Clozapine Immunoassay

Abstract

Novel conjugates and immunogens derived from clozapine and antibodies generated by these immunogens are useful in immunoassays for the quantification and monitoring of clozapine in biological fluids.

Claims

1-10. (canceled)

11. An antibody which binds selectively to clozapine and does not substantially bind to N-desmethylclozapine and clozapine-N-oxide.

12. The antibody of claim 11, wherein said antibody is derived from mice, sheep, rabbits or rats.

13. The antibody of claim 11, wherein said antibody is a monoclonal antibody.

14. The antibody of claim 11, wherein said antibody is derived from an immunogenic carrier having a reactive amino or thiol group polymer conjugated to a compound selected from the group consisting of compounds of the formula: ##STR00015## wherein B is: (CO), (CO)NH, (CO)O, CH2-; Y is an organic spacing group; X is a terminal functional group capable of binding to a polyamine polymer; and p is an integer from 0 to 1; or a thereof.

15. The antibody of claim 14, wherein the carrier contains a thiol group and X in the compound which is conjugated to the immunogenic polymer is a functional group capable of reacting with said thiol.

16. The antibody of claim 15, wherein X in said compound is ##STR00016##

17. The antibody of claim 16, wherein Y in said compound is lower alkylene.

18. The antibody of claim 17, wherein the immunogenic carrier contains as the functional group: ##STR00017## wherein v is an integer from 1 to 6.

19. The antibody of claim 18, wherein said antibody is derived from mice, sheep, rabbits or rats.

20-38. (canceled)

Description

EXAMPLES

[0086] In the examples, the following abbreviations are used for designating the following: [0087] MsCl Methanesulfonyl chloride [0088] DIPEA NN-Diisopropylethylamine [0089] CH.sub.2Cl.sub.2 chloroform [0090] MeOH methanol [0091] Na.sub.2SO.sub.4 sodium sulfate [0092] LiOH lithium hydroxide [0093] EtOAc ethyl acetate [0094] Et.sub.3N triethylamine [0095] THF tetrahydrofuran [0096] TFA trifluoroacetic acid [0097] pTSA p-Toluenesulfonic acid [0098] HATU O-(7-Azabenzotriazol-1-yl)-N,N,N,N-tetramethyluronium hexafluorophosphate [0099] DMF Dimethylformamide [0100] DMSO Dimethylsulfoxide [0101] s-NHS sulfo-N-hydroxy succinimide [0102] EDC 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride [0103] KLH Keyhole Limpet HemocyaninBSA Bovine serum albumin [0104] PBS Phosphate buffered saline [0105] NaCl sodium chloride [0106] HRP horse radish-perxidase [0107] ANS 8-Anilino-1-naphthalenesulfonic acid [0108] TMB 3,3,5,5-Tetramethylbenzidine [0109] TRIS Tris(hydroxymethyl)aminomethane hydrochloride [0110] di H.sub.2O deionized water

[0111] The phosphate buffer composition has an aqueous solution containing

[0112] 15.4 mM Sodium phosphate dibasic (Na.sub.2HPO.sub.4)

[0113] 4.6 mM Sodium phosphate monobasic (NaH.sub.2PO.sub.4)

[0114] pH=7.20.10

[0115] In the following Examples, Schemes 1-2 below set forth the specific compounds prepared and referred to by numbers in the Examples. The schemes are as follows:

##STR00012## ##STR00013## ##STR00014##

Example 1

Preparation of 6-[8-Chloro-11-(4-methyl-piperazin-1-yl)-dibenzo[b,e]-[1,4]-5-yl]-6-oxo-hexanoic acid (Scheme 1)

[0116] To a solution of compound [1] (2 & 6.12 mmol) in toluene (60 mL), pyridine (0.49 mL, 6.12 mmol) was added followed by methyl adipoyl chloride (1.3 mL, 7.95 mmol). The reaction mixture was heated at reflux for 3.5 h, cooled to ambient temperature and stirred with 10% aq. Na.sub.2CO.sub.3 (12 mL) for 20 min. The reaction mixture was diluted with ether and washed with water. The organic phase was dried (Na.sub.2SO.sub.4), filtered and evaporated. Purification by flash chromatography using CH.sub.2Cl.sub.2/MeOH 98:2 to 97.3 containing 0.1% Et.sub.3N afforded compound [2] (3.07 g, quantitative).

