US00877 1972B2 (12) United States Patent (10) Patent No.: US 8,771,972 B2 SalamOne et al. (45) Date of Patent: Jul. 8, 2014 (54) CLOZAPINE IMMUNOASSAY (56) References Cited (75) Inventors: Salvatore J. Salamone, Stockton, NJ U.S. PATENT DOCUMENTS (US); Jodi Blake Courtney, 6, 197,764 B1 3/2001 Bradley et al. Doylestown, PA (US); Howard Sard, 2006/02231.34 A1* 10, 2006 Salamone et al. ........... 435,792 Arlington, MA (US); Christopher 2006/0252744 A1 11/2006 Burstein Spedaliere, Allentown, PA (US) 2009, 0215082 A1 8/2009 Salamone et al. 2012/030 1973 A1* 11/2012 Salamone et al. ............ 436,501 (73) Assignee: Saladax Biomedical Inc., Bethlehem, OTHER PUBLICATIONS PA (US) Ming et al., “Therapeutic drug monitoring of clozapine and (*) Notice: Subject to any disclaimer, the term of this norclozapine in human serum using ultra-performance liquid chro patent is extended or adjusted under 35 matography-tandem mass spectrometry.” J. Anal. Toxicol., 2009, vol. U.S.C. 154(b) by 189 days. 33, No. 4, pp. 198-203.* Mendoza et al., “N-Desmethylclozapine: Is There Evidence for its Antipsychotic Potential?.” Clin. Neuropharm., 2009, vol. 32, issue 3, (21) Appl. No.: 13/186,147 pp. 154-157.* Gardner et al., “A Comparison of the Covalent Binding of Clozapine (22) Filed: Jul.19, 2011 and Olanzapine to Human Neutrophils. In Vitro and In Vivo.” Mol. Pharmacol., 1998, vol. 53, pp. 999-1008.* (65) Prior Publication Data Liu et al., "Clozapine is oxidized by activated human neutrophils to a reactive nitrenium ion that irreversibly binds to the cells,” J. Pharm. US 2012/O3O1974 A1 Nov. 29, 2012 Exper. Therap., 1995, vol. 275, No. 3, pp. 1476-1483.* Lai et al., “Bioactivation and Covalent Binding of Hydroxyfluperlapine in Human Neutrophils: Implications for Related U.S. Application Data Fluperlapine-Induced Agranulocytosis. Drug Metabolism Disposi tion, 2000, vol. 28, No. 3, pp. 255-263.* (63) Continuation-in-part of application No. 13/114.252, The International Search Report and Written Opinion by the Inter filed on May 24, 2011, now abandoned. national Searching Authority, issued on Jul. 31, 2012, in the related PCT application No. PCT/US 12/36257. (51) Int. Cl. GOIN33/53 (2006.01) * cited by examiner GOIN33/543 (2006.01) C07K L/10 (2006.01) Primary Examiner — Galina Yakovleva C07K 17/02 (2006.01) (52) U.S. C. (57) ABSTRACT USPC .......... 435/7.93; 435/7.1:435/7.92; 436/501; Novel conjugates and immunogens derived from clozapine 436/518; 436/523: 436/815; 530/388.9; 530/389.8; and antibodies generated by these immunogens are useful in 530/402:530/403 immunoassays for the quantification and monitoring of cloza (58) Field of Classification Search pine in biological fluids. None See application file for complete search history. 15 Claims, No Drawings US 8,771,972 B2 1. 2 CLOZAPINE IMMUNOASSAY N-desmethylclozapine has the following formula: CROSS-REFERENCE TO RELATED II APPLICATION NH This Application is a Continuation-in-Part Application of ( ) U.S. application Ser. No. 13/114,252 filed May 24, 2011 now abandoned. 10 FIELD OF THE INVENTION sco This invention relates to the field of immunoassays for determining the presence and/or quantifying the amount of 15 Clozapine-N-oxide has the following formula: clozapine in human biological fluids in order to rapidly deter mine optimal drug concentrations during treatment. III BACKGROUND OF THE INVENTION Schizophrenia is a severe psychiatric disorder affecting approximately 1% of the world's population. Clinical symp toms of schizophrenia include delusions, auditory hallucina 25 tions, disorganized thoughts and speech, social withdrawal, lack of motivation, and cognitive dysfunction Such as disor C ganized thinking and memory impairments. This disorder is N believed to be caused by a combination of neurological H defects including dopamine and serotonin levels, and inhibi 30 Plasma concentration of clozapine is effected by varying tory interneuron deficiencies. Schizophrenia can be treated cytochrome P450 activity (Chetty, M and M Murray, Curr with drugs which target neurotransmitters and receptors, Drug Metab. 8, 4, 307-13 2007; Mauri, MC, et al., Clin commonly referred to as antipsychotic or neuroleptic drugs. Pharmacokinet, 46, 5, 359-88 2007: Mendoza, M C and JP One class of antipsychotic drug termed "atypical antipsy 35 Lindenmayer, ClinNeuropharmacol, 32, 3, 154-7 2009), age, chotics' or more commonly 'second generation antipsychot sex, caffeine use, and Smoking (Rostami-Hodegan, A, et al., J Clin Psychopharmacol, 24, 1, 70-8 2004). ics’ includes the tricyclic dibenzazepine derivative, clozapine Clozapine has been shown to have high inter-patient vari (I). Clozapine, marketed under Clozaril.R by Novartis in the ability in plasma steady-state concentrations and this vari United States, interferes with the binding of dopamine at the 40 ability can impact safety and quality of life (Mauri, MC, LS dopamine receptors D, D, D, and Ds, and has a high affinity Volonteri, A Colasanti, A Fiorentini, IF De Gaspari and S R for the D receptor (Package Insert Clozaril 2009). Clozaril Bareggi, Clin Pharmacokinet, 46, 5,359-88 2007). also acts as an antagonist at adrenergic, cholinergic, histamin Since efficacy of clozapine is improved within a specific ergic, and serotonergic receptors. Clozapine is almost com range of plasma drug concentration and the drug exhibits wide intra-patient pharmacokinetic variability monitoring pletely metabolized by cytochrome P450 in humans to the 45 N-desmethyl, and N-Oxide derivatives. The N-desmethyl concentrations of this drug in blood and adjusting to target levels would be of value in increasing efficacy and minimiz metabolite has only limited activity, while the N-Oxide ing toxicity (Mauri, MC, L S Volonteri, A Colasanti, A derivative is inactive. The activity of N-desmethyl metabolite Fiorentini, IF De Gaspari and SRBareggi, Clin Pharmacoki is with receptors other than the receptors with which clozap net, 46, 5,359-88 2007) ine is active. 50 As a result of this variability, equal doses of the same drug Clozapine has the following formula: in different individuals can result in dramatically different clinical outcomes. The effectiveness of the same dosage of clozapine varies significantly based upon individual drug clearance and the ultimate serum drug concentration in the CH 55 patient. Therapeutic drug management would provide the W clinician with insight on patient variation in drug administra tion. With therapeutic drug management, drug dosages could be individualized to the patient, and the chances of effectively treating the disorder without the unwanted side effects would 60 be much higher. N In addition, therapeutic drug management of clozapine C would serve as an excellent tool to ensure compliance (Treur, M, et al., BMC Health Serv. Res., 9, 9 2009; Valenstein, M, et N al., J. Clin. Psychiatry, 67, 10, 1542-1550 2006) with admin H 65 istration and prescribed dosage to achieve effective serum concentration levels. Routine therapeutic drug management of clozapine would require the availability of simple auto US 8,771,972 B2 3 4 mated tests adaptable to general laboratory equipment. The use of liquid chromatography (LC) with ultraviolet (UV) or IV mass spectrometry detection to determine the concentration AH3 of clozapine in human blood and plasma has been described (Rosland, M, et al., Drug Dev Ind Pharm, 33, 10, 1158-66 r 2007: Rao, LV, et al., J. Clin Lab Anal, 23, 6, 394-8 2009: N Ming, D S and J Heathcote, J Anal Toxicol, 33, 4, 198-203 C NS 2009; Niederlander, HA, et al., JChromatogrB Analyt Tech nol Biomed Life Sci., 834, 1-2, 98-107 2006). These methods are labor intensive, requiring liquid-liquid or Solid phase 10 extractions, use expensive equipment and are not amenable to routine clinical laboratory use. To date, there are no immu B-(Y)-X noassays for measuring clozapine in human biological fluids of patients treated with this antipsychotic agent. As seen from the foregoing, there are no immunoassays for 15 wherein B is: (C=O)—, (C=O) NH-, determining the presence and/or quantifying the amount of —(C=O)—O , —CH2-; clozapine in human biological fluids. Routine therapeutic Y is an organic spacing group; drug management of clozapine by immunoassays would pro X is a terminal functional group capable of binding to a vide simple automated tests adapted to standard laboratory polyamine polymer, and equipment. However, in order to provide such immunoas p is an integer from 0 to 1; says, antibodies specific to clozapine must be produced. The or salts thereof, derivatives and immunogen used in this assay must impart produce antibodies which are specific for clozapine and do through these corresponding antibodies produced specific not substantially react with or bind to N-desmethylclozapine reactivity to clozapine without any Substantial cross-reactiv and clozapine-N-oxide. ity reactivity to therapeutically or pharmacologically active 25 The provision of these antibodies which substantially or inactive metabolites of clozapine which interfere with the selectively react with clozapine and do not cross react with detection of clozapine, these interfering metabolites being N-desmethylclozapine and clozapine-N-oxide allows one to N-desmethylclozapine, (the compound of formula II) and produce an immunoassay which can specifically detect and clozapine-N-oxide (the compound of formula III). In con quantify so as to monitor clozapine in the fluid samples of ducting immunoassays for monitoring clozapine, it is impor 30 patients being treated with clozapine. Also, included within tant that the antibodies used not be reactive with the pharma this invention are reagents and kits for said immunoassay. ceutically active metabolite, N-desmethylclozapine (the compound of formula II). This is true since reactivity with DETAILED DESCRIPTION N-desmethylclozapine interferes with the detection of cloza pine in the patient sample and there is no correlation between 35 In accordance with this invention, a new class of antibodies the detectable N-desmethylclozapine and clinical outcome.