Preparation of Antibodies and Development of an Enzyme-Linked

1652 Biol. Pharm. Bull. 38, 1652–1657 (2015) Vol. 38, No. 10 Note

Preparation of Antibodies and Development of an Enzyme-Linked Immunosorbent Assay for the Tyrosine Kinase Inhibitors Lapatinib and Nilotinib Tetsuya Saita,*,a Yuta Yamamoto,a Masashi Shin,a and Yukitaka Nakanob a Applied Life Science Department, Faculty of Biotechnology and Life Science, Sojo University; 4–22–1 Ikeda, Kumamoto 860–0082, Japan: and b Department of Pharmacy, Kyushu Central Hospital; 3–23–1 Shiobaru, Minami- ku, Fukuoka 815–8588, Japan. Received April 13, 2015; accepted June 23, 2015

In this paper, we describe the production of the first specific antibodies against the tyrosine kinase inhibitors lapatinib and nilotinib. Anti-lapatinib antibody was obtained by immunizing rabbits with an antigen conjugated with bovine serum albumin using 3-chloro-4-((3-fluorobenzyl)oxy)aniline. Anti-nilotinib antibody was produced by immunizing mice with an antigen conjugated with bovine serum albumin using 2-(5-amino- 2-methylanilino)-4-(3-pyridyl)pyrimidine. The generated antibodies were used to develop highly sensitive and specific enzyme-linked immunosorbent assays (ELISAs) for lapatinib and nilotinib in human serum. The assays were capable of detecting lapatinib and nilotinib at serum concentrations as low as 40 and 8 ng/mL, respectively. Using the two ELISAs, drugs levels were easily measured in the serum of rats after a single dose oral administration of lapatinib or nilotinib. The assays are therefore expected be valuable tools for therapeutic drug monitoring in the clinical setting and pharmacokinetic studies of lapatinib and nilotinib. Key words lapatinib; nilotinib; tyrosine kinase inhibitor; enzyme-linked immunosorbent assay

Small-molecule tyrosine kinase inhibitors (TKIs) are a class od for the labeling of the two drugs with horseradish peroxi- of targeted drugs for the treatment of malignant pathologies, dase (HRP) as a tracer, characterization of the specificity of and have widely been used in advanced cancer. Among TKIs, the antibodies, and the measurement of lapatinib and nilotinib lapatinib is a dual inhibitor of the tyrosine kinase functional- by ELISA is presented. ity of the epidermal growth factor receptor (EGFR or ErbB1) and HER-2/neu (erbB2) receptors of the epidermal growth MATERIALS AND METHODS factor (EGF) family,1) and has been approved for use in breast cancer treatment.2) In addition, nilotinib is a Bcr-Abl TKI Reagents Lapatinib and nilotinib were obtained from that is effective in imatinib-resistant or -intolerant patients AdooQ BioScience LLC (CA, U.S.A.). 3-Chloro-4-{(3- with Philadelphia-positive chronic myelogenous leukemia.3,4) fluorobenzyl) oxy}aniline (CFBA) was purchased from Despite their proven benefit in cancer treatment, lapatinib and Ark Pharm, Inc. (Libertyville, U.S.A.). 2-(5-Amino-2- nilotinib exhibit inter-individual pharmacokinetic variability, methylanilino)-4-(3-pyridyl) pyrimidine (AMPP) was obtained and may have drug interactions and toxic effects. Therefore, from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan). HRP to prevent treatment failure and avoid potential toxicity with and 3,3′,5,5′-tetramethylbenzidine (TMB) were purchased TKIs, therapeutic drug monitoring (TDM) on an individual from Boehringer Ingelheim Pharma GmbH (Ingelheim, Ger- patient basis has been proposed as an approach to reduce drug many). All other reagents and solvents were of the highest toxicity and resistance, and to achieve a high level of adher- grade commercially available. ence for a higher likelihood of treatment response. Current Preparation of Immunogen for Lapatinib Lapatinib im- analytical methods for TKIs include high-performance liquid munogen was prepared using a partial structure of lapatinib chromatography (HPLC)5) and liquid chromatography with (CFBA) as shown in Figs. 1 and 2. CFBA (10 mg, 40 µmol) in tandem mass spectrometry (LC-MS/MS).6,7) Although these 100 µL dimethylformamide (DMF) was acidified by the addi- methods are highly sensitive and specific, they require expen- tion of 100 µL of 1 M acetic acid and was then diazotized with sive equipment and complicated sample pretreatment process- sodium nitrite (2.8 mg, 40.5 µmol) in 50 µL distilled water at es, including purification, concentration, and/or derivatiza- 0°C for 10 min. The resulting solution was then mixed with tion steps. Thus, the development of a simple quantification bovine serum albumin (BSA) (20 mg) in 4 mL of 0.1 M borate method for TKIs is needed to facilitate TDM in the clinical buffer (pH 9.0), followed by a 1-h incubation at room tempera- setting. An attractive approach is the use of enzyme-linked ture. The reaction mixture was dialyzed successively for 48 h immunosorbent assay (ELISA), which is sensitive, specific, against 50 and 1 mM phosphate buffer (pH 7.0) and H2O. The and has the added benefits of simplicity, low cost, and capac- purified conjugate was lyophilized and used as an immunogen ity for high throughput. Recently, we developed ELISAs for for lapatinib. several TKIs and demonstrated that the assays were sensitive, Preparation of Lapatinib Antibody An aliquot of a specific and useful for TDM and pharmacokinetic studies of solution containing 1 mg CFBA–BSA conjugate (1.0 mL) was the target drugs.8,9) emulsified with an equal volume of Freund’s complete adju- This study describes a methodology for the production of vant. Two female white rabbits (Kyudo Exp. Animals, Kuma- antibodies against lapatinib and nilotinib. In addition, a meth- moto, Japan) were administered multiple subcutaneous injec-

