Antibodies to quetiapine haptens and use thereof

THE REPUBLIC OF POLAND (12) TRANSLATION OF THE EUROPEAN PATENT (19) PL (11) PL / EP 2888286

<img file="PL2888286T3_D0001.tif" />

The Patent Office of the Republic of Poland (96) Date and number of the European patent application: 20.08.2013 13830990.1 (97) The grant of the European patent was announced:

21.02.2018 European Patent Bulletin 2018/08 EP 2888286 B1 (13) T3 (51) Int.CI.

C07K 16/44 (2006.01)

C07K 14/00 (2006.01)

A61K 31/551 (2006.01)

G01N 33/94 (2006.01) (54) Title of the invention:

ANTIBODIES TARGETED AGAINST QUETIAPINE HAPTENES AND THEIR USE (3 °) "·

Priority:

08/21/2012 US 201261691598 P (43) Application announced:

2015/27 (45) in the European Patent Bulletin 2015/27 (45) The following was announced about the submission of the translation of the patent:

31.07.2018 News of the Patent Office 2018/07 (73) Authorized by the patent:

Janssen Pharmaceutica NV, Beerse, BE (72) Inventor (s):

ERIC HRYHORENKO, Hilton, US BANUMATHI SANKARAN, Pittsford, US THOMAS R. DECORY, Pittsford, US THERESA TUBBS, Rochester, US LINDA COLT, Rochester, US BART M. REMMERIE, Gent, BE RHYS SALTER, Doylestown, US

P MATTHEW GARRETT DONAHUE, Warrington, US

CD YONG GONG,

WHAT

CM

0® (74) Representative:

WHAT the thing is, Pat. Adam Kudaj

S.¹ LDS ŁAZEWSKI DEPO I WSPÓLNICY SP. K.

Q_ iii ul. Simple 70

J 00-838 Warsaw

ABOUT.

Caution:

Within nine months of the publication of the information on the grant of the European patent, any person may file an objection to the European Patent Office against the European patent granted. The objection must be made in the form of a written statement of reasons. It is considered filed only when the opposition fee has been paid (Art. 99 (1) of the Convention on the Grant of European Patents).

EP 2 888 286 B1

Z-16950/18

ANTIBODIES TARGETED AGAINST QUETIAPINE HAPTENES AND THEIR USE

Field of the Invention

The present invention relates to the field of immunoassay, and in particular to antibodies that bind to quetiapine, which can be used in immunoassays for the detection of quetiapine.

Background

Schizophrenia is a chronic and debilitating psychiatric disorder affecting approximately 0.45-1% of the world's population (van Os, J .; Kapur, S. Schizophrenia Lancet 2009, 374, 635-645). The main goals of treatment are to achieve sustained remission of psychotic symptoms, reduce the risk and consequences of relapse, and improve patient functioning and overall quality of life. Although many schizophrenic patients can achieve symptomatic stabilization with available antipsychotic drugs, poor adherence is a common cause of relapse with daily oral medications. Many studies (Abdel-Baki, A .; Ouellet-Plamondon, C .; Malla, A. Farmacotherapy Challenges in Patients with First-Episode Psychosis Journal of Affective Disorders 2012, 138, S3-S14) on non-compliance scores showed that patients with schizophrenia who do not take their medications as prescribed have a higher rate of relapse, admission to hospital and suicide, as well as increased mortality. It is estimated that 40 to 75% of patients with schizophrenia have difficulty adhering to the daily oral regimen (Lieberman, J. AND.; Stroup, TS; McEvoy, JP; Swartz, MS; Rosenheck, RA; Perkins, DO; Keefe, RSE; Davis, SM; Davis, CE; Lebowitz, BD; Severe, J .; Hsiao, JK Effectiveness of Antipyschotic Drugs in Patients with Chronic Schizophrenia New England Journal of Medicine 2005, 353 (12), 12091223).

[0003] Therapeutic drug monitoring (TDM) is the quantification of serum or plasma concentrations of drugs, including antipsychotics, for therapeutic monitoring and optimization. Such monitoring allows, for example, the identification of patients who do not adhere to their treatment regimen, who do not achieve therapeutic doses, who are non-responsive to therapeutic doses, who are suboptimal tolerance, who are experiencing pharmacokinetic drug-drug interactions, or who have abnormalities. metabolism, resulting in inadequate plasma concentrations. There is considerable individual variability in the patient's ability to absorb, distribute, metabolize, and secrete antipsychotic drugs. Such differences may be due to concurrent disease, age, concomitant medications, or genetic peculiarities. Various drug formulations may also affect the metabolism of antipsychotic drugs. TDM enables dose optimization for individual patients, improving therapeutic and functional outcomes. TDM further enables the prescribing physician to ensure compliance with prescribed dosages and achieve effective serum concentrations.

[0004] As of today, methods for determining the serum or plasma concentration levels of antipsychotics include the use of liquid chromatography (LC) with detection by UV or mass spectrometry and radioimmunoassay (see, for example, Woestenborghs et al., 1990 On the selectivity of some recently developed RIA's in Methodological Surveys in Biochemistry and Analysis 20: 241-246. Analysis of Drugs and Metabolites, Including Anti-infective Agents; Heykants et al., 1994 The Pharmacokinetics of Risperidone in Humans: A Summary, J Clin Psychiatry 55/5, Supplement: 13-17; Huang et al., 1993 Pharmacokinetics of the novel anti-psychotic agent risperidone and the prolactin response in healthy subjects, Clin Pharmacol Ther 54: 257-268). Radioimmunoassays detect one or both of risperidone and paliperidone. Salamone et al. in US Patent No. 8,088,594 disclose a competitive immunoassay for risperidone using antibodies that detect both risperidone and paliperidone, but not pharmacologically inactive metabolites. Antibodies used in competitive immunoassay are designed against a specific immunogen. ID Labs Inc. (London, Ontario, Canada) is producing an ELISA for olanzapine, another antipsychotic drug that also uses the competitive format. The instructions for use indicate that the study is designed for screening purposes and is intended for forensic or research use, and is not specifically intended for therapeutic use. The instructions recommend that all positive samples be confirmed by Gas Chromatography / Mass Spectrometry (GC-MS) and indicate that the antibody used detects both olanzapine and clozapine (see ID Labs Inc., Instructions For Use Data Sheet IDEL-F083, Check Date August 8 2011). Certain of these methods, namely HPLC and GC / MS, can be expensive and labor intensive and are generally only performed in large or specialized laboratories with appropriate equipment.

[0005] There is a need to develop other methods for determining the levels of antipsychotic drugs, in particular methods that can be performed in the prescribing physician's office (where the treatment for a particular patient can be appropriately tailored at a much better time) and in other medical facilities lacking LC or LC equipment. GC / MS or requiring rapid test results.

[0006] WO 2011/082076 describes the synthesis and use of quetiapine conjugates with standard, non-standard or artificial amino acids. WO 2011/112657 describes the synthesis of quetiapine fatty acid conjugates. WO 2011/115733 describes conjugates and immunogens derived from risperidone or paliperidone, and methods for producing antibodies that selectively bind to paliperidone and risperidone, but not to other metabolites.

[0007] Quetiapine is:

<img file="PL2888286T3_D0002.tif" />

Summary of the invention

[0008] The present invention is directed to a method of producing an antibody that binds quetiapine, the method comprising (i) selecting a non-human host for producing the antibody; and (ii) inoculating the host with a conjugate of a compound of Formula I and an immunogenic carrier, wherein the host produces an antibody that binds to quetiapine.

Formula I.

<img file="PL2888286T3_D0003.tif" />

wherein:

R¹ represents H,

<img file="PL2888286T3_D0004.tif" />

CH2NHC (O) (CH2) mCO2H;

R² represents H,

<img file="PL2888286T3_D0005.tif" />

<img file="PL2888286T3_D0006.tif" />

<img file="PL2888286T3_D0007.tif" />

.CH2NH2 or

CH2NHC (O) (CH2) mCO2H;

R³ is H; provided that either R.¹ or R.² must be H and further provided that both R.¹ and r² they cannot be H at the same time;

m is 1, 2, 3, 4, or 5; and n is 1, 2, 3, 4, or 5.

[0009] Currently preferred embodiments of the antibodies of the disclosure are the antibodies designated as 11, 89-3, 89-5, and 89-13 generated against a compound of Formula II. Another suitable immunogen for a compound of Formula III.

<img file="PL2888286T3_D0008.tif" />

<img file="PL2888286T3_D0009.tif" />

[0010] The antibodies produced by the method of the invention may be provided in test kits and devices, with a present preferred device being a lateral flow test device that provides analysis at the point of contact between the physician and the patient.

[0011] Furthermore, there is provided a method of producing a hybridoma cell line capable of producing a monoclonal antibody that binds to quetiapine. The method comprises: (i) selecting a non-human host for antibody production; (ii) inoculating the host with a conjugate of the compound of Formula I and an immunogenic carrier; (iii) fusing a cell line from the inoculated host with a continuously dividing cell to produce a fused cell capable of producing a monoclonal antibody that binds to quetiapine; and (iv) cloning the combined cell so as to obtain a hybridoma cell line.

[0012] The invention further provides a method for detecting quetiapine in a sample. The method comprises: (i) contacting the sample with an antibody produced by the method of the invention that is labeled with a detectable label wherein the labeled antibody and quetiapine present in the sample form a labeled complex; and (ii) detecting the labeled complex so as to detect quetiapine in the sample.

[0013] Further, a competitive immunoassay method for detecting quetiapine in a sample is provided. The method comprises: (i) contacting an antibody prepared according to the invention and with quetiapine or a competing partner of quetiapine, wherein one of the antibody and quetiapine or its competing partner is labeled with a detectable label and wherein the quetiapine competes in the sample. with quetiapine or its competitive binding partner; and (ii) detecting a marker so as to detect quetiapine in the sample.

[0014] Further objects, features and advantages of the present invention will be apparent to those skilled in the art from the detailed consideration of the following preferred embodiments.

Brief Description of Drawings

[0015]

Figures 1 and 2 show the results of competition ELISAs generated with different hybridoma cells;

Fig. 3 shows a competitive immunoassay format used in a lateral flow test rig;

Fig. 4 shows a typical dose response curve generated with quetiapine sub-clones 893, 89-13 and 89-5;

Fig. 5 shows a chip design for a lateral flow test apparatus in accordance with the present invention;

Fig. 6 shows a typical dose response curve for aripiprazole positive control generated with antibody 5C7 and labeled competition partner for aripiprazole;

Fig. 7 shows a typical dose response curve for olanzapine positive control generated with antibody 4G9-1 and labeled competition binding partner for olanzapine;

Figure 8 shows a typical dose response curve for quetiapine positive control generated with antibody 11 and labeled competition binding partner for quetiapine;

Fig. 9 shows a typical dose response curve for the positive control for risperidone generated with antibody 5-9 and for the labeled competition binding partner for risperidone;

Figure 10 shows a typical dose-response curve for an aripiprazole-containing sample generated with the anti-aripiprazole antibody 5C7 in the presence of a labeled competition partner for aripiprazole, no dose response curve for olanzapine, quetiapine, or risperidone in the presence of a labeled competition partner for each of them;

Figure 11 shows a typical dose response curve for a sample containing olanzapine generated with anti-olanzapine antibody 4G9-1 in the presence of a labeled competition binding partner for olanzapine, no dose response curve for aripiprazole, quetiapine or risperidone in the presence of a labeled competition binding partner. for each one of them;

Figure 12 shows a typical dose response curve for a sample containing quetiapine generated with an anti-quetiapine 11 antibody in the presence of a labeled competition partner for quetiapine, no dose response curve for aripiprazole, olanzapine or risperidone in the presence of a labeled competition partner for each of them;

Figure 13 shows a typical dose response curve for a sample containing risperidone generated with an anti-risperidone antibody 5-9 in the presence of a labeled competition partner for risperidone, no dose response curve for aripiprazole, olanzapine or quetiapine in the presence of a labeled competition partner for each one of them;

Fig. 14 shows a typical dose-response curve for an aripiprazole-containing sample generated with the anti-aripiprazole antibody 5C7 in the presence of a labeled competition partner for aripiprazole, no dose response curve for olanzapine, quetiapine, or risperidone in the presence of the antibody and labeled competition. a partner for each of them;

Figure 15 shows a typical dose response curve for a sample containing olanzapine generated with anti-olanzapine antibody 4G9-1 in the presence of a labeled competition partner for olanzapine, no dose response curve for aripiprazole, quetiapine, or risperidone in the presence of the antibody and labeled binding partner. competitively partner for each of them;

Figure 16 shows a typical dose response curve for a sample containing quetiapine generated with an anti-quetiapine 11 antibody in the presence of a labeled competition binding partner for quetiapine, no dose response curve for aripiprazole, olanzapine or risperidone in the presence of the antibody and labeled competition partner for each one of them;

Figure 17 shows a typical dose response curve for a risperidone containing sample generated with an anti-risperidone antibody 5-9 in the presence of a labeled competition partner for risperidone, no dose response curve for aripiprazole, olanzapine or quetiapine in the presence of the antibody and labeled binding partner. competitively partner for each of them;

Fig. 18 shows a comparison of the dose response curve for aripiprazole generated as a positive control for the dose response curve of aripiprazole generated in a multiplex format;

Fig. 19 shows a comparison of the dose response curve for olanzapine generated as a positive control for the dose response curve of olanzapine generated in a multiplex format;

Fig. 20 shows a comparison of the dose response curve of quetiapine generated as a positive control for the dose response curve of quetiapine generated in a multiplex format; and

Figure 21 shows a comparison of the dose response curve for risperidone generated as a positive control for the dose response curve for risperidone generated in a multiplex format.

Detailed Description of the Preferred Embodiments

[0016] The invention provides a method of producing an isolated antibody that binds to quetiapine. The invention further provides a test kit and test device comprising an antibody. Methods for generating a hybridoma cell line capable of producing an antibody are also provided. In addition, a method for detecting quetiapine in a sample is provided, including a method with a competitive immunoassay.

[0017] In one embodiment, the present disclosure is directed to an isolated antibody or binding fragment thereof which binds to quetiapine and which: (i) is generated in response to a conjugate of a compound of Formula I and an immunogenic carrier; or (ii) competes for an epitope that is the same as the antibody bound epitope of (i).

Formula I.

<img file="PL2888286T3_D0010.tif" />

Formula I where:

R¹ states

H,

<img file="PL2888286T3_D0011.tif" />

<img file="PL2888286T3_D0012.tif" />

• CH2NH2 or

CH2NHC (O) (CH2) mCO2H, or z- (y) pg;

R² represents H,

<img file="PL2888286T3_D0013.tif" />

<img file="PL2888286T3_D0014.tif" />

<img file="PL2888286T3_D0015.tif" />

• CH2NH2 or

CH2NHC (O) (CH2) mCO2H, or, Z- (Y) pG;

R³ is H or W- (Y) pG; provided that two of the R.¹, R², R³ must be H and further provided that all R1, R² and r³ they cannot be H at the same time;

wherein:

Z is selected from the group consisting of:

-N (R⁴) -, -O-, -S-, -alkyl-, -aminoalkyl-, -thioalkyl-, -heteroalkyl-, -alkylcarbonyl-,

<img file="PL2888286T3_D0016.tif" />

R⁴ is H, an alkyl group, a cycloalkyl group, an aralkyl group, or a substituted or unsubstituted aryl group; wherein:

W is selected from the group consisting of:

-C (O) -, alkyl, aminoalkyl, thioalkyl, heteroalkyl, alkylcarbonyl;

Y is an organic walk;

G is a linking functional group capable of binding to a carrier;

p is 0 or 1;

m is 1, 2, 3, 4, or 5;

n is 1, 2, 3, 4, or 5.

