Cyclosporine-aziridines and methods of making and using the same

Abstract

Compositions comprising acridinium esters of cyclosporine A or C are disclosed. Kits containing the same, as well as methods of making and using the same, are also disclosed.

Description

[0001] Cross-references to related applications / statements incorporated herein by reference

[0002] This application claims the benefit of U.S. Provisional Application No. 63 / 476,552, filed December 21, 2022, pursuant to 35 USC §119(e). The entire contents of the patent application cited above are hereby expressly incorporated herein by reference.

[0003] Statement regarding federally sponsored research or development

[0004] not applicable.

[0005] background

[0006] The body relies on a complex immune response system to distinguish itself from non-self. Sometimes, it is necessary to control the body's immune system to enhance deficient responses or suppress excessive responses. For example, when organs such as (but not limited to) kidneys, hearts, lungs, bone marrow, and livers are transplanted into the human body, the body will typically reject the transplanted tissue through a process known as allogeneic transplant rejection.

[0007] In the treatment of allogeneic transplant rejection, medication is often used to suppress the immune system in a controlled manner. Immunosuppressive drugs are carefully administered to transplant recipients to help prevent rejection of allogeneic transplants containing non-self-tissue tissue. Some of the most commonly used immunosuppressive drugs for preventing organ rejection in transplant patients are cyclosporine A (CsA), mycophenolate mofetil, FK-506 (also known as tacrolimus), sirolimus (also known as rapamycin), and everolimus.

[0008] Side effects associated with immunosuppressive drugs can be partially managed by carefully controlling the levels of the drug in the patient's body. Treatment monitoring of blood concentrations of immunosuppressive drugs and related medications is necessary to optimize dosing regimens, ensuring maximum immunosuppression with minimal toxicity. Although immunosuppressive drugs are highly effective immunosuppressants, their use must be carefully managed because the effective dose range is often narrow, and excessive doses can lead to serious side effects. On the other hand, excessively low doses of immunosuppressants can cause tissue rejection. Because the distribution and metabolism of immunosuppressive drugs can vary greatly among patients, and due to the wide range and severity of adverse reactions, accurate monitoring of drug levels is crucial.

[0009] Therefore, there is a persistent need to develop rapid and accurate diagnostic methods to measure the levels of analytes (such as immunosuppressive drugs like CsA) in patient samples. This method should be fully automated and accurate even in samples containing various interfering substances. The assay should provide an accurate measurement of the amount of analyte in the sample while minimizing inaccuracies caused by interfering substances present in the sample. Brief description of the attached diagram

[0011] Figure 1 depicts the chemical structures of cyclosporine A (CsA) and cyclosporine C (CsC).

[0012] Figure 2 depicts the prior art cyclosporine C-acridone ester (CsCAE) tracer CsC-NSP-DMAE-HEG3-CsA (C 138 H 226 N 18 O 40 The chemical structure of S; Mol.Wt 2809.43.

[0013] Figure 3 A non-limiting embodiment of the cyclosporine A assay format constructed according to this disclosure is illustrated graphically.

[0014] Figure 4 The chemical structure of a non-limiting embodiment of a composition constructed according to this disclosure and labeled CsC-DA-10-NSP-DMAE (Formula VI) is described.

[0015] Figure 5 The chemical structure of a non-limiting embodiment of a composition constructed according to this disclosure and labeled CsC-DA-10-TSPAE (Formula VII) is described.

[0016] Figure 6 The chemical structure of a non-limiting embodiment of a composition constructed according to this disclosure and labeled CsC-DA-10-HEGAE (Formula VIII) is described.

[0017] Figure 7 The chemical structure of a non-limiting embodiment of a composition constructed according to this disclosure and labeled CsC-DA-10-NSP-ZAE (Formula IX) is described.

[0018] Figure 8 The chemical structure of a non-limiting embodiment of a composition constructed according to this disclosure and labeled CsC-PEG15-ZAE (Formula X) is described.

[0019] Figure 9 The chemical structure of a non-limiting embodiment of a composition constructed according to this disclosure and labeled CsC-PEG23-ZAE (Formula XI) is described.

[0020] Figure 10 The chemical structure of a non-limiting embodiment of a composition constructed according to this disclosure and labeled CsC-PEG23-NSP-DMAE (Formula XII) is described.

[0021] Figure 11 The chemical structure of a non-limiting embodiment of a composition constructed according to this disclosure and labeled CsA-oxime-PEG3-HEGAE (Formula XIII) is described.

[0022] Figure 12 The chemical structure of a non-limiting embodiment of a composition constructed according to this disclosure and labeled CsA-oxime-PEG3-ZAE (Formula XIV) is described.

[0023] Figure 13 The chemical structure of a non-limiting embodiment of a composition constructed according to this disclosure and labeled CsA-Pent-DA-10-NSP-DMAE (Formula XV) is described.

[0024] Figure 14 The chemical structure of a non-limiting embodiment of a composition constructed according to this disclosure and labeled CsA-Pent-DA-10-ZAE (Formula XVI) is described.

[0025] Figure 15 The chemical structure of a non-limiting embodiment of a composition constructed according to this disclosure and labeled CsA-Pent-DA-10-FG-DA13-ZAE (Formula XVII) is described.

[0026] Figure 16 It is depicted in graphics Figure 4 Synthetic pattern of CsC-DA-10-NSP-DMAE.

[0027] Figure 17 It is depicted in graphics Figure 5 The synthetic diagram of CsC-DA-10-TSPAE.

[0028] Figure 18 It is depicted in graphics Figure 6 The synthetic diagram of CsC-DA-10-HEGAE.

[0029] Figure 19 It is depicted in graphics Figure 7 The synthetic pattern of CsC-DA-10-NSP-ZAE.

[0030] Figure 20 It is depicted in graphics Figure 8 Synthesis pattern of CsC-PEG15-ZAE.

[0031] Figure 21 It is depicted in graphics Figure 9 Synthesis pattern of CsC-PEG23-ZAE.

[0032] Figure 22 It is depicted in graphics Figure 10 Synthesis pattern of CsC-PEG23-NSP-DMAE.

[0033] Figure 23 It is depicted in graphics Figure 11 Synthetic pattern of CsA-oxime-PEG3-HEGAE.

[0034] Figure 24 It is depicted in graphics Figure 12 Synthesis pattern of CsA-oxime-PEG3-ZAP.

[0035] Figure 25 It is depicted in graphics Figure 13 Synthetic pattern of CsA-Pent-DA-10-NSP-DMAE.

[0036] Figure 26 It is depicted in graphics Figure 14 The synthetic pattern of CsA-Pent-DA-10-ZAE.

[0037] Figure 27 It is depicted in graphics Figure 15 The synthetic pattern of CsA-Pent-DA-10-HG-DA13-ZAE.

[0038] Figure 28 The diagram illustrates the screening of acridine esters (AEs) 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 non-preformed CsA assay formats.

