting the photosensitizer and the thiamine or a derivative thereof to photoactivating conditions at the site of or near the cancer. In an exemplary embodiment, the cancer is breast cancer. In an exemplary embodiment, the cancer is triple negative breast cancer. In an exemplary embodiment, the photosensitizer is temoporfin. In an exemplary embodiment, the photosensitizer is temoporfin, and the amount of temoporfin is as described herein (such as from about 0.25µM to about 3µM). In an exemplary embodiment, theAttorney Docket No.: UCSF-813WO thiamine or a derivative thereof is thiamine. In an exemplary embodiment, the thiamine or a derivative thereof is thiamine, and the amount of thiamine is as described herein (such as from about 100ng / ml and about 100µg / ml). In an exemplary embodiment, the photoactivating conditions are as described herein (such as a wavelength of about 652 nm and an irradiance of between about 45mW / cm2and about 150mW / cm2for between about 1 min and about 1 hour).

[0064] In an exemplary embodiment, the invention is a method of treating a scleral condition, the method comprising: (a) administering to a subject in need of treatment thereof a photosensitizer and thiamine or a derivative thereof; and (b) subjecting the photosensitizer and the thiamine or a derivative thereof to photoactivating conditions at the site of or near the scleral condition site. In an exemplary embodiment, the photosensitizer is cardiogreen or indocyanine green. In an exemplary embodiment, the thiamine or a derivative thereof is thiamine. In an exemplary embodiment, the photoactivating conditions are as described herein.

[0065] In an exemplary embodiment, for a method, composition and / or system described herein, the photosensitizer is riboflavin. In an exemplary embodiment, for a method, composition and / or system described herein, the photosensitizer is indocyanine green. In an exemplary embodiment, for a method, composition and / or system described herein, the photosensitizer is temoporfin. In an exemplary embodiment, for a method, composition and / or system described herein, the photosensitizer is verteporfin. In an exemplary embodiment, for a method, composition and / or system described herein, the photosensitizer is riboflavin. In an exemplary embodiment, for a method, composition and / or system described herein, the photosensitizer is cardiogreen. In an exemplary embodiment, for a method, composition and / or system described herein, the photosensitizer is aminolevulinic acid.

[0066] In an exemplary embodiment, for a method, composition and / or system described herein for treating an aneurysm, the photosensitizer is riboflavin. In an exemplary embodiment, for a method, composition and / or system described herein for treating an aneurysm, the photosensitizer is indocyanine green. In an exemplary embodiment, for a method, composition and / or system described herein for treating an aneurysm, the photosensitizer is temoporfin. In an exemplary embodiment, for a method, composition and / or system described herein for treating an aneurysm, the photosensitizer is verteporfin. In an exemplary embodiment, for a method, composition and / or system described herein for treating an aneurysm, the photosensitizer isAttorney Docket No.: UCSF-813WO riboflavin. In an exemplary embodiment, for a method, composition and / or system described herein for treating an aneurysm, the photosensitizer is cardiogreen. In an exemplary embodiment, for a method, composition and / or system described herein for treating an aneurysm, the photosensitizer is aminolevulinic acid.

[0067] In an exemplary embodiment, for a method of treating a condition described herein, the condition is not related to the heart. In an exemplary embodiment, for a method of treating a condition described herein, the condition is not an aneurysm. In an exemplary embodiment, for a photoactivatable drug conjugate described herein, the targeting moiety is not anakinra.

[0068] In an exemplary embodiment, for a method of treating a condition described herein, the photosensitizer is methylene blue, and the condition is not related to the heart. In an exemplary embodiment, for a method of treating a condition described herein, the photosensitizer is methylene blue, and the condition is not an aneurysm. In an exemplary embodiment, for a photoactivatable drug conjugate described herein, the photosensitizer is methylene blue, and the targeting moiety is not anakinra. III. Aneurysm Embodiments

[0069] There is a need in the art for treating aneurysms. The present disclosure shows that using a photosensitizer such as methylene blue in the presence of thiamine or a derivative thereof can suppress vascular diameter growth, maintain stability of the vasculature, treat and / or stabilize an aneurysm, and / or increase extracellular matrix (ECM) crosslinking.

[0070] The present disclosure further provides for and exemplifies the non-invasive treatment of aneurysms by two means for selectively activating a photosensitizer in the presence of thiamine or a derivative thereof at or near an aneurysm site in order to treat and / or stabilize an aneurysm. First, the photosensitizer contemplated herein is attached (e.g., conjugated) to a targeting moiety wherein the targeting moiety binds to an inflamed tissue. Aneurysms generally comprise inflammatory tissue. Inflammatory tissue preferentially expresses inflammatory markers, such as, IL1R. As such, in some embodiments, the targeting moiety binds IL1R. In one embodiment, the targeting moiety is anakinra. The ability to target a photosensitizer in the presence of thiamine or a derivative thereof allows for systemic administration (e.g., via intravenous injection or infusion) and non-invasive treatment of the aneurysm. Second, an energy source (such as a light source coupled to a catheter) is used to activate the photosensitizer in theAttorney Docket No.: UCSF-813WO presence of thiamine or a derivative thereof via energy (e.g., by shining light) at or near the site of inflammation or the aneurysm to activate the photosensitizer locally. This can be beneficial because it provides an additional control to specific activation by using light to activate the crosslinking reaction (e.g., restricting the therapeutic effect to the aneurysm). Additional disclosures as to the photosensitizers, thiamine or a derivative thereof, photoactivatable drug conjugates, and methods for using the same are provided below. Aneurysm Targeting Moieties

[0071] As described and exemplified herein, photosensitizer(s) can be attached (coupled / conjugated) to a targeting moiety in order to target the photosensitizer(s) to a site near and / or within an aneurysm, concentrate the photosensitizer(s) at the site of, near, and / or within an aneurysm, and / or reduce the overall exposure of the subject to the photosensitizer(s). A targeting moiety is preferably one that targets one or more photosensitizers to an inflammatory tissue (e.g., by binding an inflammation-associated cell surface receptor). Such inflammatory tissue can be inflammatory tissue of the vasculature, such as at a site near or of an aneurysm. In some embodiments, the targeting moiety does not selectively bind to a cancer or tumor cell. Alternatively, the targeting moiety can be one that selectively binds non-malignant cells.

[0072] Anakinra is a targeting moiety that binds an inflammatory (e.g., inflammation associated) cell surface receptor IL1R. In some embodiments, Anakinra is conjugated to a photosensitizer(s). For example, activatable drug conjugates (ADCs) having anakinra attached to one or more methylene blue photosensitizers (e.g., through an amide linkage) demonstrate the ability to target the photosensitizer(s) to a site near and / or within an aneurysm, concentrate the photosensitizer(s) at the site near and / or within an aneurysm, and / or reduce the overall exposure of the subject to the photosensitizer(s). In turn, activating a photosensitizer such as a methylene blue photosensitizer in the presence of thiamine or a derivative thereof at a site near or within an aneurysm (e.g., via an optical catheter) reduces an increase in aneurysm diameter; reduces an increase in the volume of an aneurysm, increases ECM crosslinking and / or prevents rupture of an aneurysm.

[0073] In some embodiments, a photosensitizer is attached to a targeting moiety other than anakinra, or to a targeting moiety that targets a marker other than IL1R. In such embodiments aAttorney Docket No.: UCSF-813WO targeting moiety can selectively target an inflammatory (e.g., inflammation associated) cell surface receptor such as any one of: CCR1, IL1R, IFNAR1, TNF-R2, CXCR2, CCR2, IL2R, IFNAR2, CD40, CXCR3, CCR3, IL3R, IFNGR1, CD30, CXCR4, CCR4, IL4R, IFNGR2, CD27, CXCR5, CCR5, IL5R, IFNLR1, CD28, CXCR6, CCR6, IL6R, CD95, CXCR7, CCR7, IL7R, LTbR, CXC3R1, CCR8, IL8R, OX40, CCR9, IL9R, 4-1BB, CCR10, IL10R, BAFFR, IL11R, BCMA, IL12R, TACI, IL13R, RANK, IL15R, NGFR, IL17R, TROY, IL18R, EDAR, IL20R, RELT, IL21R, Fn14, IL22R, TIM3, IL23R, IL27R, IL28R, or IL31R.

[0074] Such targeting moiety can be an antibody, an antibody fragment, a protein or peptide, a small molecule, a ligand, etc. In certain embodiments, antibody fragments described herein comprises a single chain variable fragment (scFv), a Fab, Fab2, Fab3, F(ab’)2 diabody, triabody, tetrabody, BiTE, TandAB or DART.

[0075] In some embodiments, the targeting moiety comprises anakinra. In certain embodiments, the targeting moiety comprises SEQ ID NO: 1 or an amino acid sequence having at least 85%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO: 1, and binds IL1R. In some embodiments, the targeting moiety comprises EBI-005. In certain embodiments, the targeting moiety comprises SEQ ID NO: 2 or 3 or an amino acid sequence having at least 85%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO: 2 or 3 and binds IL1R. In some embodiments, the targeting moiety comprises Rilonacept. In certain embodiments, the targeting moiety comprises SEQ ID NO: 4 or an amino acid sequence having at least 85%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO: 4, and binds IL1-beta.