[0018] In a further embodiment, the present disclosure is directed to an isolated antibody or binding fragment thereof that binds to quetiapine and which: (i) is generated in response to a conjugate of a compound of Formula I and an immunogenic carrier; or (ii) competes for an epitope that is the same as the antibody bound epitope of (i); wherein:

R¹ represents H,

<img file="PL2888286T3_D0017.tif" />

<img file="PL2888286T3_D0018.tif" />

• CH2NH2 or

CH2NHC (O) (CH2) mCO2H or Z (Y) pG;

R² represents H,

<img file="PL2888286T3_D0019.tif" />

<img file="PL2888286T3_D0020.tif" />

CH2NH2 or

CH2NHC (O) (CH2) mCO2H, or, Z- (Y) pG;

R³ represents H, provided that either R.¹ or R.² must be H and further provided that both R.¹ and r² they cannot be H at the same time;

wherein:

Z is selected from the group consisting of: -N (R⁴) -, -O-, -S-, -alkyl-, -aminoalkyl-, -thioalkyl-, oo R⁴ about

Ls-) jPj1ul) = N-NH-1 heteroalkyl-, -alkylcarbonyl-, ⁰ - ^or4 , ⁰ AA:

R⁴ is H, an alkyl group, a cycloalkyl group, an aralkyl group, or a substituted or unsubstituted aryl group;

Y is an organic walk;

G is a linking functional group capable of binding to a carrier;

p is 0 or 1;

m is 1, 2, 3, 4, or 5;

n is 1, 2, 3, 4, or 5.

[0019] In a further embodiment, the present disclosure is directed to an isolated antibody or binding fragment thereof that binds to quetiapine and which: (i) is generated in response to a conjugate of a compound of Formula I and an immunogenic carrier; or (ii) competes for an epitope that is the same as the antibody bound epitope of (i); wherein:

R¹ is H or CH2NH- (Y) pG;

R² is H or CH2NH- (Y) pG; provided that either R.¹ or R.² must be H and further provided that both R.¹ and r² they cannot be H at the same time;

R³ is H;

where:

Y is an organic walk group

G is a linking functional group capable of binding to a carrier; p is 1.

[0020] In a further embodiment, the present disclosure is directed to an isolated antibody or binding fragment thereof which binds to quetiapine and which: (i) is generated in response to a conjugate of a compound of Formula I and an immunogenic carrier; or (ii) competes for an epitope that is the same as the antibody bound epitope of (i); wherein:

OO ooo A ^^NIN^0H

R¹ represents H, ^about⁰ ° , ^about .CH2NH2 or

CH2NHC (O) (CH2) mCO2H;

R² represents H,

<img file="PL2888286T3_D0021.tif" />

.CH2NH2 or

CH2NHC (O) (CH2) mCO2H; provided that either R.¹ or R.² must be H and further provided that both R.¹ and r² they cannot be H at the same time;

R3 is H;

m is 1, 2, 3, 4, or 5;

n is 1, 2, 3, 4, or 5.

[0021] In a further embodiment, the present disclosure is directed to an isolated antibody or binding fragment thereof that binds to quetiapine and which: (i) is generated in response to a conjugate of a compound of Formula I and an immunogenic carrier; or (ii) competes for an epitope that is the same as the antibody bound epitope of (i); wherein:

R¹ represents H,

<img file="PL2888286T3_D0022.tif" />

• or CH2NH2;

R² represents H,

<img file="PL2888286T3_D0023.tif" />

or CH2NH2; provided that either R.¹ or R.² must be H and further provided that both R.¹ and r² they cannot be H at the same time;

R³ is H;

m is 1, 2, 3, 4, or 5;

n is 1, 2, 3, 4, or 5.

[0022] In a preferred embodiment, the present disclosure is directed to an isolated antibody or binding fragment thereof which binds to quetiapine and which: (i) is generated in response to a conjugate of a compound of Formula IV and an immunogenic carrier; or (ii) competes for an epitope that is the same as the antibody bound epitope of (i).

Formula IV

<img file="PL2888286T3_D0024.tif" />

[0023] In a preferred embodiment, the present disclosure is directed to an isolated antibody or binding fragment thereof which binds to quetiapine and which: (i) is generated in response to a conjugate of a compound of Formula V and an immunogenic carrier; or (ii) competes for an epitope that is the same as the antibody bound epitope of (i).

Pattern V

<img file="PL2888286T3_D0025.tif" />

[0024] In a preferred embodiment, the present disclosure is directed to an isolated antibody or binding fragment thereof which binds to quetiapine and which: (i) is

Model VI

<img file="PL2888286T3_D0026.tif" />

generated in response to a conjugate of a compound of Formula VI and an immunogenic carrier; or (ii) competes for an epitope that is the same as the antibody bound epitope of (i).

[0025] In a preferred embodiment, the present disclosure is directed to an isolated antibody or binding fragment thereof which binds to quetiapine and which: (i) is generated in response to a conjugate of a compound of Formula VII and an immunogenic carrier; or (ii) competes for an epitope that is the same as the antibody bound epitope of (i).

Formula VII

<img file="PL2888286T3_D0027.tif" />

[0026] Preferably, an antibody of the subject of the disclosure is generated in response to a conjugate of a compound selected from the compounds of Formula I, Formula IV, Formula V, Formula VI, and Formula VII; and an immunogenic carrier.

[0027] Further details of the compounds described by the above formulas and the conjugates made with the compounds and the immunogenic carrier are provided in the section entitled Compounds, Conjugates and Immunogens below.

[0028] Further details of the antibodies produced by the method of the invention are provided in the section entitled Antibodies below.

[0029] The subject of the invention further provides a test kit containing an antibody as well as a test device containing the antibody. Preferably, the test device is a lateral flow test device. Further details on the test kits and test devices are provided below in the section headed Test kits and devices.

[0030] The invention provides a method of producing an antibody that binds quetiapine, the method comprising: (i) selecting a host other than a human cell for producing the antibody; and (ii) inoculating the host with a conjugate of a compound of Formula I and an immunogenic carrier, wherein the host produces an antibody that binds to quetiapine. In additional embodiments, the conjugate used in the method may be a conjugate of a compound selected from the compounds of Formula IV, Formula V, Formula VI, and Formula VII; and an immunogenic carrier. Further details on producing the antibodies of the subject invention are provided in the section entitled Antibodies below.

[0031] Further provided is a method of producing a hybridoma cell line capable of producing a monoclonal antibody that binds to quetiapine. The method comprises: (i) selecting a non-human host for antibody production; (ii) inoculating the host with a conjugate of the compound of Formula I and an immunogenic carrier; (iii) fusing a cell line from the inoculated host with a continuously dividing cell to produce a fused cell capable of producing a monoclonal antibody that binds to quetiapine; and (iv) cloning the combined cell so as to obtain a hybridoma cell line. In additional embodiments, the conjugate used in the method may be a conjugate of a compound selected from the compounds of Formula IV, Formula V, Formula VI, and Formula VII; and an immunogenic carrier. Further details for the production of hybridoma cells according to the invention are provided in the section entitled Antibodies below.

[0032] The invention further provides a method for detecting quetiapine in a sample. The method comprises: (i) contacting the sample with an antibody produced by the method of the invention that is a labeled detectable label wherein the labeled antibody and quetiapine present in the sample form a labeled complex; and (ii) detecting the labeled complex so as to detect quetiapine in the sample. Further details of the method for detecting quetiapine according to the invention are provided in the following section entitled Immunoassays.

[0033] Furthermore, a competitive immunoassay method for detecting quetiapine in a sample is provided. The method comprises: (i) contacting an antibody produced by the method of the invention and with quetiapine or a competing partner of quetiapine, wherein one of the antibody and quetiapine or its competing partner is labeled with a detectable label and wherein quetiapine competes in the sample. with quetiapine or its competitive binding partner in binding to an antibody; and (ii) detecting a marker so as to detect quetiapine in the sample. Further details of a competitive immunoassay as a method for detecting quetiapine in accordance with the invention are provided in the following section entitled Immunoassay.

[0034] In a preferred embodiment of the invention, the detection of quetiapine is accompanied by the detection of one or more analytes determined in addition to quetiapine. Preferably said one or more analytes are antipsychotics other than quetiapine and more preferably antipsychotics other than quetiapine are selected from the group consisting of: aripiprazole, risperidone, paliperidone, olanzapine and their metabolites. [0035] As discussed above, the antibodies produced by the method of the invention may be used in studies to detect the presence and / or amount of an antipsychotic drug in patient samples. Such detection enables therapeutic monitoring of a drug, revealing all its benefits. Detecting the levels of antipsychotic drugs can be useful for a variety of purposes, each representing another embodiment of the subject matter of the invention, including: determining patient compliance or compliance with a prescribed treatment; use as a decision-making tool to determine if a patient's treatment should be switched from an oral antipsychotic regimen to a long term injectable antipsychotic regimen; use as a decision-making tool to determine whether the dose level or dosage interval of orally or injected antipsychotic drugs should be increased or decreased in order to achieve or maintain effective or safe drug levels; use as an aid in initiating antipsychotic drug therapy by providing evidence of the achievement of minimum pK levels; use to determine the bioequivalence of an antipsychotic drug in multiple formulations or from multiple sources; use to evaluate the effects of polypharmacy and potential drug-drug interactions; and use as an indication that a patient should be excluded from or included in a clinical trial and as an aid in the subsequent monitoring of drug compliance in clinical trials.

COMPOUNDS, CONJUGATES AND IMMUNOGENS

[0036] With respect to compounds and conjugates and immunogens, the following abbreviations have been used: AMAS is N- (α-maleimidoacetoxy) succinimide ester; BINAP is 2,2'bis (diphenylphosphino) -1,1'-binaphthyl; Boc or BOC is tet-butoxycarbonyl; BTG is bovine thyroglobulin; BuaN is tributylamine; DCC is dicyclohexylcarbodiimide; DCM is dichloromethane; DIEA is diisopropylethylamine; DMF is N, N-dimethylformamide; EDCl or EDC is 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride; EDTA is ethylenediaminetetraacetic acid;

HOBT or HOBt is 1-hydroxybenzotriazole hydrate; KLH is marine snail haemocyanin; Pdz (dba) 3 is tris (dibenzylideneacetone) dipalladium (0); SATA is N-succinimidyl S-acetylthioacetate; TEA or Et3N is triethylamine; THF is tetrahydrofuran; TFA is trifluoroacetic acid; rt is room temperature; DEAD is diethylazodicarboxylate; DIC is diisopropylcarbodiimide; NHS is N-hydroxysuccinimide; TFP is Tetrafluorophenyl; PNP is pnitrophenyl; TBTU is O- (Benzotriazol-1-yl) -N, N, N ', N'-tetramethyluronium tetrafluoroborate; DEPBT is 3- (diethoxyphosphoryloxy) -1,2,3-benzotrazin-4 (3H) -one; BOP-Cl is Bis (2-oxo-3-oxazolidinyl) phosphonic chloride; DTT is dithioerythritol.

[0037] Conjugate refers to any substance that results from bringing separate parts together. Representative conjugates include those made by joining together a small molecule, such as for the compounds of Formula I, and a large molecule, such as a carrier or a polyamine polymer, in particular a protein. In the conjugate, the small molecule can be attached at one or more active sites on the large molecule.

[0038] The term hapten refers to a partial or incomplete antigen. Hapten is a protein-free substance that is not capable of stimulating the formation of an antibody, but that reacts with antibodies. Antibodies are produced by conjugating a hapten to a high molecular weight immunogenic carrier and then injecting this conjugate product, i.e., an immunogen, into a human or animal subject.

[0039] The term immunogen refers to a substance capable of eliciting, producing or generating an immune response in the body.

[0040] An immunogenic carrier as used herein is an immunogenic substance, typically a protein that can bind to haptens at one or more positions, thus allowing the production of antibodies that can bind to these haptens. Examples of immunogenic carrier substances include, but are not limited to, proteins, glycoproteins, polyamine-polysaccharide complexes, particles, and nucleic acids that are recognized as foreign and thus elicit an immune response in a non-human host. The polyamine-polysaccharides can be prepared from the polysaccharides by any conventional means known for such preparation.

[0041] Various types of proteins can be used as immunogenic carriers, including, without limitation, albumin, serum protein, lipoprotein, etc. Illustrative proteins include bovine serum albumin, marine snail hemocyanin, ovalbumin, bovine thyroglobulin, human serum albumin fraction V, albumin. rabbit, pumpkin seed globulin, diphtheria toxoid, tetanus toxoid, botulinum toxin, succinylated proteins and artificial poly (amino acids) such as polylysine.

[0042] Immunogenic carriers can also include polyamino polysaccharides, which are high molecular weight polymers constructed by the refolding of monosaccharides. Examples of polysaccharides are starches, glycogen, cellulose, carbohydrate gums such as acacia, agar, and so on. The polysaccharide also contains poly (amino acid) and / or fatty residues.

[0043] The immunogenic carrier may also be a poly (nucleic acid) either alone or conjugated to one of the above-mentioned poly (amino acids) or polysaccharides.

[0044] The immunogenic carrier may also include solid particles. The particles are generally at least about 0.02 micron (µm) and no greater than about 100 µm, and typically from about 0.05 µm to 10 µm in diameter. The particle may be organic or inorganic, swellable or non-swellable, porous or non-porous, optimally with a density close to water, generally from about 0.7 to 1.5 g / ml, and composed of a material which may be transparent, partially transparent or opaque. The particles can be biological materials such as cells and microorganisms, including non-limiting examples such as erythrocytes, leukocytes, lymphocytes, hybridoma cells, streptococci, Staphylococcus aureus, E co / ii viruses. The particles can also be composed of organic and inorganic polymers, liposomes, latex, phospholipid vesicles, or lipoproteins.

[0045] The term derivative refers to a chemical compound or molecule made from a parent compound by one or more chemical reactions.

[0046] The term chemical analog refers to a chemical compound that contains a chain of carbon atoms and the same individual functional groups as the reference compound, but the carbon chain of the analog is longer or shorter than that of the reference compound.

[0047] A label, detection molecule, reporter, or detectable marker is any molecule that produces or can be induced to produce a detectable signal. The label can be conjugated to an analyte, immunogen, antibody or other molecule such as a receptor or a molecule that can bind to the receptor such as a ligand, in particular a hapten or an antibody. The tag can be attached directly or indirectly with a linking or bridge-forming moiety. Non-limiting examples of labels include radioactive isotopes (e.g., ¹²⁵I), enzymes (e.g. β-galactosidase, peroxidase), enzyme fragments, enzyme substrates, enzyme inhibitors, coenzymes, catalysts, fluorophores (e.g., rhodamine, fluorescein isothiocyanate or FITC or Dylight 649), dyes, chemiluminescent compounds and luminescent compounds (e.g., dioxetanes, luciferin) or sensitizers.