[0039] Figure 29 The binding curves of HEG3, NSP-DMAE, NSP-ZAE, TSP-AE and HEGAE to cyclosporine C-acridone ester (CsC-AE) in the non-preformed CsA assay format were plotted graphically.

[0040] Figure 30 The diagram illustrates the AE screening using CsC-DA-10-NSP-ZAE, CsC-O-PEG15-ZAE, CsC-PEG23, ZAE, and CsC-PEG23-NSP-DMAE in non-preformed CsA assay formats.

[0041] Figure 31The binding curves of -DA-10-NSP-ZAE, -PEG15-ZAE, -PEG23-ZAE and -PEG23-NSP-DMAE to CsC-AE in the non-preformed CsA assay format were plotted graphically.

[0042] Figure 32 The diagram illustrates the 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 non-preformed CsA assay formats.

[0043] Figure 33 The binding curves of cyclosporine A-acridone ester (CsA-AE) to -oxime-PEG3-ZAE, -NSP-DMAE, -NSP-ZAE and -HG-DA-13-ZAE in the non-preformed CsA assay format were plotted graphically.

[0044] Figure 34 The diagram illustrates the use of CsC AE (HEG3, DMAE, NSP-ZAE, TSPAE, and HEGAE) for ambient temperature effort (ATE) screening studies.

[0045] Figure 35 The diagram illustrates ATE studies using CsC-DA-10-NSP-ZAE, CsC-PEG23-NSP-DMAE, CsC-PEG15-ZAE, CsC-PEG23-ZAE, and CsA-Pent-ZAE.

[0046] Detailed Explanation

[0047] Before explaining at least one embodiment of this disclosure in detail with the aid of exemplary wording and results, it should be understood that this disclosure, in its application, is not limited to the details of the construction and component arrangement set forth in the following specification. This disclosure can be implemented or carried out in other embodiments or in various ways. Therefore, the wording used herein is intended to be given the broadest possible scope and meaning; and the embodiments are intended to be exemplary and not exhaustive. Furthermore, it should be understood that the terms and terminology used herein are for descriptive purposes and should not be considered limiting.

[0048] Unless otherwise defined herein, the scientific and technical terms used in connection with this disclosure should have the meanings commonly understood by one of ordinary skill in the art. Furthermore, unless the context requires otherwise, singular terms should include plural and plural terms should include singular. The foregoing techniques and procedures are generally performed according to conventional methods well known in the art and as described in the various general and more specific references cited and discussed throughout this specification. The nomenclature and laboratory procedures and techniques used in relation to analytical chemistry, synthetic organic chemistry, and medical and medicinal chemistry described herein are those well known and commonly used in the art. Standard techniques are used for chemical synthesis and chemical analysis.

[0049] All patents, published patent applications, and non-patent publications mentioned in this specification represent the technical level of a person skilled in the art to which this disclosure pertains. All patents, published patent applications, and non-patent publications cited in any part of this application are expressly incorporated herein by reference in their entirety, to the extent that each individual patent or publication is specifically and individually indicated to be incorporated herein by reference.

[0050] Given that all articles, compositions, kits, and / or methods disclosed herein can be manufactured and implemented without excessive experimentation, it will be apparent to those skilled in the art that changes can be made to these articles, compositions, kits, and / or methods and to the steps or order of steps of the methods described herein without departing from the concept, spirit, and scope of this disclosure. All such similar substitutions and modifications that are apparent to those skilled in the art are considered to be within the spirit, scope, and concept of this disclosure as defined in the appended claims.

[0051] As used in accordance with this disclosure, unless otherwise specified, the following terms shall be understood to have the following meanings:

[0052] When used in conjunction with the term "comprising" in the claims and / or description, the use of the terms "a" or "an" can refer to "one," but also is consistent with the meaning of "one or more," "at least one," and "one or more." Therefore, unless the context clearly indicates otherwise, the terms "a," "an," and "the" include plural objects. Thus, for example, reference to "a compound" may 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 "a plurality" means "two or more."

[0053] The term "at least one" will be understood to include 1 and any quantity greater than 1, including but not limited to 2, 3, 4, 5, 10, 15, 20, 30, 40, 50, 100, etc. The term "at least one" may be extended to 100 or 1000 or more, depending on the terms associated with it; furthermore, quantities of 100 / 1000 should not be considered limiting, as higher limits may also produce satisfactory results. Additionally, the term "at least one of X, Y, and Z" will be understood to include only X, only Y, and only Z, as well as any combination of X, Y, and Z. The use of ordinal terms (i.e., "first," "second," "third," "fourth," etc.) is solely for distinguishing two or more items and is not intended to imply any order or sequence, or that one item is more important than another, or any order of addition.

[0054] The term "or" used in the claims is intended to indicate an inclusive "and / or" unless explicitly stated to refer only to alternatives or unless the alternatives are mutually exclusive. For example, any of the following satisfy the condition "A or B": 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 exists), and both A and B are true (or exist).

[0055] As used herein, any reference to “an embodiment,” “an embodiment,” “some embodiments,” “an example,” “for example,” or “an instance” means that a particular element, feature, structure, or characteristic described in connection with that embodiment is included in at least one embodiment. The appearance of the phrase “some embodiments” or “an example” throughout the specification does not necessarily refer to, for example, the same embodiment. Furthermore, all references to one or more embodiments or instances should be construed as not limiting the claims.

[0056] Throughout this application, the term "about" is used to indicate that a value includes variations inherent in the composition / apparatus / equipment, the method used to determine the value, or variations present between subjects under study. For example, but not as a limitation, when using the term "about," a specified value may be varied relative to a given value by plus or minus 20%, 15%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1%, as such variations are appropriate for carrying out the disclosed method and as understood by one of ordinary skill in the art.

[0057] The terms “comprising” (and any form of “comprise” such as “comprise” and “comprises”), “having” (and any form of “have” such as “have” and “has”), “including” (and any form of “includes” and “include”), or “containing” (and any form of “contains” and “contain”) as used in this specification and claims are inclusive or open-ended and do not exclude additional, unlisted elements or method steps.

[0058] As used herein, the term "or combinations thereof" refers to all permutations and combinations of the items listed preceding the term. For example, "A, B, C, or combinations thereof" is intended to include at least one of the following: A, B, C, AB, AC, BC, or ABC, and also BA, CA, CB, CBA, BCA, ACB, BAC, or CAB if the order is significant in a particular context. Continuing from this example, it 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 otherwise apparent from the context, there is generally no limit to the number of items or terms in any combination.

[0059] As used herein, the term "basically" means that the subsequently described event or situation occurs completely, or that the subsequently described event or situation occurs to a great extent or degree. For example, when associated with a particular event or situation, the term "basically" means that the subsequently described event or situation 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 "basically adjacent" can mean that two items are 100% adjacent to each other, or that two items are closely adjacent to each other but not 100% adjacent, or that a portion of one of two items is not 100% adjacent to the other item but is closely adjacent to the other item.