[0076] In some embodiments, the targeting moiety is the antibody Nadunolimab. In certain embodiments, the targeting moiety is an antibody comprising a VH domain comprising SEQ ID NO: 9 and a VL domain comprising SEQ ID NO: 10, or an amino acid sequence having at least 85%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO: 9 and / or 10, and binds IL1R. In certain embodiments, the targeting moiety is CDX-1140 targeting CD40. In certain embodiments, the targeting moiety is AFM13 targeting CD30. In certain embodiments, the targeting moiety is SEA-CD30 targeting CD30. In certain embodiments, the targeting moiety is KW-0761 targeting C-C chemokine receptor 4 (CCR4). In certain embodiments, the targeting moiety is AMG 317 targeting Interleukin-4 receptor (IL4R). In certain embodiments, the targeting moiety is Olokizumab targeting Interleukin-6 receptor (IL6R). In certain embodiments,Attorney Docket No.: UCSF-813WO the targeting moiety is MEDI6383 targeting OX40. In certain embodiments, the targeting moiety is PF-05082566 targeting 4-1BB. In certain embodiments, the targeting moiety is AMG 139 targeting Interleukin-10 receptor (IL10R). In certain embodiments, the targeting moiety is GSK2857916 targeting B-cell activating factor receptor (BAFFR). In certain embodiments, the targeting moiety is Teclistamab targeting B-cell maturation antigen (BCMA). In certain embodiments, the targeting moiety is CC-92480 targeting B-cell maturation antigen (BCMA). In certain embodiments, the targeting moiety is JNJ-64007957 targeting B-cell maturation antigen (BCMA). In certain embodiments, the targeting moiety is Blisibimod targeting Transmembrane activator and CAML interactor (TACI). In certain embodiments, the targeting moiety is Atacicept targeting Transmembrane activator and CAML interactor (TACI). In certain embodiments, the targeting moiety is Tralokinumab targeting Interleukin-13 receptor (IL13R). In certain embodiments, the targeting moiety is Secukinumab targeting Interleukin-17 receptor (IL17R). In certain embodiments, the targeting moiety is Ixekizumab targeting Interleukin-17 receptor (IL17R). In certain embodiments, the targeting moiety is Sabatolimab targeting T-cell immunoglobulin and mucin domain 3 (TIM3). In certain embodiments, the targeting moiety is TSR-022 targeting T-cell immunoglobulin and mucin domain 3 (TIM3). In certain embodiments, the targeting moiety is Ustekinumab targeting Interleukin-23 receptor (IL23R). In some embodiments, the targeting moiety is the antibody canakinumab. In certain embodiments, the targeting moiety is an antibody comprising a VH domain comprising SEQ ID NO: 5 and a VL domain comprising SEQ ID NO: 6, or an amino acid sequence having at least 85%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO: 5 and / or 6, and binds IL1-beta. In some embodiments, the targeting moiety is the antibody gevokizumab. In certain embodiments, the targeting moiety is an antibody comprising a VH domain comprising SEQ ID NO: 7 and a VL domain comprising SEQ ID NO: 8, or an amino acid sequence having at least 85%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO: 7 and / or 8, wherein the antibody binds IL1-beta. In certain embodiments, the targeting moiety is LY2189102 targeting IL1-beta. Photosensitizer Drug Conjugates targeting aneurysms

[0077] Any of the photosensitizers herein can be coupled to a targeting moiety that targets an aneurysm. By way of example, anakinra is conjugated to one or more methylene blue photosensitizers via an amide linkage formed by reacting the methylene blue NHS ester and oneAttorney Docket No.: UCSF-813WO or more lysine residues on anakinra following the protocol provided in the Examples. Methylene blue NHS ester comprises a structure represented by the formula: N In one embodiment, blue photosensitizers via an amide linkage formed byone or more lysine residues on anakinra.

[0078] One or more photosensitizers can be attached to the targeting moiety through a linkage comprising an amide linkage, an oxime linkage, triazole linkage, or a thioether linkage. Anakinra conjugated forms of MB include a structure represented by the formula: Techniques for makingare contemplated herein include, for example: Khadieva A, Rayanov M, Shibaeva K, Piskunov A, Padnya P, Stoikov I. Towards Asymmetrical Methylene Blue Analogues: Synthesis and Reactivity of 3-N'- Arylaminophenothiazines. Molecules. 2022 May 8;27(9):3024. doi: 0.3390 / molecules27093024. PMID: 35566375.

[0079] In certain embodiments, the linkage further introduces a spacer between the linker and the photosensitizer. In some embodiments, the spacer is a heteroatom. In some embodiments, the spacer is an alkyl chain. In some embodiments, the spacer is an alkyl chain comprising one or more heteroatoms. In some embodiments, the spacer is a carbonyl group. In certain embodiments, the photosensitizer is directedly attached to the targeting moiety.Attorney Docket No.: UCSF-813WO

[0080] A linker (e.g., bifunctional linkage) can also be introduced via a linkage. In some embodiments, the linkage introduces a linker, wherein the photosensitizer is further attached to the targeting moiety via the linker. In some embodiments, a linker or a linker precursor used to form the linker comprises 6-maleimidocaproyl (MC), Maleimide-DOTA, maleimidopropanoyl (MP), alanine-phenylalanine (AP), p- aminobenzyloxycarbonyl (PAB), N-succinimidyl 4-(2- pyridylthio) pentanoate (SPP), N-succinimidyl 4-(N-maleimidomethyl) cyclohexane-1- carboxylate (SMCC), N-succinimidyl (4-iodo-acetyl) aminobenzoate (SIAB), valine-citrulline (VC), 6-maleimidocaproyl-valine-citrulline (MC-VC), 6-maleimidocaproyl-valine-citrulline-p- aminobenzyloxycarbonyl (MC-VC-PAB), N-succinimidyl-1-carboxylate-valine-citrulline-p- aminobenzyloxy-carbonyl (SC-VC-PAB), 6-maleimidocaproyl- polyethylene glycol-valine- citrulline (MC-PEG4-VC), 6-maleimidocaproyl-polyethylene glycol- valine-alanine (MC-PEG4- VA), or MC-PEG8-VC-PAB. In certain embodiments, the linker precursor is 6- maleimidocaproyl-valine-citrulline-p- aminobenzyloxycarbonyl (MC-VC-PAB), or N- succinimidyl-1-carboxylate-valine-citrulline-p- aminobenzyloxycarbonyl (SC-VC-PAB).

[0081] In certain embodiments, the linkers described herein may be attached to the targeting moieties described herein at a naturally occurring amino acid residue such as a lysine or a reduced cysteine. In certain embodiments, the photosensitizer is attached to a cysteine residue of the targeting moiety. In some embodiments, the photosensitizer is attached to a lysine residue of the targeting moiety. In certain embodiments, the linkers described herein may be attached to the targeting moieties described herein at a non-naturally occurring amino acid residue introduced into a protein sequence. In some embodiments, the one or more lysine residues is at positions K7, K10, K46, K65, K94, and K97 of FIG. 4A of SEQ ID NO: 1.

[0082] As such, in some embodiments, the PDC comprises a heterogenous composition of one or more photosensitizers conjugated to the targeting moieties. For example, in certain embodiments, the PDC comprises a heterogenous composition of one or more methylene blue photosensitizers conjugated to the anakinra.

[0083] In certain embodiments, the linker is first conjugated to the photosensitizer, and then subsequently conjugated to the targeting moiety. In certain embodiments, the linker is first conjugated to the targeting moiety, and then subsequently conjugated to the photosensitizer.Attorney Docket No.: UCSF-813WO

[0084] In some embodiments, provided herein are pharmaceutical compositions and / or formulations comprising the photosensitizer and the thiamine or a derivative thereof for treating aneurysms, or the photoactivatable drug conjugate targeting aneurysms and the thiamine or a derivative thereof. Pharmaceutical compositions include and / or refers a preparation that is in such form as to permit the biological activity of the photosensitizer alone or in the PDC to be effective, and that contains no additional components which are unacceptably toxic to a subject to which the formulation would be administered. In certain embodiments, the pharmaceutical composition comprises a pharmaceutically acceptable carrier (e.g., an ingredient in a pharmaceutical formulation, other than an active ingredient, which is nontoxic to a subject). A pharmaceutically acceptable carrier includes, but is not limited to, a buffer, excipient, stabilizer, or preservative. Systems for targeting aneurysms