[0048] As used herein, spacer refers to that part of a chemical structure that connects two or more substructures, such as haptens, carriers, immunogens, tags, or partners that bind through a connecting functional group. These spacer groups are composed of atoms normally present and assembled by methods commonly found in organic compounds and may therefore be referred to as organic spacer groups. The chemical building blocks used to assemble the spacers will be described herein in this application.

[0049] Among the preferred spacers are straight or branched, saturated or unsaturated carbon chains. These carbon chains may also contain one or more heteroatoms within the chain, one or more heteroatoms replacing one or more hydrogens of any of the carbon atoms in the chain or at the ends of the chains. By heteroatoms is meant atoms other than carbon which are selected from the group consisting of oxygen, nitrogen, phosphorus and sulfur, whereby the nitrogen, phosphorus and sulfur atoms may be in any oxidation state and may have carbon or other heteroatoms attached thereto. The spacer may also include cyclic or aromatic groups as part of the chain or as a substituent on one of the atoms in the chain.

[0050] The number of atoms in a spacer is determined by the counting of the atoms other than hydrogen.

[0051] The number of atoms in a chain within a spacer group is determined by counting the number of non-hydrogen atoms along the shortest path between the linked substructures. Preferred chain lengths are between 1 and 20 atoms.

[0052] A linking functional group refers to a reactive group that is present on a hapten and can be used to provide an accessible reactive site through which a hapten portion can be conjugated to another moiety by forming a covalent chemical bond to conjugate the hapten with another. a moiety (such as a label or carrier). The hapten may thus be linked to a moiety such as biotin to form a competitive binding partner.

Spacer groups may be used to link the hapten to a carrier. Spacers of different lengths allow the hapten to be assembled at different distances from the carrier for presentation to the immune system of the immunized animal or human to optimize the antibody formation process. Attachments at various positions in the hapten molecule make it possible to present specific hapten sites to the immune system in order to influence antibody recognition. The spacer may contain hydrophilic dissolving groups to make the hapten derivative more soluble in the aqueous medium. Examples of hydrophilic dissolving groups include, but are not limited to, polyoxyalkyloxy groups, for example, polyethylene glycol chains; hydroxyl, carboxyl and sulfonate groups.

[0054] The term nucleophilic group or nucleophile refers to species that donate an electron pair to produce a chemical bond in a reaction. The term electrophilic group or electrophile refers to species that take an electron pair from a nucleophile to form a chemical bond in a reaction.

[0055] Substituted refers to the substitution of an atom or group of atoms for a hydrogen atom on a carbon atom at any position on the parent molecule. Non-limiting examples of substituents include halogen, amino, hydroxy, carboxyl, alkyl, aryl, heteroalkyl, heteroaryl, cyano, alkoxy, nitro, aldehyde and ketone groups.

[0056] The term alkyl refers to saturated or unsaturated linear and branched chain radicals having up to 12 carbon atoms, unless otherwise indicated, and is specifically intended to include radicals of any degree or level of saturation. Alkyl includes, but is not limited to, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl, hexyl, isohexyl, heptyl, octyl, 2,2,4-trimethylpentyl, nonyl , decyl, undecyl and dodecyl.

[0057] The term cycloalkyl refers to a saturated or partially unsaturated monocyclic or bicyclic hydrocarbon ring radical of from 3 to 10 carbon atoms. Alkyl substituents may optionally be present on the ring. Examples include cyclopropyl, 1,1-dimethylcyclobutyl, 1,2,3-trimethylcyclopentyl, cyclohexyl, and cyclohexenyl.

[0058] The term heteroalkyl refers to an alkyl group that includes one or more chain heteroatoms, one or more heteroatoms replacing one or more hydrogens of any of the chain carbon atoms or at the ends of the chains.

[0059] Aminoalkyl refers to at least one primary or secondary amino group bonded to any carbon atom along the alkyl chain.

[0060] The term alkoxy refers to straight or branched chain radicals of up to 12 carbon atoms, unless otherwise indicated, bonded to an oxygen atom. Examples include, but are not limited to, methoxy, ethoxy, propoxy, isopropoxy, and butoxy.

[0061] The term alkoxyalkyl refers to at least one alkoxy group bonded to any carbon atom along the alkyl chain.

[0062] The term thioalkyl refers to at least one sulfur group bonded to any carbon atom along the alkyl chain. The sulfur group can be in any oxidation state and includes sulfoxides, sulfones and sulfates (VI).

[0063] The term carboxylic acid group includes carboxylic acids and alkyl, cycloalkyl, aryl or aralkyl esters of a carboxylate.

[0064] The term alkylcarbonyl refers to a group which has a carbonyl group bonded to any carbon atom along the alkyl chain.

[0065] The term heteroaryl refers to radicals with a 5- to 7-membered mono- or 8- to 10-membered bicyclic aromatic ring, each ring may consist of one to four heteroatoms selected from N, O or S, wherein the nitrogen and sulfur atoms may be in any acceptable oxidation state. Examples include benzimidazolyl, benzothiazolyl, benzothienyl, benzoxazolyl, furyl, imidazolyl, isothiazolyl, isoxazolyl, oxazolyl, pyrazinyl, pyrazolyl, pyridyl, pyrimidinyl, pyrrolyl, quinolinyl, thiazolyl, and thienyl.

[0066] The term aryl refers to monocyclic or bicyclic aromatic ring radicals containing from 6 to 12 ring carbons. Alkyl substituents may optionally be present on the ring. Examples include phenyl, biphenyl, and naphthalene.

[0067] The term arylalkyl refers to a C1-6 alkyl group having an aryl substituent. Examples include benzyl, phenylethyl or 2-naphthylmethyl.

[0068] The term acyl refers to the group -C (O) Ra, where Ra is hydrogen, alkyl, cycloalkyl, heteroalkyl, aryl, aralkyl and heteroaryl. An acylating agent adds a -C (O) Ra group to the molecule.

[0069] Sulfonyl refers to the group -S (O) zRb, where Rb is hydrogen, alkyl, cycloalkyl, heteroalkyl, fluoroalkyl, aryl, arylalkyl and heteroaryl. A sulfonylation agent adds a -S (O) -Ra group to the molecule.

Spacers with reactive functional linking groups for attaching haptens to carrier moieties can be made by a wide variety of methods. The spacer can be made using a molecule that is functionalized or activated with different groups at either end to allow selective, sequential reaction with the hapten and the carrier, but the same reactive moiety can also be used at both ends. The groups selected for the reaction with the hapten and the linking functional group to be bound to the support are determined by the type of functionality in the hapten and the support to which the hapten is to be bound. Spacers and methods for linking to haptens and carriers include, but are not limited to, those described by Brinkley, M., A., Bioconjugate Chem. 1992, 3: 2-13, Hermanson, Greg T., Bioconjugate Techniques,. Academic Press, London, Amsterdam, Burlington, MA USA, 2008 and Thermo Scientific Pierce Cross / inking Technica / Handbook; available for download or upon request as a hard copy from Thermo Scientific 3747 N Meridian Rd, Rockford, IL USA 61101, ph 800-874-3723 or at: http://www.piercenet.com/ and references therein. Many differentially activated spacer molecules are commercially available from vendors, for example Thermo Scientific.

[0071] For haptens with an amino group, the modes of attachment of the spacer to the hapten include reacting the amine in the hapten with a spacer building block containing an acyl halide or active ester. Active esters are defined as esters that react with a nucleophilic group, for example an amino group, under mild conditions to form a stable bond. A stable binding is defined as one that remains intact under the conditions of further use, for example subsequent synthetic steps, used as an immunogen, or in a biochemical test. A preferred example of a stable bond is an amide bond. Active esters and preparation methods are described by Benoiton, NL, in Houben-Weyl, Methods of Organic Chemistry, Tieme Stuttgart, New York, vol. E22 section 3.2: 443 and Benoiton, NL, Chemistry of Peptide Synthesis, Taylor and Francis, NY, 2006. Preferred active esters include p-nitrophenyl ester (PNP), N-hydroxysuccinimide (NHS) ester, and tetrafluorophenyl ester (TFP). The acyl halides can be prepared by a number of methods known to those skilled in the art, for example, by reacting a carboxylic acid with thionyl chloride or oxalyl chloride, see: Fieser, LF and Fieser, M. Reagents for Organic Synthesis, John Wiley and Sons, NY, 1967 and found in their references. These can be converted to other active esters such as p-nitrophenyl esters (PNP), which can also be used in active difunctional spacers, as described by Wu et al., Organic Letters, 2004, 6 (24): 4407. N-hydroxysuccinimide (NHS) esters can be prepared by reacting N, N-disuccinimidyl carbonate (CAS 74124-79-1) with a carboxylic acid of a compound in the presence of an organic base such as triethylamine or diisopropylethylamine in an aprotic solvent under anhydrous conditions as described in Example 35 WO2012012595 or by the use of N-hydroxysuccinimide and dicyclohexylcarbodiimide (DCC) or another dehydrating agent under anhydrous conditions. Tetrafluorophenyl esters (TFP) can be prepared by reacting carboxylic acids with 2,3,5,6-tetrafluorophenyltrifluoroacetate in the presence of an organic base such as triethylamine or diisopropylethylamine in an aprotic solvent under anhydrous conditions as reported by Wilbur, et al., Bioconjugate Chem., 2004, 15 (1): 203. It will be appreciated by one of skill in the art that the spacers shown in Table 1, among others, can be obtained using known methods and attached to amine-containing haptens using routine optimization of reaction conditions. These spacers allow the hapten to be attached to the thiol group on the support.

Table 1

<img file="PL2888286T3_D0028.tif" />

<img file="PL2888286T3_D0029.tif" />

[0072] Direct coupling of the amine functionality in the hapten and the carboxylic acid on the spacer building block in the presence of a coupling agent can also be used as a linking method. Preferred reagents are those that are commonly used in peptide synthesis. Peptide coupling reagents include, but are not limited to, O- (Benzotriazol-1-yl) -N, N, N ', N'-tetramethyluronium (TBTU, CAS # 125700-67-6) tetrafluoroborate, see Pruhs, S. , Org. Process. Res. Dev. 2006, 10: 441; N-Hydroxybenzotriazole (HOBT, CAS # 2592-95-2) with a carbodiimide dehydrating agent such as NN-dicyclohexylcarbodiimide (DCC), diisopropylcarbodiimide (DIC) or 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDCigydride) ., Geiger, R. Chem. Ber., 1970, 103 (3): 788; 3- (diethoxyphosphoryloxy) -1,2,3-benzotrazin-4 (3H) -one (DEPBT, CAS # 165534-43-0), see Liu, H. et al., Chinese Chemical Letters, 2002, 13 (7): 601; Bis (2-oxo-3-oxazolidinyl) phosphonic chloride; (ΒΟΡ-CI, CAS # 68641-49-6) see DiagoMeseguer, J et al. Synthesis, 1980, 7: 547-51 and others described in detail by Benoiton in Chemistry of Peptide Synthesis, CRC Press, Boca Raton, FL, 2005, Chapter 2 and a technical bulletin provided by Advanced Automated Peptide Protein Technologies (aapptec), 6309 Shepardsville Rd ., Louisville KY 40228, ph 888 692 9111; www.aapptec.com and references therein.

[0073] These methods form stable amide bonds linking the hapten to the spacer. Examples of spacers that can be obtained using known methods and attached to amine-containing haptens using routine optimization of reaction conditions using the methods described and cited above are shown but not limited to those in Table 2. These spacers allow the attachment of the hapten to the thiol group. on the carrier.

<img file="PL2888286T3_D0030.tif" />

[0074] Spacers may also be produced in a step by step process by sequentially attaching the appropriate chemical groups to the hapten, including the step of producing a linking functional group that is capable of binding to a carrier. See illustrative examples in the General Response Patterns section.

[0075] Additionally, where the hapten has a nucleophilic group, for example a thiol group, amino group, or a hydroxyl group, which will become the spacer's point of attachment, the spacer may also be formed by alkylating a thiol, amino or hydroxyl group. Any alkyl group which is suitably substituted with a moiety capable of undergoing a substitution reaction, for example, with an alkyl halide or sulfonic acid ester such as p-toluenesulfonate, may be used for the spacer attachment. Many examples of alkylation reactions are known to those skilled in the art and specific examples can be found in the general chemical literature and optimized by routine experimentation. For a discussion of the alkylation reaction with multiple references, see Chapter 10 March's Advanced Organic Chemistry, Smith, MB and March, J., John Wiley &amp; sons, Inc. NY, 2001. Other linkages may also be used, such as the reaction of a nucleophilic moiety, for example an amine, in a hapten with an isocyanate to form a urea or a reaction with an isocyanate to form a thiourea linkage, see Li, Z., et al., Phosphorus, Sulfur and Silicon and the Related Elements, 2003, 178 (2): 293-297. Spacers can be attached to haptens with hydroxyl groups by reaction with an isocyanate group to form a carbamate or carbamate bond. The spacer can be activated in a variety of ways using an iisocyanate functional group at one end and a carrier-reactable linking functional group, see Annunziato, ME, Patel, US, Ranade, M. and Palumbo, PS, Bioconjugate Chem., 1993 , 4: 212-218.

[0076] For haptens with a carboxylic acid group, the modes for attaching the spacer to the hapten include activating the carboxylic acid group as an acyl halide or active ester, examples of which are shown in Table 3, the preparation of which was described previously, followed by reaction with an amino group (-NH2 -), hydrazine (-NH-NH2-), hydrazide (-C (O) -NH-NH2-) or hydroxyl (-OH) on the spacer to form amide, hydrazide, diacylhydrazine or ester linkages or direct coupling of the carboxylic acid group to the amino group on the spacer or directly on the support with the peptide coupling reagent and / or the carbodiimide dehydrating reagent described previously, examples of which are shown in Tables 4 and 5. Procedures that can be found in the references cited previously for the preparation of activated esters and the use of peptide coupling agents can be used to attach carboxylic acid containing haptens to spacer building blocks and amino accessible protein carriers using routine optimization of reaction conditions.

Table 3

<td>o. o⁺Ν3-Ο₃5 ^ Λ O</td><td>Tl Tl 0 = 0 / \ 1 Tl Tl</td><td>-WHAT₂X</td><td>⁰ ιΓΎ^ν°²</td>

<td>Sulfo NHS and NHS</td><td>TFP</td><td>X = Cl, Br. Acyl chloride</td><td>PNP</td>

Table 4

<td>1 OH</td><td>^ = O zz b O-TJ S b m</td><td>.O \ / ^ ° WITH 1 OD = O ^- 1 _, Z L / ^ °</td><td>1 of CD o O Τ ' IIO ω ω,</td>

<td>HOBT</td><td>DEPT</td><td>BOP-CI</td><td>TBTU</td>

Table 5

<td>NCN- ^</td><td>NCN - ^^</td><td>^Cl^ 'NCN</td>

<td>diisopropylcarbodiimide (DIC)</td><td>Dicyclohexylcarbodiimide (DCC)</td><td>l-ethyl-3 (3- dimethylaminopropyl) carbodiimide HCI (EDC)</td>

Other electrophilic groups may be present on the hapten to attach a spacer, e.g.

