Cyclosporine-acridinium esters, their manufacturing methods, and uses

Abstract

Compositions are disclosed that include acridinium esters of cyclosporin A or C. Kits containing same, as well as methods of making and using same, are also disclosed.

Description

[Technical Field]

[0001] Cross-reference of related applications / Invocation by statement of reference This application claims the benefit of U.S. Provisional Application No. 63 / 476,552, filed on 21 December 2022 under Section 119(e) of the U.S. Patent Act. The entire content of the patent application referenced above is expressly incorporated herein by reference.

[0002] Description of research and development funded by the federal government. Not applicable. [Background technology]

[0003] background The body relies on a complex immune response system to distinguish self from non-self. The body's immune system sometimes needs to be regulated to enhance insufficient responses or suppress excessive ones. For example, when organs such as (but not limited to) kidneys, hearts, cardiopulmonary system, bone marrow, and liver are transplanted in humans, the body often rejects the transplanted tissue through a process called allograft rejection.

[0004] In treating allograft rejection, the immune system is frequently suppressed in a controlled manner using drug therapy. Immunosuppressants are carefully administered to transplant recipients to help prevent allograft rejection of non-self tissues. Some of the immunosuppressants most commonly administered to prevent organ rejection in transplant patients are cyclosporine A (CsA), mycophenolate, FK-506 (also known as tacrolimus), sirolimus (also known as rapamycin), and everolimus.

[0005] Side effects associated with immunosuppressive drugs can be partially controlled by carefully controlling the levels of the drugs present in the patient. Therapeutic monitoring of the concentrations of immunosuppressive drugs and related drugs in the blood is necessary to optimize the dosing regimen to ensure maximum immunosuppression with minimal toxicity. Immunosuppressive drugs are highly effective immunosuppressive agents, but often have a narrow effective dosage range, and their use must be carefully managed because excessive dosages can cause serious side effects. On the other hand, too low dosages of immunosuppressive agents can result in tissue rejection. The distribution and metabolism of immunosuppressive drugs can vary greatly among patients, and accurate monitoring of drug levels is essential due to the wide range and severity of adverse reactions. Summary of the Invention Problems to be Solved by the Invention

[0006] Therefore, there is a continuing need to develop rapid and accurate diagnostic methods for measuring the levels of analytes (e.g., immunosuppressive drugs such as CsA) in samples taken from patients. These methods should be fully automatable and accurate even when performed on samples containing various interfering substances. The assay should provide an accurate measurement of the amount of analyte in the sample while minimizing errors caused by interfering substances present in the sample. Brief Description of the Drawings

[0007] [Figure 1] Figure 1 shows the chemical structures of cyclosporin A (CsA) and cyclosporin C (CsC). [Figure 2] Figure 2 shows the chemical structure of a prior art cyclosporin C-acridinium ester (CsC AE) tracer CsC-NSP-DMAE-HEG3-CsA (C138H226N18O40S; molecular weight 2809.43). [Figure 3] Figure 3 illustrates one non-limiting embodiment of a cyclosporin A assay format constructed in accordance with the present disclosure. [Figure 4] Figure 4 shows the chemical structure of one non-limiting embodiment of a composition constructed in accordance with the present disclosure and designated CsC-DA-10-NSP-DMAE (Formula VI). [Figure 5] Figure 5 shows the chemical structure of one non-limiting embodiment of a composition constructed in accordance with the present disclosure and designated CsC-DA-10-TSPAE (Formula VII). [Figure 6] Figure 6 shows the chemical structure of one non-limiting embodiment of a composition constructed in accordance with the present disclosure and designated CsC-DA-10-HEGAE (Formula VIII). [Figure 7] Figure 7 shows the chemical structure of one non-limiting embodiment of a composition constructed in accordance with the present disclosure and designated CsC-DA-10-NSP-ZAE (Formula IX). [Figure 8] Figure 8 shows the chemical structure of one non-limiting embodiment of a composition constructed in accordance with the present disclosure and designated CsC-PEG15-ZAE (Formula X). [Figure 9] Figure 9 shows the chemical structure of one non-limiting embodiment of a composition constructed in accordance with the present disclosure and designated CsC-PEG23-ZAE (Formula XI). [Figure 10] Figure 10 shows the chemical structure of one non-limiting embodiment of a composition constructed in accordance with the present disclosure and designated CsC-PEG23-NSP-DMAE (Formula XII). [Figure 11] Figure 11 shows the chemical structure of one non-limiting embodiment of a composition constructed in accordance with the present disclosure and designated CsA-Oxime-PEG3-HEGAE (Formula XIII). [Figure 12] Figure 12 shows the chemical structure of one non-limiting embodiment of a composition constructed in accordance with the present disclosure and designated CsA-Oxime-PEG3-ZAE (Formula XIV). [Figure 13] Figure 13 shows the chemical structure of one non-limiting embodiment of a composition constructed in accordance with the present disclosure and designated CsA-Pent-DA-10-NSP-DMAE (Formula XV). [Figure 14] Figure 14 shows the chemical structure of one non-limiting embodiment of a composition constructed in accordance with this disclosure and designated as CsA-Pent-DA-10-ZAE (Formula XVI). [Figure 15] Figure 15 shows the chemical structure of one non-limiting embodiment of a composition constructed in accordance with this disclosure and designated as CsA-Pent-DA-10-FG-DA13-ZAE (Formula XVII). [Figure 16] Figure 16 illustrates the synthesis scheme for CsC-DA-10-NSP-DMAE shown in Figure 4. [Figure 17] Figure 17 illustrates the synthesis scheme for CsC-DA-10-TSPAE shown in Figure 5. [Figure 18] Figure 18 illustrates the synthesis scheme for CsC-DA-10-HEGAE shown in Figure 6. [Figure 19] Figure 19 illustrates the synthesis scheme for CsC-DA-10-NSP-ZAE shown in Figure 7. [Figure 20] Figure 20 illustrates the synthesis scheme for CsC-PEG15-ZAE shown in Figure 8. [Figure 21] Figure 21 illustrates the synthesis scheme for CsC-PEG23-ZAE shown in Figure 9. [Figure 22] Figure 22 illustrates the synthesis scheme of CsC-PEG23-NSP-DMAE shown in Figure 10. [Figure 23] Figure 23 illustrates the synthesis scheme for CsA-Oxime-PEG3-HEGAE shown in Figure 11. [Figure 24] Figure 24 illustrates the synthesis scheme for CsA-Oxime-PEG3-ZAP shown in Figure 12. [Figure 25] Figure 25 illustrates the synthesis scheme for CsA-Pent-DA-10-NSP-DMAE shown in Figure 13. [Figure 26] Figure 26 illustrates the synthesis scheme for CsA-Pent-DA-10-ZAE shown in Figure 14. [Figure 27]Figure 27 illustrates the synthesis scheme for CsA-Pent-DA-10-HG-DA13-ZAE shown in Figure 15. [Figure 28] Figure 28 illustrates acridinium ester (AE) screening using CsA-HEG3-NSP-DMAE, CsC-NSP-DA-10-NSP-DMAE, CsC-DA-10-NSP-ZAE, CsC-DA-10-TSPAE, and CsC-DA-10-HEGAE in a non-preformed CsA assay format. [Figure 29] Figure 29 illustrates the binding curves for cyclosporine C-acridinium ester (CsC-AE) of HEG3, NSP-DMAE, NSP-ZAE, TSP-AE, and HEGAE in a non-prepared CsA assay format. [Figure 30] Figure 30 illustrates AE screening using CsC-DA-10-NSP-ZAE, CsC-O-PEG15-ZAE, CsC-PEG23-ZAE, and CsC-PEG23-NSP-DMAE in a non-prepared CsA assay format. [Figure 31] Figure 31 illustrates the binding curves for CsC-AEs of -DA-10-NSP-ZAE, -PEG15-ZAE, -PEG23-ZAE, and -PEG23-NSP-DMAE in a non-prepared CsA assay format. [Figure 32] Figure 32 illustrates AE screening using CsA-Oxime-PEG3-ZAE, CsA-Pent-DA-10-NSP-DMAE, CsA-Pent-DA-10-ZAE, and CsA-Pent-DA-10-HG-DA-13-ZAE in a non-prepared CsA assay format. [Figure 33] Figure 33 illustrates the binding curves for cyclosporine A-acridinium esters (CsA-AEs) of -Oxime-PEG3-ZAE, -NSP-DMAE, -NSP-ZAE, and -HG-DA-13-ZAE in a non-prepared CsA assay format. [Figure 34]Figure 34 illustrates a screening study of ambient temperature effort (ATE) using CsC AEs (HEG3, DMAE, NSP-ZAE, TSPAE, and HEGAE). [Figure 35] Figure 35 illustrates ATE studies using CsC-DA-10-NSP-ZAE, CsC-PEG23-NSP-DMAE, CsC-PEG15-ZAE, CsC-PEG23-ZAE, and CsA-Pent-ZAE. [Modes for carrying out the invention]