* To whom correspondence should be addressed. e-mail: [email protected] © 2015 The Pharmaceutical Society of Japan Vol. 38, No. 10 (2015) Biol. Pharm. Bull. 1653 tions of the solution at bilateral sites along the back. Booster mice were euthanized and sera were collected, separated by injections were administered three times at biweekly intervals, centrifugation, heated at 55°C for 30 min, and then stored at using half the dose of the first immunization. The rabbits were −30°C. The obtained anti-nilotinib serum was directly used as bled from an ear vein 10 weeks after the initial immunization. anti-nilotinib antibody for ELISA. The experimental protocol The collected sera (5 mL) were separated by centrifugation at was approved by the Ethies Review Committee for Animal 1048×g at 4°C for 10 min and then heated at 55°C for 30 min. Experimentation of Sojo University. Fractions of immunoglobulin G (IgG) were purified using a Preparation of AMPP–HRP Conjugate AMPP was HiTrap Protein G column (GE Healthcare, Stockholm, Swe- labeled with HRP using a similar method as used for the den), with 20 mM sodium phosphate (pH 7.0) as binding buffer preparation of nilotinib immunogen (Fig. 3). Briefly, a solu- and 0.1 M glycine HCl (pH 2.7) as elution buffer, following the tion of AMPP (5 mg, 18 µmol) and succinic anhydride (1.8 mg, manufacturer’s protocol. The fraction that passed through the 18 µmol) in pyridine (0.5 mL) was stirred overnight at 60°C. column was lyophilized and used as anti-lapatinib antibody After removing pyridine by passing nitrogen through the for ELISA. The experimental protocol was approved by the reaction mixture, the obtained residue, carboxylic-modified Ethies Review Committee for Animal Experimentation of AMPP, was dissolved in DMF (100 µL) and 95% dioxane Sojo University. (1.0 mL). After the addition of 1 mL water, the resulting Preparation of CFBA–HRP Conjugate Enzyme label- mixture was extracted with ethylacetate and then added to ing of lapatinib with HRP similarly performed using CFBA EDPC (8.0 mg, 36 µmol) and N-hydroxysuccinimide (4.15 mg, (Fig. 2). A solution of CFBA (10 mg, 40 µmol) and succinic 36 µmol). The resulting solution was allowed to stand at room anhydride (4.0 mg, 40 µmol) in pyridine (0.5 mL) was stirred temperature for 2 h, and a 140-µL aliquot of the reaction overnight at 60°C. After the addition of 1 mL water, the re- mixture, containing succinimidyl AMPP, was then added di- sulting mixture was extracted with ethylacetate and then dried rectly to HRP (0.5 mg, 12.5 nmol) in 1.0 mL of 0.1 M phosphate over anhydrous sodium sulfate. After removing the solvent by buffer (pH 7.0), followed by a further 1-h incubation at room evaporation, the obtained residue, carboxylic-modified CFBA, temperature. The mixture was chromatographed on a column was dissolved in 95% dioxane (1.0 mL) and was then added to of Sephadex G-75 (2.0×30 cm) using buffer A to remove any 1-ethyl-3,3-dimethylaminopropyl carbodiimide hydrochloride remaining small molecules. Three-milliliter fractions were (EDPC) (15.2 mg, 80 µmol) and N-hydroxysuccinimide (9.2 mg, collected and fractions 12 to 14, corresponding to the main 80 µmol). The resulting solution was allowed to stand at room peaks showing enzyme activity, were combined and used as a temperature for 2 h. A 30-µL aliquot of this reaction mixture label for ELISA. containing succinimidyl CFBA was added directly to HRP ELISA for Lapatinib and Nilotinib ELISA is based (0.5 mg, 12.5 nmol) in 0.5 mL of 0.1 M phosphate buffer (pH on the principle of competition between enzyme-labeled and 7.0), followed by a 1-h incubation at room temperature. The unlabeled drugs for an immobilized antibody, followed by mixture was chromatographed on a column of Sephadex G-75 measurement of the marker enzyme activity of the immuno- (2.0×30 cm) using 20 mM phosphate buffer (pH 7.0) containing complex bound to the solid phase. To develop an ELISA for 0.15 M NaCl and 0.1% BSA (buffer A) to remove any remain- the measurement of lapatinib and nilotinib levels in serum, ing small molecules. Three-milliliter fractions were collected the wells of microtiter plates (Nunc F Immunoplates I; Nunc, and fractions 12 to 14, corresponding to the main peaks show- Reskilde, Denmark) were coated by loading 100 µL of anti- ing enzyme activity, were combined and used as a label for lapatinib antibody (2 µg/mL) or anti-nilotinib serum diluted ELISA. 1 : 5000 in 10 mM Tris–HCl buffer (pH 8.5) containing 10 mM

