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CN-122003405-A - Tolylthiazide hapten, immunogen and protein conjugate and methods of producing and using the same

CN122003405ACN 122003405 ACN122003405 ACN 122003405ACN-122003405-A

Abstract

Compositions, kits, and systems containing a benzothiazine hapten, an immunogen, and/or a protein conjugate are disclosed. Methods of producing and using the benzothiazine haptens, immunogens and conjugates are also disclosed.

Inventors

  • WILLIAM BEDZYK
  • Bogdan Draghi Xi
  • ZHENG YIFENG

Assignees

  • 美国西门子医学诊断股份有限公司

Dates

Publication Date
20260508
Application Date
20241010
Priority Date
20231012

Claims (20)

  1. 1. A composition comprising a compound having the structure of formula I: I is a kind of Provided that at least one of R 1 、R 2 and R 3 is not H.
  2. 2. The composition of claim 1, wherein at least one of R 1 、R 2 and R 3 that is not H comprises at least one of an aliphatic group, a hydrocarbyl group, a bulky organic group, a non-bulky organic group, an alkyl group, an alkenyl group, an alkynyl group, a phenyl group, an aryl group, a substituted aryl group, a carbocyclic ring, a heterocyclic ring, a hydrocarbonyloxy group, a lower alkoxy group, an arylalkyl group, a halide, an amide, an imide, a nitrate, a nitrite, a carbonyl group, a ketone, an ester, a carboxylic acid, a hydroxide, a thiol, a sulfide, a sulfate, a sulfonate, a peptide, a protein, an enzyme, a linker, an immunogenic carrier, a label, or a combination thereof.
  3. 3. The composition of claim 1, wherein the compound comprises the structure of one of formulas II, III, IV, V, XI, XII, XIII, XIV, XV, XVI, XVII, XVIII, XIX, XXII or XXIX: 。
  4. 4. The composition of claim 1, wherein the compound has the structure of one of formulas VI, VII, VIII, IX, XX, XXI, XXIII or XXX: wherein R 4 is a peptide or protein.
  5. 5. The composition of claim 4, wherein R 4 is an immunogenic carrier.
  6. 6. The composition of claim 5, wherein the immunogenic carrier is selected from the group consisting of Keyhole Limpet Hemocyanin (KLH), bovine Serum Albumin (BSA), ovalbumin (OVA), egg ovalbumin, bovine Gamma Globulin (BGG), thyroglobulin, fibrinogen, polysaccharides, glucose-6-phosphate dehydrogenase (G6 PDH), carrier proteins, marker proteins, and combinations thereof.
  7. 7. A composition comprising a compound having the structure of formula X: Wherein one of R 5 and R 6 is present and comprises at least one of an aliphatic group, a hydrocarbyl group, a bulky organic group, a non-bulky organic group, an alkyl group, an alkenyl group, an alkynyl group, a phenyl group, an aryl group, a substituted aryl group, a carbocyclic ring, a heterocyclic ring, a hydrocarbonyloxy group, a lower alkoxy group, an arylalkyl group, a halide, an amide, an imide, a nitrate, a nitrite, a carbonyl group, a ketone, an ester, a carboxylic acid, a hydroxide, a thiol, a sulfide, a sulfate, a sulfonate, a peptide, a protein, an enzyme, a linker, an immunogenic carrier, a label, or a combination thereof.
  8. 8. The composition of claim 7, wherein the compound has the structure of one of formulas XXIV, XXV, XXVI, XXVII, XXVIII, XXXI, XXXII, XXXIII, XXXIV, XXXV or XXXVI: wherein R 4 is a peptide or protein.
  9. 9. The composition of claim 7, wherein R 5 or R 6 comprises an immunogenic carrier.
  10. 10. The composition of claim 9, wherein the immunogenic carrier is selected from the group consisting of Keyhole Limpet Hemocyanin (KLH), bovine Serum Albumin (BSA), ovalbumin (OVA), egg ovalbumin, bovine Gamma Globulin (BGG), thyroglobulin, fibrinogen, polysaccharides, glucose-6-phosphate dehydrogenase (G6 PDH), carrier proteins, marker proteins, and combinations thereof.
  11. 11. The composition of any one of claims 1-10, further comprising at least one excipient.
  12. 12. A method of making an antibody comprising the steps of: Immunizing an antibody-producing animal with an immunogen comprising the composition of any one of claims 1-11; Isolating antibodies from the animal, wherein the antibodies specifically bind to the immunogen.
  13. 13. The method of claim 12, wherein the step of isolating the antibody further comprises the steps of: isolating antibody-producing spleen cells from the antibody-producing animal; selecting at least one antibody-producing spleen cell that secretes a monoclonal antibody that specifically binds to the immunogen; A hybrid cell line is prepared by fusing selected antibody-producing spleen cells from the antibody-producing animal with an appropriate fusion partner, whereby the hybrid cell line secretes the monoclonal antibody.
  14. 14. A coupling composition comprising: the composition according to any one of claims 1 to 11, and An enzyme coupled to the composition.
  15. 15. The coupling composition of claim 14, wherein the enzyme is selected from the group consisting of glucose-6-phosphate dehydrogenase (G6 PDH), alkaline phosphatase, beta-galactosidase, and horseradish peroxidase.
  16. 16. The coupling composition of claim 15, wherein the enzyme is G6PDH.
  17. 17. An antibody produced by the method of claim 12 or 13.
  18. 18. A kit comprising the antibody of claim 17.
  19. 19. The kit of claim 18, further comprising a coupling composition according to any one of claims 14-16.
  20. 20. A method of detecting a benzothiazine in a sample, the method comprising the steps of: binding a sample to an antibody according to claim 17, and Detecting the presence of xylazine in the sample based on the specific binding of the antibody thereto.