[0117] To a mixture of compound [2] (3.06 g, 6.12 mmol), THF (50 mL), MeOH (50 mL) and H.sub.2O (19 mL), LiOH.H.sub.2O (1.8 g 42.84 mmol) was added and the reaction mixture was stirred at ambient temperature under nitrogen for 3 h, after which the TLC showed the absence of starting material. The reaction mixture was diluted with EtOAc and washed with 0.1 N HCl. The aqueous layer was re-extracted with CH.sub.2Cl.sub.2 (3). The EtOAc and CH.sub.2Cl.sub.2 layers were separately washed with sat. brine, dried using amhyd. Na.sub.2SO.sub.4, filtered and evaporated. The combined product was triturated with CH.sub.2Cl.sub.2/hexane to afford compound [3], which is 6-[8-Chloro-11-(4-methyl-piperazin-1-yl)-dibenzo[b,e]-[1,4]diazepin-5-yl]-6-oxo-hexanoic acid (2.50 g, 90%).

Example 2

Preparation of 4-({6-[8-Chloro-11-(4-methyl-piperazin-1-yl)-dibenzo[b,e]-[1,4]diazepin-5-yl]6-oxo-hexanoylamino}-methyl)-benzoic acid tert-butyl ester (Scheme 2)

[0118] At 0 C., to a mixture of compound [3], 6-[8-Chloro-11-(4-methyl-piperazin-1-yl)-dibenzo[b,e]-[1,4]diazepin-5-yl]-6-oxo-hexanoic acid (600 g, 1.32 mmol), compound [6] (430 mg, 145 mmol), DIPEA (0.78 mL, 4.48 mmol) in dry DMF (13 mL), HATU (601 mg, 1.58 mmol) was added and the reaction mixture was stirred overnight. An additional amount of HATU (601 mg, 1.58 mmol) was added and the reaction mixture was stirred at ambient temperature for 1 day. The reaction mixture was partitioned between water and EtOAc, the organic layer was successively washed with 1 N HCl, sat. NaHCO.sub.3, water and sat. brine, and dried using amhyd. Na.sub.2SO.sub.4, filtered and evaporated. Purification by chromatography using CH.sub.2Cl.sub.2/MeOH/Et.sub.3N (93:7:0.1) afforded to compound [7] (638 mg, 75%).

[0119] At ambient temperature, to a solution of compound [7] (635 ng, 0.986 mmol) in CH.sub.2Cl.sub.2 (4 mL), TFA (4 mL) was added and the reaction mixture was stirred at ambient temperature under nitrogen for 4 h. The solvent was evaporated and co-evaporated with CH.sub.2Cl.sub.2 (2) and dried under vacuum. The residue on trituration with ether afforded compound [8], which is 4-({6-[8-Chloro-11-(4-methyl-piperazin-1-yl)-dibenzo[b,e]-[1,4]diazepin-5-yl] 6-oxo-hexanoylamino}-methyl)-benzoic acid tert-butyl ester (563 mg, 97%).

Example 3

General Method for Preparing s-NHS Activated Drug Derivatives from the Corresponding Acids [3] & [8]

[0120] In Examples 3a and 3b, clozapine acid derivatives [3] & [8] were activated with EDC and s-NHS to produce the s-NHS activated esters of clozapine [4] & [9] for eventual conjugation to proteins (examples 4 and 5).

Example 3a

Preparation of s-NHS Activated 6-[8-Chloro-11-(4-methyl-piperazin-1-yl)-dibenzo[b,e]-[1,4]diazepin-5-yl]-6-oxo-hexanoic acid [4]

[0121] Clozapine derivative [3], Example 1, Scheme 1, (56.8 mg) was dissolved in 5.68 mL of DMSO to which was added s-NHS (66.56 mg) and EDC (58.58 mg). The reaction mixture was stirred for 20 hours at ambient temperature under a nitrogen atmosphere to produce the s-NHS activated ester of clozapine [4]. The reaction mixture was used directly in Examples 4 and 5a.

