Cyclosporin-acridinium esters and methods for their preparation and use

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 to Related Applications / Incorporation by Reference Statement This application claims the benefit under 35 U.S.C. §119(e) of U.S. Provisional Application No. 63 / 476,552, filed December 21, 2022. The entire contents of the above-referenced patent application are expressly incorporated herein by reference.

[0002] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT Not applicable. [Background technology]

[0003] background To distinguish self from non-self, the body relies on a complex immune response system. Sometimes the body's immune system must be regulated to enhance an insufficient response or suppress an excessive response. For example, when organs such as (but not limited to) kidneys, hearts, heart-lungs, bone marrow, and livers 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 rejection of non-autologous tissue allografts. Some of the immunosuppressants most commonly administered to prevent organ rejection in transplant patients are cyclosporine A (CsA), mycophenolic acid, FK-506 (also known as tacrolimus), sirolimus (also known as rapamycin), and everolimus.

[0005] Side effects associated with immunosuppressants can be partially controlled by carefully controlling the levels of drugs present in patients. Therapeutic monitoring of blood concentrations of immunosuppressants and related drugs is necessary to optimize dosing regimens to ensure maximum immunosuppression with minimal toxicity. Although immunosuppressants are highly effective immunosuppressive drugs, their use must be carefully controlled because the effective dose range is often narrow and excessive dosages can cause serious side effects. On the other hand, too low a dosage of immunosuppressant can lead to tissue rejection. The distribution and metabolism of immunosuppressants can vary greatly between patients, and the wide range and severity of adverse reactions make accurate monitoring of drug levels essential. Summary of the Invention [Problem to be solved by the invention]

[0006] Therefore, there is a continuing need to develop rapid and accurate diagnostic methods for measuring the level of an analyte (e.g., an immunosuppressant drug such as CsA) in a sample collected from a patient. These methods should be fully automatable and accurate when performed on samples in the presence of various interfering substances. The assay should provide an accurate measurement of the amount of analyte in a sample while minimizing errors resulting from interfering substances present in the sample. [Brief explanation of the drawings]

[0007] [Figure 1] FIG. 1 shows the chemical structures of cyclosporin A (CsA) and cyclosporin C (CsC). [Figure 2] FIG. 2 shows the chemical structure of the prior art cyclosporine C-acridinium ester (CsC AE) tracer CsC-NSP-DMAE-HEG3-CsA (C138H226N18O40S; molecular weight 2809.43). [Figure 3] FIG. 3 illustrates one non-limiting embodiment of a cyclosporin A assay format constructed in accordance with the present disclosure. [Figure 4] FIG. 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] FIG. 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] FIG. 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] FIG. 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] FIG. 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] FIG. 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] FIG. 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] FIG. 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] FIG. 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] FIG. 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] FIG. 14 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-ZAE (Formula XVI). [Figure 15] FIG. 15 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-FG-DA13-ZAE (Formula XVII). [Figure 16] FIG. 16 illustrates the synthetic scheme for CsC-DA-10-NSP-DMAE of FIG. [Figure 17] FIG. 17 illustrates the synthetic scheme for CsC-DA-10-TSPAE of FIG. [Figure 18] FIG. 18 illustrates the synthetic scheme for CsC-DA-10-HEGAE of FIG. [Figure 19] FIG. 19 illustrates the synthetic scheme for CsC-DA-10-NSP-ZAE of FIG. [Figure 20] FIG. 20 illustrates the synthetic scheme for CsC-PEG15-ZAE of FIG. [Figure 21] FIG. 21 illustrates the synthetic scheme for CsC-PEG23-ZAE of FIG. [Figure 22] FIG. 22 illustrates the synthetic scheme for CsC-PEG23-NSP-DMAE of FIG. [Figure 23] FIG. 23 illustrates the synthetic scheme for CsA-Oxime-PEG3-HEGAE of FIG. [Figure 24] FIG. 24 illustrates the synthetic scheme for CsA-Oxime-PEG3-ZAP of FIG. [Figure 25] FIG. 25 illustrates the synthetic scheme for CsA-Pent-DA-10-NSP-DMAE of FIG. [Figure 26] FIG. 26 illustrates the synthetic scheme for CsA-Pent-DA-10-ZAE of FIG. [Figure 27]FIG. 27 illustrates the synthetic scheme for CsA-Pent-DA-10-HG-DA13-ZAE of FIG. [Figure 28] FIG. 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] FIG. 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-preformed CsA assay format. [Figure 30] FIG. 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-preformed CsA assay format. [Figure 31] FIG. 31 illustrates the binding curves for -DA-10-NSP-ZAE, -PEG15-ZAE, -PEG23-ZAE, and -PEG23-NSP-DMAE to CsC-AE in a non-preformed CsA assay format. [Figure 32] FIG. 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-preformed CsA assay format. [Figure 33] FIG. 33 illustrates the binding curves for -Oxime-PEG3-ZAE, -NSP-DMAE, -NSP-ZAE, and -HG-DA-13-ZAE to cyclosporin A-acridinium ester (CsA-AE) in a non-preformed CsA assay format. [Figure 34]FIG. 34 illustrates an ambient temperature effort (ATE) screening study using CsC AEs (HEG3, DMAE, NSP-ZAE, TSPAE, and HEGAE). [Figure 35] FIG. 35 illustrates ATE studies using CsC-DA-10-NSP-ZAE, CsC-PEG23-NSP-DMAE, CsC-PEG15-ZAE, CsC-PEG23-ZAE, and CsA-Pent-ZAE. DETAILED DESCRIPTION OF THE INVENTION

[0008] Detailed Description Before describing at least one embodiment of the present disclosure through exemplary language and results, it should be understood that the present disclosure is not limited to the details of construction and arrangement of components set forth in the following description of the present application. The present disclosure is capable of other embodiments or of being practiced or carried out in various ways. As such, the language used herein is intended to be accorded the widest possible scope and meaning; and the embodiments are intended to be illustrative, not all-inclusive. Also, it should be understood that the phraseology and terminology employed herein is for the purpose of description and should not be regarded as limiting.

[0009] Unless otherwise defined herein, scientific and technical terms used in connection with this disclosure shall have the meanings commonly understood by those of ordinary skill in the art. Furthermore, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. The foregoing techniques and procedures are well known in the art and are generally performed according to conventional methods as described in the various general and more specific references cited and discussed throughout this specification. The nomenclature utilized in connection with analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein, as well as laboratory procedures and techniques, are well known and commonly used in the art. Standard techniques are used for chemical syntheses and chemical analyses.