[0008] Detailed explanation Before describing at least one embodiment of the present disclosure through illustrative language and results, it should be understood that the present disclosure is not limited to the details of construction and arrangement of the components shown in the following description in the application. Other embodiments of the present disclosure are possible, or can be implemented or performed in various ways. In this manner, the language used herein is intended to give the broadest possible range and meaning; and the embodiments are intended to be illustrative and not exhaustive. It should also be understood that the expressions and terms used herein are for illustrative purposes only and should not be considered limiting.

[0009] Unless otherwise defined herein, scientific and technical terms used in connection with this disclosure shall have meanings generally understood by those skilled in the art. Furthermore, unless otherwise specified by context, singular terms shall include plural terms and plural terms shall include singular terms. The techniques and procedures described herein are well known in the art and are generally performed in accordance with conventional methods as described in the various general and more specific references cited and discussed throughout this specification. The nomenclature, testing procedures and techniques used in relation to analytical chemistry, synthetic organic chemistry, and medicinal chemistry and pharmaceutical chemistry described herein are well known in the art and are generally used. Standard techniques are used for chemical synthesis and chemical analysis.

[0010] All patents, published patent applications, and non-patent publications referenced herein represent the level of skill of a person skilled in the art to which this disclosure belongs. All patents, published patent applications, and non-patent publications referenced in any part of this application are expressly incorporated herein by reference as a whole to the same extent that each individual patent or publication is specifically and individually indicated as being incorporated by reference.

[0011] All articles, compositions, kits, and / or methods disclosed herein can be manufactured and performed without excessive experimentation, taking into consideration the present disclosure. While articles, compositions, kits, and / or methods have been described in relation to specific embodiments, it will be apparent to those skilled in the art that modifications can be applied to the articles, compositions, kits, and / or methods and the steps or sequences of steps of the methods described herein without departing from the concepts, spirit, and scope of the present disclosure. All such similar substitutions and modifications, apparent to those skilled in the art, shall be deemed to be within the spirit, scope, and concepts of the present disclosure as defined by the appended claims.

[0012] The following terms used in accordance with this disclosure shall be understood to have the following meanings unless otherwise indicated: The use of the terms "a" or "an" in relation to the term "including" in the claims and / or specification may mean "one," which also coincides with the meanings of "one or more," "at least one," and "one or more." Therefore, the terms "a," "an," and "the" include multiple referents unless the context clearly indicates otherwise. Thus, for example, a reference to "a compound" could refer to one or more compounds, two or more compounds, three or more compounds, four or more compounds, or a larger number of compounds. The term "plural" means "two or more."

[0013] The use of the term "at least one" is understood to include any quantity of one and more than one, including, but not limited to, 2, 3, 4, 5, 10, 15, 20, 30, 40, 50, 100, etc. The term "at least one" may extend up to 100 or 1000 or more, depending on the term it is attached to; furthermore, the quantity 100 / 1000 should not be considered an limitation, as a higher limitation may produce a more satisfactory result. In addition, the use of the term "at least one of X, Y, and Z" is understood to include not only X alone, Y alone, and Z alone, but also any combination of X, Y, and Z. The use of ordinal terms (i.e., "first," "second," "third," "fourth," etc.) is solely for the purpose of distinguishing two or more items and is not intended to indicate, for example, any order or sequence or importance or order of addition of one item to another item.

[0014] The use of the term “or” in the claims is used to mean an inclusive “and / or” unless it is explicitly indicated that it refers only to the options, or unless those options are mutually exclusive. For example, the condition “A or B” is satisfied by any of the following: A is true (or exists) and B is false (or does not exist), A is false (or does not exist) and B is true (or does not exist), and both A and B are true (or exist).

[0015] Any reference in this specification to “one embodiment,” “embodiment,” “several embodiments,” “an example,” “for example,” or “example” means that any particular element, feature, structure, or characteristic described in relation to that embodiment is included in at least one embodiment. For example, the appearance of the phrase “in several embodiments” or “an example” in various places in the specification does not necessarily all refer to the same embodiment. Furthermore, all references to one or more embodiments or examples should be considered as not being limited to the claims.

[0016] Throughout this application, the term “about” is used to indicate that the value includes inherent variations in error with respect to the variability present between the composition / apparatus / device, the method used to determine its value, or the test subject. Not as an limitation, but for example, where the term “about” is used, the specified value may vary from a particular value by plus or minus 20 percent, or 15 percent, or 12 percent, or 11 percent, or 10 percent, or 9 percent, or 8 percent, or 7 percent, or 6 percent, or 5 percent, or 4 percent, or 3 percent, or 2 percent, or 1 percent, such variations being suitable for the execution of the disclosed method and understandable to those skilled in the art.

[0017] The words “comprising” (and all forms of including, such as “comprise” and “comprises”), “having” (and all forms of having, such as “have” and “has”), “including” (and all forms of including, such as “includes” and “include”), or “containing” (and all forms of containing, such as “contains” and “contain”), as used in this specification and claims, are comprehensive or open-ended and do not exclude any additional undescribed elements or processes.

[0018] As used herein, the terms “or any combination thereof” refer to all reorders and combinations of the items listed before the term. For example, “A, B, C, or any combination thereof” is intended to include: A, B, C, AB, AC, BC, or ABC, and, where the order is important in particular circumstances, at least one of BA, CA, CB, CBA, BCA, ACB, BAC, or CAB. This example explicitly includes combinations containing repetitions of one or more items or terms, such as BB, AAA, AAB, BBC, AAABCCCC, CBBAAA, CABABB, etc. Those skilled in the art will understand that, unless evident from the context, there is typically no limit to the number of items or terms in any combination.

[0019] As used herein, the term “substantially” means that the event or situation described thereafter occurs entirely, or that the event or situation described thereafter occurs to a considerable or large extent. For example, in relation to a particular event or situation, the term “substantially” means that the event or situation described thereafter occurs at least 80% of the time, or at least 85% of the time, or at least 90% of the time, or at least 95% of the time. The term “substantially adjacent” means that two items are 100% adjacent to each other, or that two items are close to each other but not 100% adjacent, or that a portion of one item is close to the other item but not 100% adjacent to it.

[0020] As used herein, the phrases “combined with” and “coupled to” include both direct association / bonding of two parts to each other and indirect association / bonding of two parts to each other. Non-limiting examples of association / bonding include, for example, covalent bonding of one part to another by direct bonding or via spacer groups, non-covalent bonding of one part to another part directly or using specific bond pair members bonded to that part, incorporation of one part into another part by dissolution or synthesis of one part into another part, and coating of one part onto another part.

