Methods for diagnosing amyloid diseases

Abstract

Provided herein are methods for the detection and diagnosis of all types of amyloid-related diseases. Also provided herein are methods of treatment, including selecting a treatment based on the specific type of amyloid disease.

Description

[Technical Field]

[0001] Cross-references to related applications This application claims priority to U.S. Provisional Application No. 63 / 121,779, filed on 4 December 2020, the entire contents of which are incorporated herein by reference in their entirety.

[0002] Submission of sequence listings in ASCII text files. The following content, submitted in ASCII text file, is incorporated in its entirety herein by reference: a computer-readable (CRF) sequence listing (file name: 165992000440SEQLIST.TXT, date: November 29, 2021, size: 7,103 bytes).

[0003] field This application relates to a method for detecting and diagnosing all types of amyloid-related diseases. [Background technology]

[0004] background Amyloidosis is a fatal protein folding disorder characterized by the aggregation and deposition of proteinifibrillars and heparan sulfate proteoglycans in vital organs and tissues (Merlini, G. et al. (2003) N.Engl.J.Med. 349, 583-596; Merlini, G. et al. (2004) J.Intern.Med. 255, 159-178; DeLorenzi, E. et al. (2004) Curr.Med.Chem. 11, 1065-1084; Merlini, G. (2004) Neth.J.Med. 62, 104-105). Amyloid accumulation persists without abatement and always leads to organ failure and severe morbidity or death. Amyloid deposits can be cerebral, as in patients with Alzheimer's disease, Huntington's disease, or prion disease, or peripheral, as seen in patients with light chain (AL) amyloidosis and type 2 diabetes. Further subdivisions into localized and systemic, indicating whether precursor proteins are produced locally (at the deposit site) or circulate in the bloodstream (Westermark, P. et al. (2007) Amyloid. 14, 179-183). Amyloid can affect any organ or tissue except the kidneys, pancreas, liver, spleen, and nerve tissue, with the heart constituting a major deposit site in patients with familial or sporadic peripheral amyloid disease. Currently, more than 4 million Americans have Alzheimer's disease, a number projected to increase to over 16 million by 2050. It is by far the most common form of amyloidosis and has the greatest socioeconomic impact. In contrast, peripheral amyloidosis is a rare disorder, but it accounts for more than 5,000 new cases each year in the United States alone.

[0005] Of these, the main peripheral amyloidosis is light chain-related (AL) amyloidosis (sporadic monoclonal plasmacytosis resulting from deposits of fibrils composed of immunoglobulin light chain proteins). AL accounts for approximately two-thirds of all peripheral amyloid cases, with an estimated incidence of about 1.4 per 100,000 people per year in the United States, which is comparable to that of acute lymphoblastic leukemia and chronic myeloid leukemia (Group, USCSW (2007) United States Cancer Statistics: 1999-2003 Incidence and Mortality Web-Based Report, USD Department of Health and Human Services Centers for Disease Control and Prevention, National Cancer Institute, Atlanta). Although AL occurs at a rate of about one-fifth that of associated plasma cell dysplasia multiple myeloma, some cases are perhaps more destructive due to the rapid progression of organ destruction, the lack of effective anti-amyloid drugs, and the inability to effectively diagnose the disease before the onset of organ failure, resulting in a median survival of only 13.2 months. Less than 5% of all AL patients survive for 10 years or more from diagnosis (Comenzo, R. Let al. (2002) Blood 99, 4276-4282). In patients with cardiac AL amyloidosis, the median survival is less than 5 months. Unfortunately, there is still no effective mouse model of AL disease.

[0006] ATTR is a form of systemic amyloidosis. 25% of patients with ATTR amyloidosis die within 24 months of diagnosis (Gertz and Dispenzieri JAMA 324(1)79-89(2002)). Current treatments do not prevent organ damage. ATTR amyloidosis is caused by transthyretin (TTR) fibrils. Transthyretin is a protein produced by the liver that transports thyroid hormones and vitamin A into the bloodstream. Normally, TTR is a tetramer composed of four single-chain monomers. In hereditary ATTR amyloidosis, it is thought that mutations in the TTR gene destabilize the protein, causing tetrameric dissociation into monomers and aggregation into amyloid fibrils. In wild-type ATTR amyloidosis, normal TTR proteins become unstable, undergoing abnormal folding and forming amyloid fibrils.

[0007] LECT2 amyloidosis (ALECT2) is another common form of amyloidosis caused by the LECT2 protein. This disorder generally presents with progressive or end-stage kidney disease. Associated signs and symptoms of kidney disease may include fatigue, dehydration, blood in the urine, and / or other evidence of the presence of nephrotic syndrome or renal failure. LECT2 amyloidosis causes significant kidney disease in older adults. Individuals with this disorder are suggested to exhibit increased LECT2 production and / or decreased LECT2 catabolism. Mutations in the LECT2 gene have been identified, but have not been associated with ALECT2.

[0008] In addition to disorders for which the pathogenesis of amyloid is well established, fibrillary deposits with amyloid structural and pigment properties have been identified in other syndromes, but their association with disease status has not yet been established. For example, in type 2 diabetes, islet amyloid precursor protein (IAPP) is deposited as amyloid in the islets of Langerhans (Jaikaran, ET et al. (2001) Biochim. Biophys. Acta 1537, 179-203). IAPP aggregation leads to oligomeric structures that are toxic to pancreatic cells (Lin, CY et al. (2007) Diabetes 56, 1324-1332). Therefore, it is suggested that IAPP amyloid formation in patients with type 1 diabetes contributes to β-cell destruction and leads to insulin dependence (Jaikaran, ET et al. (2001) Biochim. Biophys. Acta 1537, 179-203). In another example, plaques containing amyloid fibrils composed of apolipoprotein AI have been identified in more than half of patients with atherosclerotic carotid arteries (Westermark, P. et al. (1995) Am. J. Pathol. 147, 1186-1192; Mucchiano, G. et al. (2001) J. Pathol. 193, 270-275). While these fibril deposits are more common in elderly patients, apoA-I is undoubtedly present early in plaque development (Vollmer, E. et al. (1991) Virchows Arch. A. Pathol. Anat. Histopathol. 419, 79-88). As a final example, Apo-AI amyloid has recently been found in knee menisci obtained from patients undergoing knee replacement surgery, suggesting it may contribute to the physical deterioration of the joint (Solomon, A. et al. (2006) Arthritis Rheum. 54, 3545-3550).

[0009] In total, more than 25 proteins have been chemically or serologically identified as components of fibrils in amyloid deposits. It is the nature of these proteins that allows us to identify diseases, determine treatments, and establish prognoses. Amyloid fibrils can form precursor proteins that are clinically associated with a heterogeneous group of diseases, are structurally identifiable, and are functionally diverse, but the deposits themselves share many very similar features, including fibril structure, fibril epitopes, and similar accessory molecules, such as heparan sulfate proteoglycans (HSPGs), which are naturally increasing. Amyloid is a heterogeneous complex containing not only profibrils but also glycosaminoglycans (GAGs) and, in particular, perlecan HSPG (Ancsin, JB (2003) Amyloid 10, 67-79; Ailles, L. et al. (1993) Lab. Invest. 69, 443-448; Kisilevsky, R. (1994) Mol. Neurobiol. 9, 23-24; Kisilevsky, R. (1990) Lab. Invest. 63, 589-591; Snow, A. et al. (1987) Lab. Invest. 56, 120-123; Li, J. et al. (2005) Proc. Natl. Acad. Sci. USA 102, 6473-6477).

[0010] In different types of systemic amyloidosis, amyloid deposits occur in multiple organs, including the kidneys, pancreas, liver, spleen, heart, and nerve tissue, making it difficult to distinguish between different types of amyloidosis. Simultaneously, promising treatments targeting specific types of amyloid are being developed. Therefore, methods for diagnosing and differentiating amyloid-related diseases are urgently needed. [Overview of the project]

[0011] This specification provides a method for diagnosing the type of amyloid disease. In some embodiments, the method includes administering an amyloid-reactive agent or a detection dye. In some embodiments, the method includes measuring the organ dispersion pattern of the amyloid-reactive agent or detection dye in one or more organs of an individual. In some embodiments, the organ dispersion pattern of the amyloid-reactive agent or detection dye indicates the type of amyloid disease.

[0012] This specification also provides methods for treating amyloid diseases. In some embodiments, the method includes administering an amyloid-reactive agent or a detection dye. In some embodiments, the method includes measuring the organ dispersion pattern of the amyloid-reactive agent or detection dye in one or more organs, wherein the organ dispersion pattern of the amyloid-reactive agent or detection dye indicates the type of amyloid disease. In some embodiments, the method includes selecting a treatment method based on the type of amyloid disease. In some embodiments, the method includes administering treatment to an individual.

[0013] This specification also provides a method for diagnosing the type of amyloid disease. In some embodiments, the method includes receiving organ dispersion pattern data relating to amyloid-reactive substances or detection dyes for an individual. In some embodiments, the method includes calculating organ:organ ratios for two or more organs and using the organ:organ ratios to diagnose the type of amyloid disease in the individual.

[0014] In some embodiments, the type of amyloid disease includes systemic amyloidosis. In some embodiments, the type of amyloid disease is selected from the group consisting of amyloid light chain amyloidosis (AL), transthyretin-associated amyloidosis (ATTR), and ALECT2.

[0015] In some embodiments, the organ is selected from the group consisting of the heart, spleen, kidneys, and liver.

[0016] In some embodiments, the amyloid-reactive agent is a detectably labeled amyloid-reactive peptide. In some embodiments, the amyloid-reactive peptide comprises the amino acid sequence described in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 13, or SEQ ID NO: 14. In some embodiments, the amyloid-reactive agent reacts with Aβ fibrils. In some embodiments, the amyloid-reactive agent is selected from the group consisting of florbetapyr, florbetaben, and flutemetamol.

[0017] In some embodiments, the detection dye is ThT. In some embodiments, the amyloid-reactive substance reacts with synthetic fibers consisting of light chains or fragments thereof.

[0018] In some embodiments, organ dispersion patterns are measured using PET / CT images. In some embodiments, amyloid-reactive substances are radiolabeled.

[0019] In some embodiments, the organ:blood ratio of amyloid-reactive substance or detection dye is calculated. In some embodiments, the organ:organ ratio is selected from the group consisting of liver:heart, spleen:heart, spleen:liver, spleen:kidney, kidney:heart, and kidney:liver. In some embodiments, the heart:spleen ratio is calculated. In some embodiments, if the heart:spleen ratio is greater than 1.4, the individual is diagnosed with ATTR amyloidosis.

[0020] This specification also provides kits for diagnosing types of amyloid disease, including amyloid-reactive agents or detection dyes and instructions for use. In some embodiments, the kits are for detecting or diagnosing amyloid light chain amyloidosis (AL), transthyretin-associated amyloidosis (ATTR), or ALECT2.

[0021] In some embodiments, the kit includes a detectably labeled amyloid-reactive peptide. In some embodiments, the amyloid-reactive peptide includes the amino acid sequence described in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 13, or SEQ ID NO: 14. In some embodiments, the amyloid-reactive agent reacts with Aβ fibers. In some embodiments, the amyloid-reactive agent reacts with synthetic fibers consisting of light chains or fragments thereof.

[0022] In some embodiments, the kit includes an amyloid-reactive substance selected from the group consisting of florbetapyr, florbetaben, and flutemetamol.

[0023] In some embodiments, the kit includes the detection dye ThT.

[0024] In some embodiments, the kit includes instructions for measuring organ distribution patterns.

[0025] In some embodiments, the kit includes a radiolabeled amyloid-reactive substance.

[0026] In some embodiments, the kit includes instructions for calculating the organ:blood ratio of amyloid-reactive substance or detection dye.

[0027] In some embodiments, the kit includes instructions for calculating the organ:organ ratio of amyloid-reactive substance or detection dye. In some embodiments, the organ:organ ratios are liver:heart, spleen:heart, spleen:liver, spleen:kidney, kidney:heart, and kidney:liver.

[0028] In some embodiments, the kit provides instructions for diagnosing ATTR amyloidosis if the heart:spleen ratio is greater than 1.4.

[0029] In some embodiments, the kit includes instructions for treating an individual based on a diagnosis.