[0085] Provided and described herein are systems useful for treating an aneurysm. In some embodiments, a system comprises: a photoactivatable drug conjugate comprising anakinra attached to methylene blue, thiamine or a derivative thereof, and an energy source that delivers light delivery unit such as an optical applicator. Such a system can also comprise a light generating unit comprising a light source. In certain embodiments, the optical applicator comprises a catheter configured to deliver light at the range required to activate the photosensitizer incorporated in the system. For example, a system comprising an anakinra- methylene blue conjugate, thiamine or a derivative thereof, and a light source configured to emit light in the range of 600 nm to 1000 nm. Preferably, the catheter is configured for delivery of light within a blood vessel at the location of or near the site of an aneurysm. In some instances, the catheter is a laser catheter and / or light-emitting diode catheter. In certain embodiments, the catheter contains optical fibers to deliver the light. In certain embodiments, the light delivery unit is attached to the light generator and facilitates the transmission of light (e.g., a laser beam). In certain embodiments, the light generating unit and / or the light delivery unit is configured to administer light at an irradiance of about 1 mW / cm2to about 600 mW / cm2. In certain embodiments, the light generating unit and / or the light delivery unit is configured to administer light at an irradiance of about 25 mW / cm2to about 100 mW / cm2.Attorney Docket No.: UCSF-813WO

[0086] In some embodiments, also provided and described herein are kits comprising one or more components of the systems described herein. For example, a kit can include all components of the photodynamic therapy systems, or only the photoactivatable drug conjugate, thiamine or a derivative thereof, and a light delivery unit. Methods for treating aneurysms

[0087] Provided and described herein are methods for treating an aneurysm. In some embodiments, the method of treating an aneurysm in a subject comprises: (a) administering to a subject in need thereof a photosensitizer and thiamine or a derivative thereof; and (b) subjecting the photosensitizer and the thiamine or the thiamine derivative to photoactivating conditions at the site of or near the aneurysm, thereby treating the aneurysm.

[0088] In some embodiments, the method of treating an aneurysm comprises: (a) administering a photoactivatable drug conjugate (e.g., anakinra linked to one or more methylene blue photosensitizers) and thiamine or a derivative thereof; and (b) subjecting the photoactivatable drug conjugate in the presence of the thiamine or the thiamine derivative at the site of or near the aneurysm using light, thereby activating the photosensitizer comprises delivering energy to the site or near the site of the aneurysm.

[0089] In an exemplary embodiment, for an aneurysm treatment method, the photosensitizer is methylene blue. In an exemplary embodiment, for an aneurysm treatment method, the photosensitizer is methylene blue, and the amount of methylene blue is as described herein (such as about 100ug / mL). In an exemplary embodiment, for an aneurysm treatment method, the thiamine or a derivative thereof is thiamine. In an exemplary embodiment, for an aneurysm treatment method, the thiamine or a derivative thereof is thiamine, and the amount of thiamine is as described herein (such as from about 0.5 mg / mL to about 20 mg / mL). In an exemplary embodiment, for an aneurysm treatment method, the photoactivating conditions are as described herein (such as light at a wavelength of about 660 nm and an irradiance of between about 45mW / cm2and about 150mW / cm2for between about 5 min and about 1 hour).

[0090] Also provided and described herein are, in certain embodiments, methods advantageous for decelerating aneurysm progression, reducing elastase-induce extracellular matrix degradation, increasing ECM crosslinking, and / or improving mechanical strength of aortic tissue. In some embodiments, the methods comprising:(a) administering an activatable agentAttorney Docket No.: UCSF-813WO (e.g., a photosensitizer); and (b) activating the activatable agent at the site of or near the aneurysm using light, wherein activating the activatable agent comprises delivering energy to the site or near the site of the aneurysm. In some embodiments, the methods comprise: (a) administering a activatable drug conjugate (e.g., anakinra linked to one or more methylene blue photosensitizers); and (b) activating the activatable agent at the site of or near the aneurysm using light, wherein activating the activatable agent comprises delivering energy to the site or near the site of the aneurysm.

[0091] In some embodiments, the photoactivatable drug conjugate localizes to regions near or within tissue inflammation or an aneurysm, as compared to a region of healthy tissue. In some embodiments, the activatable agent is a photosensitizer and activating the photosensitizer comprises photoactivation.

[0092] As described and exemplified herein, in some embodiments, activating the activatable agent (e.g., photoactivating the photosensitizer): (i) reduces or maintains the diameter of a blood vessel having an aneurysm, as compared to an untreated blood vessel having an aneurysm; (ii) decreases the volume of an aneurysm or inhibits an increase in the volume of the aneurysm, as compared to an untreated aneurysm; (iii) reduces the rate of blood vessel diameter growth in an blood vessel having an aneurysm, as compared to an untreated blood vessel having an aneurysm; and / or (iv) decreases the rate of volume increase in an aneurysm or inhibits an increase in the volume of the aneurysm, as compared to an untreated aneurysm.

[0093] The methods described herein can be used for aneurysms within different anatomical regions. In some embodiments, the aneurysm is an aortic aneurysm, a cerebral aneurysm, a thoracic aortic aneurysm, an abdominal aortic aneurysm, a popliteal artery aneurysm, a peripheral aneurysm, a fusiform aneurysm, a saccular aneurysm, or a mycotic aneurysm. In certain embodiments, the aneurysm is an aortic aneurysm In certain embodiments, the aneurysm is a cerebral aneurysm. In certain embodiments, the aneurysm is a thoracic aortic aneurysm. In certain embodiments, the aneurysm is an abdominal aortic aneurysm. In certain embodiments, the aneurysm is a popliteal artery aneurysm. In certain embodiments, the aneurysm is a peripheral aneurysm. In certain embodiments, the aneurysm is a fusiform aneurysm. In certain embodiments, the aneurysm is a saccular aneurysm, a mycotic aneurysm. In certainAttorney Docket No.: UCSF-813WO embodiments, the aneurysm is a pseudo aneurysm. In certain embodiments, the blood vessel is the aorta.

[0094] In certain embodiments, photoactivating takes place within a subject, e.g., in (b), through the use of an optical applicator. In certain embodiments, the optical applicator comprises a catheter configured to deliver light (e.g., a laser catheter and / or light-emitting diode catheter). In certain embodiments, the catheter contains optical fibers to deliver the light. In certain embodiments, the optical applicator (e.g., catheter) is inserted through an artery. In certain embodiments, the optical applicator (e.g., catheter) is inserted through the groin artery.

[0095] The photoactivatable drug conjugate can be administered by any suitable means, including parenteral, and, if desired for local treatment. Parenteral infusions include intravenous and intraarterial administration. Dosing can be by any suitable route. Various dosing schedules can be implemented, including but not limited to single or multiple administrations over various time-points, bolus administration, and pulse infusion. In certain embodiments, the photoactivatable drug conjugate is administered via parenteral infusion or injection. In certain embodiments, the photoactivatable drug conjugate is administered via intravenous infusion or injection.

[0096] The methods herein are also useful for extending the therapeutic window for treating an aneurysm. In some embodiments, administering the photosensitizer or photoactivatable drug conjugate and activating the photosensitizer can allow for delayed surgical intervention for an aneurysm due to reduced long term growth rate.

[0097] In certain embodiments, the aneurysm is an abdominal aortic aneurysm. In certain embodiments, the aneurysm is a cerebral aneurysm. In certain embodiments, the aneurysm is a thoracic aortic aneurysm. In certain embodiments, the aneurysm is an abdominal aortic aneurysm. In certain embodiments, the aneurysm is a popliteal artery aneurysm. In certain embodiments, the aneurysm is a peripheral aneurysm. In certain embodiments, the aneurysm is a fusiform aneurysm. In certain embodiments, the aneurysm is a saccular aneurysm. In certain embodiments, the aneurysm is a mycotic aneurysm. In certain embodiments, the aneurysm is a pseudo aneurysm.

[0098] In certain embodiments, the dose of the photosensitizer is less than 4 per dose. In certain instances, the targetable moiety enables a therapeutic effect (e.g., increasing an amount of tissue)Attorney Docket No.: UCSF-813WO at lower amounts of the activatable agent or photosensitizer that if administered without the targeting moiety (i.e., in a non-conjugate manner).

[0099] In certain embodiments, the light is administered by a laser catheter. In certain embodiments, the light is administered at an irradiance of about 1 mW / cm2to about 600 mW / cm2. In certain embodiments, the light is administered at an irradiance of about 25 mW / cm2to about 100 mW / cm2.

[0100] All publications and patent applications mentioned in this disclosure are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

[0101] No admission is made that any reference cited herein constitutes prior art. The discussion of the references states what their authors assert, and the Applicant reserves the right to challenge the accuracy and pertinence of the cited documents. It will be clearly understood that, although a number of information sources, including scientific journal articles, patent documents, and textbooks, are referred to herein; this reference does not constitute an admission that any of these documents forms part of the common general knowledge in the art.

[0102] The discussion of the general methods given herein is intended for illustrative purposes only. Other alternative methods and alternatives will be apparent to those of skill in the art upon review of this disclosure, and are to be included within the spirit and purview of this application.

[0103] Additional embodiments are disclosed in further detail in the following examples, which are provided by way of illustration and are not in any way intended to limit the scope of this disclosure or the claims.

