Sonodynamic therapy involving thiamine or a derivative thereof

Abstract

The present disclosure relates generally to methods of utilizing sonosensitizers in the presence of thiamine or a derivative thereof for the purpose of cross-linking animal tissue, producing singlet oxygen within or at the site of animal tissue, or treating a condition in a subject, wherein the condition is an aneurysm, cancer, and / or a scleral condition.

Description

Attorney Docket No.: UCSF-891WOSONODYNAMIC THERAPY INVOLVING THIAMINE OR A DERIVATIVE THEREOFCROSS-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 / 685,653 filed August 21, 2024, the disclosure of which is herein incorporated by reference in its entirety.BACKGROUND

[0002] Methods and compositions for enhancing sonodynamic therapies (SDT) 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 sonosensitizer 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 arc utilized, and the accompanying drawings.Attorney Docket No.: UCSF-891WO

[0006] FIG 1 shows diagnostic ultrasound causes an increase in tissue crosslinking in the presence of thiamine and a sonosensitizer.

[0007] FIG 2 shows intravascular ultrasound (IVUS) and transthoracic echocardiogram (TTE) probes cause an increase in singlet oxygen production in the presence of thiamine and a sonosensitizer.

[0008] FIG 3 shows sonodynamic therapy is enhanced by thiamine in aortic tissue and increases elastase resistance.DETAILED DESCRIPTION OF THE DISCLOSUREI. Definitions

[0009] 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.

[0010] 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”.

[0011] 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 the 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.Attorney Docket No.: UCSF-891WO

[0012] 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%.

[0013] Crosslinking, as used herein, refers to the process of chemically joining two or more molecules by a covalent bond.

[0014] MB, MetB, or Met B, as used herein, refers to methylene blue.

[0015] 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 ail, 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 appropriate 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.

[0016] 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) generallyAttorney Docket No.: UCSF-891WO 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 (rlgG) 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, multispecific 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., IgGl and IgG4), IgM, IgE, IgA, and IgD.

[0017] “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 light 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 thatAttorney Docket No.: UCSF-891WO 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., IgGl 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., IgGl and IgG4), IgM, IgE, IgA, and IgD.

[0018] 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.

[0019] “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 certain 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).Attorney Docket No.: UCSF-891WO

[0020] 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.

[0021] 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.

[0022] 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.

[0023] 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.

[0024] 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 humans 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 appeal's 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 / orAttorney Docket No.: UCSF-891WO 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.

[0025] 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 subcombination. 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

[0026] It has been found that thiamine or a derivative thereof enhances the reactions of sonosensitizers. In one aspect, the invention provides a method of producing singlet oxygen, the method comprising: subjecting a sonosensitizer in the presence of thiamine or a derivative thereof to sonoactivating conditions; thereby producing singlet oxygen. In one aspect, the invention provides a method of increasing the production of singlet oxygen from a sonosensitizer, the method comprising: subjecting a sonosensitizer in the presence of thiamine or a derivative thereof to sonoactivating conditions; thereby increasing the production of singlet oxygen from the sonosensitizer. In one aspect, the invention provides a method of stabilizing singlet oxygen, the method comprising: subjecting a sonosensitizer in the presence of thiamine or a derivative thereof to sonoactivating conditions, thereby stabilizing singlet oxygen. In one aspect, the invention provides a method of increasing the stability of singlet oxygen from a sonosensitizer, the method comprising: subjecting a sonosensitizer in the presence of thiamine or a derivative thereof to sonoactivating conditions, thereby increasing the stability of singlet oxygen from the sonosensitizer. In an exemplary embodiment, for any of the methods described herein, the sonosensitizer is part of a sonoactivatable drug conjugate described herein.Attorney Docket No.: UCSF-891WO