IIO or electrophilic phosphorus group, for example: o 1p-ci

OR_C.

See: Małachowski, William P., Coward, James K., Journal of Organic Chemistry, 1994, 59 (25): 7616 or: and ^Q

POR_C.

OR_C.

Rc is alkyl, cycloalkyl, aryl, substituted aryl, arylalkyl.

See Aliouane, L., et al., Tetrahedron Letters, 2011, 52 (28): 8681.

[0080] Haptens that have an aldehyde or ketone group can be attached to walks using methods, including but not limited to reacting with an H2N-NH-C (O) hydrazide group - on a walk to form an acylhydrazone, see Chamów, SM , Kogan, TP, Peers, DH, Hastings, RC, Byrn, RA and Askenaszi, A., J. Biol. Chem., 1992,

267 (22): 15916. Examples of difunctional spacer hydrazide groups that allow attachment to a supported thiol group are shown in Table 6.

Table 6

<img file="PL2888286T3_D0031.tif" />

[0081] Haptens can also contain thiol groups that can be reacted with a carrier provided that the carrier has been modified to provide a group that can react with a thiol. Carrier groups can be modified by methods including, but not limited to, attachment of a group containing a maleimide functional group by reaction of the supported amino group with N-Succinimidyl maleimideacetate, (AMAS, CAS # 55750-61-3), succinimidyl iodoacetate (CAS # 151199-81 -4) or any of the bifunctional spacer groups shown in Table 1 to introduce a group that can react, resulting in attachment of the hapten to the support.

[0082] A linking functional group capable of forming a bond with a carrier may be any group capable of forming a stable bond and may be reactive with a wide variety of groups on the carrier. The linking functional group can preferably react with an amino group, a carboxylic acid group, or a thiol group on the support or derivative thereof. Non-limiting examples of the linking functional group are carboxylic acid group, acyl halide, active ester (as defined previously), isocyanate, isothiocyanate, alkyl halide, amino group, thiol group, maleimide group, acrylate group (H2C = CH-C (O) -) or vinylsulfone H2C = CH-SO2-) See Park, JW, et al., Bioconjugate Chem., 2012, 23 (3): 350. Linking functional groups can be present as part of a differentially activated spacer building block which can be reacted "step by step with the hapten" and the resulting hapten derivative can then be reacted with a carrier. Alternatively, the hapten can be derivatized with 'foot which has a precursor group that can be converted to a linking functional group by subsequent reaction. When the linking functional group on the walk is an amino group or a carboxylic acid group, the coupling reaction with the carboxylic acid or amino group on the support can be carried out directly by using peptide coupling reagents according to the procedures in the references cited above for these reagents.

[0083] Particular disulfide groups, for example, pyridyl disulfides, can be used as a walking linking functional group that can undergo thiol replacement on the support to form a mixed disulfide bond, see Ghetie, V., et al., Bioconjugate Chem. , 1990, 1: 24-31. These spacers can be attached by reacting the hapten having an amino group with an active ester that is attached to the spacer with the pyridyl disulfide group, examples of which include but are not limited to those shown in Table 7.

Table 7

<img file="PL2888286T3_D0032.tif" />

[0084] Most often the carrier is a protein, and the ε-amino groups of lysine residues can be used for attachment, either directly by reaction with an amine-reactive linking functional group or after derivatization with a thiol-containing group, including V-Succinimidyl SAcetylthioacetate, (SATA, CAS 76931-93-6) or an analog thereof followed by cleavage of the acetate group with hydroxylamine to expose the thiol group for reaction with the linking functional group on the hapten. Thiol groups can also be introduced onto the support by reduction of disulfide bonds within protein carriers with mild reducing agents, including but not limited to 2-mercaptoethylamine, see Bilah, M., et al., Bioelectrochemistry, 2010, 80 (1): 49, phosphine reagents, see Kirley, TL, Analytical Biochemistry, 1989, 180 (2): 231 or dithioerythritol (DTT, CAS 3483-12-3) Cleland, W., Biochemistry, 1964, 3: 480-482.

GENERAL RESPONSE SCHEMES

[0085] Compounds useful for producing the antibodies of the invention can be synthesized according to the general synthetic methods described below. The compounds of Formula (I) can be prepared by methods known to those skilled in the art. The following reaction schemes are intended to illustrate only examples of the invention and are not intended to limit the invention in any way.

[0086] Quetiapine derivatives can be prepared in a number of ways. The primary hydroxyl group in quetiapine, the starting compound (R1 and R2 = H) shown in Scheme 1, can be acylated using, for example, succinic anhydride and the method described by Fiedler, H., et al., Langmuir, 1994, 10: 3959 .

[0087] The resulting acid may be further functionalized as described elsewhere in this disclosure, or attached immediately to the support using any number of the aforementioned methods including those shown in the following examples.

Scheme 1

<img file="PL2888286T3_D0033.tif" />

[0088] The basic hydroxyl group of quetiapine may also be alkylated to form an ether according to the procedure of US20100069356, such as shown in Scheme 2, using an alkyl halide or sulfonate ester such as 4-bromomethylpentanoate in the presence of tetrabutylammonium bisulfate and aqueous sodium hydroxide to provide the acid. which can be used as described above.

<img file="PL2888286T3_D0034.tif" />

[0089] Compounds of Formula I wherein R² then CH2NHC (O) (CH2) mCO2H can be prepared according to Scheme 3. Reaction 2- (2- (2- (aminomethyl) -4- (dibenzo [b, f] [1,4] thiazepin-11-yl) piperazin- 1-yl) ethoxy) ethanol, prepared as described in Example 1, proceeds with an anhydrous cyclic compound such as succinic anhydride or glutaric anhydride in a solvent such as pyridine at temperatures ranging from room temperature to 60 ° C for about 48 hours. It will be recognized by those skilled in the art that the same chemical processes can be used to make compounds of Formula I where R.¹ this

CH2NHC (O) (CH2) mCO2H.

Scheme 4

<img file="PL2888286T3_D0035.tif" />

according to Scheme 4. Compounds of Formula I, where R.² to CH2NHC (O) (CH2) mCO2H, prepared as described in Scheme 1, are treated with? - t -butoxycarbonylpiperazine, diethyl cyanophosphonate and a base such as diisopropylethylamine. The reaction is performed in a solvent such as dichloromethane for about 2 hours at room temperature. Deprotection of the piperazinyl group is achieved with trifluoroacetic anhydride as described in Scheme 4 followed by reaction with a suitable anhydride such as succinic anhydride or maleic anhydride in the presence of a suitable base such as diisopropylethylamine. It will be recognized by those skilled in the art that the same chemical processes can be used to make compounds of Formula I where R.¹ this

<img file="PL2888286T3_D0036.tif" />

OO

Scheme 5

<img file="PL2888286T3_D0037.tif" />

[0091] Compounds of Formula I wherein R¹ this ^{H m} o can be prepared according to Scheme 5. Maleimide can be introduced by any method known in the art. Maleimide functional groups such as 2,5-dioxopyrrolidin-1-yl 2- (2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl) acetate, where m is 1, can be used in a solvent such as DMF or CH 2 Cl 2 and a base such as tributylamine or triethylamine. Alternatively, the deprotected piperazinyl group described in Scheme 4 can be elaborated in detail with maleimide functionality as described in Scheme 5 to provide compounds of Formula I where R¹ this

<img file="PL2888286T3_D0038.tif" />

It will be recognized by those skilled in the art that the same chemical processes can be used to make compounds of Formula I where R.² this

<img file="PL2888286T3_D0039.tif" />

<img file="PL2888286T3_D0040.tif" />

<img file="PL2888286T3_D0041.tif" />

Maleimide functionalized haptens, where R.¹ or R² these o can be conjugated to proteins according to the method shown in Scheme 6. Activation of lysine protein residues by acylation of epsilon nitrogen with N-succinimidyl S-acetylthioacetate (SATA) followed by subsequent hydrolysis of the S-acetyl group with hydroxylamine produces a nucleophilic sulfohydryl group . The conjugation of the sulfohydryl activated protein with the maleimide hapten (prepared as described in general scheme 3) follows a Michael addition reaction. Suitable proteins are known to those skilled in the art and include marine snail haemocyanin, bovine thyroglobulin, and ovalbumin. The same methodology can be used for the conjugation of proteins with maleimide-functionalized haptens where R.¹ or R² this

<img file="PL2888286T3_D0042.tif" />

[0092] Carboxylic acid functionalized haptens, wherein R¹ or R² this

CH2NHC (O) (CH2) mCO2H, can be conjugated to proteins according to the method shown in Scheme 7. Reaction with N-hydroxysuccinimide and a suitable coupling agent such as dicyclohexylcarbodiimide and a base such as tributylamine in a solvent such as DMF at a temperature of equal to about 20 ° C, for about 18 hours activates the carboxylic acid with the leaving group - hydroxypyrrolidine-2,5-dione. The activated linker and hapten can then be conjugated to the protein in a solvent such as a pH 7.5 phosphate buffer at about 20 ° C for about 2.5 hours. Suitable proteins are known to those skilled in the art and include marine snail haemocyanin, bovine thyroglobulin, and ovalbumin. The same methodology can be used for conjugates of proteins with carboxylic acid-functionalized haptens, where R¹ or R² this

<img file="PL2888286T3_D0043.tif" />

ANTIBODIES

[0093] The present disclosure is directed to an isolated antibody or binding fragment thereof which binds to quetiapine and which: (i) is generated in response to a conjugate of a compound of Formula I and an immunogenic carrier; or (ii) competes for an epitope that is the same as the antibody bound epitope of (i). The term antibody refers to a specific protein capable of binding an antigen or a portion thereof (according to this invention, capable of binding an antipsychotic drug or its metabolite). The antibody is produced in response to an immunogen that may have been introduced into a non-human host, e.g., an animal, by injection. The general term antibody includes polyclonal antibodies, monoclonal antibodies, and antibody fragments.

[0094] Antigen-binding antibody or antibody fragment refers to an intact antibody or fragment thereof that competes with intact antibody for binding. Generally speaking, an antibody or antigen binding antibody fragment is said to bind specifically to an antigen when the dissociation constant is less than or equal to 1 µΜ, preferably less than or equal to 100 nM, and most preferably less than or equal to 10 nM. Binding can be measured by methods known to those skilled in the art, for example using a BIAcore ™ instrument.

[0095] The antibody fragments contain a portion of an intact antibody, most commonly the antigen binding or variable region of the intact antibody. Binding fragments include Fab, Fab ', F (ab') 2, and Fv fragments; diabodies; linear antibodies; single chain antibody molecules; and multispecific antibodies made of antibody fragments. An antibody other than a bispecific or bifunctional antibody is to be understood as having each binding site identical.

[0096] As used herein, an epitope includes any protein capable of specifically binding an immunoglobulin or a T-cell receptor. Epitope determinants typically consist of chemically active surface groupings of molecules such as amino acids or sugar side chains, and typically having a specific three-dimensional structure. as well as a specific load. Two antibodies are said to bind the same epitope if one antibody has been shown to compete with the other antibody in a competitive binding study by any method well known to those skilled in the art (such as the BIAcore ™ method mentioned above). With regard to a hapten (such as quetiapine or other antipsychotic drug), an antibody can be produced against a non-antigenic hapten molecule by conjugating the hapten to an immunogenic carrier. Then an antibody is made that recognizes the epitope defined by the hapten. Isolated when used in the context of an antibody means modified by man from any natural state; that is, which, if it occurs in nature, has been altered or removed from its original environment, or both. For example, a naturally occurring antibody naturally present in a living animal in its natural state is not isolated, but the same antibody isolated from coexisting materials in its natural state is isolated in the sense the term is used herein. The antibodies may be present in a composition, such as an immunoassay reagent, that is not a naturally occurring composition and retains isolated antibodies within the meaning of that term as used herein.

[0097] Cross-reactivity refers to the reaction of an antibody with an antigen that was not used to induce that antibody.

[0098] Preferably, the antibody produced by the method of the invention will bind to the drug and any desired pharmacologically active metabolites. By altering the site of attachment of an immunogenic carrier to the compounds of the invention, selectivity and cross-reactivity with metabolites can also be engineered in antibodies. For quetiapine, cross-reactivity with quetiapine metabolites such as N-dealkyl quetiapine (norquetiapine), quetiapine sulfoxide, O-desalkyl quetiapine, or 7-hydroxyquetiapine may or may not be desired. Antibodies can be generated that detect many of these drugs and / or metabolites, or antibodies that can detect each of them separately (thus determining the specific binding properties of the antibody) can be generated. The antibody specifically binds to one or more compounds when the binding of one or more compounds through them is equimolar or substantially equimolar.

[0099] Methods for producing such antibodies include inoculating a non-human host with a conjugate as described herein. Suitable hosts include, but are not limited to, mice, rats, hamsters, coffee houses, rabbits, chickens, donkeys, horses, monkeys, chimpanzees, orangutans, gorillas, and any species capable of producing a mature immune response. Immunization procedures are well defined in the art and are outlined in numerous treatises and publications including The Immunoassay Handbook, 2nd Edition, edited by David Wild (Nature Publishing Group, 2000).

[0100] Preferably, an immunogen containing the features of the present invention is administered to a non-human host, eg, an animal, in association with an adjuvant. Suitable adjuvants include, but are not limited to, Freund's adjuvant, powdered aluminum hydroxide (alum), aluminum hydroxide including Bordetella pertussis, and monophosphoryl lipid A-synthetic trehalose dicorinomycolate (MPL-TDM).

[0101] Typically, the immunogen or combination of immunogen and adjuvant is injected into the non-human mammalian host by one or more subcutaneous or intraperitoneal injections. Preferably, the immunization program is carried out for at least one week, and more preferably, for two or more weeks. Polyclonal antibodies produced in this manner can be isolated and purified using methods well known in the art.

[0102] Monoclonal antibodies can be made by the hybridoma methods reviewed by Kohler and Milstein, e.g., Nature 256: 495-497 (1975). Methods with hybridoma cells typically involve immunizing a non-human host or lymphocytes from a non-human host, harvesting lymphocytes that secrete or have the potential to secrete a monoclonal antibody, fuse the lymphocytes with immortalized cells, and select cells that secrete the desired monoclonal antibody.

[0103] The non-human host may be immunized to elicit lymphocytes that produce or are capable of producing immunogen-specific antibodies. Alternatively, lymphocytes can be immunized in vitro. If human cells are desired, peripheral blood lymphocytes can be used, although spleen cells or lymphocytes from other mammalian sources are preferred.

[0104] Lymphocytes can be fused with an immortalized cell line to form hybridoma cells, a process that can be facilitated by the use of a fusion agent, eg, polyethylene glycol. By way of example, mutant rodent, bovine or human myeloma cells immortalized by transformation may be used. In general, pure populations of hybridoma cells are preferred, as opposed to unfused immortalized cells. Thus, after fusion, cells can grow in a suitable medium that inhibits the growth or survival of unfused immortalized cells, for example, by using mutant myeloma cells that lack the enzyme hypoxanthine guanine phosphoribosyltransferase (HGPRT). In such a case, hypoxanthine, aminopterin, and thymidine can be added to the medium (HAT medium) to prevent growth of HGPRT deficient cells while allowing hybridoma cells to grow.