[0010] All patents, published patent applications, and non-patent publications mentioned in this specification are indicative of the level of skill of those skilled in the art to which this disclosure pertains. All patents, published patent applications, and non-patent publications referenced in any part of this application are expressly incorporated herein by reference in their entirety to the same extent as if each individual patent or publication was specifically and individually indicated to be incorporated by reference.

[0011] All of the articles, compositions, kits, and / or methods disclosed herein can be made and executed without undue experimentation in light of the present disclosure. While the articles, compositions, kits, and / or methods have been described with reference to specific embodiments, it will be apparent to those skilled in the art that variations can be applied to the articles, compositions, kits, and / or methods and to the steps or sequence of steps of the methods described herein without departing from the concept, spirit, and scope of the present disclosure. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope, and concept of the present disclosure as defined by the appended claims.

[0012] As utilized in accordance with the present disclosure, the following terms, unless otherwise indicated, shall be understood to have the following meanings: The use of the terms "a" or "an" when used in the claims and / or specification in connection with the term "comprising" can mean "one," but this is also consistent with the meanings of "one or more," "at least one," and "one or more than one." Thus, the terms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a compound" can refer to one or more compounds, two or more compounds, three or more compounds, four or more compounds, or a greater number of compounds. The term "plurality" refers to "two or more."

[0013] Use of the term "at least one" is understood to include one and any amount greater than one, including, but not limited to, 2, 3, 4, 5, 10, 15, 20, 30, 40, 50, 100, etc. The term "at least one" can extend to 100 or 1000 or more, depending on the term to which it is attached; furthermore, an amount of 100 / 1000 should not be considered limiting, as higher limits may produce satisfactory results. Furthermore, 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. Use of ordinal terms (i.e., "first," "second," "third," "fourth," etc.) is for the purpose of distinguishing between two or more items only, and is not intended to indicate, for example, any order or sequence or importance or ordering of one item relative to another item.

[0014] The use of the term "or" in the claims is used to mean an inclusive "and / or" unless expressly indicated to refer to alternatives only, or unless the alternatives 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] As used herein, any reference to "one embodiment," "embodiment," "some embodiments," "an example," "for example," or "an example" means that a particular element, feature, structure, or characteristic described in connection with that embodiment is included in at least one embodiment. For example, the appearances of the phrases "in some embodiments" or "an example" in various places throughout the specification do not necessarily all refer to the same embodiment. Moreover, all references to one or more embodiments or examples should be considered limiting of the scope of the claims.

[0016] Throughout this application, the term "about" is used to indicate that a value includes the inherent variation of error for a composition / apparatus / device, the method used to determine the value, or variation that exists among test subjects. For example, and not as a limitation, when the term "about" is used, the specified value can vary from the 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, where such variations are appropriate in the practice of the disclosed methods and would be understood by one of ordinary skill in the art.

[0017] As used in this specification and claims, 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") are inclusive or open-ended and do not exclude additional, unrecited elements or steps.

[0018] As used herein, the term "or combinations thereof" refers to all permutations and combinations of the items listed before that term. For example, "A, B, C, or combinations thereof" is intended to include at least one of: A, B, C, AB, AC, BC, or ABC, and, if order is important in the particular situation, BA, CA, CB, CBA, BCA, ACB, BAC, or CAB. Continuing with this example, combinations containing one or more repeats of an item or term are expressly included, such as BB, AAA, AAB, BBC, AAABCCCC, CBBAAA, CABABB, etc. One of skill in the art will understand that there is typically no limit to the number of items or terms in any combination unless clear from the context.

[0019] As used herein, the term "substantially" means that the subsequently described events or circumstances occur completely, or that the subsequently described events or circumstances occur to a large extent or for the most part. For example, when referring to a particular event or circumstance, the term "substantially" means that the subsequently described events or circumstances occur 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 to each other, or that a portion of one of two items is close to the other item, but not 100% adjacent to the other item.

[0020] As used herein, the phrases "associated with" and "coupled to" include both direct association / binding of two moieties to one another and indirect association / binding of two moieties to one another. Non-limiting examples of association / binding include, for example, covalent binding of one moiety to another moiety by direct bonding or through a spacer group, non-covalent binding of one moiety to another moiety either directly or using a specific binding pair member bound to the moiety, incorporation of one moiety into another moiety such as by dissolving one moiety in another moiety or by synthesis, and coating one moiety on another moiety.

[0021] The terms "analog" and "derivative" are used interchangeably herein and refer to a substance that contains in its structure the same basic carbon skeleton and carbon functional groups as a given compound, but also contains one or more substitutions therefor. As used herein, the term "substituted" is understood to refer to the replacement of at least one substituent on a compound with a residue R. In certain non-limiting embodiments, R may comprise a C1-C4 compound selected from H, hydroxyl, thiol, halide selected from fluoride, chloride, bromide, or iodide, optionally substituted linear, branched, or cyclic alkyl, and linear, branched, or cyclic alkenyl, where any substituent may be one or more of alkenylalkyl, alkynylalkyl, cycloalkyl, cycloalkenylalkyl, arylalkyl, heteroarylalkyl, heterocycloalkyl, optionally substituted heterocycloalkenylalkyl, arylcycloalkyl, and arylheterocycloalkyl. and n is 0 or 1, each of which is optionally substituted, and wherein the optional substituents are selected from one or more of alkenylalkyl, alkynylalkyl, cycloalkyl, cycloalkenylalkyl, arylalkyl, alkylaryl, heteroarylalkyl, heterocycloalkyl, optionally substituted heterocycloalkenylalkyl, arylcycloalkyl, and arylheterocycloalkyl, phenyl, cyano, hydroxyl, alkyl, aryl, cycloalkyl, cyano, alkoxy, alkylthio, amino, -NH(alkyl), -NH(cycloalkyl), carboxy, and -C(O))-alkyl.

[0022] The term "sample," as used herein, is understood to include any type of biological sample that may be utilized in accordance with the present disclosure. Examples of fluid biological samples that may be utilized include, but are not limited to, whole blood or any fraction thereof (i.e., plasma or serum), urine, saliva, sputum, cerebrospinal fluid (CSF), skin, intestinal fluid, peritoneal fluid, cyst fluid, sweat, interstitial fluid, extracellular fluid, tears, mucus, bladder washings, semen, feces, pleural effusion, nasopharyngeal fluid, combinations thereof, and the like.

[0023] The term "specific binding partner" or "analyte-specific binder" is understood to refer to any molecule capable of specifically binding to a target analyte. For example, but not by way of limitation, a binder / binding partner can be an antibody, a receptor, a ligand, an aptamer, a molecularly imprinted polymer (i.e., an inorganic matrix), any fragment thereof, and any combination or derivative thereof, as well as any other molecule capable of specifically binding to a target analyte.