[0060] As used herein, the phrases “joined with” and “coupled to” include two parts (moies) directly joined / combined with each other and two parts indirectly joined / combined with each other. Non-limiting examples of joining / coupling include one part covalently bonded to another part by a direct bond or via a spacer group; one part non-covalently bonded to another part directly or by means of specific binding pair members bound to these parts; one part incorporating into another part, such as by dissolving one part in another part or by synthesis; and one part coating another part.

[0061] The terms “analog” and “derivative” are used interchangeably herein and refer to substances that contain the same basic carbon skeleton and carbon functionality as the given compound, but may also contain one or more substitutions thereof. As used herein, the term “substitution” will be understood to mean that at least one substituent on the compound is replaced by residue R. In some non-limiting embodiments, R may include H, hydroxyl, thiol, halogen selected from fluorine, chlorine, bromine or iodine, and a C1-C4 compound selected from: optionally substituted straight-chain, branched or cyclic alkyl, and straight-chain, branched or cyclic alkenyl, wherein the optional substituent is selected from one or more alkenylalkyl, alkynylalkyl, cycloalkyl, cycloalkenylalkyl, arylalkyl, heteroarylalkyl, heterocycloalkyl, optionally substituted heterocycloalkenylalkyl, arylcycloalkyl and arylheterocycloalkyl, each of which is optionally substituted, wherein the optional substituent is 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)2, carboxyl and -C(O))-alkyl.

[0062] As used herein, the term "sample" will be understood to include any type of biological sample that may be used in accordance with this disclosure. Examples of available fluid biological 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 irrigation fluid, semen, feces, pleural fluid, nasopharyngeal fluid, combinations thereof, etc.

[0063] The terms “specific binding partner” or “analyte-specific binder” will be understood to refer to any molecule capable of specifically binding to a target analyte. For example, but not as a limitation, a binder / binding partner can be an antibody, receptor, ligand, aptamer, 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.

[0064] The term "antibody" is used in the broadest sense and specifically (but not limited to) encompasses 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, provided they exhibit the desired analyte binding biological activity. 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 an intact antibody for binding to a specific antigen. Antigen-binding fragments can be generated 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, biantibodies, single-chain antibodies, and single-domain antibodies (such as, but not limited to, those mentioned above). Antibodies can be any type or class (e.g., IgG, IgE, IgM, IgD, and IgA) or subclass (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2). See, for example, Hudson et al. (Nature Med. (2003) 9:129-134).

[0065] 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 antigens. The antigen-binding function of an antibody can be performed by fragments of the complete 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')2, Fv, scFv, disulfide-linked Fv, Fd, biantibodies, single-chain antibodies, and single-domain antibodies (such as, but not limited to, those mentioned above). (1) Separated CDRH3 and other antibody fragments retaining 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.

[0066] As used in this article, “antibody heavy chain” refers to the larger of the two types of polypeptide chains that exist in all antibody molecules in their naturally occurring conformation.

[0067] As used in this article, "antibody light chain" refers to the smaller of the two types of polypeptide chains that exist in all antibody molecules in their naturally occurring conformation. κ and λ light chains refer to the two main isotypes of antibody light chains.

[0068] The term "CDR" and its plural "CDRs" refer 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 (CDRL1, CDRL2, and CDRL3) are present in the light chain variable region, and three CDRs (CDRH1, CDRH2, and CDRH3) are present in the heavy chain variable region. CDRs contribute to the functional activity of the antibody molecule and are separated by amino acid sequences containing scaffold or framework regions. Among the various CDRs, the CDR3 sequence, and especially CDRH3, is the most diverse and therefore contributes the strongest to antibody specificity. There are at least two techniques for determining CDR: (1) a method based on cross-species sequence variability (i.e., Kabat et al., Sequences of Proteins of Immunological Interest (National Institute of Health, Bethesda, Md. (1987)), which is incorporated herein by reference in its entirety); and (2) a method based on crystallographic studies of antigen-antibody complexes (Chothia et al., Nature, 342:877 (1989), which is incorporated herein by reference in its entirety).

[0069] The term "epitope" includes any protein determinant capable of specifically binding to immunoglobulins or T-cell receptors. In some embodiments, an epitope is an antigenic region that is specifically bound by an antibody. Epitope determinants typically include chemically active surface groups of a molecule, such as amino acids, sugar side chains, phosphoryl groups, or sulfonyl groups. In some embodiments, an epitope may have specific three-dimensional structural features (e.g., "conformational epitopes") and specific charge features.

[0070] If a particular antibody specifically binds to two epitopes, one epitope is defined as "identical" to the other. In some embodiments, polypeptides with different primary amino acid sequences may contain the same epitope. In some embodiments, the same epitope may have a different primary amino acid sequence. If different antibodies compete for specific binding to that epitope, they are said to bind to that same epitope.

[0071] An antibody "specifically binds" to an antigen when it preferentially recognizes an antigen in a complex mixture of proteins and / or macromolecules. In some embodiments, the antibody contains an antigen-binding site that specifically binds to a particular epitope. In some such embodiments, the antibody is capable of binding to different antigens, provided that the different antigens contain that particular epitope or a closely related epitope. In some cases, for example, homologous proteins from different species may contain the same epitope. In some embodiments, the antibody binds at a concentration of no more than 10... -6 M, 10 -7 M, 10 -8 M or 10 -9 The dissociation constant of M specifically binds to the antigen. When an antibody specifically binds to a receptor or ligand (i.e., the antireceptor), it can substantially inhibit receptor-ligand adhesion. As used herein, when an excess of antibody reduces the amount of receptor bound to the ligand by at least approximately 20%, 40%, 60%, or 80%, 85%, or 90% (as measured in an in vitro competitive binding assay), the antibody substantially inhibits receptor-ligand adhesion.

[0072] "Isolated" antibodies are antibodies that have been isolated and / or recovered from the components of their preparation environment. The contaminating components of their preparation environment are materials that could interfere with the diagnostic or therapeutic use of the antibody and may include enzymes, hormones, and other protein or non-protein solutes. In some embodiments, the antibody will be purified, as measured by at least three different methods: 1) purified to greater than 50% by weight, such as greater than 75%, 85%, 95%, or 99% by weight, as determined by the Lowry method; 2) purified to a degree sufficient to obtain at least 10 residues of the N-terminal or internal amino acid sequence using a turn-cup sequencer, such as at least 15 residues of the sequence; or 3) purified to a degree of homogeneity as determined by SDS-PAGE using Coomassie blue or alternative silver staining under reducing or non-reducing conditions. Isolated antibodies include in situ antibodies within recombinant cells, as at least one component of the antibody preparation environment will be absent. However, isolated antibodies will typically be prepared via at least one purification step. Furthermore, "isolated antibodies" are substantially free of other antibodies with different antigen specificities. However, isolated antibodies may exhibit some cross-reactivity with other related antigens.