[0105] Advantageously, effectively immortalized cells after fusion can be isolated from mixed populations by selection in a medium such as HAT and support stable and high level expression of antibodies after fusion.

[0106] Preferred immortalized cell lines include the myeloma cell lines available from the American Type Culture Collection, Manassas, VA.

[0107] Since hybridoma cells typically produce the antibody extracellularly, the culture media can be tested for the presence of antipsychotic drug-specific monoclonal antibodies. In vitro binding studies using immunoprecipitation, for example, a radioimmunoassay (RIA) or enzyme immunoassay (ELISA), can be used to measure the binding specificity of monoclonal antibodies.

[0108] The monoclonal antibody producing hybridoma cells can be isolated as single clones by limiting the dilution procedures and sub-cultured. Suitable culture media include, but are not limited to, Dulbecco's Modified Eagle's Medium, RPMI-1640, and polypeptide free, polypeptide reduced or serum free media, e.g., Ultra DOMA PF or HL-1, available from Biowhittaker. Walkersville, MD. Alternatively, the hybridoma cells can grow in vivo as ascites.

[0109] Monoclonal antibodies can be isolated and / or purified from the culture medium or ascites fluid by standard immunoglobulin (Ig) purification procedures, including but not limited to polypeptide A-SEPHAROSE, hydroxyapatite chromatography, gel electrophoresis, dialysis, precipitation by ammonium sulfate and affinity chromatography.

[0110] Monoclonal antibodies can also be made by recombinant methods, such as described in US Patent No. 4, 166, 452. DNA-encoding monoclonal antibodies can be isolated and sequenced using standard procedures, e.g., using oligonucleotide probes that bind specifically to murine antibody heavy and light chain genes, preferably to a DNA probe isolated from monoclonal antibodies from hybridoma cell lines secreting drug-specific antibodies. antipsychotic.

[0111] Antibody fragments that contain specific binding sites for an antipsychotic drug can also be produced. Such fragments include, but are not limited to, F (ab ') 2 fragments that can be produced by pepsin digestion of the antibody molecule, and Fab fragments that can be generated by reducing the disulfide bridges of F (ab') 2 fragments. Alternatively, Fab expression libraries can be constructed to allow for the quick and easy identification of monoclonal Fab fragments with the desired specificity (Huse et al., Science 256: 1270-1281 (1989)). All Fab, Fv and ScFv antibody fragments can be expressed in and secreted by Escherichia coli, allowing the production of large amounts of these fragments.

[0112] Alternatively, Fab'-SH fragments can be directly recovered from E. coli and chemically coupled to form F (ab ') 2 fragments (Carter et al., BioTechnology 10: 163167 (1992)). Other techniques for producing antibody fragments are known to those of skill in the art. Single chain Fv (scFv) fragments are also provided (see US Patent Nos. 5,761,894 and 5,587,458). The Fv and sFv fragments are the only types with intact joining sites that are devoid of permanent regions; thus, it is likely that they will show reduced, non-specific binding. The antibody fragments can also be a linear antibody, e.g., as described in, for example, US Patent No. 5,642,870. Such linear antibody fragments can be monospecific or bispecific.

KITS AND EQUIPMENT FOR TESTING

[0113] The test kit (also referred to as a reagent kit) may also be equipped with an antibody, such as described above. A representative set of reagents may include an antibody that binds to the antipsychotic drug, quetiapine, a complex containing an antipsychotic drug analogue, or a derivative thereof conjugated to a label, and may optionally also include one or more calibrators containing a known amount of the antipsychotic drug or related template.

[0114] The term test kit refers to the pool of materials and reagents that is used to perform the test. The reagents may be supplied in packaged combination in the same or in separate containers, depending on their reactivity and cross-stability, and in liquid or lyophilized form. The amounts and ratios of the reagents provided in the kit may be selected so as to provide optimal results for a particular application. The test kit incorporating the features of the present invention comprises antibodies that bind quetiapine. The kit may additionally contain competing quetiapine partners and calibration and adjustment materials.

[0115] The term calibration and control material refers to any standard or reference material containing a known amount of the analyte. A sample suspected of containing the analyte and appropriate calibration material are tested under similar conditions. The concentration of the analyte is calculated by comparing the results obtained for the unknown sample with the results obtained for the standard. This is usually done by creating a calibration curve.

[0116] The antibodies produced in accordance with the invention may be included in a kit, container, packet, or dispenser, together with instructions for their use. When the antibodies are provided in the kit, the various components for immunoassay may be packaged in separate containers and mixed prior to use. Such packaging of the ingredients separately can allow long-term storage without significantly reducing the effect of the active ingredients. Furthermore, the reagents may be packaged under inert conditions, e.g., positive pressure of nitrogen gas, argon gas, or the like, which is particularly advantageous for reagents that are sensitive to air and / or moisture.

[0117] The reagents included in the kits incorporating the features of the present invention can be provided in all types of containers such that the activities of the various components are substantially retained while the components themselves are not substantially adsorbed or modified by the materials of the container.

[0118] Suitable containers include, but are not limited to, ampoules, bottles, test tubes, vials, flasks, syringes, envelopes, e.g., foil lined, and the like. The containers may be composed of any suitable material including, but not limited to, glass, organic polymers, e.g., polycarbonate, polystyrene, polyethylene, etc., ceramics, metal, e.g., aluminum, metal alloys, e.g., steel, cork. and the like. Additionally, the containers may include one or more sterile access ports, e.g., for needle access such as may be provided through a septum. Preferred barrier materials include rubber and polytetrafluoroethylene of the type sold under the trade name TEFLON from DuPont (Wilmington, Germany). Additionally, the containers may contain two or more compartments separated by spacers or membranes that can be removed to allow the ingredients to be mixed.

[0119] Reagent kits incorporating the features of the present invention may also be provided with materials containing instructions. The instructions may be printed, e.g., on paper, and / or provided on an electronic device readable medium. Alternatively, instructions may be provided by directing the user to a website, e.g., specified by the kit manufacturer or distributor, and / or via e-mail.

[0120] The antibody may also be provided as part of a test device. Such test equipment includes lateral flow test equipment. A common type of disposable lateral flow test equipment includes a liquid sample receiving zone or space, a conjugation zone, and a reaction zone. These test devices are usually known as lateral flow test strips. They use a porous material, e.g. nitrocellulose, defining a fluid flow path capable of supporting capillary flow. Examples include those shown in U.S. Patent Nos. 5 559 041, 5 714 389, 5 120 643, and 6 228 660.

[0121] Another type of test device is a splined non-porous test device for inducing capillary flow. Examples of such test devices include an open lateral flow device as disclosed in PCT International Publications Nos. WO 2003/103835, WO 2005/089082, WO 2005/118139 and WO 2006/137785.

[0122] In a non-porous test rig, the test rig generally has at least one sample adding zone, at least one conjugation zone, at least one reaction zone, and at least one absorption zone. The zones form the flow path through which the sample flows from the sample addition zone to the uptake zone. Also included in the reaction zone are capture elements, such as antibodies capable of binding to the analyte, optionally placed on the device (such as by coating); and the labeled conjugate material also capable of participating in reactions that allow the determination of the concentration of the analyte placed on the device in the conjugation zone, the labeled conjugate material having a marker for detection in the reaction zone.

[0123] The conigate material is dissolved as the sample flows through the conjugation zone creating a trail of dissolved labeled conjugate material and the sample that flow "downstream" into the reaction zone.

[0124] As the contrail of the conjugate enters the reaction zone, the conjugate material is captured by the capture elements, such as through a complex of conjugate material and analyte (such as in a sandwich study) or directly (as in a competition study). Unbound dissolved conjugate material will be swept out of the reaction zone into at least one wicking zone. Such devices may include projections or microcolumns in the flow path.

[0125] An apparatus such as that disclosed in US Patent Publications Nos. US20060289787A1 and US 20070231883A1 and US Patent Nos. 7,416,700 and 6,139,800 is capable of detecting bound conjugate material in the reaction zone. Common labels include fluorescent dyes that can be detected with devices that excite fluorescent dyes and include a detector capable of detecting fluorescent dyes.

IMMUNOLOGICAL TESTS

[0126] Antibodies produced in this way may be used in immunoassays to recognize / bind to an antipsychotic drug, thereby detecting the presence and / or amount of the drug in a patient sample. Preferably, the test format is a competitive immunoassay format. Such a test format and other studies are described in, inter alia, Hampton et al. (Serological Methods, A Laboratory Manual, APS Press, St. Paul, MN 1990) and Maddoks et al. (J. Exp. Med. 158: 12111,1983).

[0127] The term analyte refers to any substance or group of substances whose presence or quantity is to be determined. Representative antipsychotic drug analytes include, but are not limited to, risperidone, paliperidone, olanzapine, aripiprazole, and quetiapine.

[0128] Competitive binding partner refers to a substance or group of substances that can be used in a competitive immunoassay that behaves similar to an analyte in terms of binding affinity for an antibody.

Representative competing partner compounds include, but are not limited to, antipsychotic drug derivatives and the like.

[0129] The term detection when used with an analyte refers to any quantitative, semi-quantitative or qualitative method, as well as to all other methods of determining the analyte in general, and in particular to an antipsychotic drug. For example, a method that detects merely the presence or absence of an antipsychotic drug in a sample is within the scope of the present invention, as are methods that provide data such as the amount or concentration of an antipsychotic drug in a sample. The terms detecting, determining, identifying, and the like are used herein as synonyms and are all included within the scope of the present invention.

[0130] A preferred embodiment of the invention is a competitive immunoassay, wherein the antibodies that bind the antipsychotic drug or drug or its competitive binding partner are attached to a solid support (such as the reaction zone in a lateral flow tester) and the labeled drug or its competitive binding partner or labeled antibody, as appropriate, and a sample taken from the non-human host passes over the solid support, and the amount of label detected attached to the solid support can be related to the amount of drug in the sample.

[0131] Any sample that is suspected of containing an analyte, e.g., an antipsychotic, can be analyzed according to the methods of presently preferred embodiments. The sample may be pretreated if desired and may be prepared in any convenient medium that will not interfere with the assay. Preferably, the sample comprises an aqueous medium such as a body fluid from a non-human host, more preferably plasma or serum.

[0132] It should be understood that all immunoassay methods using antibodies are contemplated for use in accordance with the presently preferred embodiments, including studies where the antibodies are bound to a solid phase and studies where the antibodies are in a liquid medium. Immunoassay methods that may be used to detect analytes using the antibodies of the invention include, but are not limited to, competitive (reagent-limited) testing, where a labeled analyte (analyte analog) and the analyte in the sample compete for antibodies and one-sided immunometric tests where the antibody is labeled; and the like.

[0133] The present invention is further described by the following examples. The examples are provided solely to illustrate the invention by referring to specific embodiments.

[0134] All examples were performed using standard techniques that are well known and routine to those skilled in the art, unless specifically stated otherwise.

The routine molecular biology techniques of the following examples can be performed as described in standard laboratory textbooks such as Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Habor Laboratory Press, Cold Spring Harbor, New York (1989) ).

Reference is made herein to co-pending applications entitled Haptens of Aripiprazole (file number PRD3265USPSP, US provisional application No. 61 / 691,450, filed August 21, 2012), Haptens of Olanzapine (file number PRD3266USPSP, US provisional application No. 61 / 691,454, filed August 21, 2012), Haptens of Paliperidone (file number PRD3267USPSP, U.S. provisional application No. 61 / 691,459, filed August 21, 2012). Haptens of Quetiapine (file number PRD3268USPSP, US provisional application No. 61 / 691.462, filed August 21, 2012), Haptens of Risperidone and Paliperidone (file number PRD3269USPSP, US provisional application No. 61 / 691.469, filed August 21, 2012), Antibodies to Aripiprazole Haptens and Use Thereof (file number CDS5128USPSP, US provisional application No. 61 / 691,544, filed August 21, 2012), Antibodies to Olanzapine Haptens and Use Thereof (file number CDS5132USPSP, U.S. provisional application No. 61 / 691,572, filed August 21, 2012), Antibodies to Paliperidone Haptens and Use Thereof (file number CDS5126USPSP, U.S. provisional application No. 61 / 691,634, filed August 21, 2012), Antibodies to Risperidone Haptens and Use Thereof (file number CDS5130USPSP, US provisional application No. 61 / 691,615, filed August 21, 2012), Antibodies to Aripiprazole and Use Thereof (file number CDS5129USPSP, US provisional application No. 61 / 691,522, filed August 21, 2012). Antibodies to Olanzapine and Use Thereof (file number CDS5133USPSP, US provisional application No. 61 / 691.645, filed August 21, 2012), Antibodies to Paliperidone and Use Thereof (file number CDS5127USPSP, US provisional application No. 61 / 691.692, filed August 21, 2012), Antibodies to Quetiapine and Use Thereof "(File Number CDS5135USPSP, US provisional application No. 61 / 691,659, filed August 21, 2012), Antibodies to Risperidone and Use Thereof" (File Number CDS5131USPSP, US provisional application No. 61 / 691,675, filed August 21, 2012) and Antibodies to Risperidone and Use Thereof (File number CDS5145USPSP, US provisional application No. 61 / 790,880, filed March 15, 2013).

EXAMPLE 1

2- (2- (2- (aminomethyl) -4- (dibenzo [b, f] [1,4] thiazepin-11-yl) piperazin-1-yl) ethoxy) ethanol

Stage A

Piperazine-2-carbonitrile

[0136]

<img file="PL2888286T3_D0044.tif" />

[0137] To a mixed solution of tetrahydrofuran (300 ml) and ethylenediamine (108.2 g) at 30 ° C, 2-chloroacrylonitrile (105.0 g) was added dropwise over a period of 2 hours and stirred for an additional 6 hours at 30 ° C. The reaction mixture was cooled to 20 ° C and a precipitate formed. The reaction mixture was filtered and the pH of the filtrate was adjusted to 4 by adding 35% hydrochloric acid. The resulting precipitate was collected by filtration. The combined solids were dissolved in a 20% hydrochloric acid solution and then poured into a THF solution to precipitate the title compound which was dried under reduced pressure and used in the next reaction without further purification. 1H NMR: (D2O, 400 MHz): δ (ppm) 5.004.97 (m, 1H), 3.79 (d, J = 4.8 Hz, 2H), 3.62-3.44 (m, 4H ).

Stage B

Tert-Butyl 3-cyanopiperazine-1-carboxylate [0138]

<img file="PL2888286T3_D0045.tif" />

[0139] To a solution of the piperazine-2-carbonitrile compound prepared as described in the previous step (90.6 g, 0.492 mol) was added triethylamine (206 ml, 1.476 mol) and Boc2O (117 g, 0.542 mol). The reaction mixture was stirred at room temperature overnight and then concentrated. The rest was purified by silica gel chromatography to afford the title compound.

1 H NMR: (CDCl 3, 400 MHz): δ (ppm) 4.06-3.91 (m, 3H), 3.28-2.83 (m, 4H), 1.47 (s, 9H).

Stage C.