[0024] The term "antibody" is used in the broadest sense and specifically includes (but is not limited to) monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), fragments of any of the above, and conjugates of any of the above, so long as they exhibit the desired analyte-binding biological activity. Thus, the term "antibody" or "antibody peptide" refers to a full-length immunoglobulin molecule (i.e., an intact antibody) or an antigen-binding fragment thereof that competes with the intact antibody for specific antigen binding. Antigen-binding fragments can be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact antibodies. Antigen-binding fragments include Fab, Fab', F(ab'), Fv, scFv, disulfide-linked Fv, Fd, diabodies, single-chain antibodies, single-domain antibodies (e.g., but not limited to, NANOBODIES). (R) ), and other antibody fragments or conjugates thereof that retain at least a portion of the variable region of an intact antibody, antibody replacement proteins or peptides (i.e., engineered binding proteins / peptides), and combinations or derivatives thereof. See, e.g., 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 term "antigen-binding fragment" or "antigen-binding portion" of an antibody refers 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 fragments of an intact antibody. Examples of binding fragments encompassed within the term "antigen-binding fragment" of an antibody include, but are not limited to, Fab, Fab', F(ab'), Fv, scFv, disulfide-linked Fv, Fd, diabodies, single-chain antibodies, single-domain antibodies (including, but not limited to, NANOBODIES). (R) ), isolated CDRH3, and other antibody fragments that retain at least a portion of the variable region of an intact antibody. These antibody fragments are obtained using conventional recombinant and / or enzymatic techniques and are screened for antigen binding in the same manner as intact antibodies.

[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 major antibody light chain isotypes.

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

[0029] The term "epitope" includes any protein determinant capable of specific binding to an immunoglobulin or T-cell receptor. In certain embodiments, an epitope is a region of an antigen specifically bound by an antibody. Epitopic determinants usually include chemically active surface groupings of molecules such as amino acids, sugar side chains, phosphoryl, or sulfonyl groups. In certain embodiments, epitopes may have specific three-dimensional structural characteristics (e.g., "conformational epitopes") as well as specific charge characteristics.

[0030] An epitope is defined as "the same" as another epitope if a particular antibody specifically binds to both epitopes. In certain embodiments, polypeptides with 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 that antigen in a complex mixture of proteins and / or macromolecules. In certain embodiments, an antibody comprises an antigen binding site that specifically binds to a particular epitope. In certain such embodiments, an antibody can bind to different antigens, so long as the different antigens contain that particular epitope or a closely related epitope. In certain instances, for example, homologous proteins from different species may contain the same epitope. In certain embodiments, an antibody can bind to a specific antigen, such as a 10 -6 M, 10 -7 M, 10 -8 M or 10 -9 It specifically binds to an antigen with a dissociation constant of less than or equal to M. When an antibody specifically binds to a receptor or a ligand (i.e., a counter-receptor), it can substantially inhibit adhesion of the receptor to the ligand. As used herein, an antibody substantially inhibits adhesion of a receptor to a ligand if an excess of antibody reduces the amount of receptor bound to the ligand by at least about 20%, 40%, 60%, or 80%, 85%, or 90% (as measured in an in vitro competitive binding assay).

[0032] An "isolated" antibody is one that has been separated and / or recovered from components of the environment in which it was produced. Contaminating components of the production environment are substances that would interfere with diagnostic or therapeutic uses of the antibody and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. In certain embodiments, the antibody is purified, as measurable by at least three different methods: 1) to greater than 50% by weight of the antibody, e.g., greater than 75%, or greater than 85%, or greater than 95%, or greater than 99% by weight, as determined by the Lowry method; 2) to a degree sufficient to obtain at least 10 residues of N-terminal or internal amino acid sequence by use of a spinning cup sequencer, e.g., to at least 15 residues of sequence; or 3) to homogeneity by SDS-PAGE under reducing or non-reducing conditions using Coomassie blue or by silver staining. Isolated antibodies include antibodies in situ within recombinant cells, since at least one component of the environment in which the antibody was produced is absent. Ordinarily, however, isolated antibodies will be prepared by at least one purification step. Moreover, an "isolated antibody" is substantially free of other antibodies having different antigenic specificities, although an isolated antibody may have some cross-reactivity to other related 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 having at least 75% amino acid sequence identity or similarity, such as at least 80%, or at least 85%, or at least 90%, or at least 95%, with the amino acid sequence of either the heavy or light chain variable domain of the antibody, and necessarily having less than 100% sequence identity or similarity.

[0034] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies that specifically bind to the same epitope; i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. In contrast to conventional (polyclonal) antibody preparations, which typically include different antibodies specific for different determinants (epitopes), each monoclonal antibody is specific for a single determinant on the antigen. In addition to their specificity, monoclonal antibodies are advantageous in that they can be synthesized by a single production method, by a hybridoma culture, and therefore are uncontaminated by other immunoglobulins. The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and should not be construed as requiring production of the antibody by any particular method. For example, in one embodiment, monoclonal antibodies produced in accordance with the present disclosure may be produced by the hybridoma method first described by Kohler and Milstein (Nature, 256:495 (1975)).

[0035] Monoclonal antibodies utilized in accordance with the present disclosure may be produced by any method known in the art, including, but not limited to, as a result of a deliberate immunization protocol; as a result of an immune response that results in the production of natural antibodies during the course of disease or cancer; phage-derived antibodies, etc. In addition to the hybridoma production methods listed above, monoclonal antibodies of the present disclosure may be produced by a variety of other methods, such as, but not limited to, recombinant DNA methods (see, e.g., U.S. Pat. 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); as well as a variety of 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 utilized in the conjugates and methods disclosed herein or otherwise contemplated are widely commercially available, and therefore further description thereof is deemed unnecessary.

[0036] As used herein, "substantially pure" means that the target species is the predominant species present (i.e., more abundant than any other individual species in the composition on a molar basis). Generally, a substantially pure composition comprises greater than about 50%, e.g., greater than about 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, and 99% of all macromolecular species present in the composition. In one embodiment, the target species is purified to essential homogeneity (contaminating species cannot be detected in the composition by conventional detection methods), where the composition consists essentially of a single macromolecular species.

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

[0038] Certain non-limiting embodiments of the present disclosure include compounds of Formula I: [ka] a composition comprising cyclosporin C and an acridinium ester linked via a spacer, the composition having the structure:

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

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

[0041] In certain non-limiting embodiments, the acridinium ester utilized in accordance with the present disclosure has Formula III: [ka] wherein "R1" is an alkyl, alkenyl, alkynyl, or aralkyl group of 1 to 35 carbon atoms and 0 to 20 heteroatoms; a sulfopropyl or sulfobutyl group; and a group -R a -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 to C4, and "R3" is located at one or more of positions C5 to 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, each containing 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. - " is a counterion introduced to pair with, for example (but not limited to), the quaternary nitrogen of the acridinium nucleus, and "A - " is CH3SO3 - , FSO3 - , CF3SO3 - , C4F9SO3 - , CH3C6H4SO3 - , halide, CF3COO - , CH3COO - , and NO3 - selected from the group consisting of It has the following structure.