[0027] In another aspect, the invention provides a method of cross-linking animal tissue, the method comprising: (a) administering to the animal tissue a sonosensitizer and thiamine or a derivative thereof; and (b) subjecting the sonosensitizer and the thiamine or a derivative thereof to sonoactivating 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 sonosensitizer and thiamine or a derivative thereof; and (b) subjecting the sonosensitizer and the thiamine or a derivative thereof to sonoactivating 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 sonosensitizer and thiamine or a derivative thereof; and (b) subjecting the sonosensitizer and the thiamine or a derivative thereof to sonoactivating 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 sonosensitizer and thiamine or a derivative thereof; and (b) subjecting the sonosensitizer and the thiamine or a derivative thereof to sonoactivating 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 sonosensitizer and thiamine or a derivative thereof; and (b) subjecting the sonosensitizer and the thiamine or a derivative thereof to sonoactivating 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, wherein the animal tissue comprises collagen and / or elastin, and wherein the increase is compared with the sonosensitizer 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 sonosensitizer and thiamine or a derivative thereof; and (b) subjecting the sonosensitizer and the thiamine or a derivative thereof to sonoactivating conditions at the site of or near the animal tissue, therebyAttorney Docket No.: UCSF-891WO 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 sonosensitizer and thiamine or a derivative thereof; and (b) subjecting the sonosensitizer and the thiamine or a derivative thereof to sonoactivating 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 sono sensitizer 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 sonosensitizer, 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 sonosensitizer. In an exemplary embodiment, for any of the methods described herein, the animal tissue is contacted with the sonosensitizer 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 sonosensitizer is part of a sonoactivatable drug conjugate described herein.

[0028] 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 sonosensitizer and thiamine or a derivative thereof; and (b) subjecting the sonosensitizer in the presence of the thiamine or a derivative thereof to sonoactivating 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 sonoactivatable drug conjugate and thiamine or a derivative thereof; and (b) subjecting the sonosensitizer in the presence of the thiamine or a derivative thereof to sonoactivating conditions, thereby treating the condition in the subject. In an exemplary embodiment, for any of the methods described herein, the sonosensitizer 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 sonosensitizer is administered to the subject. In an exemplary embodiment, for any of the methods described herein, the sonosensitizer and theAttorney Docket No.: UCSF-891WO 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 sonosensitizer is part of a sonoactivatable drug conjugate described herein.Sonosensitizers

[0029] The sonosensitizers herein can be activated by any source of sound energy.Sonosensitizers provided and described herein are generally advantageous in that they are activated when exposed to sound at a certain wavelength. In certain embodiments, activation induces cross-linking of animal tissue. In certain embodiments, activation induces the killing of a cancer cell.

[0030] In some embodiments, the sonosensitizer 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 sound within its excitation wavelength window. In some embodiments, the sonosensitizer is selected from Table 1.Table 1Attorney Docket No.: UCSF-891WO

[0031] In some embodiments, the sonosensitizer is hematoporphyrins, naturally occurring 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 mcsotrictinylporphyrins, 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-(l- 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.

[0032] In certain embodiments, the sonosensitizer is riboflavin. In certain embodiments, the sonosensitizer is methylene blue. In certain embodiments, the sonosensitizer is indocyanine green, also known as ICG. In certain embodiments, the sonosensitizer is a cyanine dye which is cardiogreen. In certain embodiments, the sonosensitizer is a chlorin which is temoporfin. In certain embodiments, the sonosensitizer is aminolevulinic acid.

[0033] In certain embodiments, the sonosensitizer is administered in an amount of from about 1 pg / mL to about 500 pg / mL. In certain embodiments, the sonosensitizer is administered in an amount of from about 80 pg / mL to about 120 pg / mL, from about 95 pg / mL to about 105 pg / mL, or about 100 pg / mL. In certain embodiments, the sonosensitizer is administered in a concentration of from about 0.1 pM to about 3 pM. In certain embodiments, the sonosensitizer is administered in a concentration of from about 0.4 pM to about 0.6 pM, or about 0.5 pM. In certain embodiments, the sonosensitizer is administered in a concentration of from about 0.8 pM to about 1.2 pM, from about 0.95 pM to about 1.05 pM, or about 1.0 pM. In certain embodiments, the sonosensitizer is administered in a concentration of from about 1.8 pM to about 2.2 pM, from about 1.95 pM to about 2.05 pM, or about 2.0 pM.Attorney Docket No.: UCSF-891WO

[0034] In some embodiments, the sonosensitizer is one that is activatable with sound that can penetrate through blood or blood cells. In certain embodiments, the sonosensitizer is one that generates singlet oxygen when exposed to sound within the tissue window.Thiamine or a derivative thereof

[0035] 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.