Preparation of Immunogen for Nilotinib Nilotinib im- NaCl and 10 mM NaN3. After a 1-h incubation at 37°C, the munogen was prepared using a partial structure of nilotinib wells were washed twice with buffer A, and were then incu- (AMPP) as shown in Figs. 1 and 3. A solution of AMPP bated with 100 µL of 10 mM Tris–HCl buffer (pH 8.5) contain-

(10 mg, 36 µmol) and succinic anhydride (3.6 mg, 36 µmol) in ing 10 mM NaCl, 10 mM NaN3, and 1% skim milk for 30 min pyridine (0.5 mL) was stirred overnight at 60°C. After remov- at 37°C to prevent nonspecific adsorption. The anti-TKI anti- ing pyridine by passing nitrogen through the reaction mixture, body-coated wells were then filled with 25 µL of human serum the obtained residue, carboxylic-modified AMPP, was dis- samples or buffer A as a control, followed immediately by the solved in DMF (100 µL) and 95% dioxane (2.0 mL) and then addition of 75 µL of the pooled TKI–HRP conjugate (diluted added to EDPC (13.8 mg, 72 µmol) and N-hydroxysuccinimide 1 : 100 and 1 : 200 in buffer A for lapatinib and nilotinib, re- (8.3 mg, 72 µmol). The resulting solution was allowed to stand spectively). The plates were incubated for 3 h at 37°C and the at room temperature for 2 h. The reaction mixture, contain- wells were then washed thoroughly with buffer A. ing succinimidyl AMPP, was then mixed with BSA (20 mg) The activity of the enzyme conjugate bound to each well in 1 mL of 0.1 M phosphate buffer (pH 7.0) and further incu- was measured by the addition of 100 µL of 0.42 mM TMB in bated at room temperature for 2 h. The reaction mixture was 0.05 M acetate–citric acid buffer (pH 5.5) containing 3% di- dialyzed successively for 48 h against 50 and 1 mM phosphate methyl sulfoxide and 0.01% hydrogen peroxide to each well, buffer (pH 7.0) and H2O. The purified conjugate was lyophi- followed by incubation of the plates at 37°C for a suitable lized and used as an immunogen for nilotinib. period for approximately 10–30 min. The enzyme reaction was