[0104] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0105] Although various features of the disclosure can be described in the context of a single embodiment, the features can also be provided separately or in any suitable combination. Conversely, although the present disclosure can be described herein in the context of separate embodiments for clarity, the present disclosure can also be implemented in a single embodiment.Attorney Docket No.: UCSF-813WO EXAMPLES

[0106] The practice of the present invention will employ, unless otherwise indicated, conventional techniques of photochemistry, which are well known to those skilled in the art.

[0107] Additional embodiments are disclosed in further detail in the following examples, which are provided by way of illustration and are not in any way intended to limit the scope of this disclosure or the claims. EXAMPLE 1 Thiamine improves efficacy of photodynamic dynamic therapy ex vivo

[0108] Mouse aortic tissue becomes stiff with photodynamic therapy and this stiffness increases with the addition of thiamine (“B1”). 1-1.2 cm sections of abdominal aortas were harvested from mice. Sections were subjected to one of the following conditions: PBS (negative control; (“Untreated”); Glutaraldehyde (cross-linking agent, positive control; “Glut”); 100 µg / mL methylene blue (“PDT”); and 100 µg / mL methylene blue plus 10 mg / mL thiamine (“PDT + B1”). The sections were then subjected to photodynamic therapy (irradiance = 45mW / cm2; wavelength = 660nm). Stiffness was measured using a forceps bend test at 0 min, 10 min, and 20 min. Results are provided in FIG. 1. The addition of thiamine increases the stiffness of aortic tissue over methylene blue alone at both 10 min and 20 min. EXAMPLE 2 Thiamine treatment in mice improves efficacy of photodynamic therapy

[0109] Mouse aortic tissue becomes stiff with photodynamic therapy and this stiffness increases with the addition of Thiamine (VitB1) injected intravenously (IV). Mouse abdominal aortas were exposed after anesthesia was induced. Aortas were then treated with PDT after being soaked in methylene blue (PDT) or PBS (untreated). PDT was performed with 660nm light for 0, 10 and 20 minutes. Vit B1 was injected IV at different doses (0, 1 and 2 mg / ml) prior to initiation of PDT. Following treatment, aortic tissue was harvested and stiffness was measured using a forceps bend test. Vit B1 injection increased the stiffness of aortic tissue with PDT in a dose dependent manner.Attorney Docket No.: UCSF-813WO EXAMPLE 3 Treatment of human tissue with thiamine during PDT improves resistance to elastase degradation post-PDT

[0110] Human aortic tissue was obtained under an IRB approved protocol. The tissues were cut into 1-1.2 cm sections. Sections were subjected to one of the following conditions: untreated, Glutaraldehyde (cross-linking agent; “Glut”); Pentagalloyl glucose (cross-linking agent; PGG”); 100 µg / mL methylene blue (“MB”); and 100 µg / mL methylene blue plus 10 mg / mL thiamine (“MB + B1”). The sections were then subjected to photodynamic therapy (irradiance = 150mW / cm2; wavelength = 660nm). After PDT treatment, tissue was treated with 20 units Type I elastase to assess tissue breakdown. Tissue breakdown was measured at 0, 1, 2, 3, and 4 hours after elastase administration. Results are provided in FIG. 3. PDT increased tissue stiffness with MB + B1 increasing stiffness more than MB alone. PDT was protective against elastase breakdown as measured over a 4 hour time course. PDT using MB + B1 was more protective than MB alone. EXAMPLE 4 Treatment of porcine tissue with thiamine during PDT improves resistance to elastase degradation post-PDT

[0111] 1-1.2 cm sections of pig aortic tissue was harvested from pigs. Sections were subjected to one of the following conditions: Untreated without PDT; Glutaraldehyde with PDT (cross- linking agent, positive control; “Glut”); 100 µg / mL methylene blue without PDT (“MB only”); Untreated with PDT (“Light only”); 100 µg / mL methylene blue with PDT (“PDT”); and 100 µg / mL methylene blue plus 10 mg / mL thiamine (“Thiamine”). The PDT conditions were: irradiance = 150mW / cm2; wavelength = 660nm; time = 20 min. After PDT treatment, tissue was treated with 20 units Type I elastase to assess tissue breakdown. Tissue breakdown was measured 4 hours after elastase administration. Results are provided in FIG. 4. PDT was protective against elastase breakdown as measured over a 4 hour time course. PDT using MB + B1 was more protective than MB alone. To assess which sections of the aortic tissue were cross- linked, aortic tissue was split into adventitial (adventitia and media) and luminal (intima) layers. Both layers showed increased stiffness and resistance to crosslinking with the lumen side showing the greatest resistance to elastase.Attorney Docket No.: UCSF-813WO EXAMPLE 5 High-dose thiamine treatment during PDT crosslinks veins and aortic tissue

[0112] To examine the effects of PDT at high doses of light we treated aortic tissue with PDT using 150mW / cm^2 irradiance with 660nm light. When Thiamine is added to methylene blue it results in tissue shape changes that are dramatic; the tissue curls. Furthermore the tissue becomes extremely stiff. Similar changes were observed with ICG. Results are shown in FIG. 5. EXAMPLE 6 Dose-dependent increase in Singlet Oxygen Production in Photodynamic Therapy Using ICG as Photosensitizer When Combined with Thiamine

[0113] Representative graph showing an increase in fluorescence due to oxygen singlet production upon incubation of ICG with increasing concentrations of Thiamine. Solutions were treated with light for 5mins to produce singlet oxygen and measured using singlet oxygen sensor green dye at 488nm / 525nm fluorescence. Results are shown in FIG. 6. EXAMPLE 7 Thiamine enhances Riboflavin based PDT in rabbit sclera

[0114] Rabbit sclera was treated with riboflavin alone and with different concentrations of Thiamine. Increased collagenase resistance at 100 micrograms of thiamine when exposed to light at 450nm for 20 minutes each side. Collagenase resistance was measured based on tissue degradation using video capture. Results are shown in FIG. 7. EXAMPLE 8 Thiamine potentiates the toxicity of photosensitizers in triple negative breast cancer

[0115] Triple negative breast cancer cells (MDA-MB-231) were exposed to different concentrations of photosensitizer, temoporfin (0.5µM, 1µM, 2µM) and increasing concentration of thiamine (100ng / ml,1µg / ml, 10µg / ml and 100µg / ml). Cells with no thiamine and no temoporfin were considered as untreated controls. One-hour post-incubation cells were exposed to red light (660nm) for 5mins. 72h post-treatment cell death was measured using Cell Titer glo (Promega) bioluminescence assay. Results are shown in FIG. 8. The bar graph indicates an increase in temoporfin toxicity on MDA-MB-231 breast cancer cells in the presence of increasing concentrations of thiamine.Attorney Docket No.: UCSF-813WO EXAMPLE 9 Treatment of porcine tissue with photosensitizers in the presence of thiamine during PDT improves resistance to elastase-dependent degradation

[0116] Chemical crosslinking with Glutaraldehyde (Glut) was used as a positive control for tissue crosslinking. After PDT treatment, whole tissue was split to lumen and adventitia and incubated with elastase to assess tissue breakdown. Untreated tissue was used as a control for elastase degradation. Results are provided in FIG. 9. Representative images are shown of tissue treated with PDT using photosensitizers (cardiogreen and ICG) in the absence and presence of thiamine (Thiamine). PDT with both, cardiogreen and ICG, in presence of thiamine increased tissue stiffness and resistance to elastase breakdown as measured over a 4 hour time course compared to untreated tissue (non-PDT treated). To assess which sections of the aortic tissue were cross-linked, aortic tissue was split into adventitial (adventitia and media) and luminal (intima) layers. Both layers showed increased stiffness and resistance to crosslinking with the lumen side showing significant resistance to elastase indicating elastin crosslinking. EXAMPLE 10 Methylene blue photodynamic therapy using methylene blue inhibits the induction of AAA

[0117] To assess if methylene blue (MB) photodynamic therapy (PDT) can exert a protective benefit against the development of abdominal aortic aneurysms (AAAs), an aneurysm was induced via surgery in a BAPN-Elastase Mouse AAA Model. Briefly, mice were maintained on standard diet and given 0.2% β-Aminopropionitrile (BAPN) water, as previously described. Aneurysms were induced through a surgical procedure, where the infrarenal aorta was isolated and treated with porcine pancreatic elastase (PPE), then rinsed, to induce aneurysms. Immediately following aneurysm induction, the site of the aneurysm was treated with MB (unconjugated) PDT by direct topical administration of MB to the aneurysm and applying light. MB has a structure represented by the formula: Vehicle group were miceHank’s balanced salt solution (HBSS) without light and considered as AAA positive control. Mice topically treated with HBS and no light were utilized as light only controls. Sham group were mice that underwent the sameAttorney Docket No.: UCSF-813WO AAA surgery but were treated with heat-inactivated PPE are considered as healthy controls. Following treatment, mice were monitored for aneurysm growth through weekly ultrasound surveillance over a period of 9 weeks post-induction surgery (FIG 10A). For ultrasound surveillance, maximum aortic diameters were measured by averaging anterior-posterior (AP) and transverse (TRV) dimensions. MB PDT treatment significantly restricted AAA growth (FIG 10A). In stark contrast to the aneurysms growth observed in vehicle-treated group, the average increase in size of the PDT-treated group (Veh+light+MB) was significantly lower. (FIG 10B) Similar to vehicle only, no benefit was observed in the light only group. This suggests that MB PDT not only limits AAA formation but also that this protective benefit extends over the duration of the study post-treatment. Morphological images and micrometry quantifications of treated and untreated AAAs at the end of the study corroborated the ultrasound findings, with marked differences apparent between the two groups (Veh versus Veh+light+MB) (FIG 10C and 10D). These results collectively show that MB PDT (a method of treating an aneurysm in a subject, the method comprising: (a) administering a photosensitizer; and (b) photoactivating the photosensitizer using light at the site of or near the aneurysm) serves as an effective intervention to mitigate AAA development. Methods - BAPN-Elastase Mouse Model:

[0118] The animal protocols were approved by the University of California San Francisco Institutional Animal Care and Use Committee. AAA Induction protocol was taken from established methods and was chosen for its ability to consistently induce aneurysms (100% of mice) and mimic similar inflammatory pathology to human aneurysms (1,2,3). 6-8 week male C57BL / 6J wild type mice (n= x) were kept on standard chow diet throughout the course of the experiment. Starting from two days before surgery until the end of the experiment, mice were kept on 0.2% β-Aminopropionitrile (BAPN; A3134-25G) drinking water. BAPN is a lysyl oxidase inhibitor that reduces collagen crosslinking in tissue. At the time of surgery, a laparotomy is performed to expose the infrarenal aorta. The infrarenal aorta is then carefully separated from the IVC by blunt dissection. 5ul of Porcine Pancreatic Elastase (PPE; E1250- 25MG) is applied topically onto the aorta using a pipette. After 5 min, the PPE was washed away using two changes of warmed sterile saline (RGC-3290). After the wash, further treatment can be done as described in the following examples. Then, the abdominal incision is closed and animalsAttorney Docket No.: UCSF-813WO allowed to recover. Unless otherwise specified, the methods described in this section can be similarly applied to similar experiments throughout the examples. Methods - MB-PDT Treatment:

[0119] The photosensitizer dye methylene blue (M291-100) was used for photodynamic therapy treatment (MB-PDT). MB-PDT was applied at two time points during the experiment. To test the effect of MB-PDT on AAA formation, MB-PDT was done directly after the PPE saline wash (POD0). To investigate the effect of MB-PDT on AAA growth, Laparotomy and MB-PDT was performed 21 days postoperatively, covering the whole aneurysm (POD21). MB-PDT involves applying 10ul of 10:1 MB Solution in DPBS (M291-100: 21-030-CV) topically to the whole aneurysm, which was carefully dissected from surrounding tissue. Gauze was placed on either side of the aneurysm to absorb excess MB. The aneurysm was then irradiated for 25 min at an irradiance of 30mW / cm^2 and fluence of 45J / cm^2 (660nm LED, Thor M660L4). Throughout the course of irradiation, 5ul of MB was applied to the aneurysm every 2.5 min to prevent the tissue from drying out. After irradiation, the MB was washed with two changes of sterile saline. For the MB only group, the total amount of MB was applied topically to the aneurysm for five minutes and washed. For the light only group, the aneurysm was irradiated for 25 min, with 5ul of sterile saline applied every 2.5min to prevent tissue desiccation. For POD21 positive controls, a laparotomy was performed with no additional treatment. Unless otherwise specified, the methods described in this section can be similarly applied to similar experiments throughout the examples. Methods - Ultrasound Surveillance:

[0120] Aneurysm size was tracked by weekly ultrasound surveillance. Animals were anesthetized and placed on a heated pad during the procedure (2-4% isoflurane, 1L / min medical grade air). A Vevo 3100 system with a 32- to 55-MHz frequency transducer was used to collect standard B-mode images in long axes (Fujifilm VisualSonics, Toronto, ON, Canada; MS550D, 40-MHz center frequency). After obtaining the picture, the anterior to posterior diameter and the transverse diameter were both measured and recorded. Weekly growth of aneurysm size was calculated by subtracting the previous week`s measurement of maximum aortic diameter (in mm) from the current week. The first three week’s calculation was averaged to represent the average weekly growth rate of aneurysms before treatment. The next six weeks (POD 21-63) were averaged to represent the average weekly growth rate of aneurysms after treatment. UnlessAttorney Docket No.: UCSF-813WO otherwise specified, the methods described in this section can be similarly applied to similar experiments throughout the examples. Methods - Micrometry:

[0121] During the harvest procedure on POD63, mice were placed under anesthesia and opened via laparotomy. Three in vivo micrometry measurements was taken of the pressurized aneurysm at maximum diameter using the Leica X Microscope system and averaged. The averaged measurement was then normalized to total body weight of the mouse at time of harvest. Unless otherwise specified, the methods described in this section can be similarly applied to similar experiments throughout the examples. Methods - Statistics

[0122] Statistics tests were performed in GraphPad Prism version 7.0 (GraphPad Software). Unless indicated otherwise, values are presented as mean±SD. All data were tested for normality and equal variance. If the data passed those tests, Student t test was used to compare 2 groups. One-way ANOVA followed by Kruskal-Wallis test post hoc analysis was used for comparisons among >2 groups. If the data did not pass those tests, the Mann-Whitney U test was used to compare 2 groups, and the Kruskal-Wallis test for >2 groups. P<0.05 was considered statistically significant. Unless otherwise specified, the methods described in this section can be similarly applied to similar experiments throughout the examples. EXAMPLE 11 MB PDT modulates immune cell infiltration and apoptosis

[0123] The formation and progression of AAA can be characterized by the degradation of the extracellular matrix (ECM) and the concurrent infiltration of immune cells into the aortic wall. To assess if MB PDT affects the extracellular matrix (ECM) of the aortic aneurysms, cross- sections of the aneurysms of the above mice which have undergone AAA induction and concurrently treated with MB PDT and the respective controls were stained with Verhoeff-Van Gieson (VVG) for elastin, Massons Trichrome for collagen and CD3 antibody for T-cells and CD68 antibody for macrophages for immune cell infiltration (FIG 11A). Upon magnification, less elastin staining was observed (pointed by arrows) in the lumen after MB PDT treatment compared to vehicle treated controls indicative of a reduction of elastin degradation after PDT. Interestingly the staining for elastin after MB PDT was stronger (pointed by arrows) than healthyAttorney Docket No.: UCSF-813WO sham controls suggesting an increase in elastin production as a compensatory mechanism. Similarly, a compensatory effect of collagen expression into the elastin layer was observed (pointed by arrows) after PDT treatment compared to both vehicle-treated positive controls and sham controls. In line with this, less collagen degradation was also observed in the adventitia after PDT treatment compared to vehicle treated controls and sham mice. Mice treated with vehicle only had an overall decrease in collagen staining in the adventitia suggesting a breakdown of adventitial collagen. To assess if T-cells and macrophage infiltration is modulated in aneurysms upon treatment with MB PDT, the aneurysm cross-sections were stained for CD3 and CD68, markers for T-cells and macrophages, respectively. CD3 and CD68 staining were reduced after MB PDT treatment compared to vehicle treated controls (FIG 11A). Little to no staining was observed in sham surgery mice suggesting that immune cell infiltration is increased in aneurysm formation and treatment with MB PDT inhibits immune cell infiltration. To confirm if there is an improvement in elastin degradation, clinical scoring as per an established protocol was performed by blinded investigators. MB PDT treated group showed a trend towards improvement against elastin degradation (FIG 11B). To assess if this benefit on elastin degradation and immune cell infiltration was due to any systemic decrease in circulating cytokine levels known to be associated with aneurysm growth, cytokines such as IL-1B, IL-6 and IL-10, serum cytokine levels were measured. No significant differences were observed in serum cytokine levels suggesting that the improvement is due to a localized effect of MB PDT treatment on aneurysmal tissue inflammation (FIG 11C). These results collectively show that MB PDT (a method comprising: (a) administering a photosensitizer; and (b) photoactivating the photosensitizer using light) serves as an effective intervention to decease elastin degradation and reduce immune cell infiltration. Methods - Histology and IHC Staining:

[0124] Fresh tissue was fixed in 10% neutral buffered formalin (Azer Scientific, PFNBF-90) for 24 hours before being transferred to 70% EtOH. Storage in 70% EtOH did not exceed one month. After paraffin embedding, sections were cut at 5 um for staining (AML Laboratories). Sections were then stained with H&E (VWR 95057-844, 95057-848), Van Gieson (Statlab Medical Products, KTVELLT), and Masson’s Trichrome (CP Lab Chemicals, TRM-500). For IHC, standard immunohistochemistry procedures for 5 μm-thick aorta sections were used. In brief, after deparaffinization, endogenous peroxidase activity was quenched by placing the slidesAttorney Docket No.: UCSF-813WO into 3% hydrogen peroxide (H2O2) for 10 min. Sections were immersed in preheated 10 mM citric acid (VWR., Radnor, PA., USA), pH 6.0, for 30 min and cooled in cold water. Sections were then blocked in 5% goat serum (Vector Laboratories, Burlingame, Calif., USA) for 20 min before being incubated overnight at 4°C in a solution of rabbit anti-CD3 (0.4 µg / ml; ab5690; Abcam, Cambridge, MA., USA) antibody, rabbit anti-CD31 (ERP1729; 0.4 μg / ml; ab182981; Abcam) antibody, rabbit alpha smooth muscle Actin ( SMA; 0.067 µg / ml; ab5694; Abcam), rabbit anti-CD68 (ERP23917; 2.0 µg / ml; ab283667; Abcam), and rabbit anti- Interleukin-1 receptor type 1 (IL1R1; 2.0 µg / ml; PAS-98766; Thermo Fisher, Eugene, OR., USA) in PBS. The next day, the sections were exposed to the biotinylated biotinylated goat-anti-rabbit IgG secondary antibody (15 μg / ml; Vector Laboratories) in PBS for 1 h followed by avidin-biotin complex for 1 h (1:200, ABC; Vector Laboratories). The reactions were visualized with 3,3ʹ- diaminobenzidine (Millipore- Sigma) for 10 min and counterstained with Gill’s Haematoxylin (Thermo Scientific) for 30 seconds. Finally, the sections were dehydrated, cleared in xylene and cover slipped. As negative controls, staining was performed in the absence of the primary antibodies and no specific staining was identified in these preparations. In addition, a sample of mouse skin and mouse lung was used as positive controls and compared the immunoreactivity.

[0125] For quantification of elastin using Van Gieson, an elastin scoring guide was used. A score of 0 was assigned to samples with no elastin breakage, I was given to elastin break / degradation or SMC loss limited to one outer medial elastin layer, II for elastin degradation or SMC loss involving more than two medial elastin layers, or entire medial elastin layers, but limited to less than one-quarter of the aortic circumference, III for elastin degradation or SMC loss involving entire medial elastin layers, but limited to less than half the aortic circumference, and IV for elastin degradation or SMC loss involving entire medial elastin layers and expanded to more than three-quarters of aortic circumference. Unless otherwise specified, the methods described in this section can be similarly applied to similar experiments throughout the examples. Methods - ELISAs:

[0126] Complete whole blood was collected from each mouse via puncture of the left ventricle of the heart. After 5mins, the blood was centrifuged and serum supernatant was collected and stored at -80C. Tissue samples were lysed by a French press. ELISAs for Anakinra (IL-1 receptor anatagonist) IL1 receptor, IL-10, IL1B, IL6, elastin and CML were run according to the manufacturer’s instructions. Absorbance was read on a microplate reader at recommendedAttorney Docket No.: UCSF-813WO wavelengths (Molecular Devices SpectraMax M2). Unless otherwise specified, the methods described in this section can be similarly applied to similar experiments throughout the examples. Methods - Ex vivo Elastase Degradation Assay with VG Staining:

[0127] Human and porcine aorta were thawed from -80C to RT. Tissue was then cut into ~1cm x 1cm squares and briefly rinsed with HBSS. Tissues were then split into two groups: control and PDT. Control tissues were kept in HBSS at RT for one hour, and treated tissues were subjected to 150mW NIR light for 20 mins in 100ug / ml methylene blue, with the lumen side facing the light. Then each group was further split into two groups, no elastase and elastase treatment. Treatment involved a 2 hour incubation at 37C in 10% Porcine Pancreatic Elastase (E1250- 25MG). After treatment all tissues were briefly rinsed, dried on a paper towel, and frozen in OCT. Tissue was sectioned at 10um by cryotome for the cross-section and stained using Van Gieson (Statlab KTVELLT). Images were taken at 20X and 100X on a Keyence BZ-X700 Fluorescence Microscope. Unless otherwise specified, the methods described in this section can be similarly applied to similar experiments throughout the examples. EXAMPLE 12 MB PDT extends therapeutic window for reducing the growth of AAA

[0128] In certain instances, AAA often goes undiagnosed or is diagnosed late, thus it is important to know if MB PDT can not only prevent AAA formation but also reduce or arrest progressive aneurysm growth. To evaluate the efficacy of MB PDT to inhibit the progression of AAA, MB PDT was administered to mice with pre-established and progressive aneurysms 21 days after the initial aneurysm induction surgery with elastase. Vehicle group were mice which have undergone AAA induction surgery and topically treated with Hank’s balanced salt solution (HBSS) without light and considered as AAA positive controls. Sham group were mice that underwent the same AAA surgery but were treated with heat inactivated PPE and considered healthy controls. Analysis of growth curves based on weekly ultrasound measurements revealed that MB PDT significantly reduced the enlargement of established AAAs in mice (FIG 12A) compared to vehicle-treated controls. The aneurysm enlargement after week 3 for those animals treated with MB PDT was significantly less than that observed in the vehicle-treated group. Similar to the measurements done in the study of MB PDT upon AAA induction in Figure 1, theAttorney Docket No.: UCSF-813WO maximum aortic diameters were measured using ultrasound, averaging anterior-posterior (AP) and transverse (TRV) dimensions for accuracy. The decrease in size of aneurysm upon MB PDT treatment was corroborated by supporting findings from the ultrasound-derived data (FIGs 12B, 12C). The healthy sham group, vehicle-treated group and MB PDT -treated groups had similar aortic diameter on day of AAA-induction surgery. An equivalent increase in aneurysm diameter was observed on week 3 between vehicle and MB PDT groups prior to treatment, indicating similar rate of AAA growth pre-treatment. However, upon MB PDT treatment, a decrease in aneurysm diameter was observed on week 9 compared to vehicle treated group (FIG 12C), suggesting that PDT-treatment prevents an increase in aortic aneurysm size. No changes in aortic size were observed in healthy sham controls. Similar to mice treated immediately upon induction of AAA in mice, morphological images and the micrometric measurements of treated and vehicle AAAs corroborated the ultrasound findings, with marked differences apparent between the two groups (FIGs 12C and 12D), suggesting that, MB PDT slows the rate of AAA growth in mice. These results collectively show that MB PDT effectively reduces the growth of the aneurysm. EXAMPLE 13 Synthesis and characterization of anakinra conjugated to methylene blue (Ana-MB)

[0129] To develop a modality for targeted delivery of methylene blue to the AAA site, methylene blue was conjugated to IL-1 receptor antagonist, Anakinra using an NHS or maleimide linker. Methods of linking a photoactivatable agent (e.g., methylene blue) to a protein (e.g., anakinra) are known in the art and can be used to form the photoactivatable drug conjugates described herein. To prepare Anakinra-methylene blue conjugates (Ana-MB) described in the examples, methylene blue NHS ester dye (3-(N,N-dimethylamino)-7-[N-(3-(N- succinimidyl)-carboxyethyl)-N-(methyl)-amino] phenothiazin-5-ium perchlorate) was dissolved in anhydrous DMSO to a final concentration of 10 mM. An anakinra (comprising SEQ ID NO: 1) solution of approximately 3.0 mg / mL was prepared in sodium borate buffer (50 mM, pH 8.5). For the conjugation step, while the dye solution was added to the protein solution at the appropriate molar ratio. The reaction was quenched with glycine (pH 7.4) after 4h. Following the reaction, any excess dye was removed using a desalting spin column.Attorney Docket No.: UCSF-813WO

[0130] To reveal the conjugation site, LC-MS / MS was performed on Ana-MB for peptide mapping. Lysine residues at positions 94 and 97 (denoted by * in the sequence) exhibited a tendency to undergo modification in tandem (FIG 13A). The prevalence of peptides labeled at both sites was disproportionately high relative to the occurrence of peptides labeled at a single site (FIG 13B). This observation aligns with the drug-loading profile observed in the intact mass analysis, which suggests a pattern of concurrent labeling. Within the primary structure of anakinra, the N terminal amine and lysine residues K7, K10, K46, K65, K94, and K97 (each further denoted by * in the sequence), were all subject to partial modification with methylene blue. Notably, none of these lysine sites are occluded within the binding interface between anakinra and the IL1 receptor (IL1R), indicating their accessibility for modification without impeding function. EXAMPLE 14 Targeted delivery of methylene blue photosensitizer to inflammatory aneurysmal tissue improves efficacy of photodynamic therapy. Effective targeting of Ana-MB