Description

Tolylthiazide hapten, immunogen and protein conjugate and methods of producing and using the same Citation of related applications The present application claims to enjoy the benefit of U.S. provisional patent application No. 63/589977 filed on day 12 of month 10 of 2023 in accordance with 35 USC ≡119 (e). The entire contents of the above-mentioned patent application are expressly incorporated herein by reference. Background The mefenazine is a medicine for sedation, anesthesia, muscle relaxation and analgesia of animals such as horses, cattle and other non-human mammals. It is an analog of clonidine and is also an alpha 2 adrenergic receptor agonist. Tolylthiazide has become a commonly used over-the-counter drug in the united states, commonly known as "sedative (tranq)", particularly in the areas of bodores. The benzothiazine mixed with fentanyl is called "sleep cut" (sleep-cut) "," zombie drug (zombie drug) "and" sedative drug (tranq dope) ", on the street. Tolthiazines are also commonly found in "speed balls (speedball)", which are mixtures of various drugs that typically include cocaine, heroin, or morphine with fentanyl. 80% of the drug-taking devices that tested positive for fentanyl also contained tolylthiazide in the needle exchange program from month 11 of 2021 to month 8 of 2022. The development of a monoclonal antibody to xylazine is described by Chao et al (New Journal of Chemistry (2021) 45:4658-4665), wei et al (CN 116003616A) and Xu et al (CN 111454912A). However, despite the exponential increase in the number of drug abuse cases of xylazine, there is currently no commercially available diagnostic immunoassay for the detection of xylazine in biological samples. Drawings FIG. 1 contains chemical structures of various non-limiting embodiments of a benzothiazine hapten constructed according to the present disclosure, wherein the hapten contains a linker at different positions. FIG. 2 contains chemical structures of various non-limiting embodiments of a benzothiazine immunogen and protein conjugate constructed in accordance with the present disclosure. As shown, non-limiting examples of "proteins" that can be coupled to the illustrated benzothiazine include Keyhole Limpet Hemocyanin (KLH), bovine Serum Albumin (BSA), ovalbumin (ovalbumin) (OVA), glucose-6-phosphate dehydrogenase (glucose-6-phosphate dehydrogenase) (G6 PDH), any carrier or tag peptide or protein, and any combination thereof. FIG. 3 contains a synthetic scheme for certain non-limiting embodiments of XLZ A hapten and conjugate for immunogen synthesis according to the present disclosure. The labels (1) - (10) shown in FIG. 3 are equivalent to the formulas disclosed herein (1) formula XI, (2) formula II, (3) formula XII, (4) formula XIII, (5) formula XIV, (6) formula XV, (7) formula XVI, (8) formula XVII, (9) formula XVIII, and (10) formula XIX. FIG. 4 contains another protocol for the synthesis of certain non-limiting embodiments of XLZ immunogens and conjugates in accordance with the present disclosure. Non-limiting examples of "proteins" that may be coupled thereto include KLH, BSA, OVA, G PDH 6 and/or any carrier and/or marker proteins. The labels (3), (6) and (10) shown in FIG. 4 are equivalent to the formulas (3) formula XII, (6) formula XV, and (10) formula XIX disclosed herein. FIG. 5 contains a synthetic scheme of a specific (but non-limiting) embodiment of the benzothiazine conjugates XLZ-C6-3K G6PDH and XLZ-C11-3K G6PDH conjugates. The formulas disclosed herein are of upper left structure, formula XVII, upper middle structure, formula XXII, upper right structure, formula XXIII, lower left structure, formula XVII, lower middle structure, formula XXIX, and lower right structure, formula XXX. FIG. 6 contains a synthetic scheme for a specific (but non-limiting) embodiment of the benzothiazine hapten XLZ-O4-C2S 2 hapten. FIG. 7 contains another version of the synthesis of certain non-limiting embodiments of XLZ-O4-C2 SAc-immunogens and conjugates in accordance with the present disclosure. Non-limiting examples of "proteins" that may be coupled thereto include KLH, BSA, OVA, G PDH 6 and/or any carrier and/or marker proteins. The formulas disclosed on the right side of the figure are the top structure, formula XXVI, the intermediate structure, formula XXVII, and the bottom structure, formula XXVIII (wherein R 4 is G6 PDH). FIG. 8 contains yet another version of certain non-limiting embodiments for the synthesis of XLZ-O4-C8-Br and XLZ-O4-C6-Br haptens according to the present disclosure. FIG. 9 contains yet another version of certain non-limiting embodiments for the synthesis of XLZ-O4-C6-3K G6PDH and XLZ-O4-C8-3K G6PDH conjugates according to the present disclosure. Fig. 10 contains a further version of certain non-limiting embodiments of synthesizing a benzothiazine hapten/immunogen/conjugate according to the present disclosure. Non-limiting examples of "proteins" that may be coupled thereto include KLH, BSA, OVA, G PDH 6 and/or