Preparation of Nilotinib Antibody Sixty-five 5-week- stopped by the addition of 100 µL of 2.0 M H2SO4 to each well, old female BALB/c mice (Kyudo Exp. Animals, Kumamoto, and color intensity was then measured spectrophotometrically Japan) were injected intraperitoneally with 0.1 mg AMPP– at 450 nm using an ELISA analyzer (ImmunoMini NJ-2300, BSA conjugate emulsified in complete Freund’s adjuvant. Nalge Nunc Int. Co., Ltd., Tokyo, Japan). Concentrations were The mice were given 3 injections of 0.05 mg conjugate alone calculated from standard curves using semi-logarithmic graph at 2-week intervals. Seven days after the final injection, the paper. 1654 Biol. Pharm. Bull. Vol. 38, No. 10 (2015)

Pharmacokinetic Evaluation Two male Wistar rats (Kyudo Exp. Animals, Kumamoto, Japan) with a weight range of 180–200 g were used in this study. Oral formulations were prepared in suspension form by triturating an accurately weighed amount of powdered compound in methyl cellulose (0.5%, w/v water) in a gravimetric dilution pattern. Oral doses of 10 mg/kg (lapatinib) and 20 mg/kg (nilotinib) were administered using a gavage needle at 5 mL/kg to rats after an overnight fast (12 h). Blood samples were collected from cervical vein at 0.5, 1, 2, 3, 4, 6, 8, 12 and 24 h after post-administration, and the serum was stored at −30°C until assayed for TKIs concentrations. The serum samples were diluted 1 to 100 fold with buffer A to obtain TKIs concentrations appro- priate for measurement by the two ELISAs. The experimental protocol was approved by the Ethics Review Committee for Animal Experimentation of Sojo University.

RESULTS AND DISCUSSION

The area under the plasma concentration–time curve (AUC) of nilotinib and lapatinib is increased after the administration with meal.10,11) Similar AUC reductions are observed in lapatinib and nilotinib when co-administered with antacid Fig. 1. Chemical Structures of the Two TKIs drugs.12,13) Thus, the blood concentration of these TKIs is al- tered differently under the meal and antacid drugs adminis- rithmic plot between 1.6 and 5000 ng/mL. For practicality, the tration. Recently, Giles et al. reported a correlation between working range of the assay was arbitrarily set between 40 and hyperbilirubinemia and nilotinib trough concentrations in pa- 5000 ng/mL based on the precision and accuracy of the ELISA tients treated with nilotinib.14) Therefore, evaluation of blood measurements for serum levels of lapatinib (data not shown). these TKIs levels in patients will become a useful tool for The detection limit of lapatinib in the ELISA was 40 ng/mL achieving the optimum therapeutic level for patients who have (Student’s t-test, n=3, p<0.01 compared with the B0 value). experienced drug interactions or adverse side effects and for As the therapeutic plasma concentration range of lapatinib those who require dose adjustment. is approximately 550–1710 ng/mL,16) this ELISA may be suf- In this study, we aimed to produce specific antibodies ficiently sensitive to quantify lapatinib in TDM and pharma- against two TKIs, lapatinib and nilotinib. To our knowledge, cokinetic studies. there is no report describing specific antibody production The specificity of the purified anti-lapatinib antibody was against two TKIs. Chemical structures of the two TKIs are evaluated based on the displacement of bound CFBA–HRP by shown in Fig. 1. One likely reason for the difficulty in gen- similar compounds. Values of cross-reactivity were defined as erating antibodies against two TKIs is that they do not have the ratio of the test compound to CFBA for the concentration a suitable reactive structure for preparing immunogen, such required for 50% inhibition of CFBA–HRP binding to anti- as a drug–BSA conjugate. Therefore, here, immunogens of lapatinib antibody. The anti-lapatinib antibody showed 100.0% lapatinib and nilotinib were prepared using partial structures cross-reactivity with lapatinib. No detectable cross-reactivity, of each TKI. however, was found with m-fluorobenzyl alcohol or 4-amino- Anti-lapatinib antibody was obtained by immunizing rab- 2-chlorophenol (Table 1). These findings suggest that the bits with an antigen conjugated with BSA using CFBA, which anti-lapatinib antibody recognize nearly the entire structure was coupled by diazotization to tyrosine and histidine residues of CFBA used as a hapten, and thus are sufficiently specific on the carrier protein (Fig. 2). The CFBA–BSA conjugate to the structure of lapatinib. Lapatinib is metabolized mainly induced the formation of specific antibodies in both of the by P450 3A4 to form O- and N-dealkylated metabolites.17) The two immunized rabbits. We isolated anti-lapatinib IgG from cross-reactivity of these metabolites with the anti-lapatinib the obtained antiserum, and confirmed that the antibodies ef- antibody has not yet been confirmed. However, based on the fectively adsorbed to the wells of microtiter plates. However, observed specificity of the anti-lapatinib antibody, the O-deal- no useful solid phase antibodies were detected in non-purified kylated metabolite would likely show only limited cross-reac- anti-lapatinib serum. tivity. In contrast, the N-dealkylated metabolite is expected to Lapatinib conjugated to HRP as a tracer was prepared display similar cross-reactivity to that of lapatinib. However, using carboxylic-modified CFBA, by the hydroxysuccinimide as the maximal concentration of this metabolite detected in ester method15) to eliminate the production of antibodies tar- human plasma was relatively low,18) this ELISA may be suf- geting the cross-linkage region of HRP (Fig. 2). The obtained ficiently specific to quantify lapatinib for pharmacokinetic conjugate was stable for over 6 months in elution buffer (pH studies in humans. 7.0) at 4°C without any loss of enzyme or immunoreactive Anti-nilotinib antibody was obtained by immunizing mice activity. with an antigen conjugated with BSA using carboxylic-mod- The dose–response standard curve for lapatinib in serum is ified AMPP (Fig. 3), which was coupled to amino groups on shown in Fig. 4. The curve was nearly linear on a semiloga- the carrier protein by the hydroxysuccinimide ester method.15) Vol. 38, No. 10 (2015) Biol. Pharm. Bull. 1655