[0036] 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.

[0037] In certain embodiments, the thiamine or a derivative thereof is administered in an amount of from about 0.01 pg / mL to about 300 pg / 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 pg / mL to about 5 pg / mL, from about 0.5 pg / mL to about 2 pg / mL, from about 0.90 pg / mL to about 1.1 pg / mL, or about 1 pg / mL. In certain embodiments, the thiamine or a derivative thereof is administered in an amount of from about 1 pg / mL to about 30 pg / mL, from about 5 pg / mL to about 20 pg / mL, from about 8 pg / mL to about 12 pg / mL, or about 10 pg / mL. In certain embodiments, the thiamine or a derivative thereof is administered in an amount of from about 25 pg / mL to about 300 pg / mL, from about 50 pg / mL to about 200 pg / mL, from about 80 pg / mL to about 120 pg / mL, from about 95 pg / mL to about 110 pg / mL, or about 100 pg / mL.Attorney Docket No.: UCSF-891WOAnimal Tissue

[0038] 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, or 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.Sonoactivating Conditions

[0039] In an exemplary embodiment, the sonoactivating conditions involve a sound source that provides sound which excites a sonosensitizer described herein.

[0040] In an exemplary embodiment, the sonoactivating conditions comprise sound waves that are ultrasound waves. In an exemplary embodiment, the sonoactivating conditions comprise sound waves that are audible sound waves. In an exemplary embodiment, the sound waves are delivered from outside the body. In an exemplary embodiment, the sound waves are delivered from inside the body.

[0041] In an exemplary embodiment, the sonoactivating conditions occur for a period of time sufficient to crosslink animal tissue without significantly damaging the underlying animal tissue. In an exemplary embodiment, the sonoactivating conditions occur for a period of time sufficient to kill cancer cells without significantly damaging non-cancer cells. In an exemplary embodiment, the sonoactivating 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 sonoactivating conditions is from about 1 second to about 2 hours. In an exemplary embodiment, the time of the sonoactivating conditions is from about 30 seconds to about 1 hour. In an exemplary embodiment, the time of the sonoactivating conditions is from about 30 seconds to about 30 minutes. In an exemplary embodiment, the time of theAttorney Docket No.: UCSF-891WO sonoactivating conditions is from about 1 minute to about 30 minutes. In an exemplary embodiment, the time of the sonoactivating conditions is from about 5 minutes to about 30 minutes. In an exemplary embodiment, the time of the sonoactivating 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

[0042] Sonosensitizer(s) can be attached (coupled / conjugated) to a targeting moiety in order to target the sonosensitizer(s) to a site near and / or within a desired activity site, concentrate the sonosensitizer(s) at the site of near and / or within a desired activity site, and / or reduce the overall exposure of the subject to the sonosensitizer(s). A targeting moiety is preferably one that targets one or more sonosensitizers to an animal tissue described herein (e.g., by binding cell surface receptor associated with the animal tissue). In some embodiments, the targeting moiety does not selectively bind to a cancer cell. Alternatively, the targeting moiety can be one that selectively binds non-malignant cells.Sonoactivatable Drug Conjugates

[0043] Any of the sonosensitizers herein can be coupled to a targeting moiety using known conjugation technologies to form a sonoactivatable drug conjugate (SDC). Conjugation can optionally include the addition of one or more spacers or linkers into the SDC. Techniques for conjugation of an activatable agent such as a sonosensitizer 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;l l(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.Attorney Docket No.: UCSF-891WOPharmaceutical Compositions

[0044] In some embodiments, provided herein are pharmaceutical compositions and / or formulations comprising the sonosensitizer and the thiamine or a derivative thereof, or the sonoactivatable 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 sonosensitizer alone or in the SDC 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

[0045] Provided and described herein are systems useful for treating a condition described herein. In some embodiments, a system comprises: a sonoactivatable drug conjugate, thiamine or a derivative thereof, and an energy source to activate the sonosensitizer (e.g. a sound delivery unit). Such a system can also comprise a sound generating unit comprising a sound source.