[0042] In certain non-limiting embodiments, the acridinium ester utilized in accordance with the present disclosure has formula IV: [ka] [Wherein: "R1", "R2", "R3", and "A -" is as defined above with reference to Formula III; "R4" and "R8" are each independently selected from hydrogen or alkyl, alkenyl, alkynyl, alkoxyl (-OR), alkylthiol (-SR), or a substituted amino group that serves, for example and without limitation, to stabilize the -COX- linkage between the acridinium nucleus and the "Y" moiety through steric and / or electronic effects. Further, "R5", "R6", and "R7" are each independently selected from hydrogen or an alkyl, alkenyl, alkynyl, aryl, or aralkyl group, wherein each group contains 0-20 heteroatoms. Additionally, one of "R5", "R6", and "R7" further comprises a functional group that links to the spacer "B" of Formula I. Functional groups that may be utilized in accordance with the present disclosure include, but are not limited to, the following groups: [ka] Examples include: is a chemiluminescent acridinium ester having the structure:

[0043] In certain non-limiting embodiments, the acridinium ester utilized in accordance with the present disclosure has Formula V: [ka] wherein "R1" is a methyl or sulfopropyl group; "R2" and "R3" are each independently selected from hydrogen or a methoxy, sulfopropyloxyl, or poly(ethylene)glycoloxy group; and "R6" is an amide group (CONH-) connected to the spacer "B" of formula I. - " is as defined in Formula III] It is a dimethylphenyl acridinium ester having the structure:

[0044] In a specific (but non-limiting) embodiment, R1 is a sulfopropyl group, and wherein R2 and R3 are each hydrogen or a sulfopropyloxyl group.

[0045] In a specific (but non-limiting) embodiment, the composition has Formula VI: [ka] It is a cyclosporin C-acridinium ester having the structure:

[0046] In a specific (but non-limiting) embodiment, the composition has Formula VII: [ka] It is a cyclosporin C-acridinium ester having the structure:

[0047] In a specific (but non-limiting) embodiment, the composition has Formula VIII: [ka] It is a cyclosporin C-acridinium ester having the structure:

[0048] In a specific (but non-limiting) embodiment, the composition has Formula IX: [ka] It is a cyclosporin C-acridinium ester having the structure:

[0049] In a specific (but non-limiting) embodiment, the composition has the formula X: [ka] It is a cyclosporin C-acridinium ester having the structure:

[0050] In a specific (but non-limiting) embodiment, the composition has Formula XI: [ka] It is a cyclosporin C-acridinium ester having the structure:

[0051] In a specific (but non-limiting) embodiment, the composition has Formula XII: [ka] It is a cyclosporin C-acridinium ester having the structure:

[0052] In a specific (but non-limiting) embodiment, the composition has Formula XIII: [ka] It is a cyclosporin A-acridinium ester having the structure:

[0053] In a specific (but non-limiting) embodiment, the composition has Formula XIV: [ka] It is a cyclosporin A-acridinium ester having the structure:

[0054] In a specific (but non-limiting) embodiment, the composition has Formula XV: [ka] It is a cyclosporin A-acridinium ester having the structure:

[0055] In a specific (but non-limiting) embodiment, the composition has Formula XVI: [ka] It is a cyclosporin A-acridinium ester having the structure:

[0056] In a specific (but non-limiting) embodiment, the composition has Formula XVII: [ka] It is a cyclosporin A-acridinium ester having the structure:

[0057] Certain non-limiting embodiments of the present disclosure relate to immunoassay kits containing any one or more of the CsC-AE / CsA-AE compositions disclosed herein or other contemplated. The choice of acridinium ester present will depend on the particular assay format to be utilized, and such a choice is well within the skill of one in the art.

[0058] In a specific (but non-limiting) embodiment, the immunoassay kit comprises a first reagent comprising any of the CsC-AE / CsA-AE compositions described herein or otherwise contemplated, and a second reagent comprising a solid phase having an antibody bound thereto, either directly or indirectly; the antibody present in the second reagent specifically binds to the CsC or CsA present in the first reagent.

[0059] Any antibody or fragment thereof known in the art or otherwise contemplated herein can be utilized in accordance with the present disclosure, so long as the antibody / fragment thereof is capable of binding to CsC or CsA at an epitope distant from the position at which the acridinium ester is attached. Antibodies that bind to CsC / CsA are well known in the art and are commercially available. By way of example and not limitation, CsC / CsA antibodies are commercially available from 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); HyTest Ltd (Turku, Finland); and others. Accordingly, further description of the antibodies utilized in accordance with the present disclosure is deemed unnecessary.

[0060] The assay reagents present in the kit may be provided in any form that enables them to function in accordance with the present disclosure. For example, but not by way of limitation, the reagents may each be provided in liquid form and disposed in the kit collectively and / or in single aliquots. Alternatively, in certain (but non-limiting) embodiments, one or more reagents may be disposed in the kit in the form of a single aliquot of lyophilized reagent. The use of dried reagents in microfluidics devices is described in detail in U.S. Pat. No. 9,244,085 (Samproni), the entire contents of which are expressly incorporated herein by reference.

[0061] In addition to the assay reagents described in detail herein above, the kit may further contain other reagents for carrying out any of the specific assays described herein or other contemplated. For example (and not by way of limitation), the kit may further include at least one pretreatment / release agent for releasing cyclosporine from any endogenous binding proteins present in the biological sample. The nature of these additional reagents will depend on the particular assay format, and their identification is well within the skill of those skilled in the art; therefore, further description thereof is deemed unnecessary. Additionally, the components / reagents present in the kit may each be in separate containers / compartments, or, depending on the cross-reactivity and stability of the components / reagents, various components / reagents may be combined in one or more containers / compartments. Additionally, the kit may include a microfluidic device in which the components / reagents are disposed.

[0062] The relative amounts of the various components / reagents in the kit can be varied widely to provide concentrations of the components / reagents that substantially optimize the reactions required to occur during the assay method and further to substantially optimize assay sensitivity. Under appropriate circumstances, one or more of the components / reagents in the kit can be provided as a dry powder, such as a lyophilized powder, and the kit may further include an excipient for dissolving the dried reagent; in this manner, a reagent solution having an appropriate concentration for performing a method or assay in accordance with the present disclosure can be obtained from these components. Non-limiting examples of other reagents that may be included in the kit include wash solutions, diluents, excipients, interference solutions, positive controls, negative controls, calibration reagents, quality control reagents, and the like. Additionally, the kit may further include 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 contemplated.