Example 3b

Preparation of s-NHS Activated 4-({6-[8-Chloro-11-(4-methyl-piperazin-1-yl)-dibenzo[b,e]-[1,4]diazepin-5-yl] 6-oxo-hexanoylamino}-methyl)-benzoic acid [9]

[0122] Clozapine derivative [8], Example 2, Scheme 2 (25.0 mg) was dissolved in 2.5 mL of DMSO to which was added s-NHS (19.9 mg) and EDC (17.6 mg). The reaction mixture was stirred for 20 hours at ambient temperature under a nitrogen atmosphere to produce the s-NHS activated ester of clozapine [9]. The reaction mixture was used directly in Example 5b.

Example 4

Preparation of KLH Immunogen [5] with Activated Hapten [4]

[0123] A protein solution of KLH was prepared by dissolving 300 mg of KLH in 20 mL of phosphate buffer (50 mM, pH 7.5), followed by addition of 4.85 mL of s-NHS activated clozapine derivative [4] prepared in Example 3a. The reaction mixture of KLH and activated clozapine derivative [4] was allowed to stir for 20 hours at room temperature to produce the clozapine-KLH conjugate [5]. The clozapine-KLH conjugate [5] was then purified by dialysis against 30% DMSO in phosphate buffer (50 mM, pH 7.5) at room temperature. Thereafter the DMSO proportion was reduced stepwise: 20%, 10% and 0%. The last dialysis was performed against phosphate buffer at 4 C. The clozapine-KLH conjugate [5] was characterized by ultraviolet-visible (UV-VIS) spectroscopy. The conjugate was diluted to a final concentration of 2 mg/mL in phosphate buffer (50 mM, pH 7.5).

Example 5a

Preparation of BSA Conjugate [5] with Activated Hapten [4]

[0124] A protein solution of BSA was prepared by dissolving 1 g BSA in phosphate buffer (50 mM, pH 7.5) for a final concentration of 50 mg/mL To this protein solution was added 0.83 mL of s-NHS activated clozapine derivative [4] prepared in Example 3a. The amount of s-NHS activated clozapine derivative [4] added to the protein solution of BSA was calculated for a 1:1 molar ratio between the derivative of clozapine [4] and BSA. The mixture of BSA and activated clozapine derivative [4] was allowed to stir for 18 hours at room temperature to produce the conjugate of the activated clozapine ester and BSA. This conjugate was then purified by dialysis against 20% DMSO in phosphate buffer (50 mM, pH 7.5) at room temperature. Thereafter the DMSO proportion was reduced stepwise: 10% and 0%. The last dialysis was performed against phosphate buffer at 4 C. The purified clozapine-BSA [5] conjugate was characterized by UV/VIS spectroscopy.

Example 5b

Preparation of BSA Conjugate [10] with Activated Hapten [9]

[0125] A protein solution of BSA was prepared by dissolving 1 g BSA in phosphate buffer (50 mM, pH 7.5) for a final concentration of 50 mg/mL. To 10.0 mL of the protein solution of BSA while stirring on ice, was added 0.620 mL of s-NHS activated clozapine derivative [9] prepared in Example 3b. The amount of s-NHS activated clozapine derivative [9] added to the protein solution of BSA was calculated for a 1:1 molar ratio between the derivative of clozapine [9] and BSA. The mixture of BSA and activated clozapine derivative [9] was allowed to stir for 18 hours at room temperature to produce the conjugate of the activated clozapine ester and BSA [10]. This conjugate was then purified by dialysis against 15% DMSO in phosphate buffer (50 mM, pH 7.5) at room temperature. Thereafter the DMSO proportion was reduced stepwise: 10%, 5%, and 0%. The last dialysis was performed against phosphate buffer at 4 C. The purified clozapine-[9]-BSA conjugate was characterized by UV/VIS spectroscopy.