[0131] To ensure selective accumulation of methylene blue in aneurysmal tissue and by extension increase the potential therapeutic effect of PDT, a targetable photoactivatable drug conjugate (PDC) comprising IL1R antagonist, Anakinra, conjugated to methylene blue (Ana- MB) was developed. This ensured the targeted delivery and localization of methylene blue at inflammatory sites within or near the site of aneurysmal tissue where IL-1 receptor is expressed, thus facilitating targeted PDT. To assess if IL1R is expressed in aneurysmal tissue, immunochemistry of IL1R was performed in healthy aortic tissue and aortic aneurysm cross- sections. Mice having undergone AAA surgery with active PPE were considered as vehicle and those treated with heat inactivated PPE were considered the healthy Sham control group. Staining for IL1R revealed significant increase in expression density of IL1R in aneurysms tissue compared to sham controls (FIGs 14A and 14B). To assess if Ana-MB localizes to aortic aneurysmal tissue, mice with AAA and healthy sham controls with no AAA were treated with different doses of Ana-MB. Minimal anakinra accumulation was observed in aortic tissue of healthy mice treated with 50µg anakinra, while a significant dose-dependent increase in anakinra accumulation was observed in mice with AAA, suggesting enhanced accumulation of Ana-MB in aneurysmal tissue (FIG 14C). Similarly, fluorescence imaging was performed to visualize theAttorney Docket No.: UCSF-813WO localization of Ana-MB within aneurysm tissue compared to adjacent distal healthy aortic tissue. An increase in fluorescence caused by Ana-MB was observed in aneurysm tissue compared to adjacent healthy aortic tissue (FIG 14D). In the vehicle treated group, no fluorescence was observed. Similarly increased fluorescence was observed from circulating Ana-MB present in serum compared to vehicle treated control, suggesting that while significant levels of drug is bound to the aneurysm site, there remains an abundant circulating drug reservoir poised for receptor binding within aneurysms following intravenous delivery. Unless otherwise specified, the methods described in this section can be similarly applied to similar experiments throughout the examples. Efficacy of Ana-MB PDT

[0132] At 21 days post-AAA-induction surgery, mice were treated with vehicle or topical MB applied on aortic wall (PDT group), or intravenously delivered Ana-MB without PDT, or intravenously delivered Ana-MB with PDT,. The sham treated group was used as surgical controls. Treatment with Ana-MB + PDT completely inhibited the growth of AAA compared to Ana-MB without PDT treatment and vehicle treated positive controls. (FIG 14E). The inhibition of AAA progression upon PDT treatment with intravenously delivered Ana-MB (Veh+Light+MB group) was comparable to topically applied MB group (Veh+Light+MB group) as shown in Examples 1, 3 (FIG 10A, FIG 12A). Furthermore, the quantified video microscopic measurement of AAA diameter supported the quantified ultrasound-derived conclusions from the duration of the study (FIG 14F). Morphological and micrometric measurement comparisons (FIG 14G, FIG 14H) between AAAs treated with Ana-MB and photodynamic therapy 21 days post-induction and those receiving vehicle only treatment revealed a significant difference, affirming the efficacy of Ana-MB PDT in safeguarding against and decelerating AAA progression in murine models. Unless otherwise specified, the methods described in this section can be similarly applied to similar experiments throughout the examples. Methods - Localization of Anakinra Study:

[0133] Mice with aneurysms were injected with the following conditions on POD21: saline, Anakinra-50µg, Anakinra-400µg, and Anankinra-800µg (n=4). 5 mice with healthy aorta was taken as an additional control. Aneurysms were harvested 30 minutes post injection and tissue was immediately flash frozen in liquid nitrogen and stored at -80˚C. Serum was collected via cardiac puncture and stored at -80˚C. For homogenization, tissue was placed in an EppendorfAttorney Docket No.: UCSF-813WO with 2 mm beads and sample diluent buffer and lysed at 30shakes / sec for 10 minutes on the Tissue Lyser II. The eppendorfs were then centrifuged at max speed for 10mins and supernatant was collected. Both serum and tissue lysate were used in the Human IL-1Ra ELISA and performed according to manufacturer’s protocol (Ab211650). For fluorescence imaging mice were treated with 400µg of Anakinra intravenously. Aortic aneurysm tissue, aortic tissue and serum were collected 30min post-injection. Fluorescence imaging was performed using a Licor imaging device. Unless otherwise specified, the methods described in this section can be similarly applied to similar experiments throughout the examples. EXAMPLE 15 MB PDT protects extracellular matrix from elastase-induced degradation across different species

[0134] To assess if MB PDT can protect aortic tissue from elastase-induce extracellular matrix degradation, aortic tissue were procured from pigs and subjected to MB PDT (20min irradiance of 150mW / cm2) or Veh only treatment ex vivo before recombinant elastase treatment. Comparative analysis of the cross-sections of these tissues were sought to ascertain the consistency of PDT's protective effects across different biological species. Post-treatment analysis entailed staining of elastin with Verhoeff-Van Gieson (VVG) to evaluate resistance to elastin degradation in the aortic sections. Tissue treated with MB PDT showed significant resistance to elastase-induced ECM degradation as observed by an increase in the presence of elastin staining within the tissue and a decrease in tissue degradation as observed by a decrease in vacuoles and elastin breakage, suggesting that MB PDT-induced elastin crosslinking protects against elastase activity (FIG. 15A). Thus, the protective benefit of MB PDT on elastase induced - aortic degradation seems to be species independent. Ne-(carboxymethyl)lysine (CML), an elastin crosslinking modification, is a major modification caused by advanced glycation end (AGE) products in diabetes. Recombinant Tropoelastin treated with MB PDT showed a significant increase CML levels compared to untreated tropoelastin controls and tropoelastin incubated with methylene and untreated with light (FIG. 15B). Similarly, total elastin in porcine tissue showed a significant increase in CML modification levels after treatment with MB PDT compared to untreated samples and the positive control pentagalloyl glucose, which is a known potent chemical crosslinker, but less than glutaraldehyde another known chemical crosslinker (FIG. 15C). These data suggests that MB PDT induces elastin crosslinking within tissues.Attorney Docket No.: UCSF-813WO EXAMPLE 16 MB PDT improves mechanical strength of aortic tissue

[0135] To assess if one of the improvements observed in aortic aneurysms upon MB PDT is due to an effect on the mechanical strength of the tissue, the regional healthy aortic tissue from the same region as MB PDT aneurysms occur in mice during experimental AAA surgery was subjected to PDT ex vivo. A forceps test was conducted to measure stiffness of the tissue. Interestingly, MB PDT-induced vascular stiffness increased with duration of light exposure. (FIG 16A). The stiffness maximized at 120mins exposure for mouse tissue. This benefit was consistent in porcine aorta treated with MB PDT (FIG 16B), although the exposure requirement was higher and visually less striking. This maybe due to variability between elastin and collagen content within aortic tissue between different species. Additionally porcine aorta subjected to incubation with recombinant elastase was resistant to degradation after MB PDT treatment (FIG 16B), with more resistance observed with higher intensity of light (60mW versus 120mW). To assess if this benefit of MB PDT on mechanical strength is true in a pathological setting, MB PDT treatment was done of on mouse aneurysms in vivo and the stiffness was assessed ex vivo (FIG 16C). A clear improvement in the stiffness of the aneurysm was observed post-MB PDT treatment compared to vehicle treatment controls further suggesting that the observed benefit on mechanical properties of the tissue is clinically relevant.

[0136] As described above, the data collectively demonstrates that the photodynamic therapy using the activatable conjugate is able to achieve photoactivation of the photosensitizer that: (i) reduces or maintains the diameter of a blood vessel having an aneurysm, as compared to an untreated blood vessel having an aneurysm; (ii) decreases the volume of an aneurysm or inhibits an increase in the volume of the aneurysm, as compared to an untreated aneurysm; (iii) reduces the rate of blood vessel diameter growth in an blood vessel having an aneurysm, as compared to an untreated blood vessel having an aneurysm; (iv) decreases the rate of volume increase in an aneurysm or inhibits an increase in the volume of the aneurysm, as compared to an untreated aneurysm; and / or (v) is localized to regions of tissue degradation or tissue inflammation or within or near the site of an aneurysm (e.g., after parenteral administration), as compared to a region of healthy tissue, and / or increases crosslinking within the ECM.Attorney Docket No.: UCSF-813WO Methods - Tissue Rigidity Assay:

[0137] Treated tissues were tested for their rigidity and ability to resist drop due to gravity. First, a ruler (with a specificity up to mm) was taped vertically against a white background and checked with a water leveler. Next, each sample was carefully held with a forcep at 0mm on the ruler, such that the sample would, if perfectly stiff, be perpendicular to the ruler. The forcep was also held as perpendicular to the ruler, such that the end of the tissue between the tips of the forcep was parallel to the forcep. Next the drop of the sample was quantified by measuring the distance the other end of the sample reached on the ruler. Each sample was also photographed and filmed for record and display purposes. The weight and length of each sample was noted. Mouse aneurysms and aortic tissue were treated with MB PDT and vehicle in vivo and ex vivo. Unless otherwise specified, the methods described in this section can be similarly applied to similar experiments throughout the examples. SEQUENCES Number Sequence NoteAttorney Docket No.: UCSF-813WO GYFPSSVKPTITWYMGCYKIQNFNNVIPEGMNLSFLI ALISNNGNYTCVVTYPENGRTFHLTRTLTVKVVGSP h vl hAttorney Docket No.: UCSF-813WO DIQMTQSTSSLSASVGDRVTITCRASQDISNYLSWY QQKPGKAVKLLIYYTSKLHSGVPSRFSGSGSGTDYT vl h l [0, d in an embodiment can interchangeably be used in another embodiment unless such a replacement is not technically feasible. It will be appreciated by those skilled in the art that various other omissions, additions and modifications may be made to the methods and structures described above without departing from the scope of the claimed subject matter. All such modifications and changes are intended to fall within the scope of the subject matter, as defined by the appended claims.

