Borylated amino acid compositions for use in boron neutron capture therapy and methods thereof

Abstract

To provide new and improved methods of treating cancer, immune disorders, and other diseases utilizing borylated amino acids and neutron capture therapy (NCT).SOLUTION: Disclosed herein are borylated amino acid ("BAA") compositions and methods of making BAAs. Accordingly, BAAs can be administered to patients as neutron capture agents and can provide methods of treating cancer, immune disorders, and other diseases by utilizing neutron capture therapy modalities. The invention described herein relates to the field of boron neutron capture therapy (BNCT). In particular, the present invention relates to borylated amino acid ("BAA") or ("BAAs") compositions that can be used as a vehicle for neutron capture therapy in humans. The invention further relates to the treatment of cancer and other immune disorders and diseases.SELECTED DRAWING: None

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 62 / 919,156, filed March 4, 2019, the contents of which are incorporated herein by reference in their entirety.

[0002] STATEMENT OF RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED RESEARCH Not applicable.

[0003] FIELD OF THE INVENTION The invention described herein relates to the field of boron neutron capture therapy (BNCT). Specifically, the invention relates to borylated amino acid ("BAA") or ("BAAs") compositions that can be used as vehicles for neutron capture therapy in humans. The invention further relates to the treatment of cancer and other immune disorders and diseases. [Background technology]

[0004] Background of the Invention Cancer is the second leading cause of death worldwide, after coronary artery disease. Millions of people die from cancer each year, with well over 500,000 people dying from cancer each year in the United States alone, and 1,688,780 new cases of cancer were diagnosed in 2017 (American Cancer Society). While deaths from heart disease have declined significantly, deaths attributable to cancer in general are increasing. Early in the next century, cancer is predicted to become the leading cause of death unless medical advances change current trends.

[0005] Some cancers are notable for their high mortality rates. In particular, carcinomas of the lung (18.4% of all cancer deaths), breast (6.6% of all cancer deaths), colorectal (9.2% of all cancer deaths), liver (8.2% of all cancer deaths), and stomach (8.2% of all cancer deaths) are the leading causes of cancer death worldwide, across all ages, in both sexes (GLOBOCAN 2018). These and virtually all other cancers share the common fatal characteristic of metastasizing to sites distant from the primary tumor, and with very few exceptions, metastatic disease is fatal. Furthermore, even for cancer patients who initially survive their primary cancer, common experience indicates that their lives change dramatically. Many cancer patients experience intense anxiety due to the awareness of the possibility of recurrence or treatment failure. Many cancer patients also experience physical debilitation after treatment. Furthermore, many cancer patients experience disease recurrence.

[0006] Although cancer treatments have improved over the past few decades, increasing survival rates, new therapeutic strategies utilizing multiple treatment modalities remain necessary due to the heterogeneity of cancer. This is particularly true for the treatment of solid tumors in anatomically significant locations (e.g., glioblastoma, squamous cell carcinoma of the head and neck, and lung adenocarcinoma), which may be limited to standard radiation therapy and / or chemotherapy. Nevertheless, adverse effects of these therapies are chemoresistance and radioresistance, which promote locoregional recurrence, distant metastases, and the development of other primary tumors, in addition to severe side effects that reduce patients' quality of life.

[0007] Neutron capture therapy (NCT) is a promising form of radiation therapy. NCT uses boron compounds to selectively kill tumor cells while sparing normal cells. BNCT uses 0.5 keV <E n Non-radiative absorbing epithermal neutrons in the low energy range <30 keV 10 After neutron capture, the boron atom reacts with an α particle and a recoil lithium nucleus ( 7Li). 10 B+n→ 7 Li+ 4 He

[0008] Alpha particles deposit high energy, i.e., 150 keV / μm, along their short path, essentially limited to the diameter of a single cell, leading to double-stranded DNA breaks and subsequent cancer cell death by apoptosis. BNCT therefore integrates both concepts of chemotherapy, targeted therapy, and the macroscopic anatomical localization of traditional radiotherapy.

[0009] While the conceptual technology of NCT, specifically boron neutron capture therapy (BNCT), is well known, progress has been slowed by the technical limitations associated with this type of treatment. During early investigations using a research reactor at MIT in 1960, several doses of patients were treated with disodium decahydrodecaborate, which was thought to be less toxic than the simpler boron compounds previously used but capable of delivering more boron to cells. Unfortunately, due to severe brain necrosis in patients undergoing BNCT and the potential hazards of using a reactor, BNCT research was discontinued in the United States.

[0010] In 1968, Hiroshi Hatanaka reviewed the clinical application of BNCT in Japan, using sodium borocaptate (BSH) to direct light beams at surgically exposed intracranial tumors, and reported a 58% 5-year survival rate. In 1987, Japanese clinicians applied BNCT to the treatment of malignant melanoma using boronophenylalanine (BPA) as the boron compound. Thus, a gradual revival of BNCT occurred, although it was limited to countries with access to research reactor facilities capable of delivering epithermal neutrons. Currently, technological improvements in both (i) the injection and delivery of capture compounds that preferentially concentrate within the tumor, and (ii) increased and easier access to neutron beams using cyclotrons have led to a revival of BNCT treatment methods.

[0011] Proton-boron fusion reaction is required for BNCT 10 B, but abundant in nature 11 Unlike BNCT, it depends on the B isotope. 1 H) and boron ( 11 B) After the fusion reaction between nuclei, three alpha particles are emitted: p+ 11 B->3α. Proton beams have the advantage of Bragg peak characteristics that reduce normal tissue damage, and when combined with proton capture, may improve the efficacy of proton therapy alone.

[0012] Boron carriers have evolved since 1950 and are reviewed in Nedunchezhian et al., J. Clin. Diag. Res., vol. 10(12) (Dec. 2016). Briefly, the first generation of boron compounds, represented by boric acid and its derivatives, were either toxic or suffered from low tumor accumulation / retention. Both BPA and BSH are considered second-generation compounds, which emerged in the 1960s. They had significantly lower toxicity and better PK and biodistribution. BPA-fructose conjugates are considered third-generation compounds, which have been used since 1994 to treat patients with head and neck (H&N), glioblastoma, and melanoma using BNCT. While BPA-fructose and BSH are the only compounds used clinically as boron carriers to date, both low- and high-molecular-weight biomolecules, such as nucleosides, porphyrins, liposomes, nanoparticles, and mAbs, have been evaluated for tumor targeting in preclinical models. The main drawback of BPA-fructose, in addition to its relatively low solubility, is its rapid excretion, which prevents it from achieving a high blood Cmax, one of the factors that influences tumor uptake.

[0013] From the above description, one skilled in the art will readily appreciate that new treatment paradigms are needed in the treatment of cancer and immune diseases. By using modern chemical synthesis and modifying natural amino acids with boron, new disease treatments can be achieved, with the overall goals of more effective treatments, reduced side effects, and reduced manufacturing costs. In view of the current deficiencies associated with NCT, it is an object of the present invention to provide new and improved methods of treating cancer, immune disorders, and other diseases utilizing borylated amino acids and NCT. [Prior art documents] [Non-patent literature]

[0014] [Non-Patent Document 1] NEDUNCHEZHIAN et al., J.Clin.Diag.Res.,vol.10(12) Summary of the Invention [Means for solving the problem]

[0015] Summary of the Invention The present invention provides compositions comprising natural amino acids borylated via chemical synthesis for use as delivery modalities for treating human diseases such as cancer, immune disorders (including but not limited to rheumatoid arthritis, ankylosing spondylitis), and other cellular diseases (including but not limited to Alzheimer's disease). In certain embodiments, the borylated amino acids are comprised of naturally occurring amino acids such as phenylalanine, tryptophan, tyrosine, histidine, and any other naturally occurring amino acid set forth in Table I.

[0016] In a further embodiment, the present invention includes a method for concentrating boron in a cell, the method comprising: (i) synthesizing a borylated amino acid (“BAA”); (ii) administering the BAA to a patient; and (iii) irradiating the cell with neutrons.

[0017] In another embodiment, the present disclosure teaches a method for synthesizing a BAA.