[0046] 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 sonodynamic therapy systems, or only the sonoactivatable drug conjugate (e.g., any one of the sonoactivatable drug conjugates described herein), thiamine or a derivative thereof, and a sound delivery unit.Specific Embodiments

[0047] 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 sonosensitizer and thiamine or a derivative thereof; and (b) subjecting the sonosensitizer and the thiamine or a derivative thereof to sonoactivating 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 sonosensitizer is temoporfin. In an exemplary embodiment, the sonosensitizer is temoporfin, and the amount of temoporfin is as described herein (such as from about 0.25pM to about 3pM). In an exemplary embodiment, theAttorney Docket No.: UCSF-891WO 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 lOOng / ml and about lOOpg / ml). In an exemplary embodiment, the sonoactivating conditions are as described herein.

[0048] 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 sonosensitizer and thiamine or a derivative thereof; and (b) subjecting the sonosensitizer and the thiamine or a derivative thereof to sonoactivating conditions at the site of or near the scleral condition site. In an exemplary embodiment, the sonosensitizer is cardiogreen or indocyanine green. In an exemplary embodiment, the thiamine or a derivative thereof is thiamine. In an exemplary embodiment, the sonoactivating conditions are as described herein.

[0049] In an exemplary embodiment, for a method, composition and / or system described herein, the sonosensitizer is riboflavin. In an exemplary embodiment, for a method, composition and / or system described herein, the sonosensitizer is indocyanine green. In an exemplary embodiment, for a method, composition and / or system described herein, the sonosensitizer is temoporfin. In an exemplary embodiment, for a method, composition and / or system described herein, the sonosensitizer is verteporfin. In an exemplary embodiment, for a method, composition and / or system described herein, the sonosensitizer is riboflavin. In an exemplary embodiment, for a method, composition and / or system described herein, the sonosensitizer is cardiogreen. In an exemplary embodiment, for a method, composition and / or system described herein, the sonosensitizer is aminolevulinic acid.

[0050] In an exemplary embodiment, for a method, composition and / or system described herein for treating an aneurysm, the sonosensitizer is riboflavin. In an exemplary embodiment, for a method, composition and / or system described herein for treating an aneurysm, the sonosensitizer is indocyanine green. In an exemplary embodiment, for a method, composition and / or system described herein for treating an aneurysm, the sono sensitizer is temoporfin. In an exemplary embodiment, for a method, composition and / or system described herein for treating an aneurysm, the sonosensitizer is verteporfin. In an exemplary embodiment, for a method, composition and / or system described herein for treating an aneurysm, the sonosensitizer is riboflavin. In an exemplary embodiment, for a method, composition and / or system describedAttorney Docket No.: UCSF-891WO herein for treating an aneurysm, the sonosensitizer is cardiogreen. In an exemplary embodiment, for a method, composition and / or system described herein for treating an aneurysm, the sonosensitizer is aminolevulinic acid.

[0051] 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 sonoactivatable drug conjugate described herein, the targeting moiety is not anakinra.

[0052] In an exemplary embodiment, for a method of treating a condition described herein, the sonosensitizer 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 sonosensitizer is methylene blue, and the condition is not an aneurysm. In an exemplary embodiment, for a sonoactivatable drug conjugate described herein, the sonosensitizer is methylene blue, and the targeting moiety is not anakinra.III. Aneurysm Embodiments

[0053] There is a need in the art for treating aneurysms. The present disclosure shows that using a sonosensitizer 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.

[0054] The present disclosure further provides for and exemplifies the non-invasive treatment of aneurysms by two means for selectively activating a sonosensitizer 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 sonosensitizer 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 sonosensitizer 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 sound source coupled to a catheter) is used to activate the sonosensitizer in the presence of thiamine or a derivative thereof via energy (e.g., sound) at or near the site of inflammation or the aneurysmAttorney Docket No.: UCSF-891WO to activate the sonosensitizer locally. This can be beneficial because it provides an additional control to specific activation by using sound to activate the crosslinking reaction (c.g., restricting the therapeutic effect to the aneurysm). Additional disclosures as to the sonosensitizers, thiamine or a derivative thereof, sonoactivatable drug conjugates, and methods for using the same are provided below.Aneurysm Targeting Moieties

[0055] As described and exemplified herein, sonosensitizer(s) can be attached (coupled / conjugated) to a targeting moiety in order to target the sonosensitizer(s) to a site near and / or within an aneurysm, concentrate the sonosensitizer(s) at the site of, near, and / or within an aneurysm, and / or reduce the overall exposure of the subject to the sonosensitizer(s). A targeting moiety is preferably one that targets one or more sonosensitizers 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.