[0030] In some embodiments, the kit further includes a therapeutic agent for treating a certain type of amyloid. [Brief explanation of the drawing]

[0031] [Figure 1] A partial list of amyloid and amyloid-related disorders is shown below. [Figure 2A] This shows the organ-to-blood pool ratios for light chain-related (AL) amyloidosis (n=14). The organ-to-blood pool ratios were calculated from region of interest (ROI) analysis of the heart, liver, spleen, and left kidney. [Figure 2B] This shows the organ-to-blood pool ratios for transthyretin-related (ATTR) amyloidosis (n=7). The organ-to-blood pool ratios were calculated from region of interest (ROI) analyses of the heart, liver, spleen, and left kidney. [Figure 2C] This shows the organ-to-blood pool ratios for patients with leukocyte chemotactic factor 2-related (ALECT2) amyloidosis (n=2). The organ-to-blood pool ratios were calculated from region of interest (ROI) analysis of the heart, liver, spleen, and left kidney. [Figure 3] This shows the average organ-to-blood pool ratio for AL, ATTR, and ALECT2 patients for whom ROI analysis of the heart, liver, spleen, and left kidney was calculated. [Figure 4A] This shows an overview of organ:blood pool ratios for AL patients after removing outlier data points. [Figure 4B] This shows a summary of organ:blood pool ratios for ATTR patients after removing outlier data points. [Figure 4C] This shows an overview of organ:blood pool ratios for ALECT2 patients after removing outlier data points. [Figure 5] This shows the mean organ:blood pool ratios for AL, ATTR, and ALECT2 patients after removing outlier data points. [Figure 6] The receiver operating characteristic (ROC) analysis performed using single-organ standardized uptake ratio (SUVR) values ​​for ATTR is shown. The ROC curve is for the heart. [Figure 7A] This shows receiver operating characteristic (ROC) analysis performed using single-organ standard uptake ratio (SUVR) values ​​for AL. A, B, and C show the ROC curves for the liver, spleen, and kidney, respectively. The shaded areas are represented by straight lines. [Figure 7B] This shows receiver operating characteristic (ROC) analysis performed using single-organ standard uptake ratio (SUVR) values ​​for AL. A, B, and C show the ROC curves for the liver, spleen, and kidney, respectively. The shaded areas are represented by straight lines. [Figure 7C] This shows receiver operating characteristic (ROC) analysis performed using single-organ standard uptake ratio (SUVR) values ​​for AL. A, B, and C show the ROC curves for the liver, spleen, and kidney, respectively. The shaded areas are represented by straight lines. [Figure 8A] This document presents receiver operating characteristic (ROC) analysis for detecting ATTR amyloidosis. ROC curves were generated using organ-to-organ uptake ratios for ATTR amyloidosis. Analysis regarding the heart / spleen ratio is shown. [Figure 8B] This paper presents receiver operating characteristic (ROC) analysis for detecting ATTR amyloidosis. ROC curves were generated using organ-to-organ uptake ratios for ATTR amyloidosis. Analysis of the cardiac / liver ratio is shown. [Figure 8C] This paper presents receiver operating characteristic (ROC) analysis for detecting ATTR amyloidosis. ROC curves were generated using organ-to-organ uptake ratios for ATTR amyloidosis. Analysis of the cardiac / kidney ratio is shown. [Figure 8D] This document presents receiver operating characteristic (ROC) analysis for detecting ATTR amyloidosis. ROC curves were generated using organ-to-organ uptake ratios for ATTR amyloidosis. Analysis of the liver / spleen ratio is shown. [Figure 8E] This paper presents receiver operating characteristic (ROC) analysis for detecting ATTR amyloidosis. ROC curves were generated using organ-to-organ uptake ratios for ATTR amyloidosis. Analysis of the liver / kidney ratio is shown. [Figure 8F]This document presents receiver operating characteristic (ROC) analysis for detecting ATTR amyloidosis. ROC curves were generated using organ-to-organ uptake ratios for ATTR amyloidosis. Analysis regarding the kidney / spleen ratio is shown. [Figure 9A] This section presents receiver operating characteristic (ROC) analysis for AL detection. ROC curves were generated using organ-to-organ uptake ratios for AL. An analysis of the liver / heart ratio is also shown. [Figure 9B] This section presents receiver operating characteristic (ROC) analysis for AL detection. ROC curves were generated using organ-to-organ uptake ratios for AL. An analysis of the spleen / heart ratio is also shown. [Figure 9C] This section presents receiver operating characteristic (ROC) analysis for AL detection. ROC curves were generated using organ-to-organ uptake ratios for AL. An analysis of the spleen / liver ratio is also shown. [Figure 9D] This section presents receiver operating characteristic (ROC) analysis for AL detection. ROC curves were generated using organ-to-organ uptake ratios for AL. An analysis of the spleen / kidney ratio is also shown. [Figure 9E] This section presents receiver operating characteristic (ROC) analysis for AL detection. ROC curves were generated using organ-to-organ uptake ratios for AL. An analysis of the kidney / heart ratio is also shown. [Figure 9F] This section presents receiver operating characteristic (ROC) analysis for AL detection. ROC curves were generated using organ-to-organ uptake ratios for AL. An analysis of the kidney / liver ratio is also shown. [Modes for carrying out the invention]

[0032] Detailed explanation This specification provides methods for the diagnosis and treatment of amyloid diseases. In some embodiments, the method includes the administration of an amyloid-reactive agent or a detection dye. In some embodiments, the method further includes measuring the organ dispersion pattern of the amyloid-reactive agent or dye in one or more organs. In some embodiments, the method provides a method for identifying different types of systemic amyloidosis, such as AL, ATTR, and ALECT2, based on the organ dispersion pattern of the amyloid-reactive agent or detection dye. In some embodiments, the method includes providing a diagnosis of the type of amyloid disease based on the organ dispersion pattern of the amyloid-reactive agent or detection dye. In some embodiments, the method further includes selecting a treatment method based on the type of amyloid disease.

[0033] As used herein, “amino acid” or “amino acid residue” refers to any naturally occurring amino acid, any non-naturally derived amino acid, any modification thereof such as derivatized amino acids, or any amino acid mimetic known in the art. Amino acids may be written using both their common three-letter and one-letter abbreviations.

[0034] The terms amyloid, amyloid deposits, amyloid fibrils, and amyloid fibers refer to insoluble fibrous protein aggregates that share specific structural characteristics. Protein aggregates are formed, for example, by the aggregation of several different proteins and have a tertiary structure consisting of a regular arrangement of β-sheets stacked perpendicular to the fiber axis. See Sunde et al., J.Mol.Biol. (1997) 273:729-39. Abnormal accumulation of amyloid in organs can lead to amyloidosis. Although they vary in their development, all amyloids share common morphological properties in that they stain with specific dyes such as Congo red, and the polarization after staining exhibits a characteristic reddish-green birefringence. Amyloids also share common ultrastructural features and common X-ray diffraction and infrared spectra.

[0035] Amyloidosis refers to a pathological condition or disease characterized by the presence of amyloid, such as the presence of amyloid deposits. "Amyloid disease" or "amyloidosis" is a disease associated with the formation, deposition, accumulation, or persistence of amyloid fibrils. Such diseases include, but are not limited to, Alzheimer's disease, Down syndrome, hereditary cerebral hemorrhage with Dutch-type amyloidosis, and cerebral beta-amyloid vascular disease. Other amyloid diseases, such as systemic AA amyloidosis, AL amyloidosis, ATTR amyloidosis, ALECT2 amyloidosis, and IAPP amyloidosis in type II diabetes, are also amyloid diseases.

[0036] As used herein, the term “carrier” includes pharmaceutically acceptable carriers, excipients, or stabilizers that are nontoxic to cells, tissues, mammals, or subjects to which the dose and concentration used will be applied. In most cases, pharmaceutically acceptable carriers are pH-buffered aqueous solutions. Examples of pharmaceutically acceptable carriers include, but are not limited to, buffers such as phosphoric acid, citrate, and other organic acids; antioxidants, including ascorbic acid; low molecular weight (less than about 10 residues) polypeptides; proteins, e.g., serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates such as glucose, mannose, or dextrin; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and / or nonionic surfactants such as Tween®, polyethylene glycol (PEG), and Pluronics®.

[0037] As used herein, the terms “effective dose” or “appropriate dose” refer to the amount of substance sufficient to achieve a beneficial or desired clinical or biochemical outcome. An effective dose may be administered once or more times. For the purposes of the present invention, an effective dose of amyloid-reactive agent or detection is an amount sufficient to bind to amyloid and enable the detection of amyloid.

[0038] In this specification, the interchangeable terms “imaging agent” or “contrast agent” refer to any active substance that can be used in connection with methods for imaging internal regions of an object and / or diagnosing the presence or absence of disease in an object by utilizing and / or detecting an energy source. Exemplary contrast agents include contrast agents used in connection with ultrasound, magnetic resonance imaging, radionuclide imaging, or X-ray imaging (including computed tomography) of a patient, and the compositions described herein.

[0039] For the purposes of this invention, the term "mammal" as used herein refers to all animals classified as mammals, including humans, domestic animals and farm animals, as well as animals in zoos, sports animals, or pets, such as dogs, cats, cows, horses, sheep, and pigs. In some embodiments, the mammal is a human.

[0040] As used herein, the term "peptide" refers to any peptide or peptide-mimicking structure that contains or consists of two or more amino acids, and includes chemical modifications and derivatives of amino acids.

[0041] As used herein, the terms “purified” or “isolated” molecules refer to biological or synthetic molecules that have been removed, isolated, or separated from their natural environment, or that have been released from other components to which they naturally associate.

[0042] As used herein, the term "specifically binds" refers to a non-random binding reaction between two molecules, for example, between an amyloid-reactive agent or detection dye and amyloid. The term "specifically binds" may be used interchangeably with "selectively target" or "selectively associate."

[0043] With respect to amyloid, the terms “selectively targeting” or “selectively associating” as used herein refer to, for example, the selective localization or binding between an amyloid-reactive agent or detection dye and amyloid compared to non-amyloid proteins. Amyloid-reactive agents or detection dyes can selectively target multiple types of amyloid.

[0044] As used herein, the term "subject" refers to a vertebrate. A vertebrate may be a mammal, such as a human. A subject may be a human patient.

[0045] As used herein, the term "amyloid-reactive agent" refers to an agent that specifically reacts with or binds to amyloid.

[0046] I. Diagnostic Methods In some aspects of this disclosure, methods for diagnosing amyloid disease are provided. In some embodiments, a method for diagnosing the type of amyloid disease includes administering an amyloid-reactive agent or detection dye and measuring the organ-to-organ dispersion pattern of the amyloid-reactive agent or dye in one or more organs. In other embodiments, a method for diagnosing the type of amyloid disease includes administering an amyloid-reactive agent or detection dye and measuring the organ-to-organ ratio of the amyloid-reactive agent or dye in one or more organs. In some embodiments, the organ-to-organ dispersion pattern, or organ-to-organ ratio, of the amyloid-reactive agent or dye indicates the type of amyloid disease.

[0047] In some embodiments, a method for diagnosing a type of amyloid disease includes administering an amyloid-reactive agent or detection dye containing an amyloid-reactive peptide. In some embodiments, the amyloid-reactive agent or detection dye includes a peptide, a fusion protein, a small molecule compound, or an antibody or a fragment thereof.

[0048] In some embodiments, a method for diagnosing a type of amyloid disease includes administering an amyloid-reactive substance or detection dye containing an amyloid-reactive peptide. In some embodiments, the amyloid-reactive peptide includes an amino acid sequence that is at least 80%, 85%, 90% or more identical to the amino acid sequence described in any of SEQ ID NOs: 1 to 14, for example, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence described in any of SEQ ID NOs: 1 to 14. In some embodiments, the amyloid-reactive peptide used in conjunction with the method herein contains or consists of about 10 to 55 amino acids. The amyloid-reactive peptides of the present invention include, for example, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, or 55 amino acids, or consist of them. Such peptides are described, for example, in international patent application WO2016032949, which is incorporated in its entirety herein. In some embodiments, a method for diagnosing the type of amyloid disease includes an amyloid-reactive peptide having the amino acid sequence described in SEQ ID NO: 13. In some embodiments, a method for diagnosing the type of amyloid disease includes p5+14. [Table 1]

[0049] The amino acids that form all or part of the amyloid-reactive peptide used in this method include stereoisomers and modified native amino acids, unnatural amino acids, post-translationally modified amino acids, enzymatically synthesized amino acids, derivatized amino acids, and constructs or structures designed to mimic amino acids. The amino acids that form the peptide of the present invention may be one or more of the 20 common amino acids found in naturally occurring proteins, or one or more modified amino acids and unusual amino acids. The amyloid-reactive peptide used in conjunction with the method described herein can be produced by any technique known to those skilled in the art, e.g., by chemical synthesis or recombinant means using standard molecular biological techniques.

