A specific and sensitive assay for gefitinib using the enzyme-linked immunosorbent assay in human serum

Article abstract of DOI:10.1248/bpb.28.1833

The epidermal growth factor tyrosine kinase inhibitor gefitinib is a novel, molecularly targeted agent that has been approved for the treatment of advanced non-small cell lung cancer. This paper reports a specific and sensitive enzyme-linked immunosorbent

Full text of DOI:10.1248/bpb.28.1833

October 2005
Biol. Pharm. Bull. 28(10) 1833—1837 (2005)
1833
A Specific and Sensitive Assay for Gefitinib Using the Enzyme-Linked
Immunosorbent Assay in Human Serum
*
Tetsuya SAITA, Hiroshi FUJITO, and Masato MORI
Department of Pharmacy, Saga University Hospital; 5–1–1 Nabeshima, Saga 849–8501, Japan.
Received May 16, 2005; accepted July 4, 2005
The epidermal growth factor tyrosine kinase inhibitor gefitinib is a novel, molecularly targeted agent that
has been approved for the treatment of advanced non-small cell lung cancer. This paper reports a specific and
sensitive enzyme-linked immunosorbent assay (ELISA) for pharmacokinetic studies of gefitinib. Anti-gefitinib
antibody was obtained by immunizing rabbits with an antigen conjugated with bovine serum albumin using dia-
zotized 4-amino-2-methoxy-1-[3-(morpholin-4-yl)propoxy]benzene. Enzyme labeling of gefitinib with b-D-galac-
tosidase was similarly performed using a diazotized 4-amino-2-methoxy-1-[3-(morpholin-4-yl)propoxy]benzene.
A simple ELISA for gefitinib was developed using the principle of direct competition between gefitinib and the
enzyme marker for anti-gefitinib antibody which had been adsorbed to the plastic surface of a microtiter plate.
Gefitinib concentrations in serum lower than 800 pg/ml were measurable reproducibly using the ELISA. Cross-
reactivity data showed that the antibody well recognizes both the 3-morpholinopropoxy and methoxy moieties
well, and thus is sufficiently specific for the structure of gefitinib. Using this assay, drug levels were easily mea-
sured in the serum of rabbits after oral administration of gefitinib at a single dose of 5 mg/kg. The specificity and
sensitivity of the ELISA for gefitinib should provide a valuable new tool for use in pharmacokinetic and toxicity
studies of gefitinib.
Key words gefitinib; enzyme-linked immunosorbent assay; anticancer drug
netic studies of the individual drugs.11—13)
Gefitinib, N-(3-chloro-4-fluorophenyl)-7-methoxy-6-[3-
We present here the first report of an ELISA for gefitinib,
including the methodology for antibody production, labeling
of gefitinib with b-D-galactosidase (b-Gal) to act as a tracer,
characterization of antibody specificity, and technique devel-
oped for the measurement of gefitinib with the ELISA. The
initial application of the assay to the measurement of drug
levels in rabbits demonstrates its usefulness for the assess-
ment of basic pharmacokinetic distribution.
(morpholin-4-yl)propoxy] quinazolin-4-amine is an orally
active epidermal growth factor receptor tyrosine kinase in-
hibitor (EGFR-TKI) that blocks signal transduction pathways
implicated in cancer cell proliferation, survival, and host-de-
pendent processes promoting cancer growth.1,2) Gefitinib ex-
hibits a broad spectrum of antitumor activity against many
human solid tumor xenografts including breast, pancreas,
lung, colorectal, and head and neck cancer.3,4) Gefitinib is
now approved for the treatment of advanced non-small cell
lung cancer in several countries, including Australia, Japan,
and the U.S.A. However, gefitinib has also been reported to
produce interstitial lung disease in some patients.5,6) Thus
MATERIALS AND METHODS
Equipment The IR spectrum was recorded using a Hi-
clinical evaluation of gefitinib is still controversial and fur- tachi 270-30 spectrometer (Hitachi, Tokyo, Japan). The pro-
ther studies are needed.
ton nuclear magnetic resonance (1H-NMR) spectrum was
In clinical trials, it was observed that there is no relation- recorded with a JEOL-MY60FT spectrometer (JEOL, Tokyo,
ship between gefitinib blood concentration and antitumor ac- Japan) at 60 MHz using tetramethylsilane as an internal stan-
tivity.7) However, dose dependency was revealed in the onset dard. Enzymatic activity was measured using a fluorescence
of skin and gastrointestinal toxicity, suggesting a relation microplate reader (Fluoroskan Ascent, Labsystems, Helsinki,
with gefitinib blood concentration.7—9) Moreover, there are Finland).
4- to 10-fold individual differences in gefitinib blood concen-
Reagents Gefitinib was supplied by AstraZeneca Phar-
tration in the steady state.7—9) Therefore the gefitinib blood maceuticals (Macclesfield, U.K.) with a purity of 99%. 4-(3-
concentration must be monitored to minimize its side effects. Hydroxypropyl)morpholine and 4-nitroguaiacol were ob-
Previous pharmacokinetic studies of gefitinib were under- tained from Aldrich Chemical Co. (St. Louis, MO, U.S.A.).
taken with liquid chromatography-mass spectrometry/mass Catechol was obtained from Wako Pure Chemical Industries
spectrometry (LC-MS/MS).10) LC-MS/MS is a specific, ac- (Osaka, Japan). b-D-Galactosidase (b-Gal; EC 3.2.1.23) from
curate, and reproducible method. However, it requires expen- Escherichia coli and 4-methylumbelliferyl-b-D-galactopyra-
sive instrumentation and a high degree of technical expertise. noside were obtained from Boehringer Mannheim (Mannheim,
Thus the development of a simple quantification method of Germany). All other reagents and solvents were of the high-
gefitinib is needed. The enzyme-linked immunosorbent assay est grade commercially available.
(ELISA) is generally very sensitive and specific and has ad-
2-[3-(Morpholin-4-yl)propoxy]phenol To a solution of
vantages in simplicity, cost, safety, and ability to process nu- 4-(3-hydroxypropyl)morpholine (100 mg, 0.7 mmol) in dry
merous samples. If a kit and automatic method could be de- benzene (10 ml), SOCl2 (0.15 ml) was added dropwise in an
veloped, it would be used in many facilities. We have already ice bath and the mixture was then heated at 40 °C for 1 h.
developed several ELISAs for some anticancer drugs, each After evaporation of the solvent, the residue was dissolved in
of which was simple, sensitive, and useful for pharmacoki- toluene (10 ml). The toluene solution was added to a solution
∗ To whom correspondence should be addressed. e-mail: saita@cc.saga-u.ac.jp
© 2005 Pharmaceutical Society of Japan

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Vol. 28, No. 10
of catechol (80 mg, 0.7 mmol) in 2% K2CO3 (5 ml). The mix-
Preparation of Gefitinib Antibody An aliquot of a so-
ture was heated under reflux at 120 °C for 8 h. After cooling lution containing 1 mg of AMMB-BSA conjugate (1.5 ml)
to room temperature, the toluene layer was dried over anhy- was emulsified with an equal volume of Freund’s complete
drous Na2SO4, and reduced by evaporation. The residue was adjuvant. Two white female rabbits were administered multi-
purified on a silica gel 60 (70—230 mesh) column chro- ple subcutaneous injections of the solution at sites along both
matographed with CHCl3–CH3OH (20 : 1, v/v). A total of 95 sides of their backs. Booster injections were then adminis-
mg (40%) of 2-[3-(morpholin-4-yl)propoxy]phenol (MPP) as tered three times at biweekly intervals, using one-half the
a white powder was obtained: mp 102—103 °C. IR (KBr) amount of the dose of the first immunization. The rabbits
1
cmꢀ1: 2964, 1496, 1266, 1112. H-NMR (CDCl3) d: 1.97 were bled from an ear vein 10 weeks after immunization
(2H, t, Jꢁ6.1 Hz, –CH2CH2CH2–), 2.50—2.74 (6H, m, N began. The sera (10 ml) were separated by centrifugation
(CH2)3), 3.74—4.09 (6H, m, O(CH2)3), 6.86—6.99 (4H, m, and heated at 55 °C for 30 min. Fractions of IgG were ex-
ArH).
tracted from the sera with 50% saturated ammonium sulfate
3-Methoxy-4-[3-(morpholin-4-yl)propoxy]nitrobenzene and chromatographed on a column of DEAE-Sephacel
To a solution of 4-(3-hydroxypropyl)morpholine (100 mg, (2.1ꢂ23 cm) using 17.5 mM phosphate buffer (pH 6.8) as
0.7 mmol) in dry benzene (10 ml), SOCl2 (0.15 ml) was an eluent. The fraction passed through the column was
added dropwise in an ice bath and the mixture was then lyophilized and used as anti-gefitinib IgG for ELISA.
b
Preparation of the Gefitinib- -Gal Conjugate Gefi-
heated at 40 °C for 1 h. After evaporation of the solvent, the
residue was dissolved in toluene (10 ml). The toluene solu- tinib was labeled by binding to b-Gal, essentially by the
tion was added to a solution of 4-nitroguaiacol (100 mg, 0.6 same method as used for the preparation of gefitinib im-
mmol) in 2% K2CO3 (5 ml). The mixture was heated under munogen. AMMB (approximately 6 mg, 24 mmol) in DMF
reflux at 120 °C for 8 h. After cooling to room temperature, 20 ml was acidified by the addition of 100 ml of 1 M acetic
the toluene layer was washed with saturated NaCl, dried over acid and then diazotized with sodium nitrite (3.3 mg,
anhydrous Na2SO4, and reduced by evaporation. The residue 48 mmol) in 100 ml of distilled water at 0 °C for 10 min. Next,
was purified on a silica gel 60 (70—230 mesh) column chro- a 30-ml aliquot of the above reaction mixture containing dia-
matographed with CHCl3–CH3OH (20 : 