[0018] In another embodiment, the present disclosure teaches methods of treating cancer, immune disorders and other diseases in humans. In an embodiment of the present invention, for example, the following items are provided: (Item 1) The chemical structure below: [ka] In the formula, E=CO2H, CONHB 12 H 11 , B(OH)2; and X=H, B(OH)2, Bpin, (-O-CH2CH2)2-OB 12 H 11 A composition comprising: (Item 2) The composition comprises: [ka] Item 1. The composition of item 1, comprising: (Item 3) The chemical structure below: [ka] In the formula, E=CO2H, CONHB 12 H 11 , B(OH)2; and X=H, B(OH)2, Bpin, (-O-CH2CH2)2-OB 12 H 11 A composition comprising: (Item 4) The composition comprises: [ka] 4. The composition according to item 3, comprising: (Item 5) The chemical structure below: [ka] In the formula, E=CO2H, CONHB 12 H 11 , B(OH)2; and X=H, B(OH)2, Bpin, (-O-CH2CH2)2-OB 12 H 11 A composition comprising: (Item 6) The composition comprises: [ka] Item 6. The composition according to item 5, comprising: (Item 7) A kit comprising the composition according to item 1. (Item 8) A kit comprising the composition according to item 2. (Item 9) A kit comprising the composition according to item 3. (Item 10) A kit comprising the composition according to item 4. (Item 11) A kit comprising the composition according to item 5. (Item 12) A kit comprising the composition according to item 6. (Item 13) A dosage unit form comprising the composition according to item 1. (Item 14) A dosage unit form comprising the composition according to item 2. (Item 15) A dosage unit form comprising the composition according to item 3. (Item 16) 5. A dosage unit form comprising the composition according to item 4. (Item 17) Item 6. A dosage unit form comprising the composition according to item 5. (Item 18) 7. A dosage unit form comprising the composition according to item 6. (Item 19) 14. The human unit form according to item 13, wherein the human unit form is used in boron neutron capture therapy (BNCT). (Item 20) 15. The human unit form according to item 14, wherein the human unit form is used in boron neutron capture therapy (BNCT). [Brief explanation of the drawings]

[0019] [Figure 1] Chemical synthesis of BPA-BS.

[0020] [Figure 2] Chemical synthesis of BPA-BN.

[0021] [Figure 3] Chemical synthesis of TLS00192.

[0022] [Figure 4] Chemical synthesis of TLS00178.

[0023] [Figure 5] Chemical synthesis of TLS00190.

[0024] [Figure 6] LCMS purity and mass confirmation of TLS00192.

[0025] [Figure 7] LCMS purity and mass confirmation of TLS00178.

[0026] [Figure 8] LCMS purity and mass confirmation of TLS00190.

[0027] [Figure 9] Summary LCMS purity and mass confirmation.

[0028] [Figure 10] Kinetic parameters of TLS00192 and BPA-fructose.

[0029] [Figure 11] Cellular retention of TLS00192 and BPA-fructose in FaDu cells.

[0030] [Figure 12] LAT-1-mediated competition studies with TLS00192.

[0031] [Figure 13] Boron uptake of TLS00190 and TLS00178 in FaDu cells.

[0032] [Figure 14] Cellular retention of TLS00190, TLS00178, and BPA-fructose in FaDu cells. DETAILED DESCRIPTION OF THE INVENTION

[0033] Detailed Description of the Invention Section Overview I.) Definition II.) BPA III.) BSH IV.) Boron a. Boron in general V.) Naturally Occurring Amino Acids VI.) Borylated Amino Acids (BAAs) a.BPA-BS b.BPA-BN C. Amino acid composition d. BAA containing phenylalanine e. BAAs containing tryptophan f. BAAs containing tyrosine g. BAA containing histidine VII.) Boron Neutron Capture Therapy using BAA VIII.) Proton-Boron Fusion Therapy Using BAA IX.) Methods for delivering BAAs to cells X.) Kits / Manufactured Products

[0034] I.) Definition: Unless otherwise defined, all technical terms, notations, and other scientific or terminology used herein are intended to have the meaning commonly understood by those of ordinary skill in the art, unless the context clearly dictates otherwise. In some instances, for clarity and / or ease of reference, terms having a commonly understood meaning have been defined herein, and the inclusion of such definitions herein should not necessarily be construed as representing a substantial departure from what is commonly understood in the art.

[0035] When trade names are used herein, reference to a trade name also refers to the product formulation, generic drug and active pharmaceutical ingredient of that trade name product, unless the context dictates otherwise.

[0036] The terms "advanced cancer," "locally advanced cancer," "advanced disease," and "locally advanced disease" refer to cancer that has spread through the involved tissue capsule and are meant to include stage C disease under the American Urological Association (AUA) system, stages C1-C2 disease under the Whitmore-Jewett system, and stages T3-T4 and N+ disease under the TNM (tumor, node, metastasis) system. Generally, surgery is not recommended for patients with locally advanced disease, and these patients have a substantially worse prognosis compared with patients with clinically localized (organ-confined) cancer.

[0037] "Amino acid" means a simple organic compound containing both a carboxyl (-COOH) and an amino (-NH2) group.

[0038] "Borylation" refers to the reaction of forming organoboron compounds through the functionalization of aliphatic and aromatic C—H bonds.

[0039] "Borylated amino acid" (BAA) refers to a compound containing a naturally occurring amino acid, such as those shown in Table I, that has undergone a borylation reaction. BAAs can be synthesized in several ways depending on the base amino acid used.

[0040] The term "compound" refers to and includes not only the chemical compound (e.g., BAA) itself, but also, whether explicitly stated or not, unless the context makes it clear to exclude the following: amorphous and crystalline forms of the compound, including polymorphic forms, which may be part of a mixture or in isolated form; free acid and free base forms of the compound, typically those shown in the structures provided herein; isomers of the compound (refers to optical isomers and tautomers, where optical isomers include enantiomers and diastereomers, chiral and non-chiral isomers, and optical isomers include isolated optical isomers and mixtures of optical isomers, including racemic and non-racemic mixtures); isomers may be in isolated form or may be a mixture with one or more other isomers; isotopes of the compound, including deuterium- and tritium-containing compounds, including compounds containing radioactive isotopes , therapeutically and diagnostically effective radioisotopes; multimeric forms of the compounds, including dimeric, trimeric, and the like forms; salts of the compounds, preferably pharmaceutically acceptable salts, including acid addition salts and base addition salts, including salts with organic and inorganic counterions, including zwitterionic forms, where when the compound is associated with two or more counterions, the two or more counterions may be the same or different; and solvates, hemisolvates, monosolvates, disolvates, and the like of the compounds, including organic hydrates and inorganic solvates, where when the compound is associated with two or more solvent molecules, the two or more molecules may be the same or different. In some instances, reference herein to a compound of the invention will include an explicit reference to one of the above forms or, for example, a salt and / or solvate. However, this reference is for emphasis only and should not be construed as excluding other of the above forms identified above.

[0041] As used herein, the term "inhibit" or "inhibition of" means to decrease by a measurable amount or to completely suppress.

[0042] The term "mammal" refers to any organism classified as a mammal, including mice, rats, rabbits, dogs, cats, cows, horses, and humans. In one embodiment of the invention, the mammal is a mouse. In another embodiment of the invention, the mammal is a human.

[0043] The terms "metastatic cancer" and "metastatic disease" refer to cancer that has spread to regional lymph nodes or distant sites and are meant to include stage D disease by the AUA system and stage TxNxM+ by the TNM system.

[0044] "Molecular recognition" refers to a chemical event that allows a host molecule to form a complex with a second molecule (i.e., a guest). This process occurs through non-covalent chemical bonds, including but not limited to hydrogen bonding, hydrophobic interactions, and ionic interactions.

[0045] "Pharmaceutically acceptable" refers to a non-toxic, inert, and / or composition that is physiologically compatible with humans or other mammals.

[0046] The term "neutron capture agent" means a stable, non-reactive chemical isotope that produces alpha particles when activated by a neutron.

[0047] The term "neutron capture therapy" refers to a non-invasive therapeutic modality for treating locally invasive malignancies, such as primary brain tumors and recurrent head and neck cancer, as well as other immune disorders and diseases, by irradiating a neutron capture agent with neutrons.

[0048] As used herein, "treat" or "therapeutic" and grammatically related terms refer to any improvement in any outcome of a disease, such as extending survival, reducing morbidity, and / or mitigating side effects that are a by-product of alternative treatment modalities; as will be readily understood in the art, complete eradication of the disease, although preferred, is not a requirement for treatment actions.

[0049] II.) BPA

[0050] For reference, ( 10 B) -BPA, L-BPA or 4-borono-L-phenylalanine (Sigma Aldrich, St. Louis, MO) has the chemical formula CH 12 It is a synthetic compound with the structure BNO4: [ka] and is an important boronated compound useful in the treatment of cancer by BNCT. It is a widely known compound for which many syntheses have been developed (U.S. Pat. No. 8,765,997, Taiwan Biotech Co., Ltd., Taoyuan Hsein, Taiwan, and U.S. Patent Application Publication No. 2017 / 0015684, Stella Pharma Corp., Osaka Prefecture, Japan). Univ., Osaka, Japan).

[0051] III.) BSH Additionally, BSH, or Borocaptate Sodium, or BSH Borocaptate Sodium, or Borocaptate Sodium 10 B, or undecahydrododecaboranethiol, has the chemical formula NaB 12 H 11 It is a synthetic chemical compound containing SH. Its structure is as follows: [ka] The boron atom is represented by a dot at the vertex of the icosahedron. BSH is used as a capture agent in BNCT. Generally speaking, BSH is injected intravenously and concentrated in tumor cells. The patient then undergoes radiation treatment with atomic particles called neutrons. The neutrons fuse with the boron nuclei in the BSH, producing high-energy alpha particles that kill tumor cells.

[0052] IV.) Boron (a.) Boron in general Generally speaking, for purposes of this disclosure, boron is a chemical element with the symbol B and atomic number 5. Naturally occurring boron, primarily used in chemical compounds, is composed of two stable isotopes, one of which is boron-10 and the other is boron-11. The boron-10 isotope is useful for capturing epithermal neutrons, making it a promising tool in therapeutic settings using boron neutron capture therapy. Biologically, the borylated compounds disclosed herein are non-toxic to humans and animals. Based on the above, it will be apparent to those skilled in the art that improved methods for providing high concentrations of boron to cancer cells would be advantageous. It is an object of the present disclosure to provide such advantages.

[0053] V.) Naturally Occurring Amino Acids Generally speaking, for purposes of this disclosure, naturally occurring amino acids are organic compounds containing amine (-NH) and carboxyl (-COOH) functional groups, along with a side chain (R group) specific to each amino acid. The essential elements of amino acids are carbon (C), hydrogen (H), oxygen (O), and nitrogen (N), although other elements are found in the side chains of certain amino acids. Approximately 500 naturally occurring amino acids are known (although only 20 appear in the genetic code (Table I)), and can be classified in many ways. They can be classified according to the location of the core structural functional group as alpha- (α-), beta- (β-), gamma- (γ-), or delta- (δ-) amino acids; other categories relate to polarity, pH level, and type of side chain group (aliphatic, acyclic, aromatic, hydroxyl-containing, sulfur-containing, etc.). In the form of proteins, amino acid residues form the second most abundant component (water is the most abundant) of human muscle and other tissues. Beyond their role as residues in proteins, amino acids are involved in many processes such as neurotransmitter transport and biosynthesis.

[0054] The twenty (20) amino acids directly encoded by the genetic code (see Table I) can be divided into several groups based on their properties. Important factors are charge, hydrophilicity or hydrophobicity, size, and functional group. These properties are important for protein structure and protein-protein interactions. Water-soluble proteins tend to have their hydrophobic residues (Leu, Ile, Val, Phe, and Trp) buried in the center of the protein, while their hydrophilic side chains are exposed to the aqueous solvent.

[0055] Integral membrane proteins tend to have an exposed outer ring of hydrophobic amino acids that anchor them to the lipid bilayer. Somewhere between these two extremes, some peripheral membrane proteins have patches of hydrophobic amino acids on their surface that anchor them to the membrane. Similarly, proteins that must bind positively charged molecules have surfaces rich in negatively charged amino acids such as glutamic acid and aspartic acid, while proteins that bind negatively charged molecules have surfaces rich in positively charged chains such as lysine and arginine. Different hydrophobic scales of amino acid residues exist.

[0056] Some amino acids have special properties, such as cysteine, which can form covalent disulfide bonds to other cysteine ​​residues, proline, which forms rings to the polypeptide backbone, and glycine, which is more flexible than other amino acids.

[0057] VI.) Borylated Amino Acids (BAAs) As a brief introduction and to better understand the background of the inventive efforts of this disclosure, large neutral amino acid transporter 1 (LAT-1, SLC7a5) is a sodium- and pH-independent transporter that supplies essential amino acids (e.g., leucine, phenylalanine) to cells. The functional transporter is a heterodimeric disulfide-linked complex composed of the multi-membrane-spanning subunit SLC7a5 and the single-membrane-spanning subunit SLC3a2 (CD98). LAT-1 is the primary transporter for channeling essential amino acids across such compartments as the placenta or the blood-brain barrier. LAT-1 also transports the thyroid hormones T3 and T4 (see FRIESEMA et al., Endocrinology, 142(10):4339-4348 (2001)), the dopamine precursor L-DOPA, and amino acid-related exogenous compounds such as the drugs melphalan and gabapentin (see UCHINO et al., Mol. Pharmacol. 61:729-737 (2002)). Furthermore, its expression is highly upregulated in various types of human cancers characterized by a strong requirement for amino acids for metabolism and growth (see SINGH et al., Int. J. Mol. Sci. 2018, 19, 1278). Furthermore, it has been reported that the nature of the amino acid side chain affects the selectivity of LAT-1 for various amino acids in the following order of increasing transport rate: Phe > Trp > Leu > Ile > Met > His > Tyr > Val (see Kanai et al., J. Biol. Chem., vol. 273, No. 37, pp. 23629-23632 (1998)). However, the effect of boron-added modifications on amino acids was unknown in the art, and the present disclosure represents a breakthrough.

[0058] The therapeutic potential of BNCT as an effective cancer treatment depends on the presence of sufficient amounts of BNCT in cancer cells. 10 It is based on the selective accumulation of B.

[0059] Based on the above, those skilled in the art have shown that essential amino acid transporter proteins such as LAT1 are responsible for the uptake of certain natural amino acids. See SCALISE et al., Frontiers in Chem. Vol. 6, Art. 243 (June 2018). With this principle in mind, the present disclosure contemplates the synthesis of natural amino acids through borylation reactions to create borylated amino acids ("BAAs") with tumor-seeking and tumor-localizing properties for use as neutron capture agents in boron neutron capture therapy ("BNCT") and / or boron proton capture therapy, commonly known as proton-boron fusion therapy ("PBFT"). See, for example, HATTORI et al., J. Med. Chem., 55, 6980-6984 (2012).

[0060] (a) BPA-thioundecahydro-dodecaborane, i.e., BPA-BS In one embodiment, within the scope of the present disclosure are precursor compositions having the following formula: [ka]

[0061] Those skilled in the art will appreciate that the above compositions are precursors to more complex branched BAAs that use additional natural amino acids such as phenylalanine, tryptophan, tyrosine, and / or histidine. Synthesis of BPA-BS, as shown in Figure 1, can be achieved by peptide coupling conditions using Fmoc-protected BPA, followed by deprotection to reveal the target material.

[0062] (b) BPA-aminoundecahydrododecaborane, or BPA-BN. In one embodiment, within the scope of the present disclosure is a second precursor composition having the following formula: [ka]

[0063] Those skilled in the art will appreciate that the BPA-BN precursor is a modification of BPA-BS and is a further precursor to more complex branched BAAs using additional natural amino acids such as phenylalanine, tryptophan, tyrosine, and / or histidine. Synthesis is performed as shown in Figure 2. For further reference, see KIRIHATA et al., 18 th See International BNCT Conference, Taipei (October 2018). Synthesis of these compounds can be achieved by peptide coupling between Fmoc-protected BPA and undecahydrododecaborate ammonium, followed by deprotection to reveal the target material.

[0064] The precursors of the present disclosure can be utilized to synthesize BAAs with functional incorporation in specific complexes to deliver concentrated amounts of boron to cancer or other diseased cells for use in BNCT and / or other cancer treatment modalities. In the present disclosure, in one embodiment, the amino acids include valine, leucine, isoleucine, histidine, tryptophan, tyrosine, and any set of amino acids set forth in Table I.

[0065] This principle can be achieved through side-chain manipulation, peptide coupling, and decarboxylation-boronation. See LI et al., Science 356, 1045 (2017); Synlett 1996(02):167-168; and U.S. Patent Application Publication No. 2018 / 0155368 (Neuboron Medtech, Nanjing, China). The wide variety of useful reactivities inherent to boronic acids, such as cross-coupling, oxidation, amination, and homologation, has been shown to guide retrosynthetic analysis. Manipulation of solubility and lipophilicity through the use of boronic esters instead of acids is also contemplated in the present disclosure. Following the modifications disclosed herein, additional antigen complexes and transporters can be associated through selective boronation of their respective molecular substrates.

[0066] (c) Amino acid composition In one embodiment, within the scope of the present disclosure are BAAs having the following formula ("phenylalanine derivatives"): [ka] In the formula, E=CO2H, CONHB 12 H 11 , B(OH)2; and X=H, B(OH)2, Bpin, (-O-CH2CH2)2-OB 12 H 11 .

[0067] In further embodiments, BAAs having the following formula are within the scope of the present disclosure ("histidine derivatives"): [ka] In the formula, E=CO2H, CONHB 12 H 11 , B(OH)2; and X=H, B(OH)2, Bpin, (-O-CH2CH2)2-OB 12 H 11 .

[0068] In further embodiments, BAAs having the following formula are within the scope of the present disclosure ("tyrosine derivatives"): [ka] In the formula, E=CO2H, CONHB 12 H 11 , B(OH)2; and X=H, B(OH)2, Bpin, (-O-CH2CH2)2-OB 12 H 11 .

[0069] In a further embodiment, within the scope of the present disclosure are BAAs having the following formula: [ka]

[0070] (d) BAA containing phenylalanine Phenylanine has the following chemical formula: [ka] Phenylanine is an essential amino acid and a precursor to the amino acid tyrosine. Phenylanine is highly concentrated in the human brain and plasma. High plasma concentrations of phenylalanine affect blood-brain barrier transport of large neutral amino acids. High plasma phenylalanine concentrations increase the influx of phenylalanine into the brain and restrict the influx of other large neutral amino acids. Phenylanine has been found to interfere with various brain enzyme systems. Phenylanine is better absorbed than tyrosine and is known to cause fewer headaches. Certain cancers are known to utilize more phenylalanine than other types of cancer. For example, melanoma has been shown to utilize higher concentrations of phenylalanine.

[0071] Thus, the use of borylated phenylalanines as neutron capture agents in certain cancers is contemplated by the present disclosure.

[0072] In one embodiment of the present disclosure, the ferrylalanine-containing BAA has the following chemical formula: [ka] Note that the dots in structure IV represent BH. The black dot at one vertex represents B. The B12H11 cluster has a net charge of -2.

[0073] (e) BAA containing tryptophan Tryptophan has the following chemical formula: [ka] Tryptophan is an essential amino acid and a precursor to both serotonin and melatonin. Melatonin is a hormone produced by the mammalian pineal gland that regulates sleep and wakefulness. Serotonin is a neurotransmitter in the brain, a platelet clotting factor, and a neurohormone found in organs throughout the body. Many conditions or diseases are characterized by tryptophan deficiency.

[0074] For example, fructose malabsorption causes inadequate absorption of tryptophan in the intestine, lowering blood tryptophan levels and leading to depression. A high-corn or other tryptophan-deficient diet can cause pellagra, a niacin-tryptophan deficiency disorder with symptoms of dermatitis, diarrhea, and dementia. Hartnup disease is a disorder in which tryptophan and other amino acids are not properly absorbed. Symptoms of Hartnup disease include skin rash, difficulty with coordination (cerebellar ataxia), and psychiatric symptoms (such as depression or psychosis). Tryptophan plays a role in "meal-induced" sleepiness. Consumption of a carbohydrate-rich meal triggers the release of insulin. Insulin then stimulates the uptake of large, neutral, branched-chain amino acids (BCAAs) into muscles, increasing the ratio of tryptophan to BCAAs in the bloodstream. Increasing the tryptophan ratio reduces competition at the large neutral amino acid transporter (which transports both BCAAs and tryptophan), resulting in greater uptake of tryptophan across the blood-brain barrier into cerebrospinal fluid (CSF). Once in the CSF, tryptophan is converted to serotonin, which is further metabolized by the pineal gland to melatonin, which promotes sleep.

[0075] Thus, the use of borylated tryptophan as a neutron capture agent in certain cancers is contemplated by the present disclosure.

[0076] In one embodiment of the present disclosure, the tryptophan-containing BAA has the following chemical formula: [ka]

[0077] (f) BAA containing tyrosine Tyrosine has the following chemical formula: [ka] Tyrosine is an essential amino acid with a cytosolic acid and is known to readily cross the blood-brain barrier. Once in the brain, tyrosine is a precursor to the neurotransmitters dopamine, norepinephrine, and epinephrine (commonly known as adrenaline). These neurotransmitters are an important part of the body's sympathetic nervous system, and their concentrations in the body and brain depend directly on dietary tyrosine. Tyrosine is rapidly metabolized. Folate, copper, and vitamin C are cofactor nutrients for these reactions. Tyrosine is also a precursor to the hormones thyroid gland, catechol estrogens, and the major human pigment, melanin. Tyrosine is an important amino acid in many proteins, peptides, and even enkephalin, the body's natural painkiller. Valine and other branched-chain amino acids, as well as possibly tryptophan and phenylalanine, can reduce tyrosine absorption. Many genetic errors of tyrosine metabolism occur, including hawkingsinuria and tyrosinemia type I. The most common is elevated blood tyrosine levels in premature infants, which are characterized by decreased motor activity, lethargy, and poor feeding. Infections and intellectual disability can occur. Some adults also experience elevated blood tyrosine levels, indicating a need for more vitamin C. Generally speaking, tyrosine is needed under stress, and tyrosine supplements can suppress stress-induced depletion of norepinephrine and cure biochemical depression.

[0078] Thus, the use of borylated tyrosine as a neutron capture agent in certain cancers is contemplated by the present disclosure.

[0079] In one embodiment of the present disclosure, the tyrosine-containing BAA has the following chemical formula: [ka]

[0080] (g) BAA containing histidine Histidine has the following chemical formula: [ka] Histidine is an α-amino acid containing an imidazole functional group. It is one of the twenty-two (22) amino acids that make up proteins. It is an essential amino acid in humans and other mammals. Histidine is a precursor for histamine and carnosine biosynthesis. Congenital disorders of histidine metabolism, including histidinemia, maple syrup urine disease, propionic acidemia, and tyrosinemia type I, exist and are characterized by elevated blood histidine levels. Elevated blood histidine levels are associated with a wide range of symptoms, from mental and physical retardation to decreased intellectual function, emotional lability, tremor, ataxia, and psychosis. Histidine and other imidazole compounds possess antioxidant, anti-inflammatory, and antisecretory properties. The effectiveness of L-histidine in protecting inflamed tissues is attributed to the ability of the imidazole ring to scavenge reactive oxygen species (ROS) generated by cells during acute inflammatory responses. When administered in therapeutic doses, histidine can inhibit cytokines and growth factors involved in cell and tissue damage (see U.S. Patent No. 6,150,392, Thomes et al.). Histidine in medical treatment has seen its most promising results in rheumatoid arthritis, where doses of up to 4.5 g per day have been used effectively in severely affected patients. Arthritis patients have been found to have low serum histidine levels, apparently due to the very rapid removal of histidine from the blood. Other patients besides arthritis who have been found to have low serum histidine include those with chronic renal failure. Urinary histidine levels are reduced in pediatric patients with pneumonia. Asthma patients show increased serum histidine levels compared with normal controls. Lower serum histidine levels are negatively associated with inflammation and oxidative stress in obese women. Histidine supplementation has been shown to reduce insulin resistance, decrease BMI and fat mass, and suppress inflammation and oxidative stress in obese women with metabolic syndrome. Histidine appears to suppress pro-inflammatory cytokine expression in adipocytes, possibly via the NF-κB pathway. Low plasma histidine levels are associated with protein-energy wasting, inflammation, oxidative stress, and greater mortality in patients with chronic kidney disease.Histidine may have many other possible functions because it is the precursor of histamine, a ubiquitous neurohormone-neurotransmitter. Histidine increases histamine in the blood and potentially in the brain. Low serum histamine, along with low serum histidine, occurs in patients with rheumatoid arthritis. Low serum histamine also occurs in some manic, schizophrenic, high copper, and hyperactive groups of psychiatric patients.

[0081] Thus, the use of borylated histidine as a neutron capture agent in certain cancers is contemplated by the present disclosure.

[0082] In one embodiment of the present disclosure, the histidine-containing BAA has the following chemical formula: [ka]

[0083] VII.) Boron Neutron Capture Therapy using BAA One aspect of the present disclosure is the use of BAA as a modality for boron neutron capture therapy (BNCT) and / or boron proton capture therapy ("BPCT"). Briefly, BNCT is a two-component treatment modality in which neither component alone is lethal or toxic to the tumor. The two components include (i) injection or delivery of a capture compound that preferentially concentrates in the tumor, and (ii) irradiation of the tumor site with neutrons or protons. In BNCT, 10 In view of the large cross section of thermal neutron interaction with B, the result is that the boron nucleus 4 He 2+ and 7 Li + He 2+ and Li +Given the high ionization capacity of boron and the short distance traveled, cells favorably enriched with boron are killed, while healthy cells are much less damaged due to the lack of high concentrations of boron. In light of this, the advantage of BNCT is that it destroys tumor cells without highly traumatic surgical procedures. However, as will be appreciated by those skilled in the art, success depends on the specificity of the tumor cells. 10 This is based on the selective localization of B at high concentrations.

[0084] In one embodiment, 10 B is concentrated on the BAA. The BAA is then administered to the patient, and the BAA is allowed to localize in tumor cells. 10 B-containing BAAs are concentrated in the tumor, and epithermal neutrons are used to irradiate the tumor, destroying the tumor cells.

[0085] VIII. Proton-boron fusion therapy using BAA Another aspect of the present disclosure is the use of BAA as a modality for proton-boron fusion therapy (PBFT). Briefly, the proton-boron fusion reaction was introduced in the 1960s. 1 H) and boron particles ( 11After the reaction between B) and C), three α particles are released. These three α particles damage tumor cells, similar to the α particles in BNCT. Theoretically, the therapeutic efficacy per incident particle in PBFT is three times (3x) greater than that of BNCT. Furthermore, proton beams have the advantage of Bragg peak characteristics, which can reduce normal tissue damage. Generally speaking, many studies have been conducted on tumor treatment using α particles. To utilize α particles for dose delivery, two important points should be considered. First, boron uptake should be accurately labeled in the target cells. As mentioned above, α particles are generated at the site of boronate compound accumulation. If this occurs in normal tissue near the tumor area, the α particles will damage not only tumor cells but also normal tissue. Second, the number of α particles generated is also an important factor for effective treatment. PBFT can achieve more effective treatment compared to BNCT or conventional proton therapy alone.

[0086] In one embodiment, 10 B and / or 11 B is concentrated on the BAA. The BAA is then administered to the patient, and the BAA is allowed to localize in tumor cells. 10 B and / or 11 B-containing BAAs are concentrated in the tumor, and epithermal neutrons are used to irradiate the tumor, destroying the tumor cells.

[0087] IX. Methods of Delivering BAAs to Cells As will be appreciated by those skilled in the art, it is an advantage of the present invention that high concentrations of boron can be efficiently delivered to cells.

[0088] The BAA of the present disclosure has been shown to enable safe administration of larger amounts of boron to cells in mammals.Briefly, the BAA of the present disclosure is prepared as described in the present disclosure.The obtained BAA is taken up by tumor cells through the upregulated LAT-1 transporter protein.

[0089] X.) Kits / Manufactured Products Kits are within the scope of the present invention for use in the laboratory, prognostic, preventive, diagnostic, and therapeutic applications described herein. Such kits can include a carrier, package, or container compartmentalized to receive one or more containers, such as vials, tubes, etc., each containing one of the individual components to be used in the method, along with a label or insert containing instructions for use, such as those described herein. For example, a container can contain one or several BAAs of the present disclosure. The kit can include a container containing a drug unit. The kit can include all or part of the BAA and / or a diagnostic assay for detecting cancer and / or other immune disorders.

[0090] Kits of the present invention typically include the above-described container and one or more other containers associated with the above-described container with equipment desirable from a commercial and user standpoint, such as buffers, diluents, filters, needles, syringes, etc.; labels on the carrier, package, container, vial, and / or tube listing the contents and / or instructions for use; and package inserts containing the instructions for use.

[0091] A label can be present on or with the container to indicate that the composition is used for a particular therapeutic or non-therapeutic use, such as prognostic, preventative, diagnostic, or laboratory use, and can also indicate instructions for either in vivo or in vitro use, such as those described herein. Instructions and other information can also be included on an insert or label included with or on the kit. The label can be present on the container or associated with the container. The label can be present on the container if letters, numbers, or other characters forming the label are molded or etched into the container itself, or it can be associated with the container if the label is present in a receptacle or carrier that also holds the container, for example, as a package insert. The label can indicate that the composition is used to diagnose, treat, prevent, or prognose a condition, such as cancer or other immune disorder.

[0092] The terms "kit" and "article of manufacture" can be used synonymously.

[0093] In another embodiment of the present invention, one or more articles of manufacture contain a composition such as a BAA of the present disclosure. The article of manufacture typically includes at least one container and at least one label. Suitable containers include, for example, bottles, vials, syringes, and test tubes. The container can be made of various materials, such as glass, metal, or plastic. The container can hold one or more BAAs and / or one or more therapeutic doses of BAAs.

[0094] Alternatively, the container may hold a composition effective for treating, diagnosing, prognosing or preventing a condition and may have a sterile access port (for example, the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). The active agent in the composition may be a BAA of the present disclosure.

[0095] The article of manufacture may further comprise a second container comprising a pharmaceutically acceptable buffer, such as phosphate-buffered saline, Ringer's solution, and / or dextrose solution. The article of manufacture may further include other buffers, diluents, filters, stirrers, needles, syringes, and / or other devices desirable from a commercial and user standpoint, including package inserts with directions and / or instructions for use.

[0096] Further embodiments of the present disclosure include those described in the following sections. Item 1 is an embodiment of a composition comprising the following chemical structure: [ka] In the formula, E=CO2H, CONHB 12 H 11 , B(OH)2; and X=H, B(OH)2, Bpin, (-O-CH2CH2)2-OB12 H 11 .

[0097] Item 2 is the composition, [ka] An embodiment of the composition includes:

[0098] Item 3 is an embodiment of a composition comprising the following chemical structure: [ka] In the formula, E=CO2H, CONHB 12 H 11 , B(OH)2; and X=H, B(OH)2, Bpin, (-O-CH2CH2)2-OB 12 H 11 .

[0099] Item 4 is the composition, [ka] An embodiment of the composition includes:

[0100] Item 4 is an embodiment of a composition comprising the following chemical structure: [ka] In the formula, E=CO2H, CONHB 12 H 11 , B(OH)2; and X=H, B(OH)2, Bpin, (-O-CH2CH2)2-OB 12 H 11 .

[0101] Item 5 is the composition, [ka] An embodiment of the composition includes:

[0102] Item 6 is an embodiment of a kit comprising the composition of item 1.

[0103] Item 7 is an embodiment of a kit comprising the composition of item 2.

[0104] Item 8 is an embodiment of a kit comprising the composition of item 3.

[0105] Item 9 is an embodiment of a kit comprising the composition of item 4.

[0106] Item 10 is an embodiment of a kit comprising the composition of item 5.

[0107] Item 11 is an embodiment of a kit comprising the composition of item 6.

[0108] Item 12 is an embodiment of a dosage unit form comprising the composition of item 1.

[0109] Item 13 is an embodiment of a dosage unit form comprising the composition of item 2.

[0110] Item 14 is an embodiment of a dosage unit form comprising the composition of item 3.

[0111] Item 15 is an embodiment of a dosage unit form comprising the composition of item 4.

[0112] Item 16 is an embodiment of a dosage unit form comprising the composition of item 5.

[0113] Item 17 is an embodiment of a dosage unit form comprising the composition of item 6.

[0114] Item 18 is an embodiment of the human unit form of item 12, wherein the human unit form is used in boron neutron capture therapy (BNCT).

[0115] Item 19 is an embodiment of the human unit form of item 13, wherein the human unit form is used in boron neutron capture therapy (BNCT).

[0116] Item 20 is an embodiment of the human unit form of item 14, wherein the human unit form is used in boron neutron capture therapy (BNCT).

[0117] Item 21 is an embodiment of the human unit form of item 15, wherein the human unit form is used in boron neutron capture therapy (BNCT).

[0118] Item 22 is an embodiment of the human unit form of item 16, wherein the human unit form is used in boron neutron capture therapy (BNCT).

[0119] Item 23 is an embodiment of the human unit form of item 17, wherein the human unit form is used in boron neutron capture therapy (BNCT).

[0120] Item 24 is an embodiment of a method for producing the composition of item 1.

[0121] Item 25 is an embodiment of a method for producing the composition of item 2.

[0122] Item 26 is an embodiment of a method for producing the composition of item 3.

[0123] Item 27 is an embodiment of a method for producing the composition of item 4.

[0124] Item 28 is an embodiment of a method for producing the composition of item 5.

[0125] Item 29 is an embodiment of a method for producing the composition of item 6.

[0126] Term 30 is the following formula: [ka] In one embodiment, the borylated amino acid ("BAA") has the formula:

[0127] Item 31 is an embodiment of a method for producing the composition of item 30.

[0128] Item 32 is an embodiment of a method for performing boron neutron capture therapy ("BNCT") on a mammal, comprising concentrating BAAs in cells, the method comprising: (i) administering BAAs to a subject; and (iii) irradiating the cells with neutrons.

[0129] Item 33 is an embodiment of a method of performing boron neutron capture therapy ("BNCT") on a mammal, comprising concentrating a BAA in a cell, the method comprising: (i) administering a BAA to a subject; and (iii) irradiating the cell with neutrons, wherein the BAA comprises the composition of Item 1.

[0130] Item 34 is an embodiment of a method of performing boron neutron capture therapy ("BNCT") on a mammal, comprising concentrating a BAA in a cell, the method comprising: (i) administering a BAA to a subject; and (iii) irradiating the cell with neutrons, wherein the BAA comprises the composition of Item 2.

[0131] Item 35 is an embodiment of a method of performing boron neutron capture therapy ("BNCT") on a mammal, comprising concentrating a BAA in a cell, the method comprising: (i) administering a BAA to a subject; and (iii) irradiating the cell with neutrons, wherein the BAA comprises the composition of Item 3.

[0132] Item 36 is an embodiment of a method of performing boron neutron capture therapy ("BNCT") on a mammal, comprising concentrating a BAA in a cell, the method comprising: (i) administering a BAA to a subject; and (iii) irradiating the cell with neutrons, wherein the BAA comprises the composition of Item 4.

[0133] Item 37 is an embodiment of a method of performing boron neutron capture therapy ("BNCT") on a mammal, comprising concentrating a BAA in a cell, the method comprising: (i) administering a BAA to a subject; and (iii) irradiating the cell with neutrons, wherein the BAA comprises the composition of Item 5.

[0134] Item 38 is an embodiment of a method of performing boron neutron capture therapy ("BNCT") on a mammal, comprising concentrating a BAA in a cell, the method comprising: (i) administering a BAA to a subject; and (iii) irradiating the cell with neutrons, wherein the BAA comprises the composition of Item 6.

[0135] Paragraph 39 is an embodiment of a method of performing boron neutron capture therapy ("BNCT") on a mammal, comprising concentrating a BAA in a cell, the method comprising: (i) administering a BAA to a subject; and (iii) irradiating the cell with neutrons, wherein the BAA comprises the composition of paragraph 30.

[0136] Paragraph 40 is an embodiment of a method for performing neutron capture therapy in the treatment of human cancer, the method comprising: (a) synthesizing a human unit dose of a borolyated amino acid (BAA) composition; (b) injecting the BAA into a tumor, whereby the BAA accumulates intracellularly; and (c) irradiating the BAA with neutrons.

[0137] Paragraph 41 is an embodiment of a method of performing neutron capture therapy in the treatment of human cancer, comprising: (a) synthesizing a human unit dose of a borylated amino acid (BAA) composition; (b) injecting the BAA into a tumor, whereby the BAA accumulates intracellularly; and (c) irradiating the BAA with neutrons, wherein the BAA is the composition of paragraph 1.

[0138] Paragraph 42 is an embodiment of a method of performing neutron capture therapy in the treatment of human cancer, comprising: (a) synthesizing a human unit dose of a borylated amino acid (BAA) composition; (b) injecting the BAA into a tumor, whereby the BAA accumulates intracellularly; and (c) irradiating the BAA with neutrons, wherein the BAA is the composition of paragraph 2.

[0139] Paragraph 43 is an embodiment of a method of performing neutron capture therapy in the treatment of human cancer, comprising: (a) synthesizing a human unit dose of a borylated amino acid (BAA) composition; (b) injecting the BAA into a tumor, whereby the BAA accumulates intracellularly; and (c) irradiating the BAA with neutrons, wherein the BAA is the composition of paragraph 3.

[0140] Paragraph 44 is an embodiment of a method of performing neutron capture therapy in the treatment of human cancer, comprising: (a) synthesizing a human unit dose of a borylated amino acid (BAA) composition; (b) injecting the BAA into a tumor, whereby the BAA accumulates intracellularly; and (c) irradiating the BAA with neutrons, wherein the BAA is the composition of paragraph 4.

[0141] Paragraph 45 is an embodiment of a method of performing neutron capture therapy in the treatment of human cancer, comprising: (a) synthesizing a human unit dose of a borylated amino acid (BAA) composition; (b) injecting the BAA into a tumor, whereby the BAA accumulates intracellularly; and (c) irradiating the BAA with neutrons, wherein the BAA is the composition of paragraph 5.

[0142] Paragraph 46 is an embodiment of a method of performing neutron capture therapy in the treatment of human cancer, comprising: (a) synthesizing a human unit dose of a borylated amino acid (BAA) composition; (b) injecting the BAA into a tumor, whereby the BAA accumulates intracellularly; and (c) irradiating the BAA with neutrons, wherein the BAA is the composition of paragraph 6.

[0143] Paragraph 47 is an embodiment of a method of performing neutron capture therapy in the treatment of human cancer, comprising: (a) synthesizing a human unit dose of a borylated amino acid (BAA) composition; (b) injecting the BAA into a tumor, whereby the BAA accumulates intracellularly; and (c) irradiating the BAA with neutrons, wherein the BAA is the composition of paragraph 30.

[0144] Item 48 is an embodiment of the method for performing neutron capture therapy according to any one of Items 40 to 47, wherein the neutron capture therapy is boron neutron capture therapy.

[0145] Item 49 is an embodiment of the method for performing neutron capture therapy according to any one of Items 40 to 47, wherein the irradiation includes epithermal neutrons.

[0146] Paragraph 50 is an embodiment of a method for performing proton-boron fusion therapy in the treatment of human cancer, comprising: (a) synthesizing a human unit dose of a borolyated amino acid (BAA) composition; (b) injecting the BAA into a tumor, whereby the BAA accumulates intracellularly; and (c) irradiating the BAA with protons.

[0147] Paragraph 51 is an embodiment of a method for performing proton-boron fusion therapy in the treatment of human cancer, comprising: (a) synthesizing a human unit dose of a borylated amino acid (BAA) composition; (b) injecting the BAA into a tumor, whereby the BAA accumulates intracellularly; and (c) irradiating the BAA with protons, wherein the BAA comprises the composition of paragraph 1.

[0148] Paragraph 52 is an embodiment of a method for performing proton-boron fusion therapy in the treatment of human cancer, comprising: (a) synthesizing a human unit dose of a borylated amino acid (BAA) composition; (b) injecting the BAA into a tumor, whereby the BAA accumulates intracellularly; and (c) irradiating the BAA with protons, wherein the BAA comprises the composition of paragraph 2.

[0149] Paragraph 53 is an embodiment of a method for performing proton-boron fusion therapy in the treatment of human cancer, comprising: (a) synthesizing a human unit dose of a borylated amino acid (BAA) composition; (b) injecting the BAA into a tumor, whereby the BAA accumulates intracellularly; and (c) irradiating the BAA with protons, wherein the BAA comprises the composition of paragraph 3.

[0150] Paragraph 54 is an embodiment of a method for performing proton-boron fusion therapy in the treatment of human cancer, comprising: (a) synthesizing a human unit dose of a borylated amino acid (BAA) composition; (b) injecting the BAA into a tumor, whereby the BAA accumulates intracellularly; and (c) irradiating the BAA with protons, wherein the BAA comprises the composition of paragraph 4.

[0151] Paragraph 55 is an embodiment of a method for performing proton-boron fusion therapy in the treatment of human cancer, comprising: (a) synthesizing a human unit dose of a borylated amino acid (BAA) composition; (b) injecting the BAA into a tumor, whereby the BAA accumulates intracellularly; and (c) irradiating the BAA with protons, wherein the BAA comprises the composition of paragraph 5.

[0152] Paragraph 56 is an embodiment of a method for performing proton-boron fusion therapy in the treatment of human cancer, comprising: (a) synthesizing a human unit dose of a borylated amino acid (BAA) composition; (b) injecting the BAA into a tumor, whereby the BAA accumulates intracellularly; and (c) irradiating the BAA with protons, wherein the BAA comprises the composition of paragraph 6.

[0153] Paragraph 57 is an embodiment of a method for performing proton-boron fusion therapy in the treatment of human cancer, comprising: (a) synthesizing a human unit dose of a borylated amino acid (BAA) composition; (b) injecting the BAA into a tumor, whereby the BAA accumulates intracellularly; and (c) irradiating the BAA with protons, wherein the BAA comprises the composition of paragraph 30. [Example]

[0154] Working Example: Various aspects of the present invention are further described and illustrated by the following several examples, none of which are intended to limit the scope of the invention.

[0155] Example 1: Synthesis of BAA No. 1 containing phenylalanine. The phenylalanine-containing BAA No. 1 is synthesized as follows: protected BPA is subjected to decarboxylation-borylation followed by deprotection to reveal the target substance.

[0156] BAA number 1, which contains phenylalanine, has the following chemical structure: [ka]

[0157] Example 2: Synthesis of histidine-containing BAA number 2. Histidine-containing BAA number 2 is synthesized in the following manner: the protected histidine is subjected to decarboxylation-borylation, followed by deprotection to reveal the target substance.

[0158] BAA number 2, which contains histidine, has the following chemical structure: [ka]

[0159] Example 3: Synthesis of histidine-containing BAA number 3. The histidine-containing BAA number 3 is synthesized in the following manner: the protected histidine succinate is subjected to nucleophilic addition with BNH, followed by deprotection to reveal the target substance.

[0160] BAA number 3, which contains histidine, has the following chemical structure: [ka]

[0161] Example 4: Synthesis of histidine-containing BAA number 4. The histidine-containing BAA number 4 is synthesized in the following manner: the protected histidine is subjected to organolithiation, then added to trimethylborate, and then deprotected to reveal the target material.

[0162] BAA number 4, which contains histidine, has the following chemical structure: [ka]

[0163] Example 5: Synthesis of TLS00192. The BAA compound known as TLS00192 was synthesized using the following protocol (see Figure 3). First, triethylamine is added to a solution of tyrosine methyl ester in dichloromethane, followed by the addition of di-tert-butyl dicarbonate. After completion of the reaction, unreacted starting materials are removed in an acidic wash, and then the solvent is removed under reduced pressure.

[0164] The isolated boc-tyrosine methyl ester was then added to a solution of DMF and potassium carbonate, followed by the addition of methyl iodide. Upon completion of the reaction, it was diluted with water, extracted into ethyl acetate, and the organic layer was washed with saturated aqueous NaCl and dried over anhydrous sodium sulfate.

[0165] The protected tyrosine compound was then added to a mixture of iodine and silver sulfate in methanol. Upon completion of the reaction, the precipitate was removed by filtration. The filtrate was washed with 10% aqueous sodium bisulfite solution and then with water. After drying over anhydrous sodium sulfate, the solvent was removed under reduced pressure. The product was isolated by column chromatography on silica gel.

[0166] The catalyst Pd(dppf)Cl2, bis(pinacolato)diboron, and potassium acetate were then added to DMSO, and the system was flushed with nitrogen. A DMSO solution of iodo-tyrosine was then added to the reaction, and the temperature was raised to 80°C. The product was extracted into ethyl acetate with water washes, dried over magnesium sulfate, and filtered, after which the solvent was removed under reduced pressure.

[0167] The pinacol boronated tyrosine was then solubilized in acetone and NaIO4 was added. After stirring the reaction mixture for 15 minutes, 1M HCl was added and the reaction was stirred for 4 hours. The resulting mixture was extracted with EtOAc, washed with deionized water, and finally with brine. The organic layer was dried over MgSO4. After filtration, the organic solvent was removed under vacuum.

[0168] Next, a solution of the above-synthesized methyl ether tyrosine boronate in DCM was brought to -78°C and flushed with nitrogen. Boron tribromide was added dropwise to the mixture and allowed to react for 12 hours. The reaction mixture was poured into water, extracted, dried over magnesium sulfate, and then the solvent was removed under vacuum.

[0169] Finally, the methyl tyrosine boronate was placed in a 3:1 solution of THF:water. LiOH was added and the reaction was continued until only the target compound was observed. The solution was neutralized with HCl, and the solvent was removed under reduced pressure.

[0170] TLS00192 has the following chemical structure: [ka]

[0171] Example 6: Synthesis of TLS00178. The BAA compound known as TLS00178 was synthesized using the following protocol (see Figure 4). First, 1-ammine-undecahydrododecaborate was added to a DMF solution at 0 °C, followed by portionwise addition of sodium hydride. Once gas evolution ceased, a boc-protected succinimide ester of histidine was added. After concentration, the material was telescoped forward. 4 molar HCl in dioxane was added to the material, followed by 12% water by volume. Once the reaction was completely converted, as monitored by LCMS, the solvent was removed under reduced pressure. The target material was obtained by trituration with ethanol.

[0172] TLS00178 has the following chemical structure: [ka]

[0173] Example 7: Synthesis of TLS00190. The BAA compound known as TLS00190 was synthesized using the following protocol (see Figure 5). First, the boc-protected methyl ester of tyrosine was added to a solution of acetonitrile / water, 20% v / v, at 50 °C. Potassium carbonate was then added to the solution and allowed to react for 15 minutes. 1-(1,4-dioxane)-undecahydrododecaborate was obtained. The reaction was allowed to proceed overnight. The solvent was exchanged to MeOH / water, 20% v / v. Lithium hydroxide was added and the solution was refluxed. Once no more methyl ester was observed, the reaction was neutralized with HCl and the solvent was removed. This material was dissolved in 4 molar HCl in dioxane and 20% v / v water was added. The target material was obtained by preparative liquid chromatography.

[0174] TLS00190 has the following chemical structure: [ka]

[0175] Example 8: LCMS purity and mass confirmation of TLS00192, TLS00178 and TLS00190. LS / MS purity confirmation of TLS00192, TLS00178, and TLS00190 was performed by LCMS. Briefly, LCMS was performed using an Acquity H-class system (Waters, Milford, MA) equipped with either an Acquity BEH C18 column (50 × 2.1 mm, 1.7 μm) or a C18 peptide CSH column (100 × 2.1 mm, 1.7 μm) maintained at 40 °C or 60 °C in a gradient of acetonitrile containing 0.1% formic acid. Detection was performed using a QDA ESI mass spectrometer.

[0176] The results in Figures 6-8 show HPLC UV traces demonstrating the purity and mass confirmation of TLS00192 (Figure 6), TLS00178 (Figure 7), and TLS00190 (Figure 8). All compounds were manufactured with purities meeting or exceeding 90%, and the MS ionization mode was optimized for each compound. Additionally, a summary of the purity and mass confirmation of TLS00192, TLS00178, and TLS00190 is shown in Figure 9.

[0177] Example 9: Kinetic parameters of TLS00192 and BPA-fructose. To examine the ability of TLS00192 to deliver boron to FaDu nasopharyngeal squamous cell carcinoma cells, we investigated a range of concentrations encompassing hypothsized physiologically relevant concentrations for boronophenylalanine (BPA), the boron drug currently most widely studied in BNCT clinical practice.

[0178] The FaDu cell line is a human nasopharyngeal carcinoma cell line. The cell line was identified as 10801 in 2019. The cell line was obtained from the American Type Culture Collection ("ATCC"), located at University Boulevard, Manassas, VA 20110-2209 USA. This cell line has the ATCC designation HTB-43™ and lot number 70014320. FaDu cells are grown in DMEM culture medium supplemented with 10% fetal bovine serum, with routine passage by trypsinization and reseeding. The cell line is stored frozen in liquid nitrogen.

[0179] Additionally, boron measurements were performed by ICP OES on an Agilent 5110 ICP-OES. Data were analyzed using Agilent's ICP Expert Software, version 7.4.2.10790. Boron was measured axially at a wavelength of 249.772 nm, and the internal standard beryllium was measured axially at a wavelength of 313.042 nm. The beryllium internal standard was added to the solution at a flow rate of 1:5 before introduction into the spray chamber via a T-connector. Finally, the boron concentration in each sample was calculated using a standard curve using 1000, 100, 10, 1, and 0 ppb of boron.

[0180] To determine the kinetic parameters of TLS00192, boron compounds were added to FaDu cells at a final concentration of 2.5 mM in HBSS, and the cells were incubated for 2 hours with shaking at 37°C in a humidified 5% CO2 atmosphere. After 2 hours of incubation, the cells were harvested, suspended in ice-cold PBS, and lysed in RIPA buffer. Protein content was determined by BCA assay. A portion was also subjected to boron measurement using ICP-OES. Results were expressed as ng boron / mg cell protein / min. Kinetic parameters, velocity, and Km, were determined by Michaelis-Menton nonlinear regression using Prism (GraphPad) software.

[0181] The results show that uptake saturation was reached at substrate concentrations above 5 mM and plateaued at approximately 10 mM. Both TLS00192 and the BPA-fructose compound exhibited efficient, saturable, concentration-dependent cellular uptake, following typical Michaelis-Menten kinetics. Michaelis-Menten nonlinear regression curve fitting indicated that the Km of TLS00192 was approximately 50% of the Km of BPA-fructose (1.97 mM vs. 0.84 mM, respectively), suggesting that BPA may be a preferred LAT-1 substrate (Figure 10).

[0182] It is noted that this data is consistent with that reported for non-borylated amino acids. See Kanai et al., J. Biol. Chem. 1998, 273, 23629-23632. However, the apparent Vmax for TLS00192 is higher (8.91 vs. 15.87 ng boron mg -1 min -1 The higher rate of boron accumulation in the cell lines for TLS00192 indicates either (i) a higher uptake rate or (ii) slower efflux (i.e., better retention of TLS00192 compared to BPA-fructose).

[0183] Example 10: Cellular retention of TLS00192 and BPA-fructose in FaDu cells. Subsequently, cell retention studies in FaDu cells were performed to determine whether efflux mediated by either LAT1 or LAT2 or other transporters was equivalent for BPA-fructose compared to TLS00192. "Time 0" samples were collected immediately after the first 2 hours of incubation with the compounds. Cells were then harvested at the indicated times and subjected to lysis, boron determination, and protein content. Data are expressed as % of the residual boron content of the time 0 amount.

[0184] The results in Figure 11 show that boron is gradually removed from the cells at both time points. However, the removal of TLS00192 is much slower compared to BPA-fructose. It is notable that by 16 hours, there is over 40% remaining TLS00192 compared to approximately 10% remaining BPA. These results demonstrate the unique characteristics of TLS00192 compared to BPA, which improves the amount of boron delivered to cells.

[0185] Example 11: LAT-1 mediated competition studies with TLS00192. The L-system transporter 1 (LAT-1) mediates the transport of L-amino acids into cells and has been shown to play a major role in BPA uptake during BPA-based BNCT (Wongthai et al., Cancer Sci Vol. 106, pp. 279-286, 2015). To assess whether TLS00192 is transported via an LAT-1-dependent mechanism, we examined its ability to be outcompeted by L-Phe, the LAT1 substrate. FaDu cells were incubated in HBSS medium with either 0.5 mM TLS00192 or BPA-fructose for 2 hours at 37°C in the absence (Subset A) or presence (Subset B) of increasing concentrations of the competitor Phe, the L-system antagonist BCH (Subset C), or the LAT-1-specific inhibitor JPH203 (Subset D). Cells were harvested and the amount of each boronated cell-associated compound determined by ICP OES. The IC50 of the respective inhibitor for each compound is also shown. BPA was used as a positive control for LAT-1-mediated uptake.

[0186] The results in Figure 12A show the uptake of the pure compound in the absence of competitor. For this and subsequent studies, TLS00192 was maintained at 0.5 mM. Increasing the concentration of Phe in the range of 0.01 to 20 mM showed decreased uptake for both BPA and TLS00192 (see Figure 12B). The unexpected result that approximately 1 log higher concentrations of L-Phe competitor were required to inhibit LAT1-mediated transport of TLS00192 is noteworthy. The IC50 values ​​were 0.04 and 0.43 mM for L-Phe in the presence of BPA-fructose and TLS00192, respectively. The results demonstrate that TLS00192 is able to better compete with endogenous amino acids and effectively utilize LAT-1 for cellular accumulation compared to BPA.

[0187] Next, we evaluated the pan-LAT antagonist 2-aminobicyclo-(2,2,1)-heptane-2-carboxylic acid (BCH) (Figure 12C) and the specific LAT-1 inhibitor JPH203 (Figure 12D) in competition assays. Similar to the results in Figure 12B, BCH inhibited boron uptake in a concentration-dependent manner. BPA was more readily competed with BCH compared to TLS00192 (0.14 and 0.80 mM for BPA and TLS00192, respectively). JPH203 was an even more potent inhibitor of LAT1-mediated uptake of TLS00192 and BPA than BCH, e.g., 0.19 and 0.78 μM, respectively (Figure 12D). The results indicate that (i) TLS00192 uptake is LAT-1 mediated and (ii) higher concentrations of competitor are required to displace TLS00192 from the LAT-1 binding pocket.

[0188] Example 12: Boron uptake of TLS00190 and TLS00178 in FaDu cells. The boron uptake efficiency of TLS00190 and TLS00178 was determined using FaDu cells. After 2 hours of incubation, the cells were harvested and suspended in ice-cold PBS. A portion was dissolved in RIPA buffer, and the protein content was determined by BCA assay. The remaining portion was subjected to boron measurement using ICP-OES. BPA-fructose was used as a control. After 2 hours of incubation, the amount of incorporated compound was determined based on the boron measurement.

[0189] The results, expressed as ng boron per mg protein, are shown in Figure 13. TLS00190 contains B linked to a phenylalanine side chain. 12 H 11 2- TLS00178 has a boron cluster linked to the C-terminus of a histidine. 12 H 11 2- As shown, both of these compounds, modified with 12 boron clusters on their respective side chains or on the C-terminus, retain the ability to be transported into FaDu cancer cells.

[0190] We also investigated the retention of TLS00190 and TLS00178 after the first 2 hours of uptake in FaDu cells compared with BPA-fructose. As shown in Figure 14, BPA-fructose was removed from the cells to a level of approximately 8% at 20 hours, similar to that observed previously, whereas TLS00190 and TLS00178 were retained at levels of 43% and 57%. Notably, TLS00190 cellular levels decreased to 14% at 2 hours but rose again to 43% at 20 hours, suggesting their ability to reaccumulate within the cells after excretion. These characteristics of TLS00190 and TLS0078 suggest their potential for better accumulation and retention in cancer cells of BNCT patients compared with BPA-fructose.

[0191] Example 13: Human clinical trials for the treatment of human carcinoma through the use of BAAs. BAAs synthesized according to the present invention specifically accumulate in tumor cells and are used to treat certain tumors as well as other immune disorders and / or other diseases. Two clinical approaches have been successfully pursued in relation to each of these indications.

[0192] I.) Adjuvant Therapy: In adjuvant therapy, patients are treated with BAAs in combination with chemotherapeutic agents, drugs, or biologic agents, or combinations thereof. The primary cancer target is treated and then irradiated with the addition of BAAs under standard protocols. Protocol design addresses efficacy, as assessed by examples including, but not limited to, reduction in tumor burden in primary or metastatic lesions, prolonged progression-free survival, overall survival, improved patient health status, disease stabilization, and the ability to reduce conventional doses of standard chemotherapy and other biologic agents. These dose reductions allow for additional and / or prolonged treatment by reducing dose-related toxicity of chemotherapeutic or biologic agents.

[0193] II.) Monotherapy: In connection with the use of BAAs in tumor monotherapy, the BAA is administered to the patient without a chemotherapeutic agent or drug or biologic agent. In one embodiment, monotherapy is clinically implemented in terminal cancer patients with widespread metastatic disease. Protocol design addresses efficacy, as assessed by examples including, but not limited to, reduction in tumor burden in primary or metastatic lesions, prolonged progression-free survival, overall survival, improved patient health status, disease stabilization, and the ability to reduce conventional doses of standard chemotherapy and other biologic agents.

[0194] Dosage Dosage regimens can be adjusted to provide the optimum desired response. For example, a single BAA injection can be administered, several divided doses can be administered over time, or the dose can be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. As used herein, "dosage unit form" refers to a physically discrete unit suitable as a unitary dosage for a mammalian subject to be treated, each unit containing a predetermined amount of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specifications for the dosage unit form of the present invention are determined by and directly depend on (a) the unique characteristics of the BAA, the individual mechanism of the irradiation mechanism (reactor) and the specific therapeutic or prophylactic effect to be achieved, and (b) the limitations inherent in the technology of compounding such compounds to treat susceptibility in individuals.

[0195] Clinical Development Plan (CDP) CDP will pursue and develop the treatment of cancer and / or immune disorders using the BAA of the present disclosure, which will then be irradiated using neutron capture therapy in conjunction with adjuvant therapy or monotherapy.The trial will first demonstrate safety, and then confirm efficacy in repeated administration.The trial will be open-label and will compare standard treatment with standard chemotherapy and the BAA that will then be irradiated using boron neutron capture therapy.As can be understood, one non-limiting criterion that can be used in relation to patient enrollment is the concentration of BAA in tumor, which is determined by standard detection methods known in the art.

[0196] The present invention is not to be limited in scope by the embodiments disclosed herein, which are intended as single illustrations of individual aspects of the invention; any functional equivalents are within the scope of the present invention. Various modifications to the models, methods, and lifecycle methodologies of the present invention, in addition to those described herein, will be apparent to those skilled in the art from the foregoing description and teachings and are also intended to be within the scope of the present invention. Such modifications or other embodiments can be made without departing from the true scope and spirit of the present invention. [Table 1]

Claims

1. The following chemical structure: 【Chemistry 1】 where E = CONHB 12 H 11 ,B(OH) 2 ; and X=H, B(OH) 2 , Bpin, (-O-CH 2 CH 2 ) 2 -O-B 12 H 11 1. A composition comprising: 【Chemistry 2】 A composition comprising:

2. A kit comprising the composition described in claim 1.

3. A dosage unit form comprising the composition of claim 1.

4. The dosage unit form described in claim 3, characterized in that the dosage unit form is used in boron neutron capture therapy (BNCT).

5. The dosage unit form described in claim 4, characterized in that the BNCT is used to treat cancer.

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