Tert-Butyl 3-cyano-4- (2- (2-hydroxyethoxy) ethyl) piperazine-1-carboxylate

[0140]

<img file="PL2888286T3_D0046.tif" />

[0141] A tert-Butyl 3-cyanopiperazine-1-carboxylate solution, prepared as described in the previous step, (10 g, 0.047 mol) and 2- (2-hydroxyethoxy) acetaldehyde (14.8 g) (see Bodin, A., Contact Dermatitis, 2001, 44: 207) in dichloromethane was treated with formic acid (12.7 g) and the reaction mixture was stirred at room temperature overnight. Sodium cyanoborohydride (7.2 g, 0.118 mol) was added portionwise. The reaction mixture was stirred at room temperature for 3 hours, then water was added and extracted with dichloromethane. The organic layer was washed with brine, dried over sodium sulfate, filtered and concentrated. The crude product was purified by column chromatography to afford the product.

1 H NMR: (CDCl 3, 400 MHz): δ (ppm) 4.15 (s, 1H), 3.69-3.63 (m, 4H), 3.58 (d, J = 4.4 Hz, 2H ), 3.47-3.44 (m, 4H), 2.61 (d, J = 5.2 Hz, 2H), 2.51-2.48 (m, 4H), 1.43 (s, 9H).

Step D tert-butyl 3- (aminomethyl) -4- (2- (2-hydroxyethoxy) ethyl) piperazine-1-carboxylate [0142]

<img file="PL2888286T3_D0047.tif" />

[0143] To a solution of tert-butyl 3-cyano-4- (2- (2-hydroxyethoxy) ethyl) piperazine-1-carboxylate, prepared as described in the previous step, (9.9 g, 33.1 mmol) in methanol (20 ml), Raney Nickel (15 g) was added. The reaction solution was stirred at room temperature overnight under a hydrogen atmosphere (50 psi). The mixture was filtered and concentrated to provide the product which was used in the next step without further purification.

ESI-MS (M + 1): 304 calculated. for C14H29N3O4 303.

Stage E

Tert-butyl 4- (2- (2-hydroxyethoxy) ethyl) -3 - ((2,2,2-trifluoroacetamido) methyl) piperazine-1-carboxylate

[0144]

<img file="PL2888286T3_D0048.tif" />

To a solution of tert-butyl 3- (aminomethyl) -4- (2- (2-hydroxyethoxy) ethyl) piperazine-1-carboxylate prepared as described in the previous step (8.8 g) in dichloromethane (100 ml) was added triethylamine (8.8 g, 87.0 mmol) and trifluoroacetic acid anhydride (6.1 g, 29.0 mmol). The reaction mixture was stirred at room temperature for 12 hours, diluted with dichloromethane and washed with water. The organic layer was washed with brine, dried over sodium sulfate, filtered and concentrated to give the crude product which was purified by column chromatography to afford the title compound. ESI-MS (M + 1): 400 calc. for C16H28F3N3O5 399.

Stage F

2,2,2-Trifluoro-N - ((1- (2- (2-hydroxyethoxy) ethyl) piperazin-2-yl) methyl) acetamide

[0146]

<img file="PL2888286T3_D0049.tif" />

A solution of tert-butyl 4- (2- (2-hydroxyethoxy) ethyl) -3 - ((2,2,2-trifluoroacetamido) methyl) piperazine-1-carboxylate, prepared as described in the previous step, (8.6 g, crude) in a methanolic hydrogen chloride solution (20 mL) was stirred at room temperature for 1 hour then concentrated to afford the title compound which was used without further purification. ESI-MS (M + 1): 300 calcd. for C11H20F3N3O3 299.

Stage G

2 - ((2-Nitrophenyl) thio) benzoic acid [0148]

<img file="PL2888286T3_D0050.tif" />

[0149] To a solution of 2-mercapto-benzoic acid (30 g, 0.195 mol) in isopropanol (500 ml) at room temperature was added 1-fluoro-2-nitro-benzene (30.2 g, 0.214 mol), water (100 ml) and potassium hydroxide (31.1 g, 0.555 mol). The reaction mixture was stirred at room temperature overnight, quenched with water, and diluted with ethyl acetate. The aqueous phase was extracted with ethyl acetate (3 x 400 ml) and the combined organic extracts were washed with saturated aqueous sodium chloride solution (500 ml), dried over magnesium sulfate, filtered and concentrated. The crude residue was purified by silica gel flash column chromatography to afford the title compound. ESI-MS (M + 1): 276 calcd. for C13H9NO4S 275. 1 H NMR: (CDCl 2, 400 MHz): δ (ppm) 8.12-8.07 (m, 2H), 7.54-7.43 (m, 2H), 7.42-7.39 (m, 2H), 7.35-7.31 (m, 1H), 7.12-7.09 (m, 1H).

Stage H.

2 - ((2-Aminophenyl) thio) benzoic acid [0150]

<img file="PL2888286T3_D0051.tif" />

[0151] Pd ​​/ C (8 g) was added to a solution of 2 - ((2-nitrophenyl) thio) benzoic acid prepared as described in the previous step (43.3 g, 0.157 mol) in ethyl acetate (500 ml). The reaction solution was stirred at room temperature overnight under an atmosphere of hydrogen gas. The mixture was filtered and concentrated to provide the title compound. ESI-MS (M + 1): 246 calcd. for C13H11NO2S 245.¹H NMR: (CDCl 3, 400 MHz): δ (ppm) 8.20-8.17 (m, 1H), 7.51-7.48 (m, 1H), 7.36-7.30 (m, 2H), 7.21-7.17 (m, 1H), 6.88-6.80 (m, 3H).

Stage I

Dibenzo [b, f] [1,4] thiazepin-11 (10H) -one

[0152]

<img file="PL2888286T3_D0052.tif" />

[0153] To a solution of 2 - ((2-aminophenyl) thio) benzoic acid prepared as described in the previous step (30 g, 0.122 mol) in dichloromethane (300 ml) was added EDCl (35.2 g, 0.183 mol). triethylamine (51 mL, 0.366 mol) and HOBT (24.7 g, 0.183 mol). The reaction mixture was stirred at room temperature for 12 hours, washed with 1M aq. HCl, saturated aq. Sodium bicarbonate, saturated aq. Sodium chloride, and dried over MgSO4. The solution was filtered, concentrated, and purified via column chromatography to afford the title compound. ESI-MS (M + 1): 228 calculated. for C13H9NOS 227.¹H NMR: (CDCl 3, 400 MHz): δ (ppm) 7.70-7.67 (m, 1H), 7.58-7.52 (m, 2H), 7.50-7.42 (m, 2H), 7.39-7.35 (m, 1H), 7.24-7.22 (m, 1H), 7.17-7.13 (m, 1H).

Stage J

11-Chlorodibenzo [b, f] [1,4] thiazepine [0154]

<img file="PL2888286T3_D0053.tif" />

[0155] A solution of dibenzo [b, f] [1,4] thiazepin-11 (10H) -one, prepared as described in the previous step, (14.6 g, 64 mmol) in phosphorus oxychloride (20 ml) was heated under a condenser. feedback for 2 hours. The mixture was concentrated to provide the crude product which was used immediately without further purification. ESI-MS (M + 1): 246 calcd. for C13H8GNS 245.

Stage K

N - ((4- (Dibenzo [b, f] [1,4] thiazepin-11-yl) -1- (2- (2-hydroxyethoxy) ethyl) piperazin-2-yl) methyl) -2,2,2- trifluoroacetamide

[0156]

<img file="PL2888286T3_D0054.tif" />

To a solution of 11-chlorodibenzo [b, f] [1,4] thiazepine prepared as described in the previous step (2 g, crude) in dioxane (20 ml) was added Pd2 (dba) 3 (327 mg, 0.357 mmol), BINAP (225 mg, 0.357 mmol), triethylamine (6 ml, 42.9 mmol) and 2,2,2-trifluoro-N - ((1- (2- (2-hydroxyethoxy) ethyl) piperazin-2-yl ) methyl) acetamide, prepared as described in Step F, (2.4 g, crude). The resulting mixture was refluxed overnight under nitrogen, filtered through CELITE ™ and concentrated. The rest was purified by silica gel chromatography to afford the title compound. ESI-MS (M + 1): 509 calculated. for C24H27F3N4O3S 508.

Stage L.

2- (2- (2- (Aminomethyl) -4- (dibenzo [b, f] [1,4] thiazepin-11-yl) piperazin-1-yl) ethoxy) ethanol

[0158]

<img file="PL2888286T3_D0055.tif" />

A mixture of N - ((4- (dibenzo [b, f] [1,4] thiazepin-11-yl) -1- (2- (2-hydroxyethoxy) ethyl) piperazin-2-yl) methyl) -2, 2,2-trifluoroacetamide, prepared as described in the previous step, (2.0 g) and an aqueous solution of potassium carbonate (5%) (15 ml) in methanol (20 ml) were stirred at room temperature for 18 hours and extracted with ethyl acetate. The organic layers were washed with saturated aqueous sodium chloride, dried over sodium sulfate, filtered, evaporated to give the crude product which was purified by column chromatography followed by preparative HPLC to afford the title compound as a yellow solid. ESI-MS (M + 1): 413 calculated. for C22H28N4O2S 412. 1H NMR: (CDCl3, 400 MHz): δ (ppm) 7.52-7.50 (m, 1H), 7.41-7.31 (m, 4H), 7.17-7, 12 (m

1H), 7.02-7.00 (m, 1H), 6.89-6.84 (m, 1H), 3.66-3.59 (m, 5H), 3.54-3.51 ( m, 2H), 3.49-3.38 (m, 1H), 3.19-3.12 (m, 1H), 3.03-2.88 (m, 2H), 2.79-2. 53 (m, 5H).

EXAMPLE 2

N - ((4- (Dibenzo [b, f] [1,4] thiazepin-11-yl) -1- (2- (2-hydroxyethoxy) ethyl) piperazin-2-yl) methyl) -2- (2.5 -dioxo-2,5-dihydro-1H-pyrrol-1-yl) acetamide

<img file="PL2888286T3_D0056.tif" />

[0161] To a solution of 2- (2- (2- (aminomethyl) -4- (dibenzo [b, f] [1,4] thiazepin-11-yl) piperazin-1-yl) ethoxy) ethanol prepared as described in Example 1, (7.8 mg, 19.0 μmol) in 410 μΙ DMF and 8.9 μl tributylamine, 480 μl of a DMF solution of N- (amaleimidoacetoxy) succinimide ester (AMAS, 10 mg / ml, 4.8 mg, 19, 0 μmol). The resulting solution was left on the stirrer for 60 minutes at 20 ° C, then used as such in the conjugation reaction with a thiol-activated protein.

EXAMPLE 3

2- {2- [4- (3-Aminomethyl-dibenzo [b, f] [1,4] thiazepin-11-yl) -piperazin-1-yl] ethoxy} -ethanol

[0162]

<img file="PL2888286T3_D0057.tif" />

Stage A

11-oxo-10,11-dihydrodibenzo [b, f] [1,4] thiazepine-3-carboxylic acid

[0163]

<img file="PL2888286T3_D0058.tif" />

A mixture of 2-amino-benzenethiol (1.34 mL, 12.5 mmol), 2-bromo-terephthalic acid (1.54 g, 6.3 mmol), copper (I) oxide (0.50 g, 3.5 mmol), quinoline (6.3 ml) and pyridine (0.63 ml) were heated in an oil bath at 180 ° C under nitrogen for 20 hours, then cooled to room temperature. Concentrated hydrochloric acid (20 ml) was added slowly while cooling in cold water with stirring. The resulting precipitate was filtered off, washed with water and dried to obtain the crude title compound (2 g). LC-MS: m / z 270 (M-1).

Stage B

11-Chloro-dibenzo [b, f] [1,4] thiazepine-3-carbonyl chloride

[0165]

[0166] To a suspension of 11-oxo-10,11-dihydrodibenzo [b, f] [1,4] thiazepine-3-carboxylic acid prepared as described in the previous step (0.41 g) in toluene (6.5 ml ) DMF (0.125 ml) and thionyl chloride (6.5 ml) were added. The mixture was heated in an oil bath at 80 ° C under nitrogen overnight. The resulting solution was concentrated to dryness. The crude product was used in the next step.

Stage C.

11-Chloro-dibenzo [b, f] [1,4] thiazepine-3-carboxylic acid amide

[0167]

[0168] A solution of 11-chloro-dibenzo [b, f] [1,4] thiazepine-3-carbonyl chloride prepared as described in the previous step (approx. 1.5 mmol) in dichloromethane (10 mL) was treated with a solution of 1,4-dioxane in ammonia (0.5 M, 9 mL) in an ice bath. The resulting suspension was stirred at room temperature for 1 hour and the reaction mixture was quenched with water (10 mL). The resulting precipitate was filtered off, washed with water and dichloromethane and dried. The organic layer of the filtrate was washed with saturated aqueous sodium bicarbonate and concentrated to an additional off-white product that was used in the next step without further purification. LC-MS: m / z 289 (M + 1).¹H NMR (DMSO-d6, 400 MHz): δ (ppm) 8.19 (br, 1H), 8.00-7.96 (m, 2H), 7.90 (d, 1H), 7.64 ( br, 1H), 7.56 (m, 1H), 7.47 (m, 1H), 7.31 (m, 2H).

Step D Acid amide

11- {4- [2- (2-Hydroxyethoxy) ethyl] -piperazin-1-yl} dibenzo [b, f] [1,4] thiazepine-3-carboxylic acid

[0169]

<img file="PL2888286T3_D0059.tif" />

[0170] To a solution of 11-chloro-dibenzo [b, f] [1,4] thiazepine-3-carboxylic acid amide prepared as described in the previous step (0.40 g) in DMF (1.5 ml) and toluene (1.5 ml), 2- (2-piperazin-1-yl-ethoxy) ethanol (0.50 g, 2.9 mmol) was added. The solution was heated in an oil bath at 110 ° C under nitrogen for 5 hours, concentrated and purified (silica gel, 2-5% eluent: methanol-dichloromethane containing ammonia) to afford the title compound as an off-white solid. LC-MS: m / z 427 (M + 1).

Stage E

2- {2- [4- (3-Aminomethyl-dibenzo [b, f] [1,4] thiazepin-11-yl) -piperazin-1-yl] ethoxy} -ethanol

[0171]

<img file="PL2888286T3_D0060.tif" />

[0172] To a solution of 2- {2- [4- (3-aminomethyl-dibenzo [b, f] [1,4] thiazepin-11-yl) -piperazin-1-yl] -ethoxy} -ethanol prepared as described in In the previous step, (0.24 g, 0.56 mmol) in THF (15 ml) a 1 M solution of lithium aluminum hydride in THF (6 ml, 6 mmol) was added. The white suspension was heated in an oil bath at 70 ° C under nitrogen for 2 hours. The reaction slurry was quenched by slowly adding saturated aqueous sodium sulfate in an ice bath. The solution phase was separated and the precipitate was extracted with THF (5 X 10 mL). The combined organic phases were concentrated and purified (silica gel, 2-5% eluent: methanol-dichloromethane containing ammonia) to yield the title compound as an off-white solid. LC-MS: m / z 413 (M + 1). 1 H NMR (CDCl 3, 400 MHz) δ (ppm) 7.47 (s, 1H), 7.38 (m, 1H), 7.26 (m, 2H, overlap with solvent), 7.17 (m, 1H), 7.06 (m, 1H), 6.88 (m, 1H), 3.85 (s, 2H), 3.76-3.46 (m, 11H, contains exchangeable protons), 2.662.57 (m, 8H).