[0021] The terms “analog” and “derivative” are used interchangeably herein and refer to substances that contain the same basic carbon skeleton and carbon functional groups in their structure as a given compound, but also contain one or more substitutions thereto. The term “substitution” as used herein is understood to refer to replacing at least one substituent on a compound with residue R. In certain non-limiting embodiments, R may include one selected C1-C4 compound of linear, branched or cyclic alkyl, and linear, branched or cyclic alkenyl, selected from H, hydroxyl, thiol, fluoride, chloride, bromide, or iodide, optionally substituted, and any substituent being one or more alkenylalkyl, alkynylalkyl, cycloalkyl, cycloalkenylalkyl, arylalkyl, heteroarylalkyl, heterocyclic alkyl, optionally substituted heterocycloalkenylalkyl, arylcycloalkyl, and arylheterocycloalkyl These are selected, and each is optionally substituted, where any substituent is selected from one or more of the following: alkenylalkyl, alkynylalkyl, cycloalkyl, cycloalkenylalkyl, arylalkyl, alkylaryl, heteroarylalkyl, heterocyclicalkyl, optionally substituted heterocycloalkenylalkyl, arylcycloalkyl, and arylheterocycloalkyl, phenyl, cyano, hydroxyl, alkyl, aryl, cycloalkyl, cyano, alkoxy, alkylthio, amino, -NH(alkyl), -NH(cycloalkyl)2, carboxy, and -C(O))-alkyl.

[0022] As used herein, the term “sample” is understood to include any type of biological sample that may be used in accordance with this disclosure. Examples of fluid biological samples that may be used include, but are not limited to, whole blood or any part thereof (i.e., plasma or serum), urine, saliva, sputum, cerebrospinal fluid (CSF), skin, intestinal fluid, intraperitoneal fluid, cystic fluid, sweat, interstitial fluid, extracellular fluid, tears, mucus, bladder lavage fluid, semen, feces, pleural fluid, nasopharyngeal fluid, or combinations thereof.

[0023] The term “specific binding partner” or “analyte-specific binder” is understood to refer to any molecule that has the ability to specifically bind to the target analyte. For example, but not limited to, a binder / binding partner may be an antibody, receptor, ligand, aptamer, molecular imprinting polymer (i.e., inorganic matrix), any fragment thereof, and any combination or derivative thereof, as well as any other molecule that has the ability to specifically bind to the target analyte.

[0024] The term “antibody” is used in its broadest sense and specifically (but not limited to) monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), any of the above fragments, and any of the above conjugates, insofar as they demonstrate the desired biological activity for binding to the analyte. Therefore, the term “antibody” or “antibody peptide” refers to a full-length immunoglobulin molecule (i.e., an intact antibody) or its antigen-binding fragment that competes with the intact antibody for specific antigen binding. Antigen-binding fragments can be produced by recombinant DNA technology or by enzymatic or chemical cleavage of an intact antibody. Antigen-binding fragments include Fab, Fab', F(ab')2, Fv, scFv, disulfide-linked Fv, Fd, diabodies, single-chain antibodies, and single-domain antibodies (e.g., nanobodies, but not limited to these). (R) This includes other antibody fragments or conjugates thereof that retain at least a portion of the variable region of an intact antibody, as well as antibody-substituted proteins or peptides (i.e., engineered binding proteins / peptides), and combinations or derivatives thereof. See, for example, Hudson et al. (Nature Med. (2003) 9:129-134). The antibody may be of any type or class (e.g., IgG, IgE, IgM, IgD, and IgA) or subclass (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2).

[0025] As used herein, the terms “antigen-binding fragment” or “antigen-binding portion” of an antibody refer to one or more fragments of an antibody that retain the ability to bind to an antigen. The antigen-binding function of an antibody may be performed by an intact antibody fragment. Examples of binding fragments contained within the term “antigen-binding fragment” of an antibody include, but are not limited to, Fab, Fab', F(ab')2, Fv, scFv, disulfide-linked Fv, Fd, diabody, single-chain antibodies, single-domain antibodies (e.g., NANOBODIES). (R) Examples include isolated CDRH3 and other antibody fragments that retain at least a portion of the variable region of the intact antibody. These antibody fragments are obtained using conventional recombinant and / or enzymatic techniques and screened for antigen binding in the same manner as the intact antibody.

[0026] As used herein, "antibody heavy chain" refers to the larger of the two polypeptide chains present in all antibody molecules in their naturally occurring conformations.

[0027] As used herein, "antibody light chain" refers to the smaller of the two polypeptide chains present in all antibody molecules in their naturally occurring conformations. Kappa and lambda light chains refer to the two main antibody light chain isotypes.

[0028] The term "CDR," and its plural "CDR," refers to the complementarity-determining region (CDR) of an antibody or antibody fragment, which determines the binding characteristics of the antibody or antibody fragment. In most cases, three CDRs are located in the light chain variable region (CDRL1, CDRL2, and CDRL3), and three CDRs are located in the heavy chain variable region (CDRH1, CDRH2, and CDRH3). CDRs contribute to the functional activity of the antibody molecule and are separated by amino acid sequences that constitute a scaffold or framework region. Among the various CDRs, the CDR3 sequence, and especially CDRH3, is the most diverse and therefore contributes most strongly to antibody specificity. There are at least two techniques for determining CDR: (1) an approach based on interspecific sequence variability (i.e., Kabat et al., Sequences of Proteins of Immunological Interest (National Institute of Health, Bethesda, Md. (1987), incorporated as a whole by reference); and (2) an approach based on crystallographic studies of antigen-antibody complexes (Chothia et al., Nature, 342:877 (1989), incorporated as a whole by reference).

[0029] The term “epitope” includes any protein determinants that have the ability to specifically bind to an immunoglobulin or T cell receptor. In certain embodiments, an epitope is a region of an antigen that is specifically bound by an antibody. Epitope determinants typically include a group of chemically active surfaces of molecules, such as amino acids, sugar side chains, phosphoryls, or sulfonyl groups. In certain embodiments, an epitope may have specific three-dimensional structural features (e.g., “structural epitopes”), and even specific charge features.

[0030] An epitope is defined as "the same as" another epitope if a particular antibody specifically binds to both epitopes. In certain embodiments, polypeptides having different primary amino acid sequences may contain epitopes that are the same. In certain embodiments, epitopes that are the same may have different primary amino acid sequences. Different antibodies are said to bind to the same epitope if they compete for specific binding to that epitope.

[0031] An antibody "specifically binds" to an antigen when it preferentially recognizes the antigen in a complex mixture of proteins and / or macromolecules. In certain embodiments, an antibody contains an antigen-binding site that specifically binds to a particular epitope. In certain such embodiments, an antibody can bind to different antigens, insofar as different antigens contain that particular epitope or a closely related epitope. In certain examples, for instance, homologous proteins from different species may contain the same epitope. In certain embodiments, an antibody contains 10 -6 M, 10 -7 M, 10 -8 M or 10 -9 The antibody binds specifically to the antigen with a dissociation constant of M or less. When an antibody specifically binds to a receptor or ligand (i.e., a counterreceptor), this can substantially inhibit the adhesion of the receptor to the ligand. As used herein, an antibody substantially inhibits the adhesion of the receptor to the ligand if the excess antibody reduces the amount of receptor to which the ligand is bound (as measured in an in vitro competitive binding assay) by at least about 20%, 40%, 60%, or 80%, 85%, or 90%.

[0032] An “isolated” antibody is one that has been separated and / or recovered from the components of the environment in which it was produced. The contaminants of its production environment are substances that would interfere with the diagnostic or therapeutic use of the antibody and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. In certain embodiments, the antibody is purified to a degree sufficient to be measurable by at least three different methods: 1) by the Lowry method to a degree higher than 50% by mass of the antibody, e.g., more than 75% by mass, or more than 85% by mass, or more than 95% by mass, or more than 99% by mass; 2) by the use of a spinning cup sequencer to a degree sufficient to obtain at least 10 residues of the N-terminal or internal amino acid sequence, e.g., to at least 15 residues of the sequence; or 3) by SDS-PAGE under reducing or non-reducing conditions using Coomassie Blue, or to homogeneity by silver staining. Because at least one component of the environment in which the antibody was produced is absent, an isolated antibody contains the antibody in situ within recombinant cells. However, typically, isolated antibodies are produced by at least one purification step. Furthermore, the "isolated antibody" substantially does not contain other antibodies with different antigen specificities. However, the isolated antibody may exhibit some cross-reactivity to other relevant antigens.