Fig. 2. Scheme for the Preparation of Immunogen and Enzyme Conjugate in the ELISA for Lapatinib

Fig. 4. Standard Curves for Lapatinib and Nilotinib in Human Serum The curves show the amount (%) of bound enzyme activity for various doses of each drug (B) as a ratio of that bound by HRP-labeled drug alone (B0). ●, lapatinib; ▲, nilotinib. Each point represents the mean±S.D. of 3 replicates.

The AMPP–BSA conjugate induced the formation of specific antibodies in each of the five immunized mice. The obtained Fig. 3. Scheme for the Preparation of Immunogen and Enzyme Conju- anti-nilotinib serum, containing anti-nilotinib IgG, was direct- gate in the ELISA for Nilotinib ly adsorbed to the wells in microtiter plates, because useful solid-phase antibodies were present in the non-purified serum. 1656 Biol. Pharm. Bull. Vol. 38, No. 10 (2015)

Table 1. Speciﬁcity of Anti-lapatinib Antibody Table 2. Speciﬁcity of Anti-nilotinib Antibody

Compounds Cross-reactivity (%) Compounds Cross-reactivity (%)

Lapatinib 100 Nilotinib 100

AMPP 100

CFBA 100

4-(3-Pyridyl)-2-pyrimidineamine <0.1

m-Fluorobenzyl alcohol <0.3

4-Amino-2-chlorophenol <0.3

An AMPP–HRP conjugate was also prepared by the same procedure used for the nilotinib immunogen. The obtained conjugate was stable for greater than 6 months in the elution buffer (pH 7.0) at 4°C without any loss of enzyme or immunoreactive activity. The dose–response standard curve of nilotinib in serum is shown in Fig. 4. The curve was approximately linear on a semilogarithmic plot between 0.32 and 1000 ng/mL. For practicality, the working range of the assay was arbitrarily set between 8 and 1000 ng/mL based on the detection limit of nilotinib in the ELISA (8 ng/mL; Student’s t-test, n=3, p<0.01 compared with the B0 value), and the precision and accuracy of the ELISA measurements for serum levels of nilotinib (data Fig. 5. Mean Serum Concentration–Time Profiles for Lapatinib and Nilotinib in Rats Following Single Oral Administration of Lapatinib not shown). The therapeutic plasma concentration range of (10 mg/kg) or Nilotinib (20 mg/kg) 19) nilotinib is between 1123 and 1595 ng/mL ; therefore, this Two rats with a weight range of 180–200 g were injected with 10 mg/kg lapatinib ELISA may be sufficiently sensitive to quantify nilotinib in or 20 mg/kg nilotinib. At each interval, blood samples were collected and serum TDM and pharmacokinetic studies. levels of lapatinib and nilotinib were measured by the two ELISAs. The anti-nilotinib antibody showed 100% cross-reactivity with nilotinib. No detectable cross-reactivity, however, was As a demonstration of the potential of the two ELISAs, found with 4-(3-pyridyl)-2-pyrimidineamine (Table 2). These preliminary pharmacokinetic studies of lapatinib and nilotinib findings suggest that the anti-nilotinib antibody recognizes in rats were performed (Fig. 5). Lapatinib and nilotinib were nearly the entire structure of AMPP used as a hapten, and rapidly absorbed, reached peak concentrations in the serum of thus is sufficiently specific to the structure of nilotinib. In 650 ng/mL and 2300 ng/mL at 2 and 6 h after oral administra- humans, nilotinib is metabolized primarily in the liver via tion of lapatinib and nilotinib, respectively, and then slowly