[0050] Furthermore, the peptide of the present invention may contain one or more modified amino acids. The modified amino acids may be derivatized amino acids, modified amino acids, and abnormal amino acids. Examples of modified and abnormal amino acids include 2-aminoadipic acid (Aad), 3-aminoadipic acid (Baad), β-aminopropionic acid (Bala, β-alanine), 2-aminobutyric acid (Abu, piperidine acid), 4-aminobutyric acid (4Abu), 6-aminocaproic acid (Acp), 2-aminoheptanoic acid (Ahe), 2-aminoisobutyric acid (Aib), 3-aminoisobutyric acid (Baib), 2-aminopimelic acid (Apm), 2,4-diaminobutyric acid (Dbu), desmosine (Des), 2,2'-diaminopimelic acid (Dpm), 2,3-diaminopropionic acid (Dpr), N- These include, but are not limited to, ethylglycine (EtGly), N-ethylasparagine (EtAsn), hydroxylysine (Hyl), allo-hydroxylysine (AHyl), 3-hydroxyproline (3Hyp), 4-hydroxyproline (4Hyp), isodesmosine (Ide), allo-isoleucine (Alle), N-methylglycine (MeGly, sarcosine), N-methylisoleucine (Melle), 6-N-methyllysine (MeLys), N-methylvaline (MeVal), norvaline (Nva), norleucine (Nle), and ornithine (Orn).

[0051] Other examples of modified and abnormal amino acids are generally described in Synthetic Peptides: A User's Guide, Second Edition, April 2002, Edited by Gregory A. Grant, Oxford University Press; Hruby VJ, Al-obeidi F and Kazmierski W: Biochem J 268:249-262, 1990; and Toniolo C: Int J Peptide Protein Res 35:287-300, 1990, all of which are incorporated herein by reference.

[0052] The peptides of the present invention may contain at least about 15% positively charged amino acids, such as arginine and / or lysine. The peptides may contain about 15% to about 50%, about 20% to about 45%, about 25% to about 40%, or about 30% to about 35% positively charged amino acids. In some embodiments, the peptides of the present invention may include the following amino acid sequence:

[0053] XBXXBXXXBXXBXXXBXXBXXXBXXBX, (Sequence ID 15), in the formula

[0054] X represents any amino acid, including uncharged modified amino acids, while B represents positively charged amino acids.

[0055] In one embodiment, the peptide of the present invention comprises SEQ ID NO: 15, where X is alanine, valine, serine, threonine, or glycine, and B is arginine, lysine, or histidine. The peptide of the present invention comprises or may comprise SEQ ID NO: 15. The peptide of the present invention may have up to 55 amino acids and comprises the amino acid sequence described in SEQ ID NO: 15.

[0056] In another embodiment, the peptide may include the following amino acid sequence: BXZBXZXBZXBZXZBXZBXZXBZXBZ, (SEQ ID NO: 16), where B is arginine, lysine, or histidine; X is isoleucine, leucine, methionine, valine, glycine, phenylalanine, tryptophan, tyrosine, serine, threonine, asparagine, or an uncharged modified amino acid; and Z may be isoleucine, leucine, methionine, valine, glycine, phenylalanine, tryptophan, tyrosine, serine, threonine, asparagine, or an uncharged modified amino acid. In certain embodiments, the peptide of the present invention comprises or may consist of the following amino acid sequence: SRAQRAQARQARQAQRAQRAQARQARQ. (SEQ ID NO: 17)

[0057] The peptide of the present invention may be a fusion protein containing a second peptide with an amino terminus labeled with a detection agent, for example, CGGY (SEQ ID NO: 18) or GGGY (SEQ ID NO: 19) as the leader sequence. Therefore, in some embodiments, the amyloid-reactive agent may have up to 55 amino acids and include the amino acid sequence described in SEQ ID NO: 17. CGGYSRAQRAQARQARQAQRAQRAQARQARQ.(SEQ ID NO: 20)

[0058] The fusion protein may contain other leader sequences, such as cell-permeable peptides (CPPs) or blood-brain barrier (BBB) ​​translocation peptides.

[0059] Furthermore, the present invention provides other peptides and fusion proteins rich in positively charged amino acids for imaging amyloid.

[0060] The peptides of the present invention can be produced by any technique known to those skilled in the art, including chemical synthesis, recombinant methods using standard molecular biological techniques, or isolation of peptides from natural sources. These peptides can be synthesized in solution or on solid supports according to the prior art. Various automated synthesizers are commercially available and can be used according to known protocols. (See, for example, Stewart and Young, Solid Phase Peptide Synthesis, 2nd ed. Pierce Chemical Co., 1984; Tam et al., J.Am. Chem. Soc., 105:6442, 1983; Merrifield, Science, 232:341-347, 1986; and Barany and Merrifield, The Peptides, Gross and Meienhofer, eds., Academic Press, New York, pp.1-284, 1979, the entire contents of which are incorporated herein by reference.)

[0061] Alternatively, recombinant DNA technology can be used, which involves inserting the nucleotide sequence encoding the peptide of the present invention into an expression vector, transforming or transfecting suitable host cells, culturing them under conditions suitable for expression, and isolating the peptide.

[0062] In certain embodiments, the amyloid-reactive substance may be a naturally occurring peptide, or obtained by isolation or purification from its natural source. Protein purification techniques are one-step processes that include homogenization of cells, tissues, or organs, and crude fractionation into peptide and non-peptide fractions. Other protein purification techniques include precipitation using, for example, ammonium sulfate, polyethylene glycol (PEG), antibodies, or thermal denaturation, followed by: centrifugation; chromatography steps, e.g., ion exchange, gel filtration, reversed phase, hydroxyl apatite, and affinity chromatography; isoelectric focusing; gel electrophoresis, e.g., polyacrylamide gel electrophoresis; and combinations of these and other techniques.

[0063] Various chromatographic techniques include, but are not limited to, ion exchange chromatography, gel exclusion chromatography, affinity chromatography, immunoaffinity chromatography, and reversed-phase chromatography. Particularly efficient peptide purification methods include fast performance liquid chromatography (FPLC) or high performance liquid chromatography (HPLC).

[0064] The order in which various purification steps are performed can be changed, or certain steps can be omitted, and furthermore, suitable methods for preparing substantially purified peptides can be obtained. The peptides of the present invention may be part of a polypeptide or protein, and may be produced by biochemical or enzymatic fragmentation of a polypeptide or protein. Accordingly, the peptides of the present invention may be (a) natural products, (b) produced by chemical synthesis, (c) produced by recombinant DNA technology, (d) produced by biochemical or enzymatic fragmentation of macromolecules, (e) produced by a combination of the above methods a to d, or (f) produced by any other peptide production means.

[0065] During chemical synthesis, peptides are modified at their N-terminus or C-terminus to improve stability and formulation, and to increase resistance to protease degradation. Examples of amino acid modifications include pegylation, acetylation, alkylation, formylation, and amidation. Furthermore, various amino acids not naturally occurring can be introduced along the chain to improve peptide stability.

[0066] Cysteine ​​is also useful in facilitating the labeling of the peptides of the present invention by conjugation with biotin, fluorophores, or other ligands. Furthermore, cysteine ​​in the leader peptide enables the generation of covalent dimer molecules that can increase the relative affinity of the peptides to their targets.

[0067] In certain embodiments, the method comprises administering an amyloid-reactive agent or a detection dye comprising florbetapir ( 18 F-florbetapir, Amyvid®), florbetaben ( 18 F-florbetaben, Neuraceq®), or flutemetamol ( 18 F-flutemetamol, Vizamyl®). In another embodiment, a method of diagnosing the type of amyloid comprises administering an amyloid-reactive agent or a detection dye comprising thioflavin T (ThT). Other exemplary amyloid-reactive agents or dyes that may be used in the methods described herein include 3, phosphono-1, 2-propanedicarboxylic acid (DPD), hydroxyl-diphosphonate (HDP), hydroxymethylene-diphosphonate (HMDP), stannous pyrophosphate (PyP), NAV4694 ( 18 F-NAV4694, 18 F-AZD4694), thioflavin S (ThS), serum amyloid P (SAP) protein or peptide, serum amyloid A (SAA) protein or peptide, tau protein or peptide, Congo red, Congo corinth, benzopurpurin 4B, vital red, trypan blue, amide black 10B, acid fuchsin, 11 C-Pittsburgh compound B ( 11 C-PIB), 18 F-THK5317, 18 F-THK5351, 18 F-flortaucipir ( 118 F-flortaucipir, 18 F-AV-1451), 18 F-T807, 18 F-fluorodeoxyglucose ( 18 F-2-fluoro-2-deoxy-D-glucose, 18 F-FDG), 18 F-MK-6240, 18 F-PI-2620, 11 C-UCB-J, mitochondrial translocator protein (TSPO) or peptide,11 CR-PK11195, 18 F-DPA-714, and 11 C-PBR28, 11 This includes, but is not limited to, C-BF-227, or amyloid-reactive antibodies or fragments.

[0068] In some embodiments, the method for diagnosing the type of amyloid disease includes administering an amyloid-reactive substance or detection dye containing a detectable label. These include radionuclides (e.g., C- 11 , I- 125 , I- 123 , I- 131 , Zr- 89 ,Tc- 99m , Cu- 64 , Br- 76 , F- 18 Examples include, but are not limited to, enzymes (horseradish peroxidase); biotin; and fluorescent dyes. Any means known in the art for detectably labeling proteins can be used and / or adapted for use in the manner described herein. For example, amyloid-reactive peptides can be labeled with radioactive isotopes, fluorescent tags, or chemiluminescent tags. Examples of radioactive isotopes include, for example, 11 C, 18 F, 111 In, 99m Tc, and 123 I, 124 I, and 125 I is one such radioisotope. These radioisotopes, and other radioisotopes, can be incorporated into amyloid-reactive agents or detection dyes. Examples of fluorescent or chemiluminescent tags include fluorescein, Texas Red, rhodamine, Alexa dye, and luciferase, which can be incorporated into amyloid-reactive agents or detection dyes using conventional methods in the art.

[0069] In some embodiments, the method for diagnosing the type of amyloid disease includes administering a detection dye containing an amyloid-reactive substance or radiolabel. In some embodiments, the radiolabel is 11 C, 18 F, 111 In, 99m Tc, 89 Zr, and 123 I, 124 I, or 125 I. In some embodiments, the radiolabeled amyloid-reactive substance or detection dye is a radiolabeled amyloid-reactive peptide. In some embodiments, the radiolabeled amyloid-reactive peptide is 124 It is an amyloid-reactive peptide labeled with I. In other embodiments, a method for diagnosing the type of amyloid disease is: 124 This includes administering I-p5+14. In another embodiment, the radiolabeled amyloid-reactive substance or detection dye is florbetapyl, florbetaben, and flutemetamol.

[0070] In another embodiment, the method for diagnosing the type of amyloid disease includes administering an amyloid-reactive agent or detection dye containing a fluorescent label. In some embodiments, the fluorescently labeled amyloid-reactive agent or detection dye is thioflavin T (ThT).

[0071] In some embodiments, the amyloid-reactive substance or detection dye comprises an amyloid-reactive peptide conjugated to a radiolabel. In some embodiments, the amyloid-reactive substance or detection dye comprises a peptide conjugated to an extender. In some embodiments, the amyloid-reactive peptide is conjugated to PEG. In some embodiments, the amyloid-reactive peptide is conjugated to an antibody.