[0063] Certain non-limiting embodiments of the present disclosure relate to methods of making any of the herein disclosed or other contemplated CsC-AE / CsA-AE compositions, which methods include attaching a linker to CsC / CsA and then attaching an acridinium ester to the linker.

[0064] Certain non-limiting embodiments of the present disclosure relate to methods for detecting CsC / CsA in a sample utilizing any of the CsC-AE / CsA-AE compositions disclosed herein or other contemplated, in which a sample suspected of containing CsC / CsA is mixed simultaneously or wholly or partially sequentially with one or more of any of the CsC-AE compositions disclosed herein or other contemplated and one or more solid phase-bound CsC / CsA-antibodies disclosed herein or other contemplated to form a mixture, and the mixture is incubated under conditions that allow the antibodies to bind to the CsC / CsA or CsC-AE / CsA-AE present in the sample, thereby forming Cs / antibody complexes and / or Cs-AE / antibody complexes. The Cs-AE / antibody complex is then detected, and the amount of CsC / CsA present in the sample is determined based on the decrease in the amount of Cs-AE / antibody complex formed compared to a negative control (i.e., the amount of Cs-AE / antibody complex formed in the absence of sample). The concentration of CsC / CsA present in the sample can then be determined based on the decrease.

[0065] In a specific (but non-limiting) embodiment of the above method, the sample is mixed simultaneously or wholly or partially sequentially with the first and second reagents of the immunoassay kit described in detail hereinabove (i.e., a first reagent comprising a CsC / CsA-AE composition and a second reagent comprising a solid phase to which an antibody or fragment thereof that specifically binds to CsC / CsA is directly or indirectly bound), to form a mixture. This mixture is then incubated under conditions that allow 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 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. The first reagent-second reagent (Cs-AE / antibody) complex is then 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 first reagent-second reagent complex formed compared to a negative control (i.e., the amount of first reagent-second reagent complex formed in the absence of sample). The concentration of target analyte (CsC or CsA) present in the sample is then determined based on the decrease.

[0066] Any sample in which it is desired to assay for the presence of a cyclosporine target analyte (i.e., CsC or CsA) can be utilized as a sample according to the methods of the present disclosure. Non-limiting examples of samples include, but are not limited to, whole blood or any fraction thereof (i.e., plasma or serum), urine, saliva, sputum, cerebrospinal fluid (CSF), skin, intestinal fluid, peritoneal fluid, cyst fluid, sweat, interstitial fluid, extracellular fluid, tears, mucus, bladder washings, semen, feces, pleural effusion, nasopharyngeal fluid, and combinations thereof. Specific non-limiting examples include lysed whole blood cells and lysed red blood cells.

[0067] As noted above, the various components of the method are provided in combination (simultaneously or sequentially). When the various components of the method are added sequentially, the order in which the components are added can vary; one skilled in the art can determine the particular desired order in which the different components are added to the assay. Of course, the simplest order of addition is to add all of the substances simultaneously and determine the signal resulting therefrom. Alternatively, each of the components, or groups of components, can be mixed sequentially. In certain embodiments, an incubation step can be included after one or more additions. For example (and not by way of limitation), it may be desirable to mix and incubate the antibody-solid phase and sample before adding the Cs-AE composition.

[0068] The conditions under which the mixture is incubated may vary widely, so long as the antibody binds to Cs or Cs-AE to form a complex under the above conditions. Immunoassays based on the sandwich assay format are widely practiced, and immunoassay conditions are well known in the art; therefore, the selection of appropriate assay conditions is well within the skill of one in the art, and therefore further explanation thereof is deemed unnecessary.

[0069] The particular detection method utilized may vary widely, so long as the complex can be detected in the manner described above. Detection of formed complexes in sandwich assay formats is widely practiced, and detection procedures are well known in the art; therefore, selection of an appropriate detection method is well within the skill of one in the art, and therefore further description thereof is deemed unnecessary.

[0070] The above methods may further comprise one or more additional steps to increase the accuracy and / or precision of the assay. For example (but not by way of limitation), the above methods may further comprise one or more pretreatment / release steps to release the cyclosporine from any endogenous binding proteins present in the biological sample prior to mixing with the immobilized antibody. Another non-limiting example of additional steps that may be utilized in accordance with the present disclosure includes one or more wash steps to remove unbound (or non-specifically bound) reagents from the reaction mixture prior to detection of complex formation.

[0071] Certain further non-limiting embodiments of the present disclosure relate to a microfluidics device comprising any of the components of the immunoassay kit described hereinabove. In particular, certain non-limiting embodiments include a microfluidics device for detecting a target analyte (CsC or CsA) in a sample. The microfluidics device comprises (i) an inlet channel through which a sample is applied; and (ii) at least a first compartment capable of fluid communication with the inlet channel. Compartment (ii) contains the first and second reagents of the immunoassay kit described in detail hereinabove.

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

[0073] The device may be provided with any arrangement of compartments and distribution of various components therebetween that enables the device to function in accordance with the present disclosure.

[0074] Any compartment of a microfluidic device may be sealed to maintain reagents disposed therein in a substantially airtight environment until their use; for example, a compartment containing a lyophilized reagent may be sealed to prevent unintentional reconstitution of the reagent. An inlet channel and a compartment, as well as two compartments, may be described as "capable of fluid communication" with each other; this phrase indicates that each of the compartments remains sealed, but that the two compartments can allow fluid to flow between them upon piercing a seal formed in or between the compartments.

[0075] The microfluidics device of the present disclosure may include any other desirable features known in the art or otherwise contemplated herein. For example, but not by way of limitation, the microfluidics device of the present disclosure may further include a read chamber; the read chamber may be any of the compartments containing one or more of the reagents described herein above, or the read chamber may be in fluid communication with the compartments containing one or more reagents. The microfluidics device may further include one or more additional compartments containing other solutions, such as (but not limited to) wash solutions, diluents, excipients, interference solutions, positive controls, negative controls, quality controls, etc. These additional compartments may be in fluid communication with one or more of the other compartments. For example, the microfluidics device may further include one or more compartments containing wash solutions, and these compartments may be capable of fluid communication with any other compartment of the device. In another example, the microfluidics device may further include one or more compartments containing excipients for dissolving one or more dried reagents, and the compartments may be capable of fluid communication with any other compartment of the device. In yet a further example, a microfluidic device may include one or more compartments that contain a diluent, and the compartments may be capable of fluid communication with any other compartment of the device. [Example]

[0076] Examples are provided below. However, it should be understood that the present disclosure is not limited to the specific experiments, results, and test procedures disclosed herein in its application. Rather, the examples are provided merely as one of various embodiments and are intended to be illustrative rather than comprehensive.