[0033] The term "antibody variant" refers to an amino acid sequence variant of an antibody in which one or more amino acid residues have been altered. Such variants have an amino acid sequence that has at least 75% amino acid sequence identity or similarity to the amino acid sequence of either the heavy chain variable domain or the light chain variable domain of the antibody, such as at least 80%, at least 85%, at least 90%, or at least 95%, and necessarily less than 100% sequence identity or similarity.

[0034] As used herein, the term “monoclonal antibody” refers to an antibody obtained from a substantially homogeneous population of antibodies that specifically bind to the same epitope; that is, the individual antibodies constituting the population are identical except for possible naturally occurring mutations that may be present in small amounts. In contrast to conventional (polyclonal) antibody preparations, which typically contain different antibodies specific to different determinants (epitopes), each monoclonal antibody is specific to a single determinant on an antigen. In addition to their specificity, monoclonal antibodies have the advantage that they can be synthesized by hybridoma culture in a single manufacturing method, and therefore are not contaminated with other immunoglobulins. The modifier “monoclonal” indicates the characteristic of an antibody that it is obtained from a substantially homogeneous population of antibodies, and this should not be considered to require the production of the antibody by any particular method. For example, in one embodiment, the monoclonal antibody produced according to this disclosure may be produced by the hybridoma method first described by Kohler and Milstein (Nature, 256:495 (1975)).

[0035] Monoclonal antibodies used in accordance with this disclosure may be produced by any method known in the art, including, but not limited to, the result of intentional immunization protocols; the result of an immune response that produces spontaneous antibodies in the course of disease or cancer; and phage-derived antibodies. In addition to the hybridoma production methods listed above, the monoclonal antibodies of this disclosure may be produced by various other methods, including, but not limited to, recombinant DNA methods (see, e.g., U.S. Patent No. 4,816,567); isolation of antibody fragments from phage display libraries (see, e.g., Clackson et al., Nature (1991) 352:624-628; and Marks et al., J.Mol.Biol. (1991) 222:581-597); and various other monoclonal antibody production techniques (see, e.g., Harlow and Lane (1988) Antibodies: A Laboratory Manual (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY)). Furthermore, many monoclonal antibodies that can be used in conjugates and the methods disclosed herein or otherwise intended are widely available commercially and therefore no further explanation is deemed necessary.

[0036] As used herein, “substantially pure” means that the species in question is the dominant species present (i.e., in molar terms, more abundant than any other individual species in the composition). Generally, a substantially pure composition consists of more than about 50% of all polymer species present in the composition, for example, more than about 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, and 99%. In one embodiment, the species in question is purified to an essential homogeneity (where the contaminating species cannot be detected in the composition by conventional detection methods), and the composition consists of essentially a single polymer species.

[0037] The "analyte" is a molecule that has the ability to be recognized by a binding partner specific to the analyte, such as an antibody (but is not limited to this). The analyte contains at least one antigenic determinant or "epitope," which is a region of the analyte that binds to a binding partner specific to the analyte (i.e., an antibody).

[0038] Certain non-limiting embodiments of this disclosure are based on Formula I: [ka] A composition comprising cyclosporine C and acridinium ester linked via a spacer, having the structure of,

[0039] And / or Formula II: [ka] A composition comprising cyclosporine A and acridinium ester (A) linked via a spacer (B) having the structure shown above. Regarding.

[0040] In formulas I and II, "A" contains an acridinium ester, and "B" is a spacer having approximately 5 to 100 atoms, respectively, selected from the group consisting of C, H, O, N, S, and P atoms.

[0041] In certain non-limiting embodiments, the acridinium ester used in accordance with this disclosure is of formula III: [ka] [In the formula, "R1" is an alkyl, alkenyl, alkynyl, or aralkyl group having 1 to 35 carbon atoms and 0 to 20 heteroatoms; a sulfopropyl or sulfobutyl group; and the group -R a -Selected from the group consisting of Z, where R ais a divalent radical selected from alkyl, alkenyl, alkynyl, aryl, or aralkyl groups of 1 to 35 carbon atoms and 0 to 20 heteroatoms. "R2" is located at one or more of positions C1 - C4, and "R3" is located at one or more of positions C5 - C8. "R2" and "R3" are each independently selected from the group consisting of hydrogen, alkyl, OR, OH, SR, SH, NH2, and NR’R’’, where R, R’, and R’’ are each independently selected from the group consisting of alkyl, alkenyl, alkynyl, aryl, and aralkyl groups, where each group contains 0 to 20 heteroatoms. Also, "X" is a group selected from halogenated or non - halogenated, branched or straight - chain alkyl groups; substituted or unsubstituted aryl groups; and heterocyclic ring groups. The "X" group also contains 0 to 20 heteroatoms and further contains a functional group that links to the spacer "B" of formula I. Further, "A - " is a counterion introduced, for example (but not limited to), to form a counter - ion pair with the quaternary nitrogen of the above acridinium nucleus, and "A - " is CH3SO3 - 、FSO3 - 、CF3SO3 - 、C4F9SO3 - 、CH3C6H4SO3 - 、halide, CF3COO - 、CH3COO - and NO3 - selected from the group consisting of] has the structure of.

[0042] In certain non - limiting embodiments, the acridinium ester utilized in accordance with the present disclosure is of formula IV:

Chemical formula

[0043] In certain non-limiting embodiments, the acridinium ester used in accordance with this disclosure is of formula V: [ka] [In the formula, "R1" is a methyl or sulfopropyl group; "R2" and "R3" are independently selected from hydrogen, methoxy, sulfopropyloxyl, or poly(ethylene) glycoloxyl groups; and "R6" is an amide group (CONH-) connected to the spacer "B" in formula I. - This is defined in Equation III. This is a dimethylphenyl acridinium ester having the following structure.

[0044] In certain (but non-limiting) embodiments, R1 is a sulfopropyl group, where R2 and R3 are hydrogen or a sulfopropyloxyl group, respectively.

[0045] In certain (but not limited) embodiments, the composition is formula VI: [ka] This is a cyclosporine C-acridinium ester having the following structure.

[0046] In certain (but not limited) embodiments, the composition is formula VII: [ka] This is a cyclosporine C-acridinium ester having the following structure.

[0047] In certain (but not limited) embodiments, the composition is formula VIII: [ka] This is a cyclosporine C-acridinium ester having the following structure.

[0048] In certain (but not limited) embodiments, the composition is formula IX: [ka] This is a cyclosporine C-acridinium ester having the following structure.

[0049] In certain (but not limited) embodiments, the composition is of formula X: [ka] This is a cyclosporine C-acridinium ester having the following structure.

[0050] In certain (but not limited) embodiments, the composition is formula XI: [ka] This is a cyclosporine C-acridinium ester having the following structure.

[0051] In certain (but not limited) embodiments, the composition is of formula XII: [ka] This is a cyclosporine C-acridinium ester having the following structure.

[0052] In certain (but not limited) embodiments, the composition is formula XIII: [ka] This is a cyclosporine A-acridinium ester having the following structure.

[0053] In certain (but not limited) embodiments, the composition is formula XIV: [ka] This is a cyclosporine A-acridinium ester having the following structure.

[0054] In certain (but not limited) embodiments, the composition is of formula XV: [ka] This is a cyclosporine A-acridinium ester having the following structure.

[0055] In certain (but not limited) embodiments, the composition is formula XVI: [ka] This is a cyclosporine A-acridinium ester having the following structure.

[0056] In certain (but not limited) embodiments, the composition is formula XVII: [ka] This is a cyclosporine A-acridinium ester having the following structure.

[0057] Certain non-limiting embodiments of this disclosure relate to immunoassay kits containing one or more of the CsC-AE / CsA-AE compositions disclosed herein or otherwise intended. The selection of acridinium esters present depends on the specific assay format to be used, and such selections are well within the scope of those skilled in the art.

[0058] In certain (but non-limiting) embodiments, the immunoassay kit comprises a first reagent comprising one of the CsC-AE / CsA-AE compositions described herein or otherwise intended, and a second reagent comprising a solid phase having an antibody directly or indirectly conjugated; the antibody present in the second reagent specifically conjugates to CsC or CsA present in the first reagent.