[0056] 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 sonosensitizer(s). For example, activatable drug conjugates (ADCs) having anakinra attached to one or more methylene blue sonosensitizers (e.g., through an amide linkage) demonstrate the ability to target the sonosensitizer(s) to a site near and / or within an aneurysm, concentrate the sonosensitizer(s) at the site near and / or within an aneurysm, and / or reduce the overall exposure of the subject to the sonosensitizer(s). In turn, activating a sonosensitizer such as a methylene blue sonosensitizer 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.

[0057] In some embodiments, a sonosensitizer is attached to a targeting moiety other than anakinra, or to a targeting moiety that targets a marker other than IL1R. In such embodiments a 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,Attorney Docket No.: UCSF-891WOCD27, CXCR5, CCR5, IL5R, IFNLR1, CD28, CXCR6, CCR6, IL6R, CD95, CXCR7, CCR7, IL7R, LTbR, CXC3R1, CCR8, IL8R, 0X40, CCR9, IL9R, 4-1BB, CCR10, IL10R, BAFFR, IL11R, BCMA, IL12R, TACI, IL13R, RANK, IL15R, NGFR, IL17R, TROY, IL18R, ED AR, IL20R, RELT, IL21R, Fnl4, IL22R, TIM3, IL23R, IL27R, IL28R, or IL31R.

[0058] 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.

[0059] 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 ILl-beta.

[0060] 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, the targeting moiety is MEDI6383 targeting 0X40. 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 isAttorney Docket No.: UCSF-891WOGSK2857916 targeting B-cell activating factor receptor (BAFFR). In certain embodiments, the targeting moiety is Tcclistamab targeting B-ccll 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 ILl-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 ILl-beta. In certain embodiments, the targeting moiety is LY2189102 targeting ILl-beta.Sonosensitizer Drug Conjugates targeting aneurysms

[0061] Any of the sonosensitizers 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 sonosensitizers via an amide linkage formed by reacting the methylene blue NHS ester and one or more lysine residues on anakinra following the protocol provided in the Examples. Methylene blue NHS ester comprises a structure represented by the formula:Attorney Docket No.: UCSF-891WOIn one embodiment, anakinra is conjugated to one or more methylene blue sonosensitizers via an amide linkage formed by reacting the methylene blue NHS ester and one or more lysine residues on anakinra.

[0062] One or more sonosensitizers 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 making methylene blue and methylene blue derivatives are 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 of3-N'- Arylaminopheno thiazines. Molecules. 2022 May 8;27(9):3024. doi: 0.3390 / molecules27093024. PMID: 35566375.

[0063] In certain embodiments, the linkage further introduces a spacer between the linker and the sonosensitizer. 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 sonosensitizer is directedly attached to the targeting moiety.

[0064] A linker (e.g., bifunctional linkage) can also be introduced via a linkage. In some embodiments, the linkage introduces a linker, wherein the sonosensitizer is further attached to the targeting moiety via the linker. In some embodiments, a linker or a linker precursor used toAttorney Docket No.: UCSF-891WO form the linker comprises 6-maleimidocaproyl (MC), Maleimide-DOTA, maleimidopropanoyl (MP), alaninc-phcnylalaninc (AP), p- aminobcnzyloxycarbonyl (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).

[0065] 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 sonosensitizer is attached to a cysteine residue of the targeting moiety. In some embodiments, the sonosensitizer 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.

[0066] As such, in some embodiments, the SDC comprises a heterogenous composition of one or more sonosensitizers conjugated to the targeting moieties. For example, in certain embodiments, the SDC comprises a heterogenous composition of one or more methylene blue sonosensitizers conjugated to the anakinra.