[0072] In some embodiments, the amyloid-reactive agent or detection dye specifically binds to amyloid deposits. In some embodiments, the amyloid-reactive agent or detection dye can detect the presence or amount of amyloid in a subject. In some embodiments, the amyloid-reactive agent or detection dye cross-reacts with amyloid deposits formed by a number of different proteins. In some embodiments, the amyloid-reactive agent or detection dye binds to amyloid deposits formed by various proteins and / or peptides. In some embodiments, the amyloid-reactive agent or detection dye binds to amyloid deposits formed by amyloid ligand chains (AL). In some embodiments, the amyloid-reactive agent or detection dye binds to amyloid formed by transthyretin (TTR) fibers. In some embodiments, the amyloid-reactive agent or detection dye binds to amyloid formed by serum amyloid protein A (sAA). In some embodiments, the amyloid-reactive substance or detection dye binds to amyloid formed by amyloidogenesis forms of immunoglobulin heavy chain (AH), β2-microglobulin (Aβ2M), transthyretin variant (ATTR), apolipoprotein Al (AApoAI), apolipoprotein All (AApoAII), gelsolin (AGel), lysozyme (ALys), leukocyte chemotactic factor (ALECT2), fibrinogen α variant (AFib), cystatin variant (ACys), calcitonin (ACal), lactadherin (AMed), islet amyloid polypeptide (AIAPP), prolactin (APro), insulin (Alns), prion protein (APrP); α-synuclein (AaSyn), tau (ATau), atrial natriuretic factor (AANF), IAAP, ALκ4, or AIλ1.

[0073] In some embodiments, the amyloid-reactive agent or detection dye binds to heperan sulfate glycosaminoglycan (GAG). In some embodiments, the GAG ​​is associated with amyloid deposits. The binding of GAG to amyloid fibrils occurs primarily via electrostatic interactions involving the negative polyelectrolyte charge of the aggregated protein and positively charged side chain residues. Similar to reaction catalysts, GAGs are favored for aggregation, nucleation, and amyloid fibril formation, functioning as structural templates for the self-assembly of highly cytotoxic oligomeric precursors, which are rich in β-sheets and become amyloid fibrils. Furthermore, GAG amyloid-promoting activity can be enhanced through specific interactions via consensus binding sites between amyloid polypeptides and GAG molecules.

[0074] In some embodiments, the method involves administering an amyloid-reactive agent or detection dye to an individual. In some embodiments, the amyloid-reactive agent or detection dye is administered in a pharmaceutical composition. In some embodiments, the composition includes an aqueous buffer. The composition may also include a solubilizer such as lignocaine and a local anesthetic to alleviate pain at the injection site. These components are supplied separately or mixed together in unit dosage forms, for example, as a dry lyophilized powder or as a water-free concentrate in a sealed container such as an ampoule indicating the amount of the activator. When the composition is administered as an infusion, it can be dispensed in an infusion bottle containing sterile pharmaceutical-grade water or saline. When the composition is administered by injection, an ampoule of sterile water or saline for injection may be prepared and the components may be mixed before administration.

[0075] The composition may further include a carrier. The present invention also provides a pharmaceutical composition comprising one or more peptides and / or fusion peptides of the present invention. Such a pharmaceutical composition contains an effective amount of peptide or fusion peptide for binding to and detection on amyloid and pharmaceutically acceptable carriers.

[0076] Pharmaceutically acceptable carriers include solid or liquid carriers or components that can be added to enhance or stabilize the composition, or to facilitate the preparation of the composition, such as, but are not limited to, syrups, water, isotonic saline, 5% dextrose water, or buffered sodium or ammonium acetate solutions, oils, glycerin, and alcohols. Examples of oils include those derived from petroleum, animal, plant, or synthetic materials, such as peanut oil, soybean oil, mineral oil, and sesame oil. The carrier may also include sustained-release substances such as glyceryl monostearate or glyceryl distearate, either alone or in combination with waxes. Other suitable pharmaceutical carriers include, but are not limited to, starch, glucose, lactose, sucrose, gelatin, malt, rice, wheat flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk powder, propylene glycol, water, ethanol, flavoring agents, preservatives, coloring agents, diluents, granulating agents, lubricants, and binders.

[0077] Water can be a preferred carrier when a pharmaceutical composition is administered intravenously. Physiological saline, glucose aqueous solution, and glycerol solution can also be used, in particular, as liquid carriers for injectable solutions. The composition may optionally contain small amounts of wetting agents or emulsifiers, or pH buffers. Such compositions can be in the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, or sustained-release formulations. The composition may be formulated as a suppository using conventional binders such as triglycerides and carriers. Oral formulations may contain standard carriers, such as pharmaceutical-grade mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, and magnesium carbonate. Examples of other suitable pharmaceutical carriers are described in “Remington's Pharmaceutical Sciences” by EW Martin.

[0078] Methods for imaging amyloid include, but are not limited to, magnetic resonance imaging (MRI), computed tomography (CAT) scanning, positron emission tomography (PET), ultrasound imaging, X-ray imaging, radionuclide imaging, single-photon emission computed tomography (SPECT), and multiphoton microscopy.

[0079] Various contrast agents can be used to increase the sensitivity of the scan. Contrast agents for scanning may contain all molecules that attenuate X-rays. Radioisotopes can be used for positron emission tomography and imaging of radionuclides. All positron-emitting isotopes are useful for imaging radionuclides for positron emission tomography, and all gamma-photon-emitting isotopes are useful for imaging radionuclides.

[0080] As contrast agents for ultrasound imaging, there are positive and negative agents. Positive agents reflect ultrasound energy and thus produce positive (light) images. Correspondingly, negative agents increase transmittance or anechoicness and thus produce negative (dark) images. Various substances, including gases, liquids, solids, and combinations thereof, are being studied as potential contrast enhancers. Examples of solid particle contrast agents disclosed in U.S. Patent No. 5,558,854 include, but are not limited to, IDE particles and SHU454. European Patent Application 0231091 discloses an oil-in-water emulsion containing a highly fluorinated organic compound for providing enhanced contrast in ultrasound imaging. Emulsions containing octyl perfluorobromide (PFOB) are also being studied as ultrasound imaging agents. U.S. Patent No. 4,900,540 describes the use of phospholipid-based liposomes containing gas or gas precursors as contrast enhancers.

[0081] Several classes of compounds have potential as MRI contrast agents. These classes include superparamagnetic iron oxide particles, nitrogen oxides, and paramagnetic metal chelates (Mann et al., 1995). Strongly paramagnetic metals are preferred. Paramagnetic lanthanides and transition metal ions are typically toxic in vivo. Therefore, these compounds need to be incorporated into chelates with organic ligands. The peptides and fusion peptides of the present invention can be used to facilitate the targeting of amyloid by such chelated metals, otherwise allowing for a reduction in the total dose of imaging composition required.

[0082] Contrast agents can be conjugated to peptides and fusion peptides using known methods. Specific conjugation methods include, for example, the use of metal chelate complexes utilizing organic chelating agents such as DTPA. Acceptable chelates are known in the art. Such include, but are not limited to, 1,4,7,10-tetraazacyclododecane-N,N',N'',N'''-tetraacetic acid (DOTA); 1,4,7,10-tetraazacyclododecane-N,N',N''-triacetic acid (DO3A); 1,4,7-tris(carboxymethyl)-10-(2-hydroxypropyl)-1,4,7,10-tetraazacyclododecane (HP-DO3A); diethylenetriaminepentaacetic acid (DTPA); and numerous others.

[0083] Several classes of compounds have potential as MRI contrast agents. These classes include superparamagnetic iron oxide particles, nitrogen oxides, and paramagnetic metal chelates (Mann et al., 1995). Strongly paramagnetic metals are preferred. Paramagnetic lanthanides and transition metal ions are typically toxic in vivo. Therefore, these compounds need to be incorporated into chelates with organic ligands. The peptides and fusion peptides of the present invention can be used to facilitate the targeting of amyloid by such chelated metals, otherwise allowing for a reduction in the total dose of imaging composition required.

[0084] A wide range of paramagnetic metals are suitable for chelation. Suitable metals include those with atomic numbers 22-29 (inclusive), 42, 44, and 58-70 (inclusive), and those having oxidation states of 2 or 3. Examples of such metals include, but are not limited to, chromium(III), manganese(II), iron(II), cobalt(II), nickel(II), copper(II), praseodymium(III), neodymium(III), samarium(III), gadolinium(III), terbium(III), dysprosium(III), holmium(III), erbium(III), ytterbium(III), and vanadium(II). Other ions useful in situations involving X-ray imaging include, but are not limited to, lanthanum(III), gold(III), lead(II), and especially bismuth(III).

[0085] Among the radioisotopes that can be used in the labeled peptides and fusion peptides of the present invention, those suitable for localization studies are gamma emitters, positron emitters, X-ray emitters, and fluorescence emitters. Astatine is a suitable radioisotope for labeling peptides and fusion proteins. 211 ,bromine 76 , 14 carbon 、11 carbon, 51 chromium, 36 chlorine, 57 cobalt, 58 Cobalt, copper 67 ,copper 64 , 152 Europium, fluorine 18 ,gallium 67 ,gallium 68 , 3 Hydrogen, iodine 123 iodine 124 iodine 125 iodine 126 iodine 131 ,indium 111 ,indium 113m , 59 iron, 177 Lutetium, mercury 107 ,mercury 203 , 32 Phosphorus, Rhenium186 , rhenium 188 , ruthenium 95 , ruthenium 97 , ruthenium 103 , ruthenium 105 , rhenium 99m , rhenium 105 , rhenium 101 , 75 selenium, <00OO087>sulfur, technetium 99m , tellurium 121m tellurium 122m , tellurium 125m , thulium 165 , thulium 167 , thulium 168 , and yttrium 90 ) are present. Halogens can be used as labels, more or less interchangeably. Iodine, a gamma emitter 123 and technetium 99m can also be used because their radioactive metals are detectable with a gamma camera and have a favorable half-life for in vivo imaging. Positron emitters 18 -fluorine or 124 iodine, which are suitable for PET imaging and have a half-life suitable for peptide imaging, can also be used. The peptides and fusion peptides of the present invention can be labeled with indium<00001OO> or technetium 99m via a conjugated metal chelating agent, for example, DTPA (diethylenetriaminepentaacetic acid), or can be directly labeled covalently to an adjacent peptide containing a Cys residue.

[0086] The radiolabeled peptides or fusion peptides can be produced according to methods well known in the art. For example, they can be iodinated by contacting sodium or potassium iodide with a chemical oxidizing agent such as sodium hypochlorite or an enzymatic oxidizing agent such as lactoperoxidase. The peptides or fusion peptides according to the present invention can be labeled with technetium 99mThe peptide can be labeled by, for example, reducing the pertechnetium salt with a stannous solution, chelating the reduced technetium with a Sephadex column, and then adding the peptide to this column, or by direct labeling techniques, incubating, for example, the pertechnetium salt, a reducing agent such as SnCl2, a buffer such as sodium potassium phthalate solution, and the peptide. The intermediate functional groups preferred for binding radioactive isotopes present as metal ions to peptides are, as mentioned above, diethylenetriaminepentaacetic acid (DTPA) and ethylenediaminetetraacetic acid (EDTA).

[0087] Other useful labels include fluorescent labels, chromogenic labels, and biotin labels. Examples of fluorescent labels include, but are not limited to, rhodamine, fluorescein isothiocyanate, sodium fluorescein, lenographin, and tex-red sulfonyl chloride. In certain embodiments, the peptides and fusion peptides of the present invention may be linked to a secondary ligand or an enzyme (enzyme tag) that produces a colored product upon contact with a chromogenic substrate. Examples of suitable enzymes include urease, alkaline phosphatase, (horseradish) hydrogen peroxidase, and glucose oxidase. Examples of secondary ligands include biotin and avidin, or streptavidin compounds. The use of such markings is well known to those skilled in the art and is described, for example, in U.S. Patents 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149 and 4,366,241; each of which is incorporated herein by reference.

[0088] The present invention provides a method for detecting amyloids in a target. The method comprises administering a pharmaceutical composition containing an effective amount of one or more peptides or fusion peptides of the present invention to a target, and detecting the peptides or fusion peptides bound to the amyloid. The peptides can be labeled with contrast agents such as radioisotopes. The peptides have a specific binding affinity to the deposits, and this binding is detectable. Binding of the peptides or fusion peptides to amyloid can be detected by MRI, CAT scans, PET images, ultrasound images, SPECT images, X-ray images, fluorescence images, or radionuclide images.