[0059] Any antibody or fragment thereof, whether publicly known in the art or otherwise intended herein, may be used in accordance with this disclosure, insofar as the antibody / fragment can bind to CsC or CsA at an epitope distant from the site where the acridinium ester is bound. Antibodies that bind to CsC / CsA are well known in the art and commercially available. While not an exclusive limitation, CsC / CsA antibodies are commercially available from companies such as ThermoFisher Scientific (Waltham, MA), LifeSpan Biosciences (Seattle, WA), MyBioSource (San Diego, CA), Novus Biologicals (Littleton, CO), GeneTex (Irvine, CA), Enzo Life Sciences, Inc. (Farmingdale, NY), Creative Biolabs (Shirley, NY), Santa Cruz Biotechnology, Inc. (Dallas, TX), Abbexa Ltd (Cambridge, UK), Absolute Antibody (Oxford, UK), Biobyt (Cambridge, UK), and HyTest Ltd (Turku, Finland). Therefore, further explanation of the antibodies used in accordance with this disclosure is considered unnecessary.

[0060] The assay reagents present in the kit may be provided in any form that enables them to function in accordance with this disclosure. For example, but not limited to, each reagent may be provided in liquid form and arranged together and / or in single aliquot form within the kit. Alternatively, in certain (but non-limiting) embodiments, one or more reagents may be provided in the kit in the form of single aliquot lyophilized reagents. The use of lyophilized reagents in microfluidic devices is described in detail in U.S. Patent No. 9,244,085 (Samproni), the entirety of which is expressly incorporated herein by reference.

[0061] In addition to the assay reagents described in detail above, the kit may further contain other reagents for performing any of the assays described herein or other particular assays intended. For example (but not limited to), the kit may further contain at least one pretreatment / release agent for releasing cyclosporine from any endogenous binding protein present in the biological sample. The nature of these further reagents depends on the specific assay form, and their identification is well within the skill of those skilled in the art; therefore, further description is considered unnecessary. Also, the components / reagents present in the kit may each be in separate containers / compartments, or various components / reagents may be combined in one or more containers / compartments, depending on the cross-reactivity and stability of the components / reagents. Furthermore, the kit may include a microfluidic device in which the components / reagents are placed.

[0062] The relative amounts of the various components / reagents in the kit can vary widely to provide component / reagent concentrations that substantially optimize the necessary reactions occurring during the assay method, and further substantially optimize the assay sensitivity. Under appropriate circumstances, one or more components / reagents in the kit can be provided as a dry powder, such as a lyophilized powder, and the kit may further contain excipients for dissolving the dry reagents; in this way, reagent solutions of appropriate concentrations for performing the method or assay according to this disclosure can be obtained from these components. Non-limiting examples of other reagents that may be included in the kit include washing solutions, diluents, excipients, interference solutions, positive controls, negative controls, calibration reagents, quality control reagents, etc. Furthermore, the kit may further contain a set of written instructions describing how to use the kit. Kits of this nature can be used in any of the methods described herein or otherwise intended.

[0063] Certain non-limiting embodiments of this disclosure relate to a method for producing any of the CsC-AE / CsA-AE compositions disclosed herein or otherwise intended. This method includes linking a linker to CsC / CsA and then linking an acridinium ester to the linker.

[0064] Certain non-limiting embodiments of this disclosure relate to a method for detecting CsC / CsA in a sample using any of the CsC-AE / CsA-AE compositions disclosed herein or otherwise intended. In this method, a sample suspected of containing CsC / CsA is mixed simultaneously, or entirely or partially sequentially, with one or more of the CsC-AE compositions disclosed herein or otherwise intended, and one or more solid-phase-bound CsC / CsA antibodies disclosed herein or otherwise intended to form a mixture, and this mixture is incubated under conditions that allow antibodies to bind to CsC / CsA or CsC-AE / CsA-AE present in the sample, thereby forming Cs / antibody complexes and / or Cs-AE / antibody complexes. Next, the Cs-AE / antibody complex is detected, and the amount of CsC / CsA present in the sample is determined based on the decrease in the amount of the formed Cs-AE / antibody complex compared to the negative control (i.e., the amount of Cs-AE / antibody complex formed when the sample is absent). Then, the concentration of CsC / CsA present in the sample can be determined based on the amount of decrease.

[0065] In certain (but non-limiting) embodiments of the above method, the first and second reagents of the immunoassay kit described in detail above herein (i.e., the first reagent comprising a CsC / CsA-AE composition, and the second reagent comprising a solid phase to which an antibody or fragment thereof specifically bound to CsC / CsA is directly or indirectly conjugated) are mixed simultaneously, whole, or partially sequentially with the sample to form a mixture. This mixture is then incubated under conditions that allow the binding of the second reagent to any target analyte (CsC or CsA) present in the sample or to the first reagent, thereby forming a Cs / antibody complex and / or a Cs-AE / antibody complex. This mixture is then incubated under conditions that allow the binding of the second reagent to any target analyte (CsC or CsA) present in the sample or to the first reagent, thereby forming a Cs / antibody complex and / or a Cs-AE / antibody complex. Next, the reagent-second reagent (Cs-AE / antibody) complex is detected by any method known in the art, and the amount of target analyte (CsC or CsA) present in the sample is determined based on the decrease in the amount of the reagent-second reagent complex formed when compared to the negative control (i.e., the amount of reagent-second reagent complex formed when no sample is present). Then, the concentration of the target analyte (CsC or CsA) present in the sample is determined based on the amount of decrease.

[0066] Any sample for which an assay for the presence of a cyclosporine-targeted analyte (i.e., CsC or CsA) is desired can be used as a sample according to the method of this disclosure. Non-limiting examples of samples include, but are not limited to, whole blood or any part thereof (i.e., plasma or serum), urine, saliva, sputum, cerebrospinal fluid (CSF), skin, intestinal fluid, intraperitoneal fluid, cystic fluid, sweat, interstitial fluid, extracellular fluid, tears, mucus, bladder lavage fluid, semen, feces, pleural fluid, nasopharyngeal fluid, and combinations thereof. Specific non-limiting examples include lysed whole blood cells and lysed red blood cells.

[0067] As described above, the various components of the above method are provided in combination (simultaneously or sequentially). When the various components of the above method are added sequentially, the order in which the components are added may vary; those skilled in the art can determine a particular preferred order for adding different components to the assay. Naturally, the simplest addition order is to add all substances simultaneously and determine the resulting signals. Alternatively, it is possible to mix each component, or a group of components, sequentially. In certain embodiments, an incubation step may be included after one or more additions. For example (but not limited to), it may be desirable to mix the antibody-solid phase and the sample and incubate them before adding the Cs-AE composition.

[0068] The incubation conditions for the mixture can vary widely, as long as the antibody binds to Cs or Cs-AE to form a complex under the conditions described above. Immunoassays based on the sandwich assay format are widely performed, and immunoassay conditions are well known in the art; therefore, the selection of appropriate assay conditions is well within the scope of those skilled in the art, and thus further explanation is considered unnecessary.

[0069] The specific detection method used may vary considerably, as long as it is possible to detect the complex under the above method. Detection of the formed complex in sandwich assay format is widely performed, and the detection procedure is well known in the art; therefore, the selection of an appropriate detection method is well within the scope of those skilled in the art, and thus further explanation is considered unnecessary.

[0070] The above method may further include one or more additional steps to increase the accuracy and / or precision of the assay. For example (but not limited to), the above method may further include one or more pretreatment / release steps to release cyclosporine from any endogenously bound proteins present in the biological sample before mixing with the immobilized antibody. Another non-limiting example of additional steps that may be utilized pursuant to this disclosure is one or more washing steps to remove unbound (or nonspecifically bound) reagents from the reaction mixture before detecting complex formation.