[0073] The term "antibody mutant" refers to an amino acid sequence variant of an antibody in which one or more amino acid residues have been modified. Such a mutant must have less than 100% sequence identity or similarity with an amino acid sequence that has at least 75%, such as at least 80%, or at least 85%, or at least 90%, or at least 95% sequence identity or similarity with the amino acid sequence of the variable domain of the heavy or light chain of the antibody.

[0074] As used herein, the term "monoclonal antibody" refers to an antibody derived from a substantially homogeneous group of antibodies that specifically bind to the same epitope; that is, the individual antibodies constituting the group are identical except for a possible small number of naturally occurring mutations. In contrast to conventional (polyclonal) antibody formulations, which typically comprise different antibodies targeting different determinants (epitaxes), each monoclonal antibody targets a single determinant on the antigen. Besides their specificity, monoclonal antibodies are advantageous because, in one preparation method, they can be synthesized via hybridoma culture and are therefore not contaminated by other immunoglobulins. The modifier "monoclonal" indicates that the antibody is derived from a substantially homogeneous group of antibodies and should not be construed as requiring preparation by any particular method. For example, in one embodiment, the monoclonal antibody prepared according to this disclosure can be manufactured using a hybridoma method first described by Kohler and Milstein (Nature, 256:495 (1975)).

[0075] The monoclonal antibodies used according to this disclosure can be prepared by any method known in the art, including but not limited to, the result of an intentional immunization protocol; the result of an immune response that leads to the natural production of antibodies during a disease or cancer process; phage-derived antibodies; and so on. In addition to the hybridoma preparation methods listed above, the monoclonal antibodies of this disclosure can also be prepared by a variety of other methods, such as, but not limited to, recombinant DNA methods (see, for example, U.S. Patent No. 4,816,567); isolating antibody fragments from phage display libraries (see, for example, Clackson et al., Nature (1991) 352:624-628; and Marks et al., J. Mol. Biol. (1991) 222:581-597); and various other monoclonal antibody preparation techniques (see, for example, Harlow and Lane (1988) Antibodies: A Laboratory Manual (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY)). Furthermore, many of the monoclonal antibodies that can be used in the conjugates and methods disclosed herein or otherwise considered are widely commercially available, and therefore it is deemed unnecessary to describe them further.

[0076] As used herein, "substantially pure" means that the target class is the dominant class present (i.e., more abundant, on a molar basis, than any other single class in the composition). Typically, a substantially pure composition will contain greater than about 50% of all macromolecular classes present in the composition, such as greater than about 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, and 99%. In one embodiment, the target class is purified to substantially homogeneity (contaminant classes are undetectable in the composition by conventional detection methods), wherein the composition consists essentially of a single macromolecular class.

[0077] An analyte is a molecule that can be 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 the analyte region that binds to an analyte-specific binding partner (i.e., an antibody).

[0078] Some non-limiting embodiments of this disclosure relate to a composition comprising cyclosporine C and acridine ester having a structure of Formula I, linked via a spacer group:

[0079]

[0080] And / or a composition comprising cyclosporine A and acridine ester (A) linked via a spacer group (B) and having a structure of formula II:

[0081]

[0082] In each of Formulas I and II, “A” contains an acridinium ester, and “B” is a spacer group having approximately 5 to approximately 100 atoms, each atom selected from C, H, O, N, S, and P atoms.

[0083] In a particular non-limiting embodiment, the acridine ester used according to this disclosure has the structure of Formula III:

[0084]

[0085] Wherein “R1” is selected from alkyl, alkenyl, ynyl or aralkyl groups having 1 to 35 carbon atoms and 0 to 20 heteroatoms; sulfopropyl or sulfobutyl; and the group –R a –Z, where R aIt is a divalent group selected from alkyl, alkenyl, ynyl, aryl, or aralkyl groups having 1 to 35 carbon atoms and 0 to 20 heteroatoms. "R2" is located at one or more positions from C1 to C4, and "R3" is located at one or more positions from C5 to C8. Each "R2" and "R3" is independently selected from hydrogen, alkyl, OR, OH, SR, SH, NH2, and NR'R", wherein R, R', and R" are each independently selected from alkyl, alkenyl, ynyl, aryl, and aralkyl groups, and each group contains 0 to 20 heteroatoms. Furthermore, "X" is a group selected from halogenated or unhalogenated branched or straight-chain alkyl; substituted or unsubstituted aryl; and heterocyclic groups. The "X" group also contains 0 to 20 heteroatoms and further includes a functional group attached to the spacer group "B" of Formula I. Additionally, "A" - "A" is an introduced counterion, for example (but not as a limitation) to pair with the quaternary nitrogen of the acridineonium nucleus, and "A" - Selected from CH3SO3 - FSO3 - CF3SO3 - C4F9SO3 - CH3C6H4SO3 - Halide ions, CF3COO - CH3COO - and NO3 - .

[0086] In a particular non-limiting embodiment, the acridine ester used according to this disclosure is a chemiluminescent acridine ester having the structure of Formula IV:

[0087]

[0088] Among them: “R1”, “R2”, “R3” and “A” - "As defined above with reference to Formula III; "R4" and "R8" are each independently selected from hydrogen or alkyl, alkenyl, alkynyl, alkoxy (-OR), alkylthiol (-SR), or substituted amino groups, used for example (but not as a limitation) to stabilize the -COX- bond between the acridine ontium core and the "Y" moiety by steric and / or electronic effects. Furthermore, "R5", "R6", and "R7" are each independently selected from hydrogen or alkyl, alkenyl, alkynyl, aryl, or aralkyl groups, wherein each group contains 0 to 20 heteroatoms. Furthermore, "R5", "R6", and "R7" further comprise a functional group attached to the spacer group "B" of Formula I. Functional groups used according to this disclosure include, but are not limited to, the following groups:

[0089]

[0090] In a particular non-limiting embodiment, the acridine ester used according to this disclosure is a dimethylphenyl acridine ester having the structure of formula V:

[0091]

[0092] Wherein “R1” is methyl or sulfopropyl; “R2” and “R3” are each independently selected from hydrogen or methoxy, sulfopropyloxy, or poly(ethylene)glycoloxy; and “R6” is an amide group (CONH-) attached to the spacer group “B” of Formula I. “A - As defined in Equation III.

[0093] In certain specific (but not limiting) embodiments, R1 is sulfopropyl, and R2 and R3 are each hydrogen or sulfopropyloxy.

[0094] In one particular (but not limiting) embodiment, the composition is a cyclosporine C-acridone ester having the structure of formula VI:

[0095]

[0096] In one particular (but not limiting) embodiment, the composition is cyclosporine C-acridone ester having the structure of formula VII:

[0097]

[0098] In one particular (but not limiting) embodiment, the composition is cyclosporine C-acridone ester having the structure of formula VIII:

[0099]

[0100] In one particular (but not limiting) embodiment, the composition is a cyclosporine C-acridone ester having the structure of formula IX:

[0101]

[0102] In one particular (but not limiting) embodiment, the composition is cyclosporine C-acridone ester having the structure of formula X:

[0103]

[0104] In one particular (but not limiting) embodiment, the composition is a cyclosporine C-acridone ester having the structure of formula XI:

[0105]

[0106] In one particular (but not limiting) embodiment, the composition is cyclosporine C-acridone ester having the structure of formula XII:

[0107]

[0108] In one particular (but not limiting) embodiment, the composition is cyclosporine A-acridone ester having the structure of formula XIII:

[0109]

[0110] In one particular (but not limiting) embodiment, the composition is cyclosporine A-acridone ester having the structure of formula XIV:

[0111]

[0112] In one particular (but not limiting) embodiment, the composition is cyclosporine A-acridone ester having the structure of formula XV:

[0113]

[0114] In one particular (but not limiting) embodiment, the composition is cyclosporine A-acridone ester having the structure of formula XVI:

[0115]

[0116] In one particular (but not limiting) embodiment, the composition is cyclosporine A-acridone ester having the structure of formula XVII:

[0117]

[0118]

[0119] Some non-limiting embodiments of this disclosure relate to immunoassay kits containing one or more CsC-AE / CsA-AE compositions disclosed herein or otherwise considered. The choice of acridine ester present will depend on the specific assay format to be used, and such choice is entirely within the skill of one of ordinary skill in the art.

[0120] In certain specific (but not limiting) embodiments, the immunoassay kit includes a first reagent and a second reagent, the first reagent comprising any CsC-AE / CsA-AE composition described herein or otherwise considered, and the second reagent comprising a solid phase having an antibody directly or indirectly attached thereto; the antibody present in the second reagent specifically binds to CsC or CsA present in the first reagent.

[0121] Any antibody or fragment thereof known in the art or otherwise considered herein may be used in accordance with this disclosure, provided that the antibody or fragment thereof can bind to CsC or CsA at an epitope separate from the site to which the acridinium ester is linked. Antibodies that bind to CsC / CsA are well known in the art and are commercially available. For example, but not as a limitation, CsC / CsA antibodies are commercially available from Thermo Fisher 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); etc. Therefore, it is deemed unnecessary to provide further description of the antibodies used according to this disclosure.

[0122] The assay reagents present in the kit may be provided in any form that allows them to function according to this disclosure. For example, but not as a limitation, the reagents may be provided in liquid form and presented in the kit in bulk and / or as individual aliquots. Alternatively, in a particular (but not limiting) embodiment, one or more reagents may be presented in the kit as individual aliquots of lyophilized reagents. A detailed description of the use of dried reagents in microfluidic devices is in U.S. Patent No. 9,244,085 (Samproni), the entire contents of which are hereby expressly incorporated herein by reference.

[0123] In addition to the assay reagents described in detail above, the kit may further contain other reagents for performing any particular assay described herein or otherwise considered. For example (but not as a limitation), the kit may further include at least one pretreatment / release agent for releasing cyclosporine from any endogenous binding protein present in a biological sample. The properties of these additional reagents will depend on the specific assay format, and their verification is entirely within the skill of a person skilled in the art; therefore, further description of them is not considered necessary. Furthermore, the components / reagents present in the kit may each be located in a separate container / compartment, or various components / reagents may be combined in one or more containers / compartments, depending on the cross-reactivity and stability of the components / reagents. Additionally, the kit may include a microfluidic device in which the components / reagents are disposed.

[0124] The relative amounts of various components / reagents in the kit can vary considerably to provide component / reagent concentrations that are substantially optimized for the reactions required during the assay and further substantially optimized for the sensitivity of the assay. Where appropriate, one or more components / reagents in the kit may be provided as dry powders, such as lyophilized powders, and the kit may further include excipients for dissolving the dried reagents; in this way, a reagent solution with an appropriate concentration 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, diluent solutions, excipients, interfering solutions, positive controls, negative controls, calibrating reagents, quality control reagents, etc. Furthermore, the kit may further include a set of written instructions explaining how to use the kit. Kits of this nature can be used for any method described herein or otherwise considered.

[0125] Certain non-limiting embodiments of this disclosure relate to a method for preparing any CsC-AE / CsA-AE composition disclosed herein or otherwise contemplated. The method includes attaching a connector to a CsC / CsA and then attaching an acridine ester to the connector.

[0126] Certain non-limiting embodiments of this disclosure relate to a method for detecting CsC / CsA in a sample using any CsC-AE / CsA-AE composition disclosed herein or otherwise considered. In this method, a sample suspected of containing CsC / CsA is simultaneously, wholly, or partially sequentially combined with one or more of any Cs-AE compositions disclosed herein or otherwise considered, and one or more CsC / CsA antibodies as disclosed herein or otherwise considered, bound to a solid, to form a mixture. This mixture is then incubated under conditions allowing the antibodies to bind to CsC / CsA present in the sample or to CsC-AE / CsA-AE, thereby forming Cs / antibody complexes and / or Cs-AE / antibody complexes. The Cs-AE / antibody complexes are then detected, and the amount of CsC / CsA present in the sample is determined based on the reduction in the amount of Cs-AE / antibody complexes formed compared to a negative control (i.e., the amount of Cs-AE / antibody complexes formed in the absence of the sample). The concentration of CsC / CsA present in the sample can then be determined based on the reduction.

[0127] In a specific (but not limiting) embodiment of this method, the sample is simultaneously or wholly or partially sequentially combined with the first and second reagents of the immunoassay kit described in detail above (i.e., the first reagent comprises a CsC / CsA-AE composition, and the second reagent comprises a solid phase having an antibody or fragment thereof specifically binding to CsC / CsA directly or indirectly attached thereto) to form a mixture. This mixture is then incubated under conditions allowing the second reagent to bind to any target analyte (CsC or CsA) present in the sample or 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 the target analyte (CsC or CsA) present in the sample is determined based on the reduction in the amount of the first reagent-second reagent complex formed compared to a negative control (i.e., the amount of the first reagent-second reagent complex formed in the absence of the sample). The concentration of the target analyte (CsC or CsA) present in the sample is then determined based on the reduction.

[0128] According to the method of this disclosure, any sample for which the presence of a cyclosporine target analyte (i.e., CsC or CsA) needs to be determined can be used as a sample. Non-limiting examples of samples include biological samples, such as, but 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 irrigation fluid, semen, feces, pleural fluid, nasopharyngeal fluid, and combinations thereof. Specific non-limiting examples include lysed whole blood cells and lysed red blood cells.

[0129] As mentioned above, the various components of the method are provided in combination (simultaneously or sequentially). The order in which the components are added can be varied when they are added sequentially; those skilled in the art can determine the specific desired order in which the different components are added to the assay. Of course, the simplest order of addition is to add all materials simultaneously and measure the resulting signal. Alternatively, the individual components or groups can be combined sequentially. In some embodiments, an incubation step may be involved after one or more additions. For example (but not as a limitation), it may be necessary to combine the sample with the antibody-solid phase and incubate it before adding the Cs-AE composition.

[0130] The incubation conditions for this mixture can vary considerably, as long as the antibody binds to Cs or Cs-AE to form a complex under such conditions. Sandwich-based immunoassays are widely performed, and the immunoassay conditions are well known in the art; therefore, the selection of appropriate assay conditions is entirely within the skill of a person skilled in the art, and thus it is deemed unnecessary to describe them further.

[0131] The specific detection methods used can vary considerably, as long as the complex can be detected under such methods. Sandwich assays are widely used to detect the formed complexes, and the detection procedures are well known in the art; therefore, the selection of an appropriate detection method is entirely within the skill of a person skilled in the art, and thus it is deemed unnecessary to describe it further.

[0132] The method may further include one or more additional steps to improve the accuracy and / or precision of the assay. For example (but not as a limitation), the method may further include one or more pretreatment / release steps to release cyclosporine from any endogenous binding proteins present in the biological sample prior to conjugation with the immobilized antibody. Another non-limiting example of the additional steps used according to this disclosure may include one or more washing steps to remove unbound (or non-specifically bound) reagents from the reaction prior to detection of complex formation.

[0133] Certain other non-limiting embodiments of this disclosure relate to a microfluidic device comprising components of any of the immunoassay kits described above. In particular, certain non-limiting embodiments include a microfluidic device 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 fluidly communicated with said inlet channel. The compartment of (ii) contains first and second reagents of the immunoassay kit described in detail above.

[0134] In some non-limiting embodiments, the first and second reagents (and any additional ingredients as described above) of (ii) are present in the same compartment. In alternative non-limiting embodiments, the first and second reagents (and any additional ingredients as described above) are separated into two or more compartments.

[0135] The device can be provided with compartments and any arrangement of various components distributed among them to allow the device to operate in accordance with this disclosure.

[0136] Any compartment of a microfluidic device can be sealed to maintain the reagent contained therein in a substantially hermetically sealed environment until its use; for example, a compartment containing a lyophilized reagent can be sealed to prevent any accidental remodeling of the reagent. Inlet channels and compartments, as well as two compartments, can be described as “fluidly connected” to each other; this phrase implies that each compartment can still be sealed, but that the two compartments are capable of fluid flow between them after the seal formed therein or between them is punctured.

[0137] The microfluidic device of this disclosure may have any other desired features known in the art or otherwise considered herein. For example, but not as a limitation, the microfluidic device of this disclosure may further include a reading chamber; the reading chamber may be any compartment containing one or more of the reagents described above, or the reading chamber may be in fluid communication with said compartment containing one or more reagents. The microfluidic device may further include one or more additional compartments containing other solutions, such as (but not limited to) washing solutions, diluents, excipients, interfering solutions, positive controls, negative controls, quality control, etc. These additional compartments may be in fluid communication with one or more other compartments. For example, the microfluidic device may further include one or more compartments containing washing solutions, and these compartments may be in fluid communication with any other compartment of the device. In another example, the microfluidic device may further include one or more compartments containing excipients for dissolving one or more drying reagents, and these compartments may be in fluid communication with any other compartment of the device. In yet another example, the microfluidic device may include one or more compartments containing diluents, and these compartments may be in fluid communication with any other compartment of the device. Example

[0138] Examples are provided below. However, this disclosure should be understood as not being limited to the specific experimental methods, results, and laboratory procedures disclosed herein in its application. Rather, the examples are provided merely as one of various implementations and are intended to be exemplary rather than exhaustive.

[0139] Cyclosporin A AIP assay is currently used for... and The IM analyzer (Siemens Healthcare Diagnostics Inc., Tarrytown, NY) quantifies cyclosporine in human whole blood (EDTA) for in vitro diagnostic applications. This assay is designed to aid 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 Cyclosporine (CsA) assays were commercialized in 2008, and this original immunoassay used a complex CsC-acridone ester (CsC-AE) conjugate (Figure 2) to detect CsA. However, the supplier discontinued the use of CsC-succinate-NHS, the raw material for preparing this conjugate. Therefore, a new AE tracer designed for CsA II assays was needed.

[0140] Furthermore, a novel Cs assay has been designed to address the EU REACH (Registration, Evaluation, Authorisation, and Restriction of Chemicals) requirements for triton reduction, as well as the US FDA's Ambient Temperature Efforts (ATE) and biotin interference mitigation requirements. This novel Cs assay employs a novel tracer, and the design process emphasizes the identification of novel tracers with simplified chemical structures that can be prepared using convenient synthetic methods.

[0141] Based on this need, a series of novel and simple cyclosporine (A and C)-acridinyl esters (CsA-AE and CsC-AE) were designed and synthesized for use in acridine ester (AE) technology (Natrajan et al., Ann Biochem (2010) 406:204; and Natrajan et al., Org Biomol Chem (2011) 9:5092). A study in which CSA II assays were performed (Siemens Healthcare Diagnostics Inc., Tarrytown, NY). These CsA-AEs and CsC-AEs were assessed using a competitive assay format on the system (Siemens Healthcare Diagnostics Inc., Tarrytown, NY). Figure 3 A key tracer, CsC-DA-10-NSP-ZAE (Formula IX), is an important tracer. Figure 7It exhibits superior performance relative to assay requirements (ATE, sensitivity, and accuracy) and has been selected as the primary candidate tracer for CsAII immunoassay.

[0142] Reagent design: The CsAII reagent consists of a solid phase and a lite reagent. The solid phase contains magnetic particles labeled with a biotinylated monoclonal anti-CsA antibody. The lite reagent contains a CsC-AE tracer. In the absence of CsA in the test sample, the lite reagent is bound to the antibody located on the surface of the solid phase. Figure 3 The bound AEs retained in the reaction vessel after magnetic separation and particle washing emit light upon activation by the addition of acid and base. Adding CsA from a patient sample to this reaction interfered with the binding of the Cs-AE conjugates to the solid phase, resulting in reduced signal generation. This signal attenuation was a direct function of the amount of CsA in the sample when measured against a calibration curve.

[0143] Considering the above assay design, a solid-phase reagent and a novel CsC-AE tracer were developed and evaluated. The preparation of biotinylated monoclonal antibodies and the pre-binding of these antibodies to the surface of commercially available streptavidin-coated particles are well-known and straightforward processes. Therefore, the remainder of this embodiment focuses on the synthesis of the novel CsC-AE and CsA-AE tracers and their performance in the prototype ADVIA Centaur CsA II assay.

[0144] In the initial assay study, a hydroxyl group from the CsC molecule (Figure 1) was selected for NSP-DMAE conjugation. Early data from this example indicated that the connection of this hydroxyl group to CsC-DA-10-NSP-DMAE produced a promising assay profile shape. Considering this initial performance, conjugates were also prepared at the same connection site using HEGAE, TSPAE, and ZAE to determine whether different AE structures would provide any additional benefit to the assay. Furthermore, the terminal bifunctionality and aldehyde functionality of CsA (Figure 1) served as the basis for introducing NSP-DMAE and ZAE. The chemical structures of the CsA-AE and CsC-AE tracers are shown in [Figure 1]. Figure 4-15 In the middle, and the synthesis of these CsA-AE and CsC-AE tracers showed that Figure 16-27 middle.

[0145] Cyclosporin Immunoassay Performance: Uses products available from Siemens Healthcare Diagnostics Inc., Tarrytown, NY. A series of immunoassay analyzers were used to evaluate CsA-AE and CsC-AE tracers. The prototype was used. Cs immunoassay ( Figure 28-33 ) Determination of CsA-AE and CsC-AE tracers ( Figure 4-15 Screening for binding to available monoclonal antibodies. Environmental temperature effort (ATE) studies using different CsC tracers also showed... Figures 34-36 middle.

[0146] Conclusion: A series of novel CsA-AE and CsC-AE tracers were successfully designed, synthesized, and evaluated in the prototype CsA immunoassay (respectively...). Figure 4-15 ; Figure 16-27 ;and Figure 28 , 30 32 and 34-36). Novel tracers were selected as excellent candidates to meet FDA ambient temperature effort requirements and overall assay performance / assay specifications. Figure 30 and 35 Compared to the currently used composite tracer CsA-HEG3-NSP-DMAE (Figure 2), this tracer CsC-DA-10-NSP-ZAE (Formula IX) is superior. Figure 7 It possesses a novel, simplified chemical structure that can be easily synthesized. Figure 19 This saves preparation time and cost, and overcomes the problem of raw material interruption in current tracers. Therefore, the novel tracer developed according to this disclosure is a key component for developing new Cs immunoassays.

[0147] Non-limiting exemplary embodiments of the inventive concept

[0148] Exemplary Embodiment 1. A composition comprising: cyclosporine C and acridine ester having a structure of Formula I, linked via a spacer group:

[0149]

[0150] Wherein: “A” contains an acridinium ester and “B” is a spacer group having approximately 5 to approximately 100 atoms, each atom selected from C, H, O, N, S and P atoms.

[0151] Exemplary Embodiment 2. The composition of Exemplary Embodiment 1, wherein "A" of Formula I is an acridine ester having the structure of Formula III:

[0152]

[0153] in:

[0154] “R1” is selected from: alkyl, alkenyl, ynyl, or aralkyl groups having 1 to 35 carbon atoms and 0 to 20 heteroatoms; sulfopropyl or sulfobutyl groups; and the group –R. a –Z, where R aIt is a divalent group selected from alkyl, alkenyl, ynyl, aryl or aralkyl groups having 1 to 35 carbon atoms and 0 to 20 heteroatoms;

[0155] "R2" is located at one or more positions from C1 to C4, and each "R2" is independently selected from hydrogen, alkyl, OR, OH, SR, SH, NH2 and NR'R", wherein R, R' and R" are each independently selected from alkyl, alkenyl, alkynyl, aryl and aralkyl, wherein each group contains 0 to 20 heteroatoms;

[0156] “R3” is located at one or more positions from C5 to C8, and each “R3” is independently selected from hydrogen, alkyl, OR, OH, SR, SH, NH2 and NR'R”, wherein R, R' and R” are each independently selected from alkyl, alkenyl, alkynyl, aryl and aralkyl, wherein each group contains 0 to 20 heteroatoms;

[0157] "X" is a group selected from halogenated or unhalogenated branched or straight-chain alkyl groups; substituted or unsubstituted aryl groups; and heterocyclic groups; wherein the "X" group comprises 0 to 20 heteroatoms, and wherein the "X" group further comprises a functional group attached to a spacer group "B" of Formula I; and

[0158] A - "It is selected from CH3SO3" - FSO3 - CF3SO3 - C4F9SO3 - CH3C6H4SO3 - Halogen ions, CF3COO - CH3COO - and NO3 - Counterions.

[0159] Exemplary Embodiment 3. The composition of Exemplary Embodiment 2, wherein “A” of Formula I is an acridine ester having the structure of Formula IV:

[0160]

[0161] Wherein: “R4” and “R8” are each independently selected from hydrogen or alkyl, alkenyl, alkynyl, alkoxy (-OR), alkyl thiol (-SR) or substituted amino; “R5”, “R6” and “R7” are each independently selected from hydrogen or alkyl, alkenyl, alkynyl, aryl or aralkyl, wherein each group contains 0 to 20 heteroatoms; and one of “R5”, “R6” and “R7” further comprises a functional group connected to the spacer group “B” of Formula I.

[0162] Exemplary Embodiment 4. The composition of Exemplary Embodiment 3, wherein "A" of Formula I is a dimethylphenyl acridine ester having the structure of Formula V:

[0163]

[0164] Wherein: "R1" is methyl or sulfopropyl; "R2" and "R3" are each independently selected from hydrogen or methoxy, sulfopropoxy, or poly(ethylene glycol)oxy; and

[0165] “R6” is an amide group (CONH-) attached to the spacer group “B” of Formula I.

[0166] Exemplary Embodiment 5. The composition of Exemplary Embodiment 4, wherein R1 is sulfopropyl, and wherein R2 and R3 are each hydrogen or sulfopropyloxy.

[0167] Exemplary Embodiment 6. A composition of any one of Exemplary Embodiments 1-5, further defined as cyclosporine C-acridone ester having the structure of Formula VI:

[0168]

[0169] Exemplary Embodiment 7. A composition of any one of Exemplary Embodiments 1-5, further defined as cyclosporine C-acridone ester having the structure of Formula VII:

[0170]

[0171] Exemplary Embodiment 8. A composition of any one of Exemplary Embodiments 1-5, further defined as cyclosporine C-acridone ester having the structure of Formula VIII:

[0172]

[0173] Exemplary Embodiment 9. A composition of any one of Exemplary Embodiments 1-5, further defined as cyclosporine C-acridone ester having the structure of Formula IX:

[0174]

[0175] Exemplary Embodiment 10. A composition of any one of Exemplary Embodiments 1-5, further defined as cyclosporine C-acridone ester having the structure of Formula X:

[0176]

[0177] Exemplary Embodiment 11. A composition of any one of Exemplary Embodiments 1-5, further defined as cyclosporine C-acridone ester having the structure of Formula XI:

[0178]

[0179] Exemplary Embodiment 12. A composition of any one of Exemplary Embodiments 1-5, further defined as cyclosporine C-acridone ester having the structure of Formula XII:

[0180]

[0181] Exemplary Embodiment 13. A composition comprising: cyclosporine A and acridine ester (A) linked via a spacer group (B) and having a structure of Formula II:

[0182]

[0183] Wherein: “A” contains acridinium ester; and “B” is a spacer group having approximately 5 to approximately 100 atoms, each atom selected from C, H, O, N, S and P atoms.

[0184] Exemplary Embodiment 14. The composition of Exemplary Embodiment 13, wherein “A” of Formula II is an acridine ester having the structure of Formula III:

[0185]

[0186] in:

[0187] “R1” is selected from: alkyl, alkenyl, ynyl, or aralkyl groups having 1 to 35 carbon atoms and 0 to 20 heteroatoms; sulfopropyl or sulfobutyl groups; and the group –R. a –Z, where R a It is a divalent group selected from alkyl, alkenyl, ynyl, aryl or aralkyl groups having 1 to 35 carbon atoms and 0 to 20 heteroatoms;

[0188] "R2" is located at one or more positions from C1 to C4, and each "R2" is independently selected from hydrogen, alkyl, OR, OH, SR, SH, NH2 and NR'R", wherein R, R' and R" are each independently selected from alkyl, alkenyl, alkynyl, aryl and aralkyl, wherein each group contains 0 to 20 heteroatoms;

[0189] “R3” is located at one or more positions from C5 to C8, and each “R3” is independently selected from hydrogen, alkyl, OR, OH, SR, SH, NH2 and NR'R”, wherein R, R' and R” are each independently selected from alkyl, alkenyl, alkynyl, aryl and aralkyl, wherein each group contains 0 to 20 heteroatoms;

[0190] "X" is a group selected from halogenated or unhalogenated branched or straight-chain alkyl groups; substituted or unsubstituted aryl groups; and heterocyclic groups; wherein the "X" group comprises 0 to 20 heteroatoms, and wherein the "X" group further comprises a functional group attached to a spacer group "B" of Formula I; and

[0191] A - "It is selected from CH3SO3" - FSO3 - CF3SO3 - C4F9SO3 - CH3C6H4SO3 - Halogen ions, CF3COO - CH3COO - and NO3 - Counterions.

[0192] Exemplary Embodiment 15. The composition of Exemplary Embodiment 14, wherein “A” of Formula II is an acridine ester having the structure of Formula IV:

[0193]

[0194] Wherein: “R4” and “R8” are each independently selected from hydrogen or alkyl, alkenyl, alkynyl, alkoxy (-OR), alkyl thiol (-SR) or substituted amino; “R5”, “R6” and “R7” are each independently selected from hydrogen or alkyl, alkenyl, alkynyl, aryl or aralkyl, wherein each group contains 0 to 20 heteroatoms; and one of “R5”, “R6” and “R7” further comprises a functional group connected to the spacer group “B” of Formula II.

[0195] Exemplary Embodiment 16. The composition of Exemplary Embodiment 15, wherein "A" of Formula II is a dimethylphenyl acridine ester having the structure of Formula V:

[0196]

[0197] Wherein: "R1" is methyl or sulfopropyl; "R2" and "R3" are each independently selected from hydrogen or methoxy, sulfopropoxy, or poly(ethylene glycol)oxy; and

[0198] “R6” is an amide group (CONH-) attached to the spacer group “B” of Formula II.

[0199] Exemplary embodiment 17. The composition of exemplary embodiment 16, wherein R1 is sulfopropyl, and wherein R2 and R3 are each hydrogen or sulfopropyloxy.

[0200] Exemplary Embodiment 18. A composition of any one of Exemplary Embodiments 13-17, further defined as cyclosporine A-acridone ester having the structure of Formula XIII:

[0201]

[0202] Exemplary Embodiment 19. The composition of any one of Exemplary Embodiments 13-17, further defined as cyclosporine A-acridone ester having the structure of Formula XIV:

[0203]

[0204] Exemplary Embodiment 20. The composition of any one of Exemplary Embodiments 13-17, further defined as cyclosporine A-acridone ester having the structure of Formula XV:

[0205]

[0206] Exemplary Embodiment 21. The composition of any one of Exemplary Embodiments 13-17, further defined as cyclosporine A-acridone ester having the structure of Formula XVI:

[0207]

[0208] Exemplary Embodiment 22. The composition of any one of Exemplary Embodiments 13-17, further defined as cyclosporine A-acridone ester having the structure of Formula XVII:

[0209]

[0210]

[0211] Exemplary Implementation 23. An immunoassay kit comprising: a first reagent comprising a composition of any one of Exemplary Implementations 1-22; and a second reagent comprising a solid phase having an antibody specifically binding to cyclosporine A directly or indirectly attached thereto.

[0212] Exemplary Implementation 24. An immunoassay kit of Exemplary Implementation 23, further comprising at least one pretreatment agent.

[0213] Exemplary Implementation 25. A method for detecting cyclosporine A in a sample, comprising the steps of: (a) simultaneously or entirely or partially sequentially combining the following to form a mixture: (i) a sample suspected of containing cyclosporine A; (ii) a first reagent comprising a composition of any one of Exemplary Implementations 1-22; and (iii) a second reagent comprising a solid phase having an antibody specifically binding to cyclosporine A directly or indirectly attached thereto; (b) incubating the mixture under conditions allowing the antibody to bind 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 by the first and second reagents; and (d) determining the amount of cyclosporine A present in the sample based on a reduction in the amount of the first reagent / second reagent complex formed compared to a determination performed in the absence of a sample.

[0214] Exemplary Implementation 30. The method of Exemplary Implementation 29 further includes a step of treating the sample with a pretreatment agent prior to step (a).

[0215] Therefore, according to this disclosure, compositions, kits, and devices that fully satisfy the purposes and advantages set forth above, as well as methods of preparation and use thereof, are provided. Although this disclosure has been described in conjunction with the specific figures, experimental methods, results, and wording set forth above, it will be apparent to those skilled in the art that many alternatives, modifications, and variations will be readily apparent. Therefore, it is intended to cover all such alternatives, modifications, and variations that fall within the spirit and broad scope of this disclosure.

Claims

1. A composition defined as cyclosporine C-acridone ester having the structure of formula IX: Formula IX.

2. An immunoassay kit, comprising: A first reagent, the first reagent comprising the composition according to claim 1; and The second reagent comprises a solid phase having antibodies specifically binding cyclosporine A and cyclosporine C directly or indirectly attached thereto.

3. The immunoassay kit according to claim 2, further comprising at least one pretreatment agent.

4. A method for detecting cyclosporine A in a sample, comprising the steps of: (a) Combining the following simultaneously or in whole or in part sequentially to form a mixture: (i) Samples suspected of containing cyclosporine A; (ii) A first reagent comprising the composition according to claim 1; and (iii) A second reagent comprising a solid phase having antibodies specifically binding cyclosporine A and cyclosporine C directly or indirectly attached thereto; (b) The mixture is incubated under conditions that allow the antibody to bind 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 by the first and second reagents; and (d) The amount of cyclosporine A present in the sample is determined based on the reduction in the amount of the first reagent / second reagent complex formed when the determination is performed in the absence of the sample.

5. The method of claim 4, further comprising the step of treating the sample with a pretreatment agent prior to step (a).

Images