[0139] In at least some of the previously described embodiments, one or more elements used in an embodiment can interchangeably be used in another embodiment unless such a replacement is not technically feasible. It will be appreciated by those skilled in the art that various other omissions, additions and modifications may be made to the methods and structures described above without departing from the scope of the claimed subject matter. All such modifications and changes are intended to fall within the scope of the subject matter, as defined by the appended claims.

[0140] It will be understood by those within the art that, in general, terms used herein, and especially in the appended claims (e.g., bodies of the appended claims) are generally intended as “open” terms (e.g., the term “including” should be interpreted as “including but not limited to,” the term “having” should be interpreted as “having at least,” the term “includes” should be interpreted as “includes but is not limited to,” etc.). It will be further understood by those within the art that if a specific number of an introduced claim recitation is intended, such an intent willAttorney Docket No.: UCSF-813WO be explicitly recited in the claim, and in the absence of such recitation no such intent is present. For example, as an aid to understanding, the following appended claims may contain usage of the introductory phrases “at least one” and “one or more” to introduce claim recitations. However, the use of such phrases should not be construed to imply that the introduction of a claim recitation by the indefinite articles “a” or “an” limits any particular claim containing such introduced claim recitation to embodiments containing only one such recitation, even when the same claim includes the introductory phrases “one or more” or “at least one” and indefinite articles such as “a” or “an” (e.g., “a” and / or “an” should be interpreted to mean “at least one” or “one or more”); the same holds true for the use of definite articles used to introduce claim recitations. In addition, even if a specific number of an introduced claim recitation is explicitly recited, those skilled in the art will recognize that such recitation should be interpreted to mean at least the recited number (e.g., the bare recitation of “two recitations,” without other modifiers, means at least two recitations, or two or more recitations). Furthermore, in those instances where a convention analogous to “at least one of A, B, and C, etc.” is used, in general such a construction is intended in the sense one having skill in the art would understand the convention (e.g., “ a system having at least one of A, B, and C” would include but not be limited to systems that have A alone, B alone, C alone, A and B together, A and C together, B and C together, and / or A, B, and C together, etc.). In those instances where a convention analogous to “at least one of A, B, or C, etc.” is used, in general such a construction is intended in the sense one having skill in the art would understand the convention (e.g., “ a system having at least one of A, B, or C” would include but not be limited to systems that have A alone, B alone, C alone, A and B together, A and C together, B and C together, and / or A, B, and C together, etc.). It will be further understood by those within the art that virtually any disjunctive word and / or phrase presenting two or more alternative terms, whether in the description, claims, or drawings, should be understood to contemplate the possibilities of including one of the terms, either of the terms, or both terms. For example, the phrase “A or B” will be understood to include the possibilities of “A” or “B” or “A and B.”

[0141] In addition, where features or aspects of the disclosure are described in terms of Markush groups, those skilled in the art will recognize that the disclosure is also thereby described in terms of any individual member or subgroup of members of the Markush group.Attorney Docket No.: UCSF-813WO

[0142] As will be understood by one skilled in the art, for any and all purposes, such as in terms of providing a written description, all ranges disclosed herein also encompass any and all possible sub-ranges and combinations of sub-ranges thereof. Any listed range can be easily recognized as sufficiently describing and enabling the same range being broken down into at least equal halves, thirds, quarters, fifths, tenths, etc. As a non-limiting example, each range discussed herein can be readily broken down into a lower third, middle third and upper third, etc. As will also be understood by one skilled in the art all language such as “up to,” “at least,” “greater than,” “less than,” and the like include the number recited and refer to ranges which can be subsequently broken down into sub-ranges as discussed above. Finally, as will be understood by one skilled in the art, a range includes each individual member. Thus, for example, a group having 1-3 articles refers to groups having 1, 2, or 3 articles. Similarly, a group having 1-5 articles refers to groups having 1, 2, 3, 4, or 5 articles, and so forth.

[0143] Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it is readily apparent to those of ordinary skill in the art in light of the teachings of this invention that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims.

[0144] Accordingly, the preceding merely illustrates the principles of the invention. It will be appreciated that those skilled in the art will be able to devise various arrangements which, although not explicitly described or shown herein, embody the principles of the invention and are included within its spirit and scope. Furthermore, all examples and conditional language recited herein are principally intended to aid the reader in understanding the principles of the invention and the concepts contributed by the inventors to furthering the art, and are to be construed as being without limitation to such specifically recited examples and conditions. Moreover, all statements herein reciting principles, aspects, and embodiments of the invention as well as specific examples thereof, are intended to encompass both structural and functional equivalents thereof. Additionally, it is intended that such equivalents include both currently known equivalents and equivalents developed in the future, i.e., any elements developed that perform the same function, regardless of structure. Moreover, nothing disclosed herein is intended to be dedicated to the public regardless of whether such disclosure is explicitly recited in the claims.Attorney Docket No.: UCSF-813WO

[0145] The scope of the present invention, therefore, is not intended to be limited to the exemplary embodiments shown and described herein. Rather, the scope and spirit of present invention is embodied by the appended claims. In the claims, 35 U.S.C. §112(f) or 35 U.S.C. §112(6) is expressly defined as being invoked for a limitation in the claim only when the exact phrase "means for" or the exact phrase "step for" is recited at the beginning of such limitation in the claim; if such exact phrase is not used in a limitation in the claim, then 35 U.S.C. § 112 (f) or 35 U.S.C. §112(6) is not invoked.

Claims

Attorney Docket No.: UCSF-813WO WHAT IS CLAIMED IS:

1. A method of cross-linking animal tissue, the method comprising: (a) administering to the animal tissue a photosensitizer and thiamine or a derivative thereof; and (b) subjecting the photosensitizer and the thiamine or a derivative thereof to photoactivating conditions at the site of or near the animal tissue, thereby cross-linking the animal tissue, wherein the animal tissue comprises collagen and / or elastin.

2. A method of producing singlet oxygen within or at the site of animal tissue, the method comprising: (a) administering to the animal tissue a photosensitizer and thiamine or a derivative thereof; (b) subjecting the photosensitizer and the thiamine or a derivative thereof to photoactivating conditions at the site of or near the animal tissue, thereby producing singlet oxygen within or at the site of animal tissue, wherein the animal tissue comprises collagen and / or elastin.

3. The method of a previous claim, wherein the animal tissue is mammalian.

4. The method of a previous claim, wherein the animal tissue is human, porcine, or murine.

5. The method of a previous claim, wherein the animal tissue is vascular or ocular tissue.

6. The method of a previous claim, wherein the animal tissue is muscle, ligament, bone, or skin.

7. The method of a previous claim, wherein the animal tissue is heart tissue.

8. The method of a previous claim, wherein the animal tissue comprises a cancer cell.Attorney Docket No.: UCSF-813WO 9. A method of treating a condition in a subject, the method comprising: (a) administering to a subject in need of treatment thereof a photosensitizer and thiamine or a derivative thereof; and (b) subjecting the photosensitizer and the thiamine or a derivative thereof to photoactivating conditions at the site of or near the condition, wherein the condition is an aneurysm, cancer, and / or a scleral condition.

10. The method of claim 9, wherein the condition is the aneurysm, and photoactivating the photosensitizer: (i) reduces or maintains the diameter of a blood vessel having an aneurysm, as compared to an untreated blood vessel having an aneurysm; (ii) decreases the volume of an aneurysm or inhibits an increase in the volume of the aneurysm, as compared to an untreated aneurysm; (iii) reduces the rate of blood vessel diameter growth in a blood vessel having an aneurysm, as compared to an untreated blood vessel having an aneurysm; and / or (iv) decreases the rate of volume increase in an aneurysm or inhibits an increase in the volume of the aneurysm, as compared to an untreated aneurysm.

11. The method of any one of claims 9-10, wherein the aneurysm is an aortic aneurysm, a cerebral aneurysm, a thoracic aortic aneurysm, an abdominal aortic aneurysm, a popliteal artery aneurysm, a peripheral aneurysm, a fusiform aneurysm, a saccular aneurysm, a mycotic aneurysm, or a pseudo aneurysm.

12. The method of claim 9, wherein the condition is cancer, and the cancer is breast cancer.

13. The method of a preceding claim, wherein the photosensitizer is methylene blue, indocyanine green, cardiogreen, temoporfin, verteporfin, aminolevulinic acid, or riboflavin.

14. The method of a preceding claim, wherein the thiamine or a derivative thereof is thiamine or benfotiamine.

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