[0089] In some cases, a method for diagnosing a type of amyloid disease involves administering a detectable amount of an amyloid-reactive reagent or dye to an individual. The detectable amount administered may depend on the type of detection being performed. For example, in some embodiments, the detectable amount of the amyloid-reactive reagent or dye may be sufficient to be imaged and detectable when administered to a subject. The detectable amount of amyloid-reactive substance or detection dye administered to an individual may vary depending on factors such as the individual's age, sex, and weight, the individual's specific response, the dosimetry method, the formulation, and the equipment-related factors. Optimization of such factors is well within the scope of the skill of the art. The detectable amount of amyloid-reactive substance or detection dye may also vary depending on the method of administration of the amyloid-reactive substance or detection dye.

[0090] In some cases, amyloid-reactive agents or detection dyes are administered parenterally, near the tumor, transmucosa, percutaneously, intramuscularly, intravenously, intradermally, subcutaneously, intraperitoneally, intraventricularly, or intracranially. In some cases, amyloid-reactive agents or detection dyes are administered intravenously. In other cases, amyloid-reactive agents or detection dyes are administered intraperitoneally.

[0091] Furthermore, those skilled in the art will understand that an effective amount of amyloid-reactive substance or detection dye can be administered in a single dose or by administering multiple doses. In some cases, the administration of the amyloid-reactive substance or detection dye may further include administration by flushing a solution. For example, the flushing solution, such as saline, may be administered immediately after the administration of the amyloid-reactive substance or detection dye, or a certain period after the administration of the amyloid-reactive substance or detection dye. In other cases, the amyloid-reactive substance or detection dye may be administered and then metabolized and excreted for a certain period.

[0092] In some embodiments, a method for diagnosing a type of amyloid disease includes detecting amyloid having an amyloid-reactive substance or a detection dye. Examples of amyloids detectable as part of this method include, but are not limited to, immunoglobulin heavy chain (AH), β2-microglobulin (Aβ2M), transthyretin variant (ATTR), amyloid beta (Aβ), apolipoprotein AI (AApoAI), apolipoprotein AII (AApoAII), gelsolin (AGel), lysozyme (ALys), leukocyte chemotactic factor (ALect2), fibrinogen α variant (AFib), cystatin variant (ACys), calcitonin (ACal), lactadherin (AMed), islet amyloid polypeptide (AIAPP), prolactin (APro), insulin (Alns), prion protein (APrP); α-synuclein (AαSyn), tau (ATau), atrial natriuretic factor (AANF), or IAAP, and other amyloidogenesis forms of amyloid-producing peptides. In some embodiments of this disclosure, a method for diagnosing a type of amyloid disease includes detecting ATTR, AL, and / or ALECT2 amyloid. In other embodiments, a method for diagnosing a type of amyloid disease includes distinguishing between ATTR, AL, and ALect2 amyloid.

[0093] In some embodiments, a method for diagnosing the type of amyloid disease includes measuring the organ distribution pattern of an amyloid-reactive agent or detection dye, and the organ distribution pattern of the amyloid-reactive agent or detection dye indicates the type of amyloid disease. The type of amyloid disease may be sporadic amyloidosis or may have a genetic component, such as hereditary amyloidosis. Some examples of amyloid diseases include, but are not limited to, AA amyloidosis, AL amyloidosis, AH amyloidosis, Aβ amyloidosis, ATTR amyloidosis, ALect2 amyloidosis, and IAPP amyloidosis in type II diabetes, Alzheimer's disease, Down syndrome, hereditary cerebral hemorrhage with Dutch type amyloidosis, cerebral beta-amyloid vascular disease, cavernous encephalopathy, thyroid tumors, Parkinson's disease, Lewis body dementia, tauopathy, Huntington's disease, senile systemic amyloidosis, familial hemodialysis, senile systemic aging, senile pituitary disorders, iatrogenic syndromes, cavernous encephalopathy, reactive chronic inflammation, thyroid tumors, myeloma, or other forms of cancer. In some embodiments, the type of amyloid disease is systemic amyloid disease. In some embodiments, the type of amyloid disease is AL amyloidosis, ATTR amyloidosis, or ALect2 amyloidosis.

[0094] In some embodiments, a method for diagnosing the type of amyloid disease includes measuring the organ distribution pattern of amyloid-reactive agents or detection dyes in one or more organs. While not bound by theory, it is thought that the anatomical distribution of amyloid in each form of the disease may have a specific pattern. For example, in ATTR amyloidosis, amyloid deposition is widespread in the heart and peripheral nerves, while in AL amyloidosis, another common amyloidosis, the pattern of amyloid deposition is not constant, and amyloidosis is found, for example, in the heart, spleen, liver, kidneys, peripheral nerves, gastrointestinal tract, muscles, lungs, and lymph nodes. In some embodiments, a method for diagnosing the type of amyloid disease includes measuring the organ distribution pattern of amyloid-reactive agents or detection dyes in one or more of the heart, spleen, liver, kidneys, peripheral nerves, gastrointestinal tract, muscles, lungs, brain, and lymph nodes. In some embodiments, the one or more organs are thoracic and abdominal organs. In some embodiments, one or more organs are the heart, spleen, liver, or kidney.

[0095] In some embodiments, similar to the present method, the step of measuring the organ dispersion pattern of amyloid-reactive substances or detection dyes in one or more organs includes determining the organ uptake value for each organ. Organ uptake can be determined by methods known to those skilled in the art. For example, organ uptake may represent the relative or absolute level of amyloid-reactive substances or detection dyes detected in each organ in an individual. In some embodiments, organ uptake is a standard uptake value for each organ. Those skilled in the art will understand that the standard uptake value can be determined by measuring the amount of amyloid-reactive substances or detection dyes detected in a reactive organ, e.g., the heart, relative to the amount of amyloid-reactive substances or detection dyes detected in a non-reactive tissue or sample, e.g., blood. The amount of amyloid-reactive substances or detection dyes in an organ can be determined, for example, by quantifying the detectable signal from the amyloid-reactive substances or detection dyes in the organ and calculating, for example, pixel values ​​in an image. In some embodiments, the standard uptake value is determined as the ratio of the amount of amyloid-reactive substance or detection dye detected in an organ to the amount of amyloid-reactive substance or detection dye detected in the blood. In some embodiments, the organ uptake value serves as an indicator of the organ dispersion pattern of the amyloid-reactive substance or detection dye.

[0096] In other cases, the method for determining the type of amyloid disease includes administering an amyloid-reactive substance or detection dye and calculating the organ:organ ratio for two or more organs. In some cases, the step of calculating the organ:organ ratio for two or more organs includes calculating the ratio between the organ uptake value for the first organ and the organ uptake value for the second organ. In some cases, the organ:organ ratio is selected from the group consisting of liver:heart, spleen:heart, spleen:liver, spleen:kidney, kidney:heart, and kidney:liver. In some cases, the organ:organ ratio is the heart:spleen ratio. In some cases, the organ:organ ratio is between 0 and 1, 1, or greater than 1. In some cases, the organ:organ ratio serves as an indicator of the type of amyloid disease in an individual.

[0097] In some embodiments, organ uptake values ​​or organ:organ ratios indicate the type of amyloid disease. In some embodiments, organ uptake values ​​or organ:organ ratios are indicators of the type of amyloid disease if they exceed a cutoff value or threshold. For example, in some embodiments, if the organ:organ ratio for amyloid disease type is 1.4, and the organ:organ ratio for the individual is calculated to be 1.4 or greater, a diagnosis of the type of amyloid disease is made. In another embodiment, but not intended to be limiting, if the cutoff for organ uptake values ​​for amyloid disease type is 1.4, and the organ:organ ratio for the individual is calculated to be less than 1.4, it is not appropriate to diagnose the type of amyloid disease. Specific cutoffs or thresholds for diagnosing the type of amyloid disease vary depending on the type of amyloid disease, the stage of disease progression, the patient population, the amyloid-reactive agent or detection dye administered, and the detection method used. In some embodiments, organ uptake values, or organ-to-organ cutoffs, or thresholds are calculated from data on the organ distribution of amyloid-reactive agents or detection dyes. In some embodiments, organ uptake values, or organ-to-organ cutoffs, or thresholds are calculated from data on populations with a specific type of amyloid disease.

[0098] Preferably, the organ uptake value or organ:organ uptake rate cutoff or threshold for diagnosing the type of amyloid disease is determined using a receiver operating characteristic curve. As understood in the art, a receiver operating characteristic curve, or ROC curve, is a plot of the performance of a particular feature that distinguishes two populations, namely patients with amyloid disease and controls, e.g., patients without amyloid disease. Data spanning the entire population (i.e., patients and controls) are sorted in ascending order based on the numerical value of a single feature (e.g., organ uptake value). For each value of that feature, the true positive rate and false positive rate of the data are determined. The true positive rate (sensitivity) is determined by counting the number of cases exceeding the value of the feature under consideration and dividing by the total number of patients. The false positive rate (specificity) is determined by counting the number of controls exceeding the value of the feature under consideration and dividing by the total number of controls.

[0099] ROC curves can be generated for single features as well as other single output values. For example, a combination of two or more features can be mathematically added (addition, division, multiplication, etc.) to provide a single sum, which can then be plotted on the ROC curve. Furthermore, any combination of multiple features, and the single output value resulting from such combination, can be plotted on the ROC curve. These feature combinations can include tests. The ROC curve is a plot of the true positive rate (sensitivity) of a test against the false positive rate (1-specificity) of the test. The area under the ROC curve is the performance index of a given sample population, with a range of 1 to 0 representing a complete test that gives a completely random response when classifying test subjects. As with any diagnostic application, the area under the ROC curve indicates the predictive power of a model and can be used to compare the predictive power of one model with another. Using ROC curves, cutoff values ​​can be selected to diagnose an individual's amyloid disease and / or amyloid type with high confidence.

[0100] In some embodiments, the step of measuring the organ dispersion pattern of amyloid-reactive agents or detection dyes in one or more organs, or the step of calculating the organ:organ ratio of amyloid-reactive agents or detection dyes, includes analyzing image data. Image data can be generated by any procedure known in the art that can enable imaging of amyloid-reactive reagents or dyes. For example, amyloid-reactive agents or detection dyes can be detected by positron emission tomography (PET), computed tomography (CT), magnetic resonance imaging (MRI), or single-photon emission computed tomography (SPECT). In certain embodiments, amyloid-reactive agents or detection dyes can be detected by combined imaging methods such as PET / CT (PET with simultaneous computed tomography) or PET / MRI (PET with simultaneous magnetic resonance imaging). The imaging procedure can acquire one or more images of an observation area of ​​an individual. In certain embodiments, imaging provides multiple images, which are combined, superimposed, added, removed, color-coded, or otherwise merged, and mathematically manipulated in any way known in the art. The resulting image may be a digital or analog image that can be displayed as a “hard” image on, for example, printer paper, photographic paper, or film, or an image on a screen, for example, a video or LCD screen.

[0101] In some embodiments, PET images are analyzed using the region of interest (ROI) method. In some embodiments, the images are planar images. In some embodiments, the images are coronal, axial, or sagittal images.

[0102] In some embodiments, the method includes obtaining organ distribution data relating to amyloid-reactive agents or detection dyes. In some embodiments, the organ distribution data is an image. Images generated using the imaging procedure embodied in the present invention can be analyzed in any way known in the art. For example, in some embodiments, image data obtained from a PET or SPECT scan can be input to a processor that identifies individual pixels or groups of pixels whose brightness is greater than a predetermined threshold or average background, and the identified pixels may be characterized by indicating the presence of amyloid-reactive agents or detection dyes. In another embodiment, an image can be obtained from an image input to a processor by scanning. In such embodiments, the location of the amyloid-reactive reagent or dye in the image can be determined using a similar process that identifies bright spots in the image. In certain embodiments, the analysis of the image further includes determining the brightness, density, intensity, or a combination thereof of the output brightness, and this analysis may correlate with the amount of radiolabeled protein in the image, which is an area or region of the image, or a specific spot on the image. While not bound by theory, areas or spots on an image with greater intensity than other areas or spots may, for example, hold higher concentrations of radiolabeled protein than the target amyloid deposition. Therefore, the radiolabeled amyloid-reactive reagent or dye attached to the area where the amyloid-reactive reagent or dye is localized may be at a high concentration.