oxidation and hydroxylation pathways mediated by CYP3A4 decreased. The nilotinib levels in the serum tested were al- enzyme.20) The cross-reactivity of these metabolites with most consistent with those reported by Xia et al.21) by means the anti-nilotinib antibody produced here has not yet been of LC-MS/MS for nilotinib. Using the two ELISAs, these examined. However, the maximal concentration of these me- TKIs levels were easily measured in the serum of rats after a tabolites detected in human plasma was relatively low, and single dose oral administration of lapatinib or nilotinib. unchanged nilotinib is the main circulating component in In conclusion, we have described the production of the first plasma.20) Based on these finding, this ELISA may be suf- antibodies against the TKIs lapatinib and nilotinib by prepar- ficiently specific to quantify nilotinib for pharmacokinetic ing immunogens using partial structures of each TKI. The studies in humans. generated antibodies against lapatinib and nilotinib were used Vol. 38, No. 10 (2015) Biol. Pharm. Bull. 1657 to develop two ELISAs, which were shown to be sensitive, enzyme-linked immunosorbent assay for the quantification of ima- specific and adaptable for high-throughput analyses. The as- tinib. Biol. Pharm. Bull., 36, 1964–1968 (2013). says are therefore expected be valuable tools in studies of the 10) Koch KM, Reddy NJ, Cohen RB, Lewis NL, Whitehead B, Mackay K, Stead A, Beelen AP, Lewis LD. Effects of food on the relative TDM and pharmacokinetics of lapatinib and nilotinib. bioavailability of lapatinib in cancer patients. J. Clin. Oncol., 27, 1191–1196 (2009). Conflict of Interest The authors declare no conflict of 11) Tanaka C, Yin OQ, Sethuraman V, Smith T, Wang X, Grouss K, interest. Kantarjian H, Giles F, Ottmann OG, Galitz L, Schran H. Clinical pharmacokinetics of the BCR-ABL tyrosine kinase inhibitor nilo- REFERENCES tinib. Clin. Pharmacol. Ther., 87, 197–203 (2010). 12) Ohgami M, Homma M, Shimada M, Kohda Y. Influence of meal 1) Xia W, Mullin RJ, Keith BR, Liu LH, Ma H, Rusnak DW, Owens and antacid drugs on the blood concentration of tyrosine kinase G, Alligood KJ, Spector NL. Anti-tumor activity of GW572016: inhibitors. Jpn. J. Ther. Drug Monit., 30, 125–133 (2013). a dual tyrosine kinase inhibitor blocks EGF activation of EGFR/ 13) Yin OQ, Gallagher N, Fischer D, Demirhan E, Zhou W, Golor G, erbB2 and downstream Erk1/2 and AKT pathways. Oncogene, 21, Schran H. Effect of the proton pump inhibitor esomeprazole on the 6255–6263 (2002). oral absorption and pharmacokinetics of nilotinib. J. Clin. Pharma- 2) Geyer CE, Forster J, Lindquist D, Chan S, Romieu CG, Pienkowski col., 50, 960–967 (2010). T, Jagiello-Gruszfeld A, Crown J, Chan A, Kaufman B, Skarlos 14) Giles FJ, Yin OQ, Sallas WM, le Coutre PD, Woodman RC, Ott- D, Campone M, Davidson N, Berger M, Oliva C, Rubin SD, Stein mann OG, Baccarani M, Kantarjian HM. Nilotinib population S, Cameron D. Lapatinib plus capecitabine for HER2-positive ad- pharmacokinetics and exposure-response analysis in patients with vanced breast cancer. N. Engl. J. Med., 355, 2733–2743 (2006). imatinib-resistant or -intolerant chronic myeloid leukemia. Eur. J. 3) Golemovic M, Verstovsek S, Giles F, Cortes J, Manshouri T, Man- Clin. Pharmacol., 69, 813–823 (2013). ley PW, Mestan J, Dugan M, Alland L, Griffin JD, Arlinghaus RB, 15) Hosoda H, Sakai Y, Nambara T. A direct enzyme immunoassay Sun T, Kantarjian H, Beran M. AMN107, a novel aminopyrimidine of 6β-hydroxycortisol in human urine. Chem. Pharm. Bull., 29, inhibitor of Bcr-Abl, has in vitro activity against imatinib-resistant 170–175 (1981). chronic myeloid leukemia. Clin. Cancer Res., 11, 4941–4947 (2005). 