EXAMPLE 4

2- (2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl) -N - ((11- (4- (2- (2-hydroxyethoxy) ethyl) piperazin-1-yl) dibenzo [b , f] [1,4] thiazepin-3-yl) methyl) acetamide

[0173]

<img file="PL2888286T3_D0061.tif" />

[0174] To a solution of 2- {2- [4- (3-aminomethyl-dibenzo [b, f] [1,4] thiazepin-11-yl) -piperazin-1-yl] -ethoxy} -ethanol prepared as described in Example 3, (5.6 mg, 13.6 μmol) in 295 μL DMF and 6.4 μL tributylamine were added 340 μL DMF solution of N- (amaleimidoacetoxy) succinimide ester (AMAS, 10 mg / mL, 3.4 mg, 13.6 μmol). The resulting solution was left on the stirrer for 60 minutes at 20 ° C, then used as such in the conjugation reaction with a thiol-activated protein.

EXAMPLE 5

Conjugate N - ((4- (dibenzo [b, f] [1,4] thiazepin-11-yl) -1- (2- (2-hydroxyethoxy) ethyl) piperazin-2-yl) methyl) -2- (2, 5-dioxo-2,5-dihydro-1H-pyrrol-1-yl) acetamide-bovine thyroglobulin

Stage A

Bovine thyroglobulin (BTG) reaction with SATA:

[0175] To 3.0 ml of a solution of bovine thyroglobulin (BTG, 20.0 mg, 0.03 μmol) in 100 mM phosphate buffer pH 7.5 was added 276.0 μl of a DMF solution of N-succinimidyl S-acetylthioacetate (SATA, 25 mg / ml, 6.9 mg, 30.0 µmol). The resulting solution was incubated at 20 ° C for 1 hour on a roller mixer. The reaction mixture was purified on a Sephadex G-25 column using 100 mM phosphate buffer, 5 mM EDTA, pH 6.0. To 6.0 ml of BTG-SATA (18.0 mg, 0.027 µmol) was added 600 µl of 2.5 M hydroxylamine, 50 mM EDTA, pH 7.0. The resulting solution was incubated at 20 ° C for 1 hour on a roller mixer.

Stage B

[0176] To a sample of the BTG-SH solution prepared as described in the previous step, 6.6ml, 0.027 µmol) was added a sample of the solution prepared in Example 2 (898.9 µl, 19.0 µmol). The resulting cloudy mixture was incubated for 3 hours at 20 ° C on a roller mixer. The reaction mixture was filtered through a 0.45 µm syringe filter and then purified on a Sephadex G-25 column using 100 mM phosphate buffer, 0.14M sodium chloride, pH 7.4.

EXAMPLE 6

2- (2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl) -N - ((11- (4- (2- (2-hydroxyethoxy) ethyl) piperazin-1-yl) dibenzo [ b, f] [1,4] thiazepin-3-yl) methyl) acetamide-bovine thyroglobulin

[0177] To a sample of the BTG-SH solution, prepared as described in Step A of Example 5, (3.4mL, 0.014 pmol) was added 641.4 μΙ of 2- (2,5-dioxo-2,5-dihydro-1H-). pyrrol-1-yl) -N - ((11- (4- (2 (2-hydroxyethoxy) ethyl) piperazin-1-yl) dibenzo [b, f] [1,4] thiazepin-3-yl) methyl) acetamide, prepared as described in Example 4, (13.6 µmol). The resulting cloudy mixture was incubated for 3 hours at 20 ° C on a roller mixer. The reaction mixture was purified on a Sephadex G-25 column using 100 mM phosphate buffer, 0.14M sodium chloride, pH 7.4.

EXAMPLE 7

Conjugate N - ((4- (dibenzo [b, f] [1,4] thiazepin-11-yl) -1- (2- (2-hydroxyethoxy) ethyl) piperazin-2-yl) methyl) -2- (2, Marine snail 5-dioxo-2,5-dihydro-1H-pyrrol-1-yl) acetamide-haemocyanin

Stage A

Keyhole limpethemocyanin (KLH) reaction with SATA

[0178] To 3.18 ml of a marine snail haemocyanin (KLH, 15.6 mg, 0.156 μmol) solution in 100 mM phosphate buffer, 0.46M sodium chloride, at pH 7.4 was added 72.1 μl of DMF S-acetylthioacetate solution N-succinimidyl (SATA, 25 mg / mL, 1.8 mg, 7.80 µmol). The resulting solution was incubated at 20 ° C for 1 hour on a roller mixer. The reaction mixture was purified on a Sephadex G-25 column using 100 mM phosphate buffer, 0.46 M sodium chloride, 5 mM EDTA at pH 6.0. To 6.27 ml of the obtained KLH-SATA solution (13.3 mg, 0.133 μmol) was added 627 μl of 2.5M hydroxylamine, 50 mM EDTA at pH 7.0. The resulting solution was incubated at 20 ° C for 1 hour on a roller mixer. The reaction mixture was used as is in a maleimide-activated conjugation reaction.

Stage B

[0179] To a sample of the KLH-SH solution prepared as described in the previous step (6.9 ml, 0.133 µmol) was added a sample of the solution prepared in Example 2 (624.3 µl, 13.3 µmol). The resulting cloudy mixture was incubated for 3 hours at 20 ° C on a roller mixer. The reaction mixture was filtered through a 0.45 µm syringe filter and then purified on a Sephadex G-25 column using 100 mM phosphate buffer, 0.46M sodium chloride, pH 7.4.

EXAMPLE 8

2- (2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl) -N - ((11- (4- (2- (2-hydroxyethoxy) ethyl) piperazin-1-yl) dibenzo [ b, f] [1,4] thiazepin-3-yl) methyl) acetamide-hemocyanin of the sea snail

[0180] To a sample of the KLH-SH solution, prepared as described in Step A of Example 7 (3.2 ml, 0.061 pmol), was added a sample of the solution prepared in Example 4 (283.0 µL, 6.10 pmol). The resulting cloudy mixture was incubated for 3 hours at 20 ° C on a roller mixer. The reaction mixture was purified on a Sephadex G-25 column using 100 mM phosphate buffer, 0.46M sodium chloride, pH 7.4.

EXAMPLE 9

Competitive immunoassay for quetiapine and multiplex, competitive immunoassay for Aripiprazole, Ola for Pins, Quetiapine and Risperidone / Paliperidone

[0181] Following a series of immunizations with quetiapine immunogens, blood from the tail of the mice was tested for reactivity using an ELISA assay. Hybridoma cell supernatants were also tested and the ELISA data shown in Tables 8 and 9 below show reactivity for several hybridoma cell lines (NSO cells were the fusion partner).

Table 8

<td>Dilution</td><td> 9</td><td> 10</td><td> 11</td><td> 12</td><td></td>

<td> 400</td><td rowspan="8"> 79</td><td rowspan="8"> 89</td><td rowspan="8"> 90</td><td rowspan="8"> 95</td><td rowspan="8">Relationship # 9</td>

<td> 400</td>

<td> 1200</td>

<td> 1200</td>

<td> 3600</td>

<td> 3600</td>

<td> 10800</td>

<td> 10800</td>

<td></td><td></td><td></td><td></td><td></td><td></td>

<td>BI Sub</td><td> 1,5858</td><td> 1,3168</td><td> 1,4302</td><td> 0,0533</td><td rowspan="2">Relationship # 9</td>

<td></td><td> 1,5111</td><td> 1,0627</td><td> 1,2186</td><td> 0,0427</td>

<td>Dilution</td><td> 9</td><td> 10</td><td> 11</td><td> 12</td><td rowspan="7"></td>

<td></td><td> 0,5578</td><td> 0,4213</td><td> 0,598</td><td> 0,0219</td>

<td></td><td> 0 554</td><td> 0,4447</td><td> 0,5353</td><td> 0,0233</td>

<td></td><td> 0,1932</td><td> 0,1582</td><td> 0,1868</td><td> 0,0154</td>

<td></td><td> 0,171</td><td> 0,2111</td><td> 0,1838</td><td> 0,0132</td>

<td></td><td> 0,0736</td><td> 0,0722</td><td> 0,0733</td><td> 0,0107</td>

<td></td><td> 0,0884</td><td> 0,0774</td><td> 0,086</td><td> 0,0107</td>

Table 9

<td>dilution</td><td>4C12</td><td>1A4</td><td>4G12</td><td>1F6</td>

<td> 400</td><td> 0.5467</td><td> 0.2002</td><td> 0.0144</td><td> 0.1308</td>

<td> 1200</td><td> 0.1793</td><td> 0.0619</td><td> 0.01035</td><td> 0.03905</td>

<td> 3600</td><td> 0.06655</td><td> 0.026</td><td> 0.00825</td><td> 0.0192</td>

<td> 10800</td><td> 0.02755</td><td> 0.0132</td><td> 0.00765</td><td> 0.01035</td>

<td> 400</td><td> 3.7296</td><td> 0.24275</td><td> 0.22585</td><td> 0.00615</td>

<td> 1200</td><td> 2.4516</td><td> 0.08695</td><td> 0.0763</td><td> 0.00685</td>

<td> 3600</td><td> 1.1575</td><td> 0.0282</td><td> 0.02875</td><td> 0.00615</td>

<td> 10800</td><td> 0.4622</td><td> 0.0147</td><td> 0.0145</td><td> 0.00645</td>

<td>dilution</td><td>5E9</td><td>2F2</td><td>3E2</td><td></td>

[0182] The supernatant was then tested by a competition ELISA to determine if the signals were specific for quetiapine. Figures 1 and 2 show the results for representative hybridoma cells. The data show specific reactivity for quetiapine.

[0183] Figure 3 shows a competition immunoassay format used in a lateral flow device in which the capture antibody, a quetiapine clone, was placed on a chip along with a detection conjugate consisting of a fluorophore-conjugated quetiapine. In this competitive format, shown in Fig. 3, a low level of the analyte (quetiapine) results in a high signal, while a high level of the analyte (quetiapine) results in a low signal. The amount of quetiapine in the sample can be calculated from the decrease in fluorescence compared to the drug-free control. A typical dose-response curve generated with the quetiapine sub-clones 89-3, 89-13 and 89-5 is shown in Figure 4.

[0184] Fig. 5 shows a chip design for a lateral flow test apparatus in accordance with one embodiment of the invention. The device comprises a sample receiving zone or space, a conjugation zone (which contains the desired labeled competitive binding partner (s)) and a reaction zone (eight spaces within the reaction zone are indicated; each space may contain a separate desired antibody). The sample flows from the sample zone through the conjugation zone and into the reaction zone.

[0185] Figures 6-9 show typical dose response curves for aripiprazole positive control (aripiprazole containing sample) generated with 5C7 antibody positioned in reaction zone 2 and labeled aripiprazole competition binding partner in the conjugation zone (Fig. 6), positive control for olanzapine (sample containing olanzapine) generated with antibody 4G9-1 placed in reaction zone 4 and labeled, competing partner for olanzapine in the conjugation zone (Fig. 7), positive control for quetiapine (sample containing quetiapine) generated with antibody 11 placed in reaction zone 6 and labeled, competitively binding partner for quetiapine in the conjugation zone (Fig. 8) and a positive control for risperidone (sample containing risperidone) generated with antibody 5-9 placed in reaction zone 8 and labeled, competitively binding partner for risperidone in the conjugation zone (Fig. 9). Labeled, competitively binding partners in the conjugation zone compete with drugs present in the samples for binding to the antibody. The amount of label is detected and is an indication of the amount of drug present in the sample (the amount of signal is inversely proportional to the amount of drug in the sample - see Fig. 3).

[0186] To confirm that the conjugates of the labeled, competitively binding partners did not bind to antibodies placed in the reaction zones, negative controls were performed using drug-free samples. With reference to Table 10, the sample containing no aripiprazole is placed in the sample zone and moves by capillary action through the conjugation zone (this time containing labeled olanzapine, labeled quetiapine, and labeled risperidone but unlabeled aripiprazole) and into the reaction zone. Reaction zone again contains the anti-aripiprazole (5C7) antibody in reaction zone 2. Table 10 below shows the results confirming that there is no dose response and that olanzapine, quetiapine and risperidone conjugates that capillary travel through the reaction zone do not bind to the anti-aripiprazole antibody.

Table 10

<td colspan="4">Aripiprazole Clone 5C7-Mathematical Model 1 (O ng / ml Conc.)</td><td></td><td></td><td></td>

<td>Research- MM</td><td>Conjugate</td><td>Reaction zone</td><td>Reading position</td><td>Average peak area</td><td>Mean peak height</td><td>Medium background</td>

<td>ARIP-MM1</td><td>OLAN, QUET, RISP</td><td>ARIP</td><td> 2</td><td> 0,77</td><td> 1,56</td><td> 3,99</td>

<td>ARIP-MM1</td><td>OLAN, QUET, RISP</td><td></td><td> 4</td><td> -0,02</td><td> 0,06</td><td> 4,14</td>

<td colspan="4">Aripiprazole Clone 5C7-Mathematical Model 1 (0 ng / ml Conc.)</td><td></td><td></td><td></td>

<td>Research- MM</td><td>Conjugate</td><td>Reaction zone</td><td>Reading position</td><td>Average peak area</td><td>Mean peak height</td><td>Medium background</td>

<td>ARIP-MM1</td><td>OLAN, QUET, RISP</td><td></td><td> 6</td><td> 0,09</td><td> 0,10</td><td> 4,29</td>

<td>ARIP-MM1</td><td>OLAN, QUET, RISP</td><td></td><td> 8</td><td> 0,13</td><td> 0,12</td><td> 4,61</td>

<td colspan="7">The other conjugates do not bind to Aripiprazole</td>

[0187] With reference to Table 11, a sample containing no olanzapine is placed in the sample zone and moves by capillary action through the conjugation zone (this time containing labeled aripiprazole, labeled quetiapine, and labeled risperidone but unlabeled olanzapine) and into reaction zones. The reaction zone again contains the anti-olanzapine antibody (4G9-1) in reaction zone 4. Table 11 below shows the results confirming that there is no dose response and that the aripiprazole quetiapine and risperidone conjugates that capillary action through the reaction zone do not bind to the anti-olanzapine antibody.