[0077] The Cyclosporine A AIP Assay is available from ADVIA (R) CENTAUR (R) and ATELLICA (R) It 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 hydroxy group on the CsC molecule (Figure 1). The original ADVIA (R) CENTAUR (R) The cyclosporine adenosine (CsA) assay was commercialized in 2008, and this original immunoassay used a complex CsC-acridinium ester (CsC-AE) conjugate (Figure 2) for the detection of CsA. However, the supplier discontinued the raw material CsC-succinate-NHS utilized to manufacture this conjugate. Therefore, there was a need for a new AE tracer designed for the CsA II assay.

[0078] Additionally, a novel Cs assay was designed that addresses European REACH (Registration, Evaluation, Authorization, and Restriction of Chemicals) requirements for Triton reduction, as well as the U.S. Food and Drug Administration's Ambient Temperature Action (ATE) requirements and biotin interference mitigation. The novel Cs assay of this disclosure utilizes a new tracer, and the design process emphasized the identification of a new tracer that has a simplified chemical structure and can be manufactured using conventional synthetic processes.

[0079] Based on this need, a series of novel and uncomplicated cyclosporine (A and C)-acridinium esters (CsA-AE and CsC-AE) were designed and developed as ADVIA inhibitors based on acridinium ester (AE) technology (Natrajan et al., Ann Biochem (2010) 406:204; and Natrajan et al., Org Biomol Chem (2011) 9:5092). (R) CENTAUR (R) These CsA-AE and CsC-AE were synthesized for testing in the CSA II assay (Siemens Healthcare Diagnostics Inc., Tarrytown, NY). (R) CENTAUR (R) The assays were evaluated using a competitive assay format (Figure 3) on a Siemens Healthcare Diagnostics Inc. system (Tarrytown, NY). One key tracer, CsC-DA-10-NSP-ZAE (Formula IX; Figure 7), demonstrated superior performance relative to the assay requirements (ATE, sensitivity, and precision) and was selected as a potential 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 biotinylated monoclonal anti-CsA antibodies. The lite reagent contained a CsC-AE tracer. In the absence of CsA 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 emitted light when activated by the addition of acid and base. Addition of CsA from a patient sample to the reaction mixture prevented the binding of the Cs-AE conjugate to the solid phase, resulting in a decrease in signal generation. This signal decrease is a direct function of the amount of CsA in the sample, as measured against a calibration curve.

[0081] With the above assay design in mind, both solid-phase reagents and novel CsC-AE tracers were developed and evaluated. The production of biotinylated monoclonal antibodies and the prebinding of these antibodies to the surface of commercially available streptavidin-coated particles are well-known and straightforward processes. Therefore, the remainder of this example focuses on the synthesis of new 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 chosen for NSP-DMAE conjugation in initial assay studies. Initial data in this example indicated that conjugation of CsC-DA-10-NSP-DMAE to this hydroxyl resulted in promising assay curve shapes. Given this initial performance, conjugates were also prepared using HEGAE, TSPAE, and ZAE at the same conjugation position to determine whether different AE structures would further benefit this assay. Additionally, the terminal bifunctional and aldehyde functional groups of CsA (Figure 1) were used as a basis to introduce NSP-DMAE and ZAE. The chemical structures of the CsA-AE and CsC-AE tracers are shown in Figures 4-15, and the syntheses of these CsA-AE and CsC-AE tracers are shown in Figures 16-27.

[0083] Cyclosporine Immunoassay Performance: Evaluation of CsA-AE and CsC-AE tracers was performed on the ADVIA immunoassay analyzer available from Siemens Healthcare Diagnostics Inc., Tarrytown, NY. (R) CENTAUR (R) Screening of CsA-AE and CsC-AE tracers (Figures A.4-15) for binding to available monoclonal antibodies was performed using the prototype ADVIA family. (R) CENTAUR (R) CsC was determined using an 18Cs immunoassay (Figures 28-33). Ambient temperature approach (ATE) studies using different CsC tracers are also shown in Figures 34-36.

[0084] Conclusion: A series of new CsA-AE and CsC-AE tracers were successfully designed, synthesized, and evaluated in a prototype CsA immunoassay (Figures 4-15; Figures 16-27; and Figures 28, 30, 32, and 34-36, respectively). The novel tracers were selected as excellent candidates to meet FDA ambient temperature commitment 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 (Figure 19) compared to the currently used, complex tracer, CsA-HEG3-NSP-DMAE (Figure 2). This saves manufacturing time and cost and overcomes the issue of discontinued raw materials for current tracers. Therefore, the new tracers developed according to this disclosure are critical components for the development of new Cs immunoassays.

[0085] Non-Limiting Exemplary Embodiments of the Inventive Concept Exemplary Embodiment 1. Formula I: [ka] wherein "A" comprises an acridinium ester; and "B" is a spacer having from about 5 atoms to about 100 atoms each selected from the group consisting of C, H, O, N, S, and P atoms. A composition comprising cyclosporin C and an acridinium ester linked via a spacer, the composition having the structure:

[0086] Exemplary Embodiment 2 "A" in Formula I is Formula III: [ka] [In formula: "R1" is: an alkyl, alkenyl, alkynyl, or aralkyl group of 1 to 35 carbon atoms and 0 to 20 heteroatoms; a sulfopropyl or sulfobutyl group; and a group -R a -Z, where R a is 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 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 of 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 straight chain alkyl groups; substituted or unsubstituted aryl groups; and heterocyclic ring groups; wherein the "X" group contains 0 to 20 heteroatoms, and wherein the "X" group further contains a functional group that links to spacer "B" of formula I; and "A - " is CH3SO3 - , FSO3 - , CF3SO3 - , C4F9SO3 - , CH3C6H4SO3 - , halide, CF3COO - , CH3COO - , and NO3 - and the counter ion is selected from the group consisting of The composition of exemplary embodiment 1, wherein the compound is an acridinium ester having the structure:

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

[0088] Exemplary embodiment 4. "A" in formula I is a group represented by formula V: [ka] wherein "R1" is a methyl or sulfopropyl group; "R2" and "R3" are each independently selected from hydrogen or a methoxy, sulfopropyloxyl, or poly(ethylene)glycoloxy group; and "R6" is an amide group (CONH-) connected to spacer "B" of Formula I. The composition of exemplary embodiment 3, wherein the compound is a dimethylphenyl acridinium ester having the structure:

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

[0090] Exemplary Embodiment 6. Formula VI: [ka] 6. The composition of any one of Exemplary Embodiments 1-5, further defined as a cyclosporin C-acridinium ester having the structure:

[0091] Exemplary Embodiment 7. Formula VII: [ka] 6. The composition of any one of Exemplary Embodiments 1-5, further defined as a cyclosporin C-acridinium ester having the structure:

[0092] Exemplary Embodiment 8. Formula VIII: [ka] 6. The composition of any one of Exemplary Embodiments 1-5, further defined as a cyclosporin C-acridinium ester having the structure:

[0093] Exemplary Embodiment 9. Formula IX: [ka] 6. The composition of any one of Exemplary Embodiments 1-5, further defined as a cyclosporin C-acridinium ester having the structure:

[0094] Exemplary embodiment 10. Formula X: [ka] 6. The composition of any one of Exemplary Embodiments 1-5, further defined as a cyclosporin C-acridinium ester having the structure:

[0095] Exemplary Embodiment 11. Formula XI: [ka] 6. The composition of any one of Exemplary Embodiments 1-5, further defined as a cyclosporin C-acridinium ester having the structure:

[0096] Exemplary Embodiment 12. Formula XII: [ka] 6. The composition of any one of Exemplary Embodiments 1-5, further defined as a cyclosporin C-acridinium ester having the structure:

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

[0098] Exemplary Embodiment 14. "A" in Formula II is Formula III: [ka] [In formula: "R1" is an alkyl, alkenyl, alkynyl, or aralkyl group of 1 to 35 carbon atoms and 0 to 20 heteroatoms; a sulfopropyl or sulfobutyl group; and a group -R a -Z, where R a is 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 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 of 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 straight chain alkyl groups; substituted or unsubstituted aryl groups; and heterocyclic ring groups; wherein the "X" group contains 0 to 20 heteroatoms, and wherein the "X" group further contains a functional group that links to spacer "B" of formula I; and "A - " is CH3SO3 - , FSO3 - , CF3SO3 - , C4F9SO3 - , CH3C6H4SO3 - , halide, CF3COO - , CH3COO - , and NO3 - and the counter ion is selected from the group consisting of The composition of exemplary embodiment 13, wherein the compound is an acridinium ester having the structure:

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

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

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

[0102] Exemplary Embodiment 18. Formula XIII: [ka] 18. The composition of any one of Exemplary Embodiments 13-17, further defined as a cyclosporin A-acridinium ester having the structure:

[0103] Exemplary Embodiment 19. Formula XIV: [ka] 18. The composition of any one of Exemplary Embodiments 13-17, further defined as a cyclosporin A-acridinium ester having the structure:

[0104] Exemplary Embodiment 20. Formula XV: [ka] 18. The composition of any one of Exemplary Embodiments 13-17, further defined as a cyclosporin A-acridinium ester having the structure:

[0105] Exemplary Embodiment 21. Formula XVI: [ka] 18. The composition of any one of Exemplary Embodiments 13-17, further defined as a cyclosporin A-acridinium ester having the structure:

[0106] Exemplary Embodiment 22. Formula XVII: [ka] 18. The composition of any one of Exemplary Embodiments 13-17, further defined as a cyclosporin A-acridinium ester having the structure:

[0107] Exemplary Embodiment 23. An immunoassay kit comprising: a first reagent comprising the composition of any one of Exemplary Embodiments 1-22; and a second reagent comprising a solid phase having attached thereto, directly or indirectly, an antibody that specifically binds to cyclosporine A.

[0108] Exemplary Embodiment 24 The immunoassay kit of 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) simultaneously or wholly or partially sequentially mixing (i) a sample suspected of containing cyclosporine A; (ii) a first reagent comprising the composition of any one of Exemplary Embodiments 1 to 22; and (iii) a second reagent comprising a solid phase having an antibody that specifically binds to cyclosporine A bound directly or indirectly to the solid phase to form a mixture; (b) incubating the mixture under conditions that allow binding of the antibody to cyclosporine A present in the sample or to the first reagent, thereby forming a cyclosporine A / second reagent complex and / or a first reagent / second reagent complex; and (c) detecting the complex 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 complex formed compared to an assay performed in the absence of sample.

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

[0111] Thus, in accordance with the present disclosure, compositions, kits, and devices, as well as methods for making and using the same, are provided that fully satisfy the objects and advantages set forth hereinabove. While the present disclosure has been described in conjunction with the specific figures, experiments, results, and language set forth hereinabove, it is evident that many alternatives, modifications, and variations will be apparent to those skilled in the art. Accordingly, it is intended to embrace all such alternatives, modifications, and variations that fall within the spirit and broad scope of the present disclosure.

Claims

1. Formula I: 【Chemistry 1】 [In the formula: "A" comprises an acridinium ester; and "B" is a spacer having from about 5 atoms to about 100 atoms each selected from the group consisting of C, H, O, N, S, and P atoms. Cyclosporin C and an acridinium ester linked via a spacer, having the structure A composition comprising:

2. "A" in Formula I is a compound of Formula III: 【Chemistry 2】 [In the formula: "R1" is: alkyl, alkenyl, alkynyl, or aralkyl groups of 1 to 35 carbon atoms and 0 to 20 heteroatoms; sulfopropyl or sulfobutyl groups; and Base-R ａ -Z is selected from the group consisting of Here, R ａ is a divalent radical selected from an alkyl, alkenyl, alkynyl, aryl, or aralkyl group of 1 to 35 carbon atoms and 0 to 20 heteroatoms; "R2" is located at one or more of positions C1 to C4, and each "R2" is selected from hydrogen, alkyl, OR, OH, SR, SH, NH ２ 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 of positions C5 to C8, and each "R3" is selected from the group consisting of hydrogen, alkyl, OR, OH, SR, SH, NH ２ 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 straight chain alkyl groups; substituted or unsubstituted aryl groups; and heterocyclic ring groups; wherein the "X" group contains 0 to 20 heteroatoms, and wherein the "X" group further contains a functional group that links to spacer "B" of formula I; and "A － " is CH ３ SO ３ － , FSO ３ － , C.F. ３ SO ３ － , C ４ F ９ SO ３ － , C.H. ３ C ６ H ４ SO ３ － , halide, CF ３ COO － , C.H. ３ COO － , and NO ３ － and the counter ion is selected from the group consisting of 2. The composition of claim 1, wherein the acridinium ester has the structure:

3. "A" in Formula I is a group represented by Formula IV: 【Transformation 3】 [In the formula: "R ４ " and "R ８ " are each independently selected from hydrogen or an alkyl, alkenyl, alkynyl, alkoxyl (-OR), alkylthiol (-SR), or substituted amino group; "R ５ "," "R ６ " and "R ７ are each independently selected from hydrogen or an alkyl, alkenyl, alkynyl, aryl, or aralkyl group, where each group contains 0 to 20 heteroatoms; and "R ５ "," "R ６ " and "R ７ " further comprises a functional group that links to spacer "B" of Formula I. is an acridinium ester having the structure The composition of claim 2.

4. "A" in Formula I is a group represented by Formula V: 【Chemistry 4】 [In the formula: "R １ " is a methyl or sulfopropyl group; "R ２ " and "R ３ are each independently selected from hydrogen or a methoxy, sulfopropyloxyl, or poly(ethylene)glycoloxy group; and "R ６ " is the amide group (CONH-) connected to the spacer "B" of Formula I. is a dimethylphenyl acridinium ester having the structure The composition of claim 3.

5. R １ is a sulfopropyl group, and where R ２ and R ３ The composition of claim 4 , wherein each is hydrogen or a sulfopropyloxyl group.

6. Formula VI: 【Transformation 5】 2. The composition of claim 1, further defined as a cyclosporin C-acridinium ester having the structure:

7. Formula VII: 【Transformation 6】 2. The composition of claim 1, further defined as a cyclosporin C-acridinium ester having the structure:

8. Formula VIII: 【Transformation 7】 2. The composition of claim 1, further defined as a cyclosporin C-acridinium ester having the structure:

9. Formula IX: 【Transformation 8】 2. The composition of claim 1, further defined as a cyclosporin C-acridinium ester having the structure:

10. Formula X: 【Chemistry 9】 2. The composition of claim 1, further defined as a cyclosporin C-acridinium ester having the structure:

11. Formula XI: 【Chemistry 10】 2. The composition of claim 1, further defined as a cyclosporin C-acridinium ester having the structure:

12. Formula XII: 【Chemistry 11】 2. The composition of claim 1, further defined as a cyclosporin C-acridinium ester having the structure:

13. Formula II: 【Chemistry 12】 [In the formula: "A" comprises an acridinium ester; and "B" is a spacer having from about 5 atoms to about 100 atoms each selected from the group consisting of C, H, O, N, S, and P atoms. Cyclosporin A and acridinium ester (A) linked via a spacer (B) having the structure A composition comprising:

14. "A" in Formula II is a group represented by Formula III: 【Chemistry 13】 [In the formula: "R1" is: alkyl, alkenyl, alkynyl, or aralkyl groups of 1 to 35 carbon atoms and 0 to 20 heteroatoms; sulfopropyl or sulfobutyl groups; and Base-R ａ -Z is selected from the group consisting of Here, R ａ is a divalent radical selected from an alkyl, alkenyl, alkynyl, aryl, or aralkyl group of 1 to 35 carbon atoms and 0 to 20 heteroatoms; "R2" is located at one or more of positions C1 to C4, and each "R2" is hydrogen, alkyl, OR, OH, SR, SH, NH ２ 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 of positions C5 to C8, and each "R3" is selected from the group consisting of hydrogen, alkyl, OR, OH, SR, SH, NH ２ 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 straight chain alkyl groups; substituted or unsubstituted aryl groups; and heterocyclic ring groups; wherein the "X" group contains 0 to 20 heteroatoms, and wherein the "X" group further contains a functional group that links to spacer "B" of formula I; and "A － " is CH ３ SO ３ － , FSO ３ － , C.F. ３ SO ３ － , C ４ F ９ SO ３ － , C.H. ３ C ６ H ４ SO ３ － , halide, CF ３ COO － , C.H. ３ COO － , and NO ３ － and the counter ion is selected from the group consisting of 14. The composition of claim 13, wherein the acridinium ester has the structure:

15. "A" in Formula II is a group represented by Formula IV: 【Chemistry 14】 [In the formula: "R ４ " and "R ８ " are each independently selected from hydrogen or an alkyl, alkenyl, alkynyl, alkoxyl (-OR), alkylthiol (-SR), or substituted amino group; "R ５ "," "R ６ " and "R ７ are each independently selected from hydrogen or an alkyl, alkenyl, alkynyl, aryl, or aralkyl group, where each group contains 0 to 20 heteroatoms; and "R ５ "," "R ６ " and "R ７ " further comprises a functional group linking to spacer "B" of Formula II.

15. The composition of claim 14, wherein the acridinium ester has the structure:

16. "A" in Formula II is a group represented by Formula V: 【Chemistry 15】 [In the formula: "R １ " is a methyl or sulfopropyl group; "R ２ " and "R ３ are each independently selected from hydrogen or a methoxy, sulfopropyloxyl, or poly(ethylene)glycoloxy group; and "R ６ " is the amide group (CONH-) connected to the spacer "B" of Formula II.

16. The composition of claim 15, wherein the acridinium ester is a dimethylphenyl acridinium ester having the structure:

17. R １ is a sulfopropyl group, and where R ２ and R ３ The composition of claim 16, wherein each is hydrogen or a sulfopropyloxyl group.

18. Formula XIII: 【Chemistry 16】 14. The composition of claim 13, further defined as a cyclosporin A-acridinium ester having the structure:

19. Formula XIV: 【Chemistry 17】 14. The composition of claim 13, further defined as a cyclosporin A-acridinium ester having the structure:

20. Formula XV: [Chemistry 18] 14. The composition of claim 13, further defined as a cyclosporin A-acridinium ester having the structure:

21. Formula XVI: 【Chemistry 19】 14. The composition of claim 13, further defined as a cyclosporin A-acridinium ester having the structure:

22. Formula XVII: 【Chemistry 20】 14. The composition of claim 13, further defined as a cyclosporin A-acridinium ester having the structure:

23. A first reagent comprising the composition of any one of claims 1 to 22; and a second reagent comprising a solid phase to which is attached, directly or indirectly, an antibody that specifically binds to cyclosporin A; An immunoassay kit comprising:

24. 24. The immunoassay kit of claim 23, further comprising at least one pretreatment agent.

25. 1. A method for detecting cyclosporin A in a sample, comprising: (a): (i) a sample suspected of containing cyclosporine A; (ii) a first reagent comprising at least one composition according to any one of claims 1 to 22; and (iii) a second reagent comprising a solid phase to which an antibody that specifically binds to cyclosporin A is directly or indirectly bound; simultaneously or wholly or partially consecutively to form a mixture; (b) incubating the mixture under conditions that allow 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) detecting a complex formed from the first and second reagents; and (d) determining the amount of cyclosporine A present in the sample based on the decrease in the amount of first reagent / second reagent complex formed compared to an assay performed in the absence of sample. The above method, comprising:

26. 26. The method of claim 25, further comprising the step of treating the sample with a pretreatment agent prior to step (a).

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