16) Fumoleau P, Koch KM, Brain E, Lokiec F, Rezai K, Awada A, Hay- 4) Weisberg E, Manley PW, Breitenstein W, Brüggen J, Cowan-Jacob ward L, Werutsky G, Bogaerts J, Marréaud S, Cardoso F. A phase SW, Ray A, Huntly B, Fabbro D, Fendrich G, Hall-Meyers E, Kung I pharmacokinetics study of lapatinib and tamoxifen in metastatic AL, Mestan J, Daley GQ, Callahan L, Catley L, Cavazza C, Azam breast cancer (EORTC 10053 Lapatam study). Breast, 23, 663–669 M, Neuberg D, Wright RD, Gilliland DG, Griffin JD. Characteriza- (2014). tion of AMN107, a selective inhibitor of native and mutant Bcr-Abl. 17) Teng WC, Oh JW, New LS, Wahlin MD, Nelson SD, Ho HK, Chan Cancer Cell, 7, 129–141 (2005). EC. Mechanism-based inactivation of cytochrome P450 3A4 by 5) Pirro E, De Francia S, De Martino F, Fava C, Ulisciani S, Cambrin lapatinib. Mol. Pharmacol., 78, 693–703 (2010). GR, Racca S, Saglio G, Di Carlo F. A new HPLC-UV validated 18) Castellino S, O'Mara M, Koch K, Borts DJ, Bowers GD, method for therapeutic drug monitoring of tyrosine kinase inhibi- MacLauchlin C. Human metabolism of lapatinib, a dual kinase tors in leukemic patients. J. Chromatogr. Sci., 49, 753–757 (2011). inhibitor: implications for hepatotoxicity. Drug Metab. Dispos., 40, 6) Götze L, Hegele A, Metzelder SK, Renz H, Nockher WA. Develop- 139–150 (2012). ment and clinical application of a LC-MS/MS method for simulta- 19) Larson RA, Yin OQ, Hochhaus A, Saglio G, Clark RE, Nakamae neous determination of various tyrosine kinase inhibitors in human H, Gallagher NJ, Demirhan E, Hughes TP, Kantarjian HM, le plasma. Clin. Chim. Acta, 413, 143–149 (2012). Coutre PD. Population pharmacokinetic and exposure-response 7) Lankheet NA, Hillebrand MJ, Rosing H, Schellens JH, Beijnen JH, analysis of nilotinib in patients with newly diagnosed Ph+ chronic Huitema AD. Method development and validation for the quantifica- myeloid leukemia in chronic phase. Eur. J. Clin. Pharmacol., 68, tion of dasatinib, erlotinib, gefitinib, imatinib, lapatinib, nilotinib, 723–733 (2012). sorafenib and sunitinib in human plasma by liquid chromatography 20) DeRemer DL, Ustun C, Natarajan K. Nilotinib: a second-generation coupled with tandem mass spectrometry. Biomed. Chromatogr., 27, tyrosine kinase inhibitor for the treatment of chronic myelogenous 466–476 (2013). leukemia. Clin. Ther., 30, 1956–1975 (2008). 8) Saita T, Fujito H, Mori M. A specific and sensitive assay for ge- 21) Xia B, Heimbach T, He H, Lin TH. Nilotinib preclinical pharmaco- fitinib using the enzyme-linked immunosorbent assay in human kinetics and practical application toward clinical projections of oral serum. Biol. Pharm. Bull., 28, 1833–1837 (2005). absorption and systemic availability. Biopharm. Drug Dispos., 33, 9) Saita T, Shin M, Fujito H. Development of a specific and sensitive 536–549 (2012).