[0067] In certain embodiments, the linker is first conjugated to the sonosensitizer, 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 sonosensitizer.

[0068] In some embodiments provided herein are pharmaceutical compositions and / or formulations comprising the sonosensitizer and the thiamine or a derivative thereof for treating aneurysms, or the sonoactivatable drug conjugate targeting aneurysms and the thiamine or a derivative thereof. Pharmaceutical compositions include and / or refers a preparation that is inAttorney Docket No.: UCSF-891WO such form as to permit the biological activity of the sonosensitizer alone or in the SDC to be effective, and that contains no additional components which arc 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

[0069] Provided and described herein are systems useful for treating an aneurysm. In some embodiments, a system comprises: a sonoactivatable drug conjugate comprising anakinra attached to methylene blue, thiamine or a derivative thereof, and an energy source that delivers sound. Such a system can also comprise a sound generating unit comprising a sound source.

[0070] In some embodiments, also provided and described herein arc kits comprising one or more components of the systems described herein. For example, a kit can include all components of the sonodynamic therapy systems, or only the sonoactivatable drug conjugate, thiamine or a derivative thereof, and a sound delivery unit.Methods for treating aneurysms

[0071] 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 sonosensitizer and thiamine or a derivative thereof; and (b) subjecting the sonosensitizer and the thiamine or the thiamine derivative to sonoactivating conditions at the site of or near the aneurysm, thereby treating the aneurysm.

[0072] In some embodiments, the method of treating an aneurysm comprises: (a) administering a sonoactivatable drug conjugate (e.g., anakinra linked to one or more methylene blue sonosensitizers) and thiamine or a derivative thereof; and (b) subjecting the sonoactivatable drug conjugate in the presence of the thiamine or the thiamine derivative at the site of or near the aneurysm using sound, thereby activating the sonosensitizer comprises delivering energy to the site or near the site of the aneurysm.Attorney Docket No.: UCSF-891WO

[0073] In an exemplary embodiment, for an aneurysm treatment method, the sonosensitizer is methylene blue. In an exemplary embodiment, for an aneurysm treatment method, the sonosensitizer is methylene blue, and the amount of methylene blue is as described herein (such as about lOOug / 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).

[0074] 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 agent (e.g., a sonosensitizer); and (b) activating the activatable agent at the site of or near the aneurysm using sound, 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 an activatable drug conjugate (e.g., anakinra linked to one or more methylene blue sonosensitizers); and (b) activating the activatable agent at the site of or near the aneurysm using sound, wherein activating the activatable agent comprises delivering energy to the site or near the site of the aneurysm.

[0075] In some embodiments, the sonoactivatable 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 sonosensitizer and activating the sonosensitizer comprises sonoactivation.

[0076] As described and exemplified herein, in some embodiments, activating the activatable agent (e.g., sonoactivating the sonosensitizer): (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.Attorney Docket No.: UCSF-891WO

[0077] 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 certain embodiments, the aneurysm is a pseudo aneurysm. In certain embodiments, the blood vessel is the aorta.

[0078] The sonoactivatable 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 sonoactivatable drug conjugate is administered via parenteral infusion or injection. In certain embodiments, the sonoactivatable drug conjugate is administered via intravenous infusion or injection.

[0079] The methods herein are also useful for extending the therapeutic window for treating an aneurysm. In some embodiments, administering the sonosensitizer or sonoactivatable drug conjugate and activating the sonosensitizer can allow for delayed surgical intervention for an aneurysm due to reduced long term growth rate.

[0080] 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 certainAttorney Docket No.: UCSF-891WO embodiments, the aneurysm is a mycotic aneurysm. In certain embodiments, the aneurysm is a pseudo aneurysm.

[0081] In certain embodiments, the dose of the sonosensitizer is less than 4 per dose. In certain instances, the targetable moiety enables a therapeutic effect (e.g., increasing an amount of tissue) at lower amounts of the activatable agent or sonosensitizer that if administered without the targeting moiety (i.e., in a non-conjugate manner).

[0082] 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.