[0103] In some embodiments, a method for diagnosing the type of amyloid disease involves analyzing images at the spatial location of an administered amyloid-reactive agent or detectable dye targeting a region of interest. In other embodiments, analysis of the pharmacokinetics of an administered amyloid-reactive reagent or dye may provide information regarding the appropriate timing of injection of the amyloid-reactive reagent or dye. The presence or absence of amyloid can be determined by identifying areas, regions, or spots on the image that correlate with the presence of radiolabeled proteins. For example, in some embodiments, identifying regions or spots of amyloid-reactive agent or detection dye concentrate indicates the presence of amyloid. In some embodiments, images correlated with the presence of amyloid-reactive agent or detection dye are used to diagnose amyloid disease in an individual.

[0104] In some embodiments, the method further includes providing a diagnosis of amyloid disease type based on organ distribution patterns. In some embodiments, a specific organ distribution pattern serves as an indicator of a specific type of amyloid disease. For example, in some embodiments, the ratio of heart:spleen, heart:liver, spleen:liver, spleen:kidney, kidney:heart, or kidney:liver is used to diagnose ATTR. In some embodiments, the ratio of heart:spleen, heart:liver, spleen:liver, spleen:kidney, kidney:heart, or kidney:liver is used to diagnose ALECT2. In some embodiments, the ratio of heart:spleen, heart:liver, spleen:liver, spleen:kidney, kidney:heart, or kidney:liver is used to diagnose AL.

[0105] In some embodiments, different types of amyloid diseases have different organ-to-organ ratios. In some embodiments, a particular type of amyloid disease may have a larger liver:heart ratio than another amyloid disease. In some embodiments, the heart:spleen ratio for individuals diagnosed with AATR is greater than the heart:spleen ratio for individuals diagnosed with AL. In some embodiments, the heart:spleen ratio for individuals diagnosed with ATTR is greater than the heart:spleen ratio for individuals diagnosed with ALECT2. In some embodiments, individuals with a heart:spleen ratio greater than 1 are diagnosed with ATTR. In some embodiments, individuals are diagnosed with ATTR if their heart:spleen ratio is greater than 1.2, greater than 1.3, greater than 1.4, or greater than 1.5. In some embodiments, individuals are diagnosed with AL or ALECT2 if their heart:spleen ratio is less than 1.5. In some embodiments, individuals are diagnosed with AL or ALECT2 if their heart:spleen ratio is less than 1.4, less than 1.3, less than 1.3, or less than 1.

[0106] In some embodiments, the heart:kidney ratio is used to diagnose the type of amyloid disease. In some embodiments, the heart:kidney ratio of individuals diagnosed with ATTR is greater than that of individuals diagnosed with AL. In some embodiments, the heart:kidney ratio is greater than that of individuals diagnosed with ALECT2. In some embodiments, an individual is diagnosed with ATTR if the heart:kidney ratio is greater than 1. In some embodiments, an individual is diagnosed with ATTR if the heart:kidney ratio is greater than 1.2, greater than 1.3, greater than 1.4, greater than 1.5, greater than 1.6, or greater than 1.8. In some embodiments, an individual is diagnosed with AL or ALECT2 if the heart:kidney ratio is less than 1.8. In some embodiments, an individual is diagnosed with AL or ALECT2 if the heart:kidney ratio is less than 1.6, less than 1.5, less than 1.4, or less than 1.

[0107] In some embodiments, the heart:liver ratio is used to diagnose the type of amyloid disease. In some embodiments, the heart:liver ratio of individuals diagnosed with ATTR is greater than the heart:kidney ratio of individuals diagnosed with AL. In some embodiments, the heart:liver ratio is greater than that of individuals diagnosed with ALECT2. In some embodiments, an individual is diagnosed with ATTR if the heart:liver ratio is greater than 1. In some embodiments, an individual is diagnosed with ATTR if the heart:liver ratio is greater than 1.6, greater than 1.8, greater than 2.0, greater than 2.2, or greater than 2.3. In some embodiments, an individual is diagnosed with AL or ALECT2 if the heart:liver ratio is less than 2.3. In some embodiments, an individual is diagnosed with AL or ALECT2 if the heart:liver ratio is less than 2.2, less than 2.0, less than 1.8, or less than 1.6.

[0108] In some embodiments, the liver:spleen ratio is used to diagnose the type of amyloid disease. In some embodiments, if the liver:spleen ratio is greater than 0.7, the individual is diagnosed with ATTR. In some embodiments, if the liver:spleen ratio is greater than 0.8, greater than 0.9, greater than 1.0, or greater than 1.2, the individual is diagnosed with ATTR. In some embodiments, if the liver:spleen ratio is less than 1.2, the individual is diagnosed with AL or ALECT2. In some embodiments, if the heart:liver ratio is less than 0.9, less than 0.8, or less than 1.7, the individual is diagnosed with AL or ALECT2.

[0109] In some embodiments, an individual is diagnosed with AL if the liver:heart ratio is greater than 0.3. In some embodiments, an individual is diagnosed with AL if the liver:heart ratio is greater than 0.4, greater than 0.5, greater than 0.6, or greater than 0.7. In some embodiments, an individual is diagnosed with ATTR or ALECT2 if the liver:heart ratio is less than 0.3. In some embodiments, an individual is diagnosed with ATTR or ALECT2 if the liver:heart ratio is less than 0.4, less than 0.6, or less than 0.7.

[0110] In some embodiments, an individual is diagnosed with AL if the spleen:heart ratio is greater than 0.5. In some embodiments, an individual is diagnosed with AL if the spleen:heart ratio is greater than 0.6, greater than 0.7, or greater than 0.8. In some embodiments, an individual is diagnosed with ATTR or ALECT2 if the spleen:heart ratio is less than 0.5. In some embodiments, an individual is diagnosed with ATTR or ALECT2 if the spleen:heart ratio is less than 0.6, less than 0.7, or less than 0.8.

[0111] In some embodiments, an individual is diagnosed as AL if the spleen:liver ratio is greater than 9. In some embodiments, an individual is diagnosed as AL if the spleen:liver ratio is greater than 10, greater than 11, or greater than 12. In some embodiments, an individual is diagnosed as ATTR or ALECT2 if the spleen:liver ratio is less than 9. In some embodiments, an individual is diagnosed as ATTR or ALECT2 if the spleen:liver ratio is less than 10, less than 11, or less than 12.

[0112] In some embodiments, an individual is diagnosed with AL if the kidney:heart ratio is greater than 0.5. In some embodiments, an individual is diagnosed with AL if the kidney:heart ratio is greater than 0.6, greater than 0.7, or greater than 0.8. In some embodiments, an individual is diagnosed with ATTR or ALECT2 if the kidney:heart ratio is less than 0.5. In some embodiments, an individual is diagnosed with ATTR or ALECT2 if the kidney:heart ratio is less than 0.6, less than 0.7, or less than 0.8.

[0113] In some embodiments, the organ-to-organ ratio is based on the standard uptake ratio (SUVR). In some embodiments, the SUVR is calculated using a blood pool as the reference tissue. In some embodiments, the SUVR is calculated for each organ by dividing the amount of amyloid detector or dye in the organ by the blood pool ratio. In some embodiments, the blood pool is a vein or artery. In some embodiments, the blood pool is the lumen of the thoracic aorta.

[0114] In some embodiments, the levels of amyloid-reactive agents or detection dyes in the heart are highest in individuals with ATTR. In some embodiments, the levels of amyloid-reactive agents or detection dyes in the liver are highest in individuals with AL. In some embodiments, the levels of amyloid-reactive agents or detection dyes in the spleen are highest in individuals with ALECT2. In some embodiments, the levels of amyloid-reactive agents or detection dyes in the kidneys are highest in individuals with ALECT2.

[0115] In some embodiments, the levels of amyloid-reactive agents or detection dyes in the heart are lowest in individuals with ALECT2. In some embodiments, the levels of amyloid-reactive agents or detection dyes in the liver are lowest in individuals with ATTR. In some embodiments, the levels of amyloid-reactive agents or detection dyes in the spleen are lowest in individuals with ATTR. In some embodiments, the levels of amyloid-reactive agents or detection dyes in the kidneys are lowest in individuals with ATTR.

[0116] In some embodiments, the cutoff value for diagnosing a particular type of amyloid disease is selected based on a specific p-value. In some embodiments, the cutoff is selected to provide p-values ​​less than 0.1, less than 0.05, less than 0.01, less than 0.005, or less than 0.001.

[0117] In some embodiments, the cutoff value for diagnosing a specific type of amyloid disease is selected based on the desired sensitivity. In some embodiments, this cutoff is selected to provide a sensitivity of at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95%.

[0118] In some embodiments, the cutoff value is selected based on the desired specificity (i.e., the ability to distinguish between different types of amyloid diseases). In some embodiments, this cutoff is selected to provide a specificity of at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95%.

[0119] Those skilled in the art will understand that each of the ratios described herein can be easily converted to its reciprocal. For example, the heart:spleen ratio of 2:1 (2) is the same as the spleen:heart ratio of 1:2 (0.5).

[0120] II. Treatment Methods Some aspects of the present invention provide a method for treating amyloid disease based on the organ distribution pattern of an amyloid-reactive agent or detection dye. In some embodiments, the present invention provides a method for treating amyloid disease, which includes administering an amyloid-reactive agent or detection dye, measuring the organ distribution of the amyloid-reactive agent or detection dye, and selecting a treatment method based on the type of disease.

[0121] In some embodiments, a method for treating amyloid disease includes administering an amyloid-reactive agent or detection dye and measuring the organ-dispersion pattern of the amyloid-reactive agent or detection dye with respect to one or more organs. In some embodiments, the organ-dispersion pattern of the amyloid-reactive agent or dye indicates the type of amyloid disease. In some embodiments, the method further includes selecting a treatment method based on the type of amyloid disease.

[0122] In some embodiments, the method includes obtaining an organ dispersion pattern of an amyloid-reactive substance or detection dye, the organ dispersion pattern indicating a specific type of amyloid disease, and treatment based on the amyloid disease.

[0123] In some embodiments, a method for treating amyloid disease involves administering an amyloid-reactive agent or detection dye containing an amyloid-reactive peptide. In some embodiments, the amyloid-reactive agent or detection dye includes a peptide, a fusion protein, a small molecule compound, or an antibody or fragment.

[0124] In some embodiments, a method for treating amyloid disease includes administering an amyloid-reactive substance or detection dye containing an amyloid-reactive peptide. In some embodiments, the amyloid-reactive peptide is a peptide having the amino acid sequence described in any one of SEQ ID NOs: 1 to 14. In some embodiments, a method for treating amyloid disease includes an amyloid-reactive peptide having the amino acid sequence described in SEQ ID NO: 13.

[0125] In some embodiments, a method for treating amyloid disease includes administering an amyloid-reactive agent or detection dye containing a detectable label that determines an organ dispersion pattern. In some embodiments, the amyloid-reactive agent or detection dye includes a fluorescent label, a chemiluminescent tag, or a radiolabel. In some embodiments, the amyloid-reactive agent or detection dye includes a radiolabel. In some embodiments, the radiolabeled amyloid-reactive agent or detection dye is 124 It is I-p5+14. In another embodiment, the radiolabeled amyloid-reactive substance or detection dye is flolbetapyl, flolbetaben, or flutemetamol. In some embodiments, the method for treating amyloid disease includes p5+14. In some embodiments, the amyloid-reactive substance is radiolabeled. In some embodiments, the radiolabeling is 11 C, 18 F, 111 In, 99m Tc, and 123 I, 124 I, or 125I. In some embodiments, the radiolabeled amyloid-reactive substance or detection dye is a radiolabeled amyloid-reactive peptide. In some embodiments, the radiolabeled amyloid-reactive peptide is 124 It is an I-labeled amyloid-reactive peptide. In another embodiment, a method for diagnosing the type of amyloid disease is: 124 This includes administering I-p5+14. In another embodiment, the radiolabeled amyloid-reactive substance or detection dye is florbetapyl, florbetaben, or flutemetamol.

[0126] In certain other embodiments, the amyloid-reactive agent or detection dye includes a fluorescent label. In some embodiments, the fluorescently labeled amyloid-reactive agent or detection dye is ThT. In some embodiments, the amyloid-reactive agent or detection dye is administered parenterally, near the tumor, transmucosa, percutaneously, intramuscularly, intravenously, intradermally, subcutaneously, intraperitoneally, intraventricularly, or intracranially. In some cases, the amyloid-reactive agent or detection dye is administered intravenously or intraperitoneally.