Example 6a

Preparation of Polyclonal Antibodies to Clozapine-KLH Conjugate [5]

[0126] Ten female BALB/c mice were immunized i.p. with 100 g/mouse of clozapine-KLH immunogen [5], as prepared in Example 4, emulsified in Complete Freund's adjuvant. The mice were boosted once, four weeks after the initial injection with 100 g/mouse of the same immunogen emulsified in Incomplete Freund's Adjuvant. Twenty days after the boost, test bleeds containing polyclonal antibodies from each mouse were obtained by orbital bleed. The anti-serum from these test-bleeds containing clozapine antibodies are evaluated in Examples 8 and 9.

Example 6b

Preparation of Monoclonal Antibodies to Clozapine-KLH [5]

[0127] Mice from Example 6a that were immunized with clozapine-[5]-KLH prepared in example 4 were used to produce monoclonal antibodies. For monoclonal antibodies starting three days before the fusion, the mice were injected i.p. with either 400 g (3 days before fusion), 200 g (2 days before fusion), and 200 g (1 day before fusion) or 100 g (3 days before fusion), 100 g (2 days before fusion), and 100 g (1 day before fusion) of clozapine-KLH conjugate [5] in PBS/DMSO prepared in example 4. Spleen cells were isolated from the selected mice and fused with 2107 SP.sub.2/0 cells with 50% polyethylene glycol 1500 according to the method of Coligan, J. E. et al., eds., Current Protocols in Immunology, 2.5.1-2.5.8, (1992), Wiley & Sons, NY. The fused cells were plated on ten 96-well plates in DMEM/F.sub.12 supplemented with 20% FetalClone I, 2% L-glutamine (100 mM) and 2% 50 HAT. Two to three weeks later, the hybridoma supernatant was assayed for the presence of anti-clozapine antibodies by ELISA (as in example 8). Cells from the wells that gave positive ELISA results (example 8) were expanded to 24 well plates. Clones positive by ELISA were subcloned twice by limiting dilution according to the method disclosed in Coligan, J. E. et al., eds., Current Protocols in Immunology, 2.5.8-2.5.17, 1992, Wiley & Sons, NY. Hybridoma culture supernatants containing monoclonal antibody from selected subclones were confirmed for clozapine binding by a competitive ELISA (Example 9). These monoclonal antibodies were tested for clozapine binding and cross-reactivity to the major clozapine metabolites, N-desmethylclozapine and clozapine-N-oxide, by indirect competitive microtiter plate assay as described in Example 9.

Example 7a

Microtiter Plate Sensitization Procedure with Clozapine-BSA Conjugate [5]

[0128] The ELISA method for measuring clozapine concentrations was performed in polystyrene microtiter plates (Nunc MaxiSorp F8 Immunomodules) optimized for protein binding and containing 96 wells per plate. Each well was coated with Clozapine-BSA Conjugate [5] (prepared as in Example 5a) by adding 300 L of Clozapine-BSA Conjugate [5] at 10 g/mL in 0.05M sodium carbonate, pH 9.6, and incubating for three hours at room temperature. The wells were washed with 0.05M sodium carbonate, pH 9.6 and then were blocked with 375 L of 5% sucrose, 0.2% sodium caseinate solution for 30 minutes at room temperature. After removal of the post-coat solution the plates were dried at 37 C. overnight.

Example 7b

Microtiter Plate Sensitization Procedure with Clozapine-BSA Conjugate [10]

[0129] The ELISA method for measuring clozapine concentrations was performed in polystyrene microtiter plates (Nunc MaxiSorp F8 Immunomodules) optimized for protein binding and containing 96 wells per plate. Each well was coated with Clozapine-BSA Conjugate [10] (prepared as in Example 5b) by adding 300 L of Clozapine-BSA Conjugate [10] at 10 g/mL in 0.05M sodium carbonate, pH 9.6, and incubating for three hours at room temperature. The wells were washed with 0.05M sodium carbonate, pH 9.6 and then were blocked with 375 L of 5% sucrose, 0.2% sodium caseinate solution for 30 minutes at room temperature. After removal of the post-coat solution the plates were dried at 37 C. overnight.