[0071] Certain further non-limiting embodiments of this disclosure relate to microfluidic devices comprising components of any of the immunoassay kits described above herein. In particular, certain non-limiting embodiments include microfluidic devices for detecting a target analyte (CsC or CsA) in a sample. The microfluidic device comprises (i) an inlet channel through which a sample is applied; and (ii) at least a first compartment that can fluidly communicate with the inlet channel. The compartment in (ii) contains the first and second reagents of the immunoassay kits described in detail above herein.

[0072] In certain non-limiting embodiments, the first and second reagents of (ii) (and any further elements as described above herein) are located in the same compartment. In alternative non-limiting embodiments, the first and second reagents (and any further elements as described above herein) are separated between two or more compartments.

[0073] The above-mentioned device may be provided with any arrangement of compartments and the distribution of various components between them that enables the device to function in accordance with this disclosure.

[0074] Any compartment of a microfluidic device may be sealed to maintain the reagents contained therein in a substantially airtight environment until use; for example, a compartment containing lyophilized reagents may be sealed to prevent unintended reconstitution of the reagents. The inlet channel and compartment, and any two additional compartments, may be described as "fluidically connected" to one another; this phrase indicates that each of the compartments remains sealed, but two compartments can allow fluid to flow between them when a seal formed in or between the compartments is punctured.

[0075] The microfluidic devices of this disclosure may have any other desirable features known in the art or otherwise contemplated herein. For example, but not limited thereto, the microfluidic devices of this disclosure may further include a reading chamber; the reading chamber may be any compartment containing one or more of the reagents described herein, or the reading chamber may be in fluid communication with the compartment containing one or more reagents. The microfluidic devices may further include one or more further compartments containing other solutions such as (but not limited to) cleaning solutions, diluents, excipients, interference solutions, positive controls, negative controls, quality controls, etc. These further compartments may be in fluid communication with one or more of the other compartments. For example, the microfluidic device may further include one or more compartments containing cleaning solutions, and these compartments may be in fluid communication with any other compartments of the device. In another example, the microfluidic device may further include one or more compartments containing excipients for dissolving one or more dry reagents, and these compartments may be in fluid communication with any other compartments of the device. In further examples, the microfluidic device may include one or more compartments containing diluted solutions, and these compartments may be able to fluidly communicate with any other compartments of the device. [Examples]

[0076] Examples are provided herein and thereafter. However, this disclosure should be understood as not being limited to the specific experiments, results, and test procedures disclosed herein in the application. Rather, the examples are provided merely as one of various embodiments and are intended to be illustrative, not comprehensive.

[0077] The cyclosporine A AIP assay is performed by ADVIA (R) CENTAUR (R) and ATELLICA (R) This assay is currently used for in vitro diagnostic applications in the quantitative measurement of cyclosporine in human whole blood (EDTA) using an IM analyzer (Siemens Healthcare Diagnostics Inc., Tarrytown, NY). This assay is intended for use as an adjunct in the management of cyclosporine therapy in kidney, heart, and liver transplant patients. The chemical structures of cyclosporine A (CsA) and cyclosporine C (CsC) are shown in Figure 1. The only structural difference between CsA and CsC is the additional hydroxyl group on the CsC molecule (Figure 1). Original ADVIA (R) CENTAUR (R) The cyclosporine (CsA) assay was commercialized in 2008, and this original immunoassay used the complex CsC-acridinium ester (CsC-AE) conjugate (Figure 2) for the detection of CsA. However, the supplier discontinued CsC-succinate-NHS, the raw material used to manufacture this conjugate. Therefore, there was a need for a new AE tracer designed for the CsA II assay.

[0078] Furthermore, novel Cs assays were designed to address European REACH (Registration, Evaluation, Authorisation, and Restriction of Chemicals) requirements for Triton reduction, as well as the U.S. Food and Drug Administration's requirements for addressing ambient temperature (ATE) and mitigating biotin interference. The novel Cs assays of this disclosure utilize new tracers, and the design process focused on identifying novel tracers that have simplified chemical structures and can be manufactured using conventional synthesis processes.

[0079] Based on this need, a series of novel and simple cyclosporine (A and C)-acridinium esters (CsA-AE and CsC-AE) were designed, and based on aridinium ester (AE) technology (Natrajan et al., Ann Biochem (2010) 406:204; and Natrajan et al., Org Biomol Chem (2011) 9:5092), ADVIA (R) CENTAUR (R) These CsA-AEs and CsC-AEs were synthesized for use in the CSA II assay (Siemens Healthcare Diagnostics Inc., Tarrytown, NY). (R) CENTAUR (R) The system (Siemens Healthcare Diagnostics Inc., Tarrytown, NY) was evaluated using a competitive assay format (Figure 3). One key tracer, CsC-DA-10-NSP-ZAE (Formula IX; Figure 7), demonstrated superior performance compared to the assay requirements (ATE, sensitivity, and precision) and was selected as a promising candidate tracer for the CsA II immunoassay.

[0080] Reagent design: ADVIA (R) CENTAUR (R) / ATELLICA (R)The CsA II reagent contained one solid phase and one lite reagent. The solid phase reagent contained magnetic particles labeled with a biotinylated monoclonal anti-CsA antibody. The lite reagent contained a CsC-AE tracer. When CsA was not present in the test sample, the lite reagent bound to the antibody located on the surface of the solid phase (Figure 3). The bound AE remaining in the reaction vessel after magnetic separation and particle washing fluoresced when activated by the addition of acid and base. When CsA from a patient sample was added to the reaction mixture, the binding of the Cs-AE conjugate to the solid phase was inhibited, resulting in a decrease in signal generation. This decrease in signal is a direct function of the amount of CsA in the sample when measured against the calibration curve.

[0081] Considering the assay design described above, both solid-phase reagents and novel CsC-AE tracers were developed and evaluated. The production of biotinylated monoclonal antibodies and the pre-binding of these antibodies to the surface of commercially available streptavidin-coated particles are well-known and simple processes. Therefore, the remainder of this example focuses on the synthesis of novel CsC-AE and CsA-AE tracers and their performance in the prototype ADVIA Centaur CsA II assay.

[0082] The hydroxyl group from the CsC molecule (Figure 1) was selected for NSP-DMAE binding in the initial assay study. Early data in this example showed that the binding of CsC-DA-10-NSP-DMAE to this hydroxyl group produced a promising assay curve shape. Considering this initial performance, the conjugate was also prepared using HEGAE, TSPAE, and ZAE at the same binding site to determine whether different AE structures would provide further benefit to this assay. Furthermore, NSP-DMAE and ZAE were introduced using the terminal double and aldehyde functional groups of CsA (Figure 1) as a base. The chemical structures of the CsA-AE and CsC-AE tracers are shown in Figures 4–15, and the synthesis of these CsA-AE and CsC-AE tracers is shown in Figures 16–27.

[0083] Cyclosporine immunoassay performance: Evaluation of CsA-AE and CsC-AE tracers using the ADVIA immunoassay analyzer, available from Siemens Healthcare Diagnostics Inc., Tarrytown, NY. (R) CENTAUR (R) The family was used for screening the binding of CsA-AE and CsC-AE tracers (Figures A.4-15) to available monoclonal antibodies using the prototype ADVIA. (R) CENTAUR (R) The determination was made using Cs immunoassays (Figures 28-33). Attenuation to ambient temperature (ATE) studies using different CsC tracers are also shown in Figures 34-36.

[0084] Conclusion: A series of novel CsA-AE and CsC-AE tracers were successfully designed, synthesized, and evaluated in prototype CsA immunoassays (Figures 4–15; 16–27; and Figures 28, 30, 32, and 34–36, respectively). A novel tracer was selected as a superior candidate that met the FDA's ambient temperature tolerance requirements and overall assay performance / assay specifications (Figures 30 and 35). This tracer, CsC-DA-10-NSP-ZAE (Formula IX; Figure 7), has a novel, simplified chemical structure and can be conveniently synthesized compared to the complex tracer CsA-HEG3-NSP-DMAE (Figure 2) currently in use (Figure 19). This saves manufacturing time and cost and overcomes the problem of discontinued raw materials for the current tracer. Therefore, the novel tracer developed in accordance with this disclosure is a crucial component for the development of new Cs immunoassays.