[0127] In some embodiments, a method for treating amyloid disease involves measuring the organ dispersion pattern of amyloid-reactive agents or detection dyes in one or more organs. In some embodiments, a method for treating a type of amyloid disease includes measuring the organ dispersion pattern of amyloid-reactive agents or detection dyes in one or more of the following organs: heart, spleen, liver, kidneys, peripheral nerves, gastrointestinal tract, muscles, lungs, brain, and lymph nodes. In some embodiments, the one or more organs are thoracic and abdominal organs. In some embodiments, the one or more organs are the heart, spleen, liver, or kidneys. In some embodiments, the step of measuring the organ dispersion pattern of amyloid-reactive agents or detection dyes in one or more organs includes determining the organ uptake value in each organ. In some embodiments, the organ uptake value is a standard uptake value for each organ. In some embodiments, the standard uptake value is determined as the ratio of the amount of amyloid-reactive agents or detection dye detected in the organ to the amount of amyloid-reactive agents or detection dye detected in the blood. In some embodiments, organ uptake values ​​indicate the organ dispersion pattern of amyloid-reactive substances or detection dyes.

[0128] In another embodiment, the step of measuring the organ dispersion pattern of an amyloid-reactive substance or detection dye in one or more organs includes calculating an organ:organ ratio for two or more organs. In some embodiments, the step of calculating an organ:organ ratio for two or more organs includes calculating a ratio between the organ uptake value for a first organ and the organ uptake value for a second organ. In some cases, the organ:organ ratio is selected from the group consisting of liver:heart, spleen:heart, spleen:liver, spleen:kidney, kidney:heart, and kidney:liver. In some cases, the organ:organ ratio is the heart:spleen ratio. In some embodiments, this ratio is the reciprocal of any of these ratios.

[0129] In some embodiments, measuring the organ dispersion pattern of an amyloid-reactive substance or detection dye involves analyzing image data generated by PET, CT, MRI, SPECT, PET / CT, PET / MRI, or other imaging methods. In some embodiments, the step of measuring the organ dispersion pattern of an amyloid-reactive reagent or detection dye involves analyzing images at the spatial location in the region of interest.

[0130] In some embodiments, the organ distribution pattern indicates the type of amyloid disease. In some embodiments, the organ distribution pattern is used to select a specific treatment based on the type of amyloid disease. In some embodiments, the method further includes providing a diagnosis of the type of amyloid disease based on the organ distribution pattern. In some embodiments, a specific organ distribution pattern indicates a specific type of amyloid disease. For example, in some embodiments, ATTR is diagnosed using the ratios heart:spleen, heart:liver, spleen:liver, spleen:kidney, kidney:heart, or kidney:liver. In some embodiments, ALECT2 is diagnosed using the ratios heart:spleen, heart:liver, spleen:liver, spleen:kidney, kidney:heart, or kidney:liver. In some embodiments, AL is diagnosed using the ratios heart:spleen, heart:liver, spleen:liver, spleen:kidney, kidney:heart, or kidney:liver.

[0131] In some embodiments, the method includes treating systemic amyloidosis and selecting a treatment method. Examples of some amyloid diseases that can be diagnosed and / or treated by the method described herein include, but are not limited to, AA amyloidosis, AL amyloidosis, AH amyloidosis, Aβ amyloidosis, ATTR amyloidosis, ALECT2 amyloidosis, and IAPP amyloidosis in type II diabetes, Alzheimer's disease, thyroid tumors, Parkinson's disease, tauopathy, senile systemic amyloidosis, familial hemodialysis, senile systemic aging, senile pituitary disorders, iatrogenic syndromes, reactive chronic inflammation, thyroid tumors, myeloma, or other forms of cancer. In some embodiments, the method for treating amyloid disease includes selecting a treatment method for systemic amyloidosis. In some embodiments, the method for treating amyloid disease includes selecting a treatment method for AL amyloidosis, ATTR amyloidosis, or ALECT2 amyloidosis. In some embodiments, the treatment is a targeted therapy for ATTR amyloidosis, AL amyloidosis, or ALECT2 amyloidosis.

[0132] In some embodiments, the treatment is a small molecule, antibody, peptide, protein, nucleic acid, and / or gene therapy. In some embodiments, the treatment is a targeted therapy specific to a particular type of amyloid disease.

[0133] In some embodiments, the treatment is a targeted therapy for ATTR amyloidosis, AL amyloidosis, or ALECT2 amyloidosis. In some embodiments, the treatment is a targeted therapy for ATTR amyloidosis. In some embodiments, the treatment comprises a TTR tetramer stabilizer. In some embodiments, the TTR tetramer stabilizer is epigallocatechin-3-gallate (EGCG), AG-10, CHF5074, tafamidis, or diflunisal. In some embodiments, the treatment comprises an antibody or fragment that binds to TTR in an abnormally folded structure. In some embodiments, the antibody is PRX004. In some embodiments, the treatment comprises an oligonucleotide. In some embodiments, the oligonucleotide is a TTR silencer. In some embodiments, the TTR silencer is patisirane (ALN-TTR02), vitricirane, inotercene, or AKCEA-TTR-LRx. In some embodiments, the treatment comprises an ATTR amyloid disruptor. In some embodiments, the treatment comprises doxycycline, tauroursodeoxycholic acid, or serum amyloid-P (SAP). In some embodiments, the treatment comprises organ transplantation. In some embodiments, the treatment comprises liver transplantation.

[0134] In another embodiment, the treatment is targeted therapy for AL amyloidosis. In some embodiments, the treatment comprises bortemodib, ixazomib, or cazilfomib. In some embodiments, the treatment comprises an antibody or fragment. In some embodiments, the treatment comprises daratumab, CAEL-101, elotuzumab, or verantamab mafodotin. In some embodiments, the treatment comprises stem cell therapy. In some embodiments, the treatment comprises a corticosteroid. In some embodiments, the corticosteroid is dexamethasone.

[0135] In some embodiments, this method is used to discontinue potential treatments for patients with amyloidosis. In some embodiments, this method is used to diagnose one type of amyloidosis and discontinue treatments for other types of amyloidosis. In some embodiments, this method is used to diagnose ALECT2 and discontinue treatments for AL or ATTR amyloidosis.

[0136] In some embodiments, this method is used to identify types of amyloidosis and develop therapies specific to particular types of amyloidosis. For example, in some embodiments, this method is used to identify individuals with ALECT2 amyloidosis and develop therapies specific to ALECT2 amyloidosis.

[0137] III. Kit Some aspects of the present invention provide a kit for diagnosing or detecting the type of amyloid disease in an individual using the method described herein.

[0138] In some embodiments, the kit includes an amyloid-reactive agent or detection dye and instructions for use. In some embodiments, the amyloid-reactive agent or detection dye includes a detectable label. In some embodiments, the amyloid-reactive agent or detection dye is 124 In another embodiment, the amyloid-reactive agent or detection dye is florbetapyl, florbetaben, or flutemetamol. In some embodiments, the amyloid-reactive agent or detection dye is ThT.

[0139] In some embodiments, the radioactive label is 11 C, 18 F, 111 In, 99m Tc, and 123 I, 124 I, or 125I. In some embodiments, the radiolabeled amyloid-reactive substance or detection dye is a radiolabeled amyloid-reactive peptide. In some embodiments, the radiolabeled amyloid-reactive peptide is 124 It is an I-labeled amyloid-reactive peptide. In another embodiment, a method for diagnosing the type of amyloid disease is: 124 This includes administering I-p5+14. In another embodiment, the radiolabeled amyloid-reactive substance or detection dye is florbetapyl, florbetaben, or flutemetamol.

[0140] In some embodiments, the instructions for use include instructions for detecting amyloid-reactive substances or detection dyes in one or more organs. In some embodiments, the amyloid-reactive substances or detection dyes are detected in the blood, heart, lungs, kidneys, or spleen.

[0141] In some embodiments, the kit includes instructions for calculating the SUVR ratio for one or more organs. In some embodiments, the SUVR is calculated using a blood pool, which is a standard tissue. In some embodiments, the SUVR for each organ is calculated by dividing the amount of amyloid-detecting agent or dye in the organ by the ratio in the blood pool. In some embodiments, the blood pool is a vein or artery. In some embodiments, the blood pool is the lumen of the thoracic aorta.

[0142] In some embodiments, the instructions for use include instructions for determining organ-to-organ ratios. In some embodiments, the organ-to-organ ratios are liver:heart, spleen:heart, spleen:liver, spleen:kidney, kidney:heart, kidney:liver, or any reciprocal of these ratios.

[0143] In some embodiments, the kit further includes instructions that provide a diagnosis based on organ:organ ratio.

[0144] In some embodiments, the kit includes a therapeutic agent for treating amyloid disease. [Examples]

[0145] Example 1 - Patient organs imaged using PET / CT 124 Quantitative analysis of I-p5+14 intake values. In this embodiment, amyloid type differentiation is explained using data obtained from PET images.

[0146] Imaging of amyloid deposition in patients Positron emission tomography / X-ray computed tomography (PET / CT) images 124 The data was obtained from patients with light chain-related (AL) amyloidosis, tranthyretin-related (ATTR) amyloidosis, and leukocyte chemotactic factor 2-related (ALECT2) amyloidosis enrolled in the first 26-patient cohort of the I-p5+14 Phase 1 / 2 trial. 124 I-p5+14 contrast agent is a radiolabeled amyloid-reactive polypeptide that can be used to image target amyloid in PET / CT scans. 124 I-p5+14 can be used to detect amyloid deposits in the heart, liver, spleen, and kidneys, and, 124 Data obtained from PET / CT images using I-p5+14 can be easily quantified.

[0147] Organ determination: Blood uptake PET images were manually analyzed using the Region of Interest (ROI) method. Using planar images, ROIs were placed in the organ of interest using CT data to achieve precise anatomical placement in the coronal, axial, or sagittal direction. Care was taken to avoid areas of organs where major blood vessels were present. ROIs were large enough to cover the mean area of ​​the tissue, or they focused on a specific area, anatomical area, or region of interest. Data from the ROIs were recorded, and the mean radioactivity per unit volume (Bq / cc) was determined.

[0148] The standard uptake ratio (SUVR) was calculated using the blood pool as the reference tissue. In this study, the lumen of the thoracic aorta was identified immediately distal to the aortic arch using CT imaging and used as the blood pool ROI. The radioactivity ROI of the blood pool was determined by careful placement (Bq / cc). Next, the SUVR for each organ (heart, spleen, liver, and left kidney) was calculated by dividing the tissue radioactivity by the blood pool radioactivity to obtain the organ:blood pool ratio.

[0149] Organs: responsible for blood uptake, 124 Calculations were performed for AL, ATTR, and ALECT2 patients enrolled in the first 26-patient cohort of the Phase 1 / 2 trial of I-p5+14. Organ:blood uptake values ​​for each patient cohort are summarized in Figure 2. The analysis of mean values ​​for the entire population is shown in the relationship described in Figure 3. The analysis revealed that for each of these patient cohorts, 124 It has become clear that there are distinct differences in organ-specific uptake of I-p5+14, namely: Heart - ATTR>AL>ALECT2 Liver - AL>ALECT2>>ATTR Spleen - ALECT2>AL>>ATTR Kidney - ALECT2 > AL >> ATTR

[0150] These relationships enabled the development of algorithms to distinguish between AL and ATTR amyloidosis.

[0151] Distinguishing between AL and ATTR As the first step, the distribution of each organ was analyzed for normality using skewness and kurtosis indices. Next, outliers with a standard deviation greater than 3.29 above the mean were removed from each list. Figure 4 shows the dataset after the removal of outlier data points. Re-analysis of the mean using the data after the outliers were removed revealed the organ-specific relationships described in Figure 5. After removing outliers from the population, the following relationships were observed: Heart - ATTR>AL>ALECT2 Liver-ALECT2>ATTR>ATTR Spleen - ALECT2 >> AL > ATTR Kidney - ALECT2 > AL > ATTR

[0152] As the second step, the ability to distinguish between AL and ATTR, and between ATTR and AL, was evaluated using receiver operating characteristic (ROC) analysis. ROC curves for AL and ATTR were generated using the SUVR values ​​for each individual's organs and the ratio of one organ to the other (based on the data originally described in Figure 5). The area under the curve (AUC) was determined, and the significance and 95% confidence interval (95% CI) of the AUC were determined. The sensitivity and specificity values ​​of the optimal cutoff number for distinguishing between AL and ATTR were also determined using the current dataset.