Table 11

<td colspan="4">OLAN clone 4G9-1-Mathematical Model 1 (Ong / ml Conc.)</td><td></td><td></td><td></td>

<td>Study MM</td><td>Conjugate</td><td>Reaction zone</td><td>Reading position</td><td>Average peak area</td><td>Mean peak height</td><td>Medium background</td>

<td>OLAN-MM1</td><td>ARIP, QUET, RISP</td><td></td><td> 2</td><td> -0,03</td><td> 0,05</td><td> 4,38</td>

<td>OLAN-MM1</td><td>ARIP, QUET, RISP</td><td>OLAN</td><td> 4</td><td> 0,74</td><td> 1,10</td><td> 4,56</td>

<td>OLAN-MM1</td><td>ARIP, QUET, RISP</td><td></td><td> 6</td><td> 0,06</td><td> 0,09</td><td> 4,79</td>

<td>OLAN-MM1</td><td>ARIP, QUET, RISP</td><td></td><td> 8</td><td> 0,11</td><td> 0,13</td><td> 5,17</td>

<td colspan="7">The other conjugates do not bind to Olanzapine</td>

[0188] Referring to Table 12, a sample free of quetiapine is placed in the sample zone and moves by capillary action through the conjugation zone (this time containing labeled aripiprazole, labeled olanzapine and labeled risperidone but unlabeled quetiapine) and reaction zones. The reaction zone again contains the anti-quetiapine antibody (11) in reaction zone 6. Table 12 below shows the results confirming that there is no dose response and that the aripiprazole olanzapine and risperidone conjugates that capillary move through the reaction zone do not bind to the anti-quetiapine antibody.

Table 12

<td colspan="4">Quetiapine Clone 11-Mathematical Model 1 (0 ng / ml Conc.)</td><td></td><td></td><td></td>

<td>Research- MM</td><td>Conjugate</td><td>Reaction zone</td><td>Reading position</td><td>Average peak area</td><td>Mean peak height</td><td>Medium background</td>

<td>QUET-MM1</td><td>ARIP, OLAN, RISP</td><td></td><td> 2</td><td> -0,01</td><td> 0,07</td><td> 3,85</td>

<td>QUET-MM1</td><td>ARIP, OLAN, RISP</td><td></td><td> 4</td><td> 0,01</td><td> 0,12</td><td> 4,01</td>

<td>QUET-MM1</td><td>ARIP, OLAN, RISP</td><td>QUET</td><td> 6</td><td> 0,03</td><td> 0,08</td><td> 4,24</td>

<td>QUET-MM1</td><td>ARIP, OLAN, RISP</td><td></td><td> 8</td><td> 0,04</td><td> 0,07</td><td> 4,56</td>

<td colspan="7">The other conjugates do not bind to Quetiapine</td>

[0189] Referring to Table 13, a sample not containing risperidone is placed in the sample zone and moves by capillary action through the conjugation zone (this time containing labeled aripiprazole, labeled olanzapine, and labeled quetiapine but unlabeled risperidone) and into the zone. reaction. The reaction zone again contains the anti-risperidone antibody (5-9) in reaction zone 8. Table 13 below shows the results confirming that there is no dose response and that the aripiprazole olanzapine and quetiapine conjugates that move by capillary forces through the reaction zone do not bind to the anti-risperidone antibody.

Table 13

<td colspan="4">Risperidone Clone 5-9-Mathematical Model 1 (0 ng / ml Conc.)</td><td></td><td></td><td></td>

<td>Study MM</td><td>Conjugate</td><td>Reaction zone</td><td>Reading position</td><td>Average peak area</td><td>Mean peak height</td><td>Medium background</td>

<td>RISP-MM1</td><td>ARIP, OLAN, QUET</td><td></td><td> 2</td><td> 0,02</td><td> 0,11</td><td> 7,43</td>

<td>RISP-MM1</td><td>ARIP, OLAN, QUET</td><td></td><td> 4</td><td> 0,05</td><td> 0,14</td><td> 7,73</td>

<td>RISP-MM1</td><td>ARIP, OLAN, QUET</td><td></td><td> 6</td><td> 0,20</td><td> 0,19</td><td> 8,11</td>

<td>RISP-MM1</td><td>ARIP, OLAN, QUET</td><td>RISP</td><td> 8</td><td> 1,97</td><td> 3,23</td><td> 8,85</td>

<td colspan="4">Risperidone Clone 5-9-Mathematical Model 1 (0 ng / ml Conc.)</td><td></td><td></td><td></td>

<td>Research- MM</td><td>Conjugate</td><td>Reaction zone</td><td>Reading position</td><td>Average peak area</td><td>Mean peak height</td><td>Medium background</td>

<td colspan="7">The other conjugates do not bind to Risperidone</td>

[0190] To confirm that the labeled, competitive binding partner conjugates only bind to the corresponding antibodies placed in the reaction zones, additional negative controls were performed reusing drug-free samples. With reference to Table 14, a sample containing no aripiprazole is placed in the sample zone and moves by capillary action through the conjugation zone (this time containing labeled aripiprazole) and into the reaction zone. The reaction zone again contains the anti-aripiprazole antibody (5C7) in reaction zone 2, as well as the anti-olanzapine antibody (4G9-1) in reaction zone 4, the anti-quetiapine antibody (11) in reaction zone 6, and the anti-risperidone antibody (5 -9) in reaction zone 8. Table 14 below shows the results confirming that there is no dose response beyond that of the anti-aripiprazole antibody 5C7 (in reaction zone 2).

Table 14

<td colspan="4">Aripiprazole Clone 5C7-Mathematical Model 1 (0 ng / ml Conc.)</td><td></td><td></td><td></td>

<td>Study MM</td><td>Conjugate</td><td>Reaction zone</td><td>Reading position</td><td>Average peak area</td><td>Mean peak height</td><td>Medium background</td>

<td>ARIP-MM1</td><td>ARIP, OI_AN, QUET, RISP</td><td>ARIP</td><td> 2</td><td> 60,34</td><td> 97,53</td><td> 5,44</td>

<td>ARIP-MM1</td><td>ARIP, OI_AN, QUET, RISP</td><td></td><td> 4</td><td> 2,86</td><td> 3,91</td><td> 11,66</td>

<td>ARIP-MM1</td><td>ARIP, OI_AN, QUET, RISP</td><td></td><td> 6</td><td> 1,12</td><td> 1,23</td><td> 11,03</td>

<td>ARIP-MM1</td><td>ARIP, OI_AN, QUET, RISP</td><td></td><td> 8</td><td> 3,14</td><td> 4,19</td><td> 12,94</td>

<td colspan="7">Only the Aripiprazole Reaction Zone is binding</td>

[0191] Referring to Table 15, a sample containing no olanzapine is placed in the sample zone and moves by capillary action through the conjugation zone (this time containing labeled olanzapine) and into the reaction zone. The reaction zone again contains the anti-aripiprazole antibody (5C7) in reaction zone 2, as well as the anti-olanzapine antibody (4G9-1) in reaction zone 4, the anti-quetiapine antibody (11) in reaction zone 6, and the anti-risperidone antibody (5 -9) in reaction zone 8. Table 15 below shows the results confirming that there is no dose response beyond that of the anti-olanzapine antibody 4G91 (in reaction zone 4).

Table 15

<td colspan="4">OLAN clone 4G9-1-Mathematical Model 1 (0 ng / ml Conc.)</td><td></td><td></td><td></td>

<td>Research- MM</td><td>Conjugate</td><td>Reaction zone</td><td>Reading position</td><td>Average peak area</td><td>Mean peak height</td><td>Medium background</td>

<td>OLAN-MM1</td><td>ARIP, OLAN, QUET, RISP</td><td></td><td> 2</td><td> 0,02</td><td> 0,08</td><td> 4,86</td>

<td>OLAN-MM1</td><td>ARIP, OLAN, QUET, RISP</td><td>OLAN</td><td> 4</td><td> 34,23</td><td> 51,80</td><td> 5,39</td>

<td>OLAN-MM1</td><td>ARIP, OLAN, QUET, RISP</td><td></td><td> 6</td><td> 0,22</td><td> 0,32</td><td> 5,39</td>

<td>OLAN-MM1</td><td>ARIP, OLAN, QUET, RISP</td><td></td><td> 8</td><td> 0,15</td><td> 0,17</td><td> 5,59</td>

<td colspan="7">Only the Olanzapine Reaction Zone is binding</td>

[0192] Referring to Table 16, a sample free of quetiapine is placed in the sample zone and moves by capillary action through the conjugation zone (this time containing labeled quetiapine) and into the reaction zone. The reaction zone again contains the anti-aripiprazole antibody (5C7) in reaction zone 2, as well as the anti-olanzapine antibody (4G9-1) in reaction zone 4, the anti-quetiapine antibody (11) in reaction zone 6, and the anti-risperidone antibody (5 -9) in reaction zone 8. Table 16 below shows the results confirming that there is no dose response beyond that of the anti-quetiapine 11 antibody (in reaction zone 6).

Table 16

<td colspan="4">Quetiapine Clone 11-Mathematical Model 1 (0 ng / ml Conc.)</td><td></td><td></td><td></td>

<td>Study MM</td><td>Conjugate</td><td>Reaction zone</td><td>Reading position</td><td>Average peak area</td><td>Mean peak height</td><td>Medium background</td>

<td>QUET-MM1</td><td>ARIP, OLAN, QUET, RISP</td><td></td><td> 2</td><td> 0,13</td><td> 0,41</td><td> 10,02</td>

<td>QUET-MM1</td><td>ARIP, OLAN, QUET, RISP</td><td></td><td> 4</td><td> 0,08</td><td> 0,23</td><td> 10,47</td>

<td>QUET-MM1</td><td>ARIP, OLAN, QUET, RISP</td><td>QUET</td><td> 6</td><td> 140,35</td><td> 181,33</td><td> 7,91</td>

<td>QUET-MM1</td><td>ARIP, OLAN, QUET, RISP</td><td></td><td> 8</td><td> 1,58</td><td> 2,61</td><td> 11,53</td>

<td colspan="4">Quetiapine Clone 11-Mathematical Model 1 (O ng / ml Conc.)</td><td></td><td></td><td></td>

<td>Research- MM</td><td>Conjugate</td><td>Reaction zone</td><td>Reading position</td><td>Average peak area</td><td>Mean peak height</td><td>Medium background</td>

<td colspan="7">Only the Quetiapine Reaction Zone is binding</td>

[0193] Referring to Table 17, a sample containing no risperidone is placed in the sample zone and moves by capillary action through the conjugation zone (this time containing labeled risperidone) and into the reaction zone. The reaction zone again contains the anti-aripiprazole antibody (5C7) in reaction zone 2, as well as the anti-olanzapine antibody (4G9-1) in reaction zone 4, the anti-quetiapine antibody (11) in reaction zone 6, and the anti-risperidone antibody (5 -9) in reaction zone 8. Table 17 below shows the results confirming that there is no dose response beyond that for anti-risperidone antibody 5-9 (in reaction zone 8).

Table 17

<td colspan="4">Risperidone Clone 5-9-Mathematical Model 1 (O ng / ml Conc.)</td><td></td><td></td><td></td>

<td>Study MM</td><td>Conjugate</td><td>Reaction zone</td><td>Reading position</td><td>Average peak area</td><td>Mean peak height</td><td>Medium background</td>

<td>RISP-MM1</td><td>ARIP, OLAN, QUET, RISP</td><td></td><td> 2</td><td> 1,03</td><td> 1,51</td><td> 9,07</td>

<td>RISP-MM1</td><td>ARIP, OLAN, QUET, RISP</td><td></td><td> 4</td><td> 0,65</td><td> 0,91</td><td> 9,60</td>

<td>RISP-MM1</td><td>ARIP, OLAN, QUET, RISP</td><td></td><td> 6</td><td> 2,61</td><td> 6,39</td><td> 10,48</td>

<td>RISP-MM1</td><td>ARIP, OLAN, QUET, RISP</td><td>RISP</td><td> 8</td><td> 55,98</td><td> 100,91</td><td> 11,58</td>

<td colspan="7">Only the Risperidone Reaction Zone is binding</td>

[0194] The results presented above confirm that the conjugates of the labeled, competitively binding partners only bind to their corresponding antibodies in the reaction zone.

[0195] Figures 10-13 show typical dose response curves in reaction zones for a specific antibody and evidence of low / high concentration dose response for each specific study in the presence of other conjugates. In Fig. 10, the aripiprazole-containing sample is placed in the sample zone and moves by capillary action through the conjugation zone (this time containing labeled aripiprazole, labeled olanzapine, labeled quetiapine, and labeled risperidone) and into the reaction zone. The reaction zone again contains an anti-aripiprazole antibody (5C7) in reaction zone 2. A typical dose-response curve was generated as shown in Fig. 10 only for aripiprazole and not for olanzapine, quetiapine or risperidone.

[0196] In Fig. 11, the olanzapine-containing sample is placed in the sample zone and moves by capillary action through the conjugation zone (this time containing labeled aripiprazole, labeled olanzapine, labeled quetiapine, and labeled risperidone) and into the reaction zone. The reaction zone again contains an anti-olanzapine antibody (4G9-1) in reaction zone 4. A typical dose-response curve was generated as shown in Fig. 11 only for olanzapine, not for aripiprazole, quetiapine or risperidone.

[0197] In Fig. 12, the quetiapine-containing sample is placed in the sample zone and moves by capillary action through the conjugation zone (this time containing labeled aripiprazole, labeled olanzapine, labeled quetiapine, and labeled risperidone) and into the reaction zone. The reaction zone again contains the anti-quetiapine antibody (11) in reaction zone 6. A typical dose-response curve was generated as shown in Fig. 12 for quetiapine only, not for aripiprazole, olanzapine or risperidone.

[0198] In Figure 13, the sample containing risperidone is placed in the sample zone and moves by capillary action through the conjugation zone (this time containing labeled aripiprazole, labeled olanzapine, labeled quetiapine and labeled risperidone) and into the reaction zone. The reaction zone again contains an anti-risperidone antibody (5-9) in reaction zone 8. A typical dose-response curve was generated as shown in Fig. 13 only for risperidone and not for aripiprazole, olanzapine or quetiapine.

[0199] Figures 14-17 show typical dose response curves for each study in the presence of other conjugates and antibodies. In Fig. 14, a sample containing aripiprazole is placed in the sample zone and moves by capillary action through the conjugation zone (again containing labeled aripiprazole, labeled olanzapine, labeled quetiapine, and labeled risperidone) and into the reaction zone. The reaction zone again contains the anti-aripiprazole antibody (5C7) in reaction zone 2, as well as the anti-olanzapine antibody (4G9-1) in reaction zone 4, the anti-quetiapine antibody (11) in reaction zone 6, and the anti-risperidone antibody (5 -9) in reaction zone 8. A typical dose-response curve was generated for aripiprazole as shown in Fig. 14. When a sample containing olanzapine was placed in the sample zone of this chip, a typical dose response curve was generated for olanzapine as shown in Fig. 15. When a sample containing quetiapine was placed in the sample zone of this chip, a typical dose response curve for quetiapine was generated as shown. in Fig. 16. When a sample containing risperidone was placed in the sample zone of this chip, a typical dose-response curve for risperidone was generated as shown in Fig. 17.

[0200] Figures 18-21 show comparison of dose response curves generated as positive controls (Figures 6-9) with dose response curves generated in multiplex format (Figures 14-17). A comparison to aripiprazole is shown in Fig. 18; for olanzapine in Fig. 19; for quetiapine in Fig. 20; and for risperidone in Fig. 21. These figures show that the positive control curves are similar to the multiplex curves.

[0201] These data show that the lateral flow testing device of the invention can be used to detect multiple antipsychotic drugs from a single patient sample in one portable device at the point of care.

Janssen Pharmaceutica NV, Belgium

Proxy:

EP 2 888 286 B1

Z-16950/18