[0085] Non-limiting exemplary embodiments of the concept of the invention Exemplary Embodiment 1. Formula I: [ka] [In the formula: "A" contains an acridinium ester; and "B" is a spacer having approximately 5 to 100 atoms, each selected from the group consisting of C, H, O, N, S, and P atoms.] A composition comprising cyclosporine C and acridinium ester linked via a spacer, having the structure shown.

[0086] Exemplary Embodiment 2 In formula I, "A" is formula III: [ka] [In formula: "R1" is: an alkyl, alkenyl, alkynyl, or aralkyl group consisting of 1 to 35 carbon atoms and 0 to 20 heteroatoms; a sulfopropyl or sulfobutyl group; and the group -R a -Selected from the group consisting of Z, where R a This is a divalent radical selected from alkyl, alkenyl, alkynyl, aryl, or aralkyl groups, comprising 1 to 35 carbon atoms and 0 to 20 heteroatoms; "R2" is located at one or more positions C1-C4, and each "R2" is independently selected from the group consisting of hydrogen, alkyl, OR, OH, SR, SH, NH2, and NR'R'', where R, R', and R'' are independently selected from the group consisting of alkyl, alkenyl, alkynyl, aryl, and aralkyl groups, where each group contains 0-20 heteroatoms; "R3" is located at one or more positions C5-C8, and each "R3" is independently selected from the group consisting of hydrogen, alkyl, OR, OH, SR, SH, NH2, and NR'R'', where R, R', and R'' are each independently selected from the group consisting of alkyl, alkenyl, alkynyl, aryl, and aralkyl groups, where each group contains 0-20 heteroatoms; "X" is a group selected from halogenated or non-halogenated, branched or linear alkyl groups; substituted or unsubstituted aryl groups; and heterocyclic ring groups; where the "X" group contains 0 to 20 heteroatoms, and where the "X" group further contains functional groups linked to the spacer "B" of formula I; and "A - " is CH3SO3 - FSO3 - CF3SO3 - , C4F9SO3 - CH3C6H4SO3 - , Halide, CF3COO - CH3COO - , and NO3 - It is a counterion selected from the group consisting of the following: The composition according to exemplary embodiment 1, which is an acridinium ester having the structure.

[0087] Exemplary Embodiment 3. In formula I, "A" is: formula IV: [ka] [In the formula: "R4" and "R8" are each independently selected from hydrogen or alkyl, alkenyl, alkynyl, alkoxyl (-OR), alkylthiol (-SR), or substituted amino groups; "R5", "R6", and "R7" are each independently selected from hydrogen or alkyl, alkenyl, alkynyl, aryl, or aralkyl groups, each containing 0 to 20 heteroatoms; and one of "R5", "R6", and "R7" further contains a functional group linked to spacer "B" in formula I] The composition according to exemplary embodiment 2, which is an acridinium ester having the structure.

[0088] Exemplary Embodiment 4. In formula I, "A" is equal to formula V: [ka] [In the formula: "R1" is a methyl or sulfopropyl group; "R2" and "R3" are independently selected from hydrogen, methoxy, sulfopropyloxyl, or poly(ethylene) glycoloxyl groups; and "R6" is an amide group (CONH-) connected to the spacer "B" in formula I.] The composition according to exemplary embodiment 3, which is a dimethylphenyl acridinium ester having the structure.

[0089] Exemplary Embodiment 5. The composition according to Exemplary Embodiment 4, wherein R1 is a sulfopropyl group, and R2 and R3 are each hydrogen or a sulfopropyloxyl group.

[0090] Exemplary Embodiment 6. Formula VI: [ka] A composition according to any one of the exemplary embodiments 1 to 5, further defined as a cyclosporine C-acridinium ester having the structure.

[0091] Exemplary Embodiment 7. Formula VII: [ka] A composition according to any one of the exemplary embodiments 1 to 5, further defined as a cyclosporine C-acridinium ester having the structure.

[0092] Exemplary Embodiment 8. Formula VIII: [ka] A composition according to any one of the exemplary embodiments 1 to 5, further defined as a cyclosporine C-acridinium ester having the structure.

[0093] Exemplary Embodiment 9. Formula IX: [ka] A composition according to any one of the exemplary embodiments 1 to 5, further defined as a cyclosporine C-acridinium ester having the structure.

[0094] Exemplary Embodiment 10. Formula X: [ka] A composition according to any one of the exemplary embodiments 1 to 5, further defined as a cyclosporine C-acridinium ester having the structure.

[0095] Exemplary Embodiment 11. Formula XI: [ka] A composition according to any one of the exemplary embodiments 1 to 5, further defined as a cyclosporine C-acridinium ester having the structure.

[0096] Exemplary Embodiment 12. Formula XII: [ka] A composition according to any one of the exemplary embodiments 1 to 5, further defined as a cyclosporine C-acridinium ester having the structure.

[0097] Exemplary Embodiment 13. Formula II: [ka] [In the formula: "A" contains an acridinium ester; and "B" is a spacer having approximately 5 to 100 atoms, each selected from the group consisting of C, H, O, N, S, and P atoms.] A composition comprising cyclosporine A and acridinium ester (A) linked via a spacer (B) having the structure shown.

[0098] Exemplary Embodiment 14. In Formula II, "A" is: Formula III: [ka] [In formula: "R1" is an alkyl, alkenyl, alkynyl, or aralkyl group consisting of 1 to 35 carbon atoms and 0 to 20 heteroatoms; a sulfopropyl or sulfobutyl group; and the group -R a -Selected from the group consisting of Z, where R a This is a divalent radical selected from alkyl, alkenyl, alkynyl, aryl, or aralkyl groups, comprising 1 to 35 carbon atoms and 0 to 20 heteroatoms; "R2" is located at one or more positions C1-C4, and each "R2" is independently selected from the group consisting of hydrogen, alkyl, OR, OH, SR, SH, NH2, and NR'R'', where R, R', and R'' are each independently selected from the group consisting of alkyl, alkenyl, alkynyl, aryl, and aralkyl groups, where each group contains 0-20 heteroatoms; "R3" is located at one or more positions C5-C8, and each "R3" is independently selected from the group consisting of hydrogen, alkyl, OR, OH, SR, SH, NH2, and NR'R'', where R, R', and R'' are independently selected from the group consisting of alkyl, alkenyl, alkynyl, aryl, and aralkyl groups, where each group contains 0-20 heteroatoms; "X" is a group selected from halogenated or non-halogenated, branched or linear alkyl groups; substituted or unsubstituted aryl groups; and heterocyclic ring groups; where the "X" group contains 0 to 20 heteroatoms, and where the "X" group further contains functional groups linked to the spacer "B" of formula I; and "A - " is CH3SO3 - FSO3 - CF3SO3 - , C4F9SO3 - CH3C6H4SO3 - , Halide, CF3COO - CH3COO - , and NO3 - It is a counterion selected from the group consisting of the following: The composition according to exemplary embodiment 13, which is an acridinium ester having the structure.

[0099] Exemplary Embodiment 15. In Formula II, "A" is: Formula IV: [ka] [In the formula: "R4" and "R8" are each independently selected from hydrogen or alkyl, alkenyl, alkynyl, alkoxyl (-OR), alkylthiol (-SR), or substituted amino groups; "R5", "R6", and "R7" are each independently selected from hydrogen or alkyl, alkenyl, alkynyl, aryl, or aralkyl groups, where each group contains 0 to 20 heteroatoms; and one of "R5", "R6", and "R7" further comprises a functional group linked to spacer "B" in formula II] The composition according to exemplary embodiment 14, which is an acridinium ester having the structure.

[0100] Exemplary Embodiment 16. In formula II, "A" is in formula V: [ka] [In the formula: "R1" is a methyl or sulfopropyl group; "R2" and "R3" are independently selected from hydrogen, methoxy, sulfopropyloxyl, or poly(ethylene) glycoloxyl groups; and "R6" is an amide group (CONH-) connected to spacer "B" in formula II.] It is a dimethylphenyl acridinium ester having the structure, The composition described in Exemplary Embodiment 15.