[0083] 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.

[0084] 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.

[0085] 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.

[0086] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0087] 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-891WOEXAMPLES

[0088] The practice of the present invention will employ, unless otherwise indicated, conventional techniques of sonochemistry, which are well known to those skilled in the art.

[0089] Additional embodiments arc 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 1Diagnostic ultrasound causes an increase in tissue crosslinkins in presence of thiamine and sonosensitizer

[0090] A 1-1.2 cm section of an abdominal aorta was harvested from mice. The section was subjected to 100 pg / mL methylene blue plus 10 mg / mL thiamine. The sections were then subjected to sonodynamic therapy. Stiffness was measured using a forceps bend test at 0 min and 30 min. Results are provided in FIG. 1.EXAMPLE 2Intravascular ultrasound (IVUS) and transthoracic echocardio ram (TIE) probes cause an increase in sin let oxy en production in presence of thiamine and sonosensitizer

[0091] Representative graph showing an increase in fluorescence due to oxygen singlet production upon incubation of IVUS and TTE probes in solutions containing thiamine and methylene blue. Solutions were treated with ultrasound for 5mins to produce singlet oxygen and measure using singlet oxygen sensor green dye at 488nm / 525nm fluorescence.EXAMPLE 3Sonodynamic therapy is enhanced by thiamine in aortic tissue and increases elastase resistance

[0092] Representative images of pig aortic tissue were treated with sonodynamic therapy (SDT) using methylene blue with and without Thiamine. The presence of thiamine resulted in significant structural changes to the tissue. Mouse aortic tissue was treated with SDT in the presence of MB and Thiamine. After SDT mouse tissue showed increased rigidity. Pig tissue shows resistance to elastase treatment after SDT.

[0093] As described above, the data collectively demonstrates that the sonodynamic therapy using the activatable conjugate is able to achieve sonoactivation of the sono sensitizer that: (i) reduces or maintains the diameter of a blood vessel having an aneurysm, as compared to anAttorney Docket No.: UCSF-891WO 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.EXAMPLE 4Synthesis and characterization of anakinra conjugated to methylene blue (Ana-MB)

[0094] Methylene blue was conjugated to IL-1 receptor antagonist, Anakinra using an NHS or maleimide linker. Methods of linking a sonoactivatable agent (e.g., methylene blue) to a protein (e.g., anakinra) are known in the art and can be used to form the sonoactivatable 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.Methods - Tissue Rigidity Assay:

[0095] 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.Attorney Docket No.: UCSF-891WOUnless otherwise specified, the methods described in this section can be similarly applied to similar experiments throughout the examples.SEQUENCESAttorney Docket No.: UCSF-891WO

[0096] 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 isAttorney Docket No.: UCSF-891WO 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.

[0097] 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.

[0098] 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 will 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 whereAttorney Docket No.: UCSF-891WO 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.”

[0099] 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.

[0100] 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 ail 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.Attorney Docket No.: UCSF-891WO

[0101] 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.

[0102] Accordingly, the preceding merely illustrates the principles of the invention. It will be appreciated that those skilled in the ail 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 constmed 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.

[0103] 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-891WOWHAT IS CLAIMED IS:

1. A method of cross-linking animal tissue, the method comprising:(a) administering to the animal tissue a sonosensitizer and thiamine or a derivative thereof; and(b) subjecting the sonosensitizer and the thiamine or a derivative thereof to sonoactivating 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 sonosensitizer and thiamine or a derivative thereof;(b) subjecting the sonosensitizer and the thiamine or a derivative thereof to sonoactivating 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-891WO9. A method of treating a condition in a subject, the method comprising:(a) administering to a subject in need of treatment thereof a sono sensitizer and thiamine or a derivative thereof; and(b) subjecting the sonosensitizer and the thiamine or a derivative thereof to sonoactivating 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 sonoactivating the sonosensitizer:(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 sonosensitizer 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.Attorney Docket No.: UCSF-891WO15. The method of a preceding claim, wherein the sonoactivating conditions comprise sound waves that arc ultrasound waves or audible sound waves.

16. The method of claim 15, wherein the sound waves are delivered from outside the body or from inside the body.

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