Example 8

Antibody Screening ProcedureTiter

[0130] This procedure is to find the dilution of antibody to be tested for displacement as in Example 9. The ELISA method for screening clozapine antibodies was performed with the microtiter plates that were sensitized with clozapine-BSA conjugate prepared in Examples 7a and 7b. The antibody screening assay was performed by diluting the murine serum from test bleeds containing polyclonal clozapine antibodies to 1:1, 1:100, 1:10,00 and 1:10,000 (volume/volume) in phosphate buffered saline containing 0.1% BSA and 0.01% thimerosal. To each well of clozapine-BSA sensitized wells (prepared in Examples 7a and 7b) 50 L phosphate buffered saline containing 0.1% BSA and 0.01% thimerosal and 50 L of diluted antibody were added and incubated for 10 minutes at room temperature with shaking. During this incubation antibody binds to the clozapine-SA conjugate passively absorbed in the wells (Examples 7a and 7b). The wells of the plates were washed three times with 0.02 M TRIS, 0.9% NaCl, 0.5% Tween-80 and 0.001% thimerosal, pH 7.8 to remove any unbound antibody. To detect the amount of clozapine antibody bound to the clozapine-BSA conjugate in the wells, 100 L of a goat anti-mouse antibodyHRP enzyme conjugate (Jackson ImmunoResearch) diluted to a specific activity (approximately 1/3000) in PBS with 0.1% BSA, 0.05% ANS, 0.01% thimerosal, capable of binding specifically with murine immunoglobins and producing a colored product when incubated with a substrate, in this example TMB, were added to each well. After an incubation of 10 minutes at room temperature with shaking, during which the goat anti-mouse antibodyHRP enzyme conjugate binds to clozapine antibodies in the wells, the plates were again washed three times to remove unbound goat anti-mouse antibodyHRP enzyme conjugate. To develop a measurable color in the wells washing was followed by the addition of 100 L of TMB (TMB Substrate, BioFx), the substrate for HRP, to develop color during a 10 minute incubation with shaking at room temperature. Following the incubation for color development, 50 L of stop solution (1.5% sodium fluoride in di H.sub.2O) was added to each well to stop the color development and after 20 seconds of shaking the absorbance was determined at 650 nm (Molecular Devices Plate Reader). The amount of antibody in a well was proportional to the absorbance measured and was expressed as the dilution (titer) resulting in an absorbance of 1.5. Titers were determined by graphing antibody dilution of the antibody measured (x-axis) IVs. absorbance 650 nm (y-axis) and interpolating the titer at an absorbance of 1.5. The titer which produced absorbance of 1.5 determined the concentration (dilution) of antibody used in the indirect competitive microtiter plate assay described in Example 9.

Example 9

Indirect Competitive Microtiter Plate Immunoassay Procedure Determining IC.SUB.50 .and Cross-Reactivity for Antibodies to Clozapine

[0131] The ELISA method for determining IC.sub.50 values and cross-reactivity was performed with the microtiter plates that were sensitized with clozapine-BSA conjugates as described in Examples 7a and 7b. The analytes were diluted as follows: clozapine was serially diluted in DMSO and further diluted to 1% DMSO over a concentration range of 0.01 to 50 ng/mL for clozapine-[5]-BSA and clozapine-[10]-BSA microtiter plates, N-desmethylclozapine was serially diluted in DMSO and further diluted to 1% DMSO over a concentration range of 0.24 to 1,000 ng/mL for clozapine-BSA conjugate [5] and clozapine-BSA conjugate [10] microtiter plates, and clozapine-oxide was serially diluted in DMSO and further diluted to 1% DMSO over a concentration range of 0.24 to 1,000 ng/mL for clozapine-BSA conjugate [5] and clozapine-BSA conjugate [10] microtiter plates. Each of the assays were performed by incubating 50 L of the analyte solution with 50 L of one of the antibodies selected from the polyclonal antibodies produced in Example 6 with the immunogen of Example 4. The assays were all performed by diluting the concentration of the antibodies in each of the wells to the titer determined in Example 8. During the 10 minute incubation (at room temperature with shaking) there is a competition of antibody binding for the clozapine-BSA conjugate in the well (produced in Examples 7a and 7b) and the analyte in solution. Following this incubation the wells of the plate were washed three times with 0.02 M TRIS, 0.9% NaCl, 0.5% Tween-80 and 0.001% thimerosal, pH 7.8 to remove any material that was not bound. To detect the amount of clozapine antibody bound to the clozapine-BSA conjugate in the wells (produced in Examples 7a and 7b), 100 L of a goat anti-mouse antibodyHRP enzyme conjugate (Jackson ImmunoResearch) diluted to a predetermined specific activity (approximately 1/3000) in PBS with 0.1% BSA, 0.05% ANS, 0.01% thimerosal, capable of binding specifically with murine immunoglobulins and producing a colored product when incubated with a substrate, in this example TMB, were added to each well. After an incubation of to minutes at room temperature with shaking, during which the goat anti-mouse antibodyHRP enzyme conjugate binds to clozapine antibodies in the wells, the plates were again washed three times to remove unbound secondary conjugate. To develop a measurable color in the wells washing was followed by the addition of 100 L of TMB (TMB Substrate, BioFx), the substrate for HRP, to develop color in a to minute incubation with shaking at room temperature. Following the incubation for color development, the absorbance was determined at 650 nm (Molecular Devices Plate Reader). The amount of antibody in a well was proportional to the absorbance measured and inversely proportional to the amount of clozapine in the sample. The IC.sub.50 values of clozapine were determined by constructing dose-response curves with the absorbance in the wells plotted versus analyte concentration in the wells. The absorbance of the color in the wells containing analyte was compared to that with no analyte and a standard curve was generated. The IC.sub.50 value for a given analyte was defined as the concentration of analyte that was required to have 50% of the absorbance of the wells containing no analyte. The cross-reactivity was calculated as the ratio of the IC.sub.50 for clozapine to the IC.sub.50 value for either N-desmethylclozapine or clozapine-N-oxide and expressed as a percent. After screening the library of monoclonal antibodies using this method, the monoclonal antibodies were chosen. These chosen antibodies were classified according to their plate and well number as follows: 5B1-24-30, 5H2-6-14, 5H2-6-30, 5G10-19-19, 1G9-22-5, and 20A5-25-24. When measured, the percent cross-reactivities of these antibodies to their reactivity with clozapine for N-desmethylclozapine (NDMC) and clozapine-N-oxide were 2%. The results for monoclonal antibodies to clozapine are given in Tables I & II below.

TABLE-US-00001 TABLE I Cross-reactivity of competitive immunoassay using monoclonal antibodies to clozapine. Plates coated with Clozapine-BSA conjugate [5] (Example 7A) Clozapine- Clozapine NDMC N-oxide % cross- % cross- IC50 IC50 IC50 reactivity reactivity Subclone # (ng/mL) (ng/mL) (ng/mL) NDMC N-oxide 5B1-24-30 10 760 >1000 1.3 0.99 5H2-6-14 5 920 880 0.53 0.56 5H2-6-30 5 >1000 620 <0.45 0.73 5G10-19-19 0.8 150 170 0.49 0.44 1G9-22-5 0.6 280 40 0.22 1.39 20A5-25-24 0.7 >1000 100 <0.07 0.72

TABLE-US-00002 TABLE II Cross-reactivity of competitive immunoassay using monoclonal antibodies to clozapine. Plates coated with Clozapine-BSA conjugate [10] (Example 7B) Clozapine- Clozapine NDMC N-oxide % cross- % cross- IC50 IC50 IC50 reactivity reactivity Subclone # (ng/mL) (ng/mL) (ng/mL) NDMC N-oxide 5B1-24-30 7 600 200 1.1 3.3 5H2-6-14 3 970 620 0.30 0.47 5H2-6-30 3 >1000 480 <0.27 0.57 5G10-19-19 2 180 280 1.2 0.78 1G9-22-5 0.5 220 60 0.23 0.86 20A5-25-24 2 >1000 150 <0.24 1.56

[0132] As seen from these tables, the antibodies of this invention are substantially selectively reactive with the active form of clozapine and are not substantially cross-reactive with the active metabolites N-desmethylclozapine and clozapine-N-oxide.