[0101] Exemplary Embodiment 17. The composition according to Exemplary Embodiment 16, wherein R1 is a sulfopropyl group, and R2 and R3 are hydrogen or a sulfopropyloxyl group, respectively.

[0102] Exemplary Embodiment 18. Formula XIII: [ka] A composition according to any one of the exemplary embodiments 13 to 17, further defined as a cyclosporine A-acridinium ester having the structure of .

[0103] Exemplary Embodiment 19. Formula XIV: [ka] A composition according to any one of the exemplary embodiments 13 to 17, further defined as a cyclosporine A-acridinium ester having the structure of .

[0104] Exemplary Embodiment 20. Formula XV: [ka] A composition according to any one of the exemplary embodiments 13 to 17, further defined as a cyclosporine A-acridinium ester having the structure of .

[0105] Exemplary Embodiment 21. Formula XVI: [ka] A composition according to any one of the exemplary embodiments 13 to 17, further defined as a cyclosporine A-acridinium ester having the structure of .

[0106] Exemplary Embodiment 22. Formula XVII: [ka] A composition according to any one of the exemplary embodiments 13 to 17, further defined as a cyclosporine A-acridinium ester having the structure of .

[0107] Exemplary Embodiment 23. An immunoassay kit comprising a first reagent containing the composition described in any one of Exemplary Embodiments 1 to 22; and a second reagent containing a solid phase to which an antibody specifically binding to cyclosporine A is directly or indirectly conjugated.

[0108] Exemplary Embodiment 24. The immunoassay kit according to Exemplary Embodiment 23, further comprising at least one pretreatment agent.

[0109] Exemplary Embodiment 25. A method for detecting cyclosporine A in a sample, comprising: (a) a step of simultaneously, whole or partially, mixing together, (i) a sample suspected to contain cyclosporine A; (ii) a first reagent comprising a composition described in any one of Exemplary Embodiments 1 to 22; and (iii) a second reagent comprising a solid phase to which an antibody specifically binding to cyclosporine A is directly or indirectly bound, to form a mixture; (b) incubating the mixture under conditions that enable the binding of the antibody to cyclosporine A or the first reagent present in the sample, thereby forming a cyclosporine A / second reagent complex and / or a first reagent / second reagent complex; (c) detecting the complexes formed from the first and second reagents; and (d) determining the amount of cyclosporine A present in the sample based on a decrease in the amount of first reagent / second reagent complexes formed compared to an assay performed in the absence of a sample.

[0110] Exemplary Embodiment 30. The method according to Exemplary Embodiment 29, further comprising the step of treating the sample with a pretreatment agent before step (a).

[0111] Accordingly, compositions, kits, and devices, as well as methods for manufacturing and using them, are provided in accordance with this disclosure, which fully satisfy the purposes and benefits set forth above herein. Although this disclosure has been described in conjunction with the specific drawings, experiments, results, and language set forth above herein, it is obvious that many alternatives, modifications, and variations will be apparent to those skilled in the art. Accordingly, it is intended to encompass all such alternatives, modifications, and variations that fall within the spirit and broad scope of this disclosure.

Claims

1. Formula I: 【Chemistry 1】 [In the formula: "A" contains acridinium ester; and "B" is a spacer having approximately 5 to 100 atoms, each selected from the group consisting of C, H, O, N, S, and P atoms. Cyclosporine C and acridinium ester having the structure of being connected via a spacer. A composition containing the following:

2. "A" in equation I is given by equation III: 【Chemistry 2】 [In the formula: "R1" is: Alkyl, alkenyl, alkynyl, or aralkyl groups comprising 1 to 35 carbon atoms and 0 to 20 heteroatoms; Sulfopropyl or sulfobutyl group; and Base-R a -Z Selected from the group consisting of, Here R a R2 is a divalent radical selected from alkyl, alkenyl, alkynyl, aryl, or aralkyl groups, comprising 1 to 35 carbon atoms and 0 to 20 heteroatoms; R2 is located at one or more positions C1 to C4, and each R2 is hydrogen, alkyl, OR, OH, SR, SH, or NH 2 A group independently selected from the group consisting of , and NR'R'', where R, R', and R'' are each independently selected from the group consisting of alkyl, alkenyl, alkynyl, aryl, and aralkyl groups, where each group contains 0 to 20 heteroatoms; "R3" is located at one or more positions C5 to C8, and each "R3" is hydrogen, alkyl, OR, OH, SR, SH, NH 2 A group independently selected from the group consisting of , and NR'R'', where R, R', and R'' are each independently selected from the group consisting of alkyl, alkenyl, alkynyl, aryl, and aralkyl groups, where each group contains 0 to 20 heteroatoms; "X" is a group selected from halogenated or non-halogenated, branched or linear alkyl groups; substituted or unsubstituted aryl groups; and heterocyclic ring groups; where the "X" group contains 0 to 20 heteroatoms, and where the "X" group further contains functional groups linked to the spacer "B" of formula I; and "A" - is a counter ion selected from the group consisting of CH 3 SO 3 - , FSO 3 - , CF 3 SO 3 - , C 4 F 9 SO 3 - , CH 3 C 6 H 4 SO 3 - , halide, CF 3 COO - , CH 3 COO - , and NO 3 - ​ The composition according to claim 1, which is an acridinium ester having the structure.

3. "A" in equation I is given by equation IV: 【Transformation 3】 [In the formula: "R 4 " and "R 8 Each of these is independently selected from hydrogen or alkyl, alkenyl, alkynyl, alkoxyl (-OR), alkylthiol (-SR), or substituted amino groups; "R 5 "R" 6 " and "R 7 Each of these is independently selected from hydrogen or alkyl, alkenyl, alkynyl, aryl, or aralkyl groups, where each group contains 0 to 20 heteroatoms; and "R 5 "R" 6 " and "R 7 One of them is a sensory element connected to spacer "B" in formula I. [Further includes the base] It is an acridinium ester having the structure, The composition according to claim 2.

4. "A" in equation I is given by equation V: 【Chemistry 4】 [In the formula: "R 1 " is a methyl or sulfopropyl group; "R 2 " and "R 3 Each of these is independently selected from hydrogen or methoxy, sulfopropyl oxyl, or poly(ethylene) glycol oxy group; and "R 6 This is the amide group (CONH-) connected to the spacer "B" in formula I. It is a dimethylphenyl acridinium ester having the structure, The composition according to claim 3.

5. R 1 is a sulfopropyl group, and here R 2 and R 3 Each is either hydrogen or sulfopropyl The composition according to claim 4, wherein the group is a ropyloxyl group.

6. Formula VI: 【Transformation 5】 The composition according to claim 1, further defined as a cyclosporine C-acridinium ester having the structure.

7. A first reagent comprising the composition according to any one of claims 1 to 6; and A second reagent comprising a solid phase to which an antibody specifically binding to cyclosporine A is directly or indirectly conjugated. An immunoassay kit including...

8. The immunoassay kit according to claim 7, further comprising at least one pretreatment agent.

9. A method for detecting cyclosporine A in a sample: (a): (i) Samples suspected of containing cyclosporine A; (ii) A first reagent comprising at least one composition according to any one of claims 1 to 6; and (iii) A second reagent comprising a solid phase to which an antibody that specifically binds to cyclosporine A is directly or indirectly conjugated. A process of mixing simultaneously, or entirely or partially, in succession to form a mixture; (b) Incubating the mixture under conditions that enable the binding of the antibody to cyclosporine A or the first reagent present in the sample, thereby forming a cyclosporine A / second reagent complex and / or a first reagent / second reagent complex; and (c) A step of detecting a complex formed from the first and second reagents; and (d) Compared to an assay performed when no sample was present, A step to determine the amount of cyclosporine A present in the sample based on the decrease in the amount of the first reagent / second reagent complex. The above method, including.

10. The method according to claim 9, further comprising the step of treating the sample with a pretreatment agent before step (a).

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