[0153] Table 2 summarizes the ROC analysis results for ATTR, while Figure 6 shows the ROC curve for cardiac uptake, and Figures 7A-7F show the ROC curves for organ-to-organ uptake ratios in ATTR. Table 3 summarizes the ROC analysis results for AL, while Figures 7A-7C show the ROC curves for individual organ uptake, and Figures 8A-8F show the ROC curves for organ-to-organ uptake ratios in AL. [Table 2] [Table 3]

[0154] For detecting ATTR patients, calculating the cardiac:splenic SUVR ratio provided the optimal method with p=0.002 and an AUC of 0.96. Using a cutoff value of 1.40, a sensitivity of 100 and specificity of 75% were obtained for the diagnosis of ATTR. The cardiac:renal SUVR ratio provided similarly good predictive values ​​with slightly higher specificity.

[0155] The mean heart:spleen SUVR ratio in ATTR patients is higher than that in AL patients. In addition, based on the limited data from ALECT2 patients, this ratio is expected to be much lower in ALECT2 than in AL, suggesting that AL and ALECT2 can be distinguished at more data points. Based on the current dataset for AL, ATTR, and ALECT2 patients, using a one-way ANOVA with multiple comparisons, the spleen SUVR in ALECT2 patients is significantly higher than in both AL (p=0.02) and ATTR (p=0.002) patients.

[0156] The results of these analyses show that ATTR amyloidosis and AL amyloidosis can be distinguished using either the SUVR value of individual organs, or more precisely, the organ-to-organ ratio, for example, the heart:spleen SUVR ratio or the heart:kidney SUVR ratio, and that ALECT2 amyloidosis can potentially be distinguished from both AL and ATTR amyloidosis.

[0157] conclusion The obtained image data is used to determine the cardiac amyloid:splenic amyloid ratio, which can be used to differentiate between AL, ATTR, and ALECT2 amyloid. In AL patients (as a population), both the heart and spleen are involved, and the cardiac amyloid:splenic amyloid ratio is approximately 1. In ATTR patients, cardiac amyloid is involved, but the spleen is not, so the ratio is greater than 1. In ALECT2 patients, there is a large amount of splenic amyloid and (if any) very little cardiac amyloid, so the ratio is much less than 1. Based on the current patient population, calculating the cardiac:splenic ratio can be used to determine what type of amyloid a patient has with a certain level of statistical certainty (e.g., >90%, >80%).

Claims

1. 1. A pharmaceutical composition comprising an amyloid-reactive agent or detector dye for use in a method for diagnosing a type of amyloid disease, the method comprising: administering to the individual an amyloid-reactive agent or a detection dye; and Measuring the organ distribution pattern of the amyloid-reactive agent or detector dye in one or more organs of said individual. Including, The pharmaceutical composition, wherein the organ distribution pattern of the amyloid-reactive agent or detectable dye is indicative of a type of amyloid disease.

2. 1. A pharmaceutical composition comprising an amyloid-reactive agent or a detection dye for use in a method of treating an amyloid disease, the method comprising: administering to the individual an amyloid-reactive agent or a detection dye; and Measuring an organ distribution pattern of the amyloid-reactive agent or detectable dye in one or more organs of the individual, the organ distribution pattern of the amyloid-reactive agent or detectable dye being indicative of a type of amyloid disease; and Selecting treatment options based on the type of amyloid disease The pharmaceutical composition comprising:

3. 1. A pharmaceutical composition comprising an amyloid-reactive agent or detector dye for use in a method for diagnosing a type of amyloid disease, the method comprising: receiving organ distribution pattern data for an amyloid reactive agent or a detection dye for the individual; and Calculating organ:organ ratios for two or more organs Including, The pharmaceutical composition, wherein the organ:organ ratio is used to diagnose the type of amyloid disease in the individual.

4. Organ:blood ratio and / or organ of said amyloid-reactive agent or detection dye The pharmaceutical composition according to any one of claims 1 to 3, wherein the ratio of the amount of the protein to the amount of the organ is calculated.

5. 5. The pharmaceutical composition of claim 4, wherein the organ:organ ratio is selected from the group consisting of liver:heart, spleen:heart, spleen:liver, spleen:kidney, kidney:heart, and kidney:liver.

6. 6. The pharmaceutical composition of claim 5, wherein the organ:organ ratio is heart:spleen ratio.

7. 7. The pharmaceutical composition of claim 6, wherein the individual is diagnosed with ATTR amyloidosis if the heart:spleen ratio is greater than 1.

4.

8. The pharmaceutical composition according to any one of claims 1 to 3, wherein the type of amyloid disease comprises systemic amyloidosis.

9. The pharmaceutical composition according to any one of claims 1 to 3, wherein the type of amyloid disease is selected from the group consisting of amyloid light chain amyloidosis (AL), transthyretin-related amyloidosis (ATTR), and ALECT2.

10. The pharmaceutical composition according to any one of claims 1 to 3, wherein the organ is selected from the group consisting of the heart, the spleen, the kidney, and the liver.

11. The pharmaceutical composition of any one of claims 1 to 3, wherein the amyloid-reactive agent is a detectably labeled amyloid-reactive peptide.

12. 1. A pharmaceutical composition comprising an amyloid-reactive agent for use in a method of diagnosing transthyretin-related amyloidosis (ATTR) in an individual, the method comprising: administering to the individual an amyloid-reactive agent; and Measuring the organ distribution pattern of the amyloid-reactive agent in one or more organs, including the heart, spleen, kidney, and / or liver, of said individual. Including, the organ distribution pattern of the amyloid-reactive agent is indicative of whether the individual has ATTR, the organ distribution pattern of the amyloid-reactive agent being an organ:organ ratio of the amyloid-reactive agent, the organ:organ ratio being spleen:heart, kidney:heart, or liver:heart; i) if the heart:spleen ratio is greater than 1, the individual is diagnosed with ATTR; ii) if the heart:kidney ratio is greater than 1, the individual is diagnosed with ATTR; or iii) if the heart:liver ratio is greater than 1, the individual is diagnosed with ATTR; The pharmaceutical composition, wherein the amyloid-reactive agent comprises an amyloid-reactive peptide comprising an amino acid sequence set forth in any one of SEQ ID NOs: 1-14, and the amyloid-reactive peptide is detectably labeled.

13. 1. A pharmaceutical composition comprising an amyloid-reactive agent for use in a method of treating transthyretin-related amyloidosis (ATTR) in an individual, the method comprising: administering to the individual an amyloid-reactive agent; and Measuring the organ distribution pattern of the amyloid-reactive agent in one or more organs, including the heart, spleen, kidney, and / or liver, of said individual. Including, The organ distribution pattern of the amyloid-reactive agent indicates whether the individual has ATTR, and the organ distribution pattern of the amyloid-reactive agent indicates whether the individual has ATTR. an organ:organ ratio of thyroid reactive agents, said organ:organ ratio being spleen:heart, kidney:heart, or liver:heart; i) if the heart:spleen ratio is greater than 1, the individual is diagnosed with ATTR; ii) if the heart:kidney ratio is greater than 1, the individual is diagnosed with ATTR; or iii) if the heart:liver ratio is greater than 1, the individual is diagnosed with ATTR; the amyloid-reactive agent comprises an amyloid-reactive peptide comprising an amino acid sequence set forth in any one of SEQ ID NOs: 1-14, the amyloid-reactive peptide being detectably labeled; The pharmaceutical composition further comprises administering a treatment based on the diagnosis.

14. 1. A pharmaceutical composition comprising an amyloid-reactive agent for use in a method of diagnosing transthyretin-related amyloidosis (ATTR) in an individual, the method comprising: receiving organ distribution pattern data for the amyloid-reactive agent administered to the individual; and calculating an organ:organ ratio of the amyloid reactive agent for two or more organs including the heart, spleen, kidney, and / or liver; the organ:organ ratio is used to diagnose ATTR in an individual, the organ:organ ratio being spleen:heart, kidney:heart, or liver:heart; i) if the heart:spleen ratio is greater than 1, the individual is diagnosed with ATTR; ii) if the heart:kidney ratio is greater than 1, the individual is diagnosed with ATTR; or iii) if the heart:liver ratio is greater than 1, the individual is diagnosed with ATTR; The pharmaceutical composition, wherein the amyloid-reactive agent comprises an amyloid-reactive peptide comprising an amino acid sequence set forth in any one of SEQ ID NOs: 1-14, and the amyloid-reactive peptide is detectably labeled.

15. The pharmaceutical composition according to any one of claims 1 to 3 and 12 to 14, wherein the amyloid-reactive peptide comprises the amino acid sequence set forth in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:13, or SEQ ID NO:

14.

16. The pharmaceutical composition of any one of claims 1 to 3 and 12 to 14, wherein the amyloid-reactive agent reacts with Aβ fibrils.

17. The pharmaceutical composition of any one of claims 1 to 3 and 12 to 14, wherein the amyloid-reactive agent reacts with synthetic fibrils comprised of light chains or fragments thereof.

18. The pharmaceutical composition according to any one of claims 1 to 3 and 12 to 14, wherein the amyloid-reactive peptide comprises the amino acid sequence set forth in SEQ ID NO:

13.

19. The amyloid-reactive peptide is １２４ The pharmaceutical composition according to any one of claims 1 to 3 and 12 to 14, comprising the amino acid sequence set forth in SEQ ID NO: 13 labeled with I radioisotope.

20. The pharmaceutical composition of any one of claims 1 to 3, wherein the amyloid-reactive agent is selected from the group consisting of florbetapir, florbetaben and flutemetamol.

21. The pharmaceutical composition according to any one of claims 1 to 3, wherein the detection dye is ThT.

22. The pharmaceutical composition according to any one of claims 1 to 3 and 12 to 14, wherein the organ distribution pattern is measured using PET / CT images.

23. The pharmaceutical composition of any one of claims 1 to 3 and 12 to 14, wherein the amyloid-reactive agent is radiolabeled.

24. The pharmaceutical composition of any one of claims 1 to 3 and 12 to 14, wherein the method further comprises administering said treatment to said individual.

25. A kit for diagnosing transthyretin-related amyloidosis (ATTR) comprising an amyloid-reactive agent for use according to any one of claims 12 to 14.

26. A kit for diagnosing a type of amyloid disease comprising an amyloid-reactive agent or a detection dye and instructions for use.

27. The kit according to claim 26, wherein the instructions for use include the use according to any one of claims 1 to 3 and 12 to 14.

28. 28. The kit of claim 26 or claim 27, wherein the kit is for detecting or diagnosing systemic amyloidosis.

29. 27. The kit of claim 26, wherein the kit is for detecting or diagnosing amyloid light chain amyloidosis (AL), transthyretin-related amyloidosis (ATTR), or ALECT2.

30. 27. The kit of claim 26, wherein the amyloid-reactive agent is a detectably labeled amyloid-reactive peptide.

31. The kit of claim 30, wherein the amyloid-reactive peptide comprises the amino acid sequence set forth in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:13, or SEQ ID NO:

14.

32. 27. The kit of claim 26, wherein the amyloid-reactive agent reacts with Aβ fibrils.

33. 27. The kit of claim 26, wherein the amyloid-reactive agent reacts with a synthetic fiber comprised of a light chain or a fragment thereof.

34. 27. The kit of claim 26, wherein the amyloid-reactive agent is selected from the group consisting of florbetapir, florbetaben, and flutemetamol.

35. 27. The kit of claim 26, wherein the detection dye is ThT.

36. 27. The kit of claim 26, comprising instructions for measuring said organ distribution pattern.

37. 27. The kit of claim 26, wherein the amyloid-reactive agent is radiolabeled.

38. 27. The kit of claim 26, comprising instructions for calculating organ:blood and / or organ:organ ratios of said amyloid-reactive agent or detectable dye.

39. 39. The kit of claim 38, wherein the organ:organ ratio is selected from the group consisting of liver:heart, spleen:heart, spleen:liver, spleen:kidney, kidney:heart, and kidney:liver.

40. 40. The kit of claim 39, comprising instructions providing a diagnosis of ATTR amyloidosis if the heart:spleen ratio is greater than 1.

4.

41. 27. The kit of claim 26, further comprising instructions for administering a treatment to the individual based on the diagnosis.

42. 27. The kit of claim 26, further comprising a therapeutic agent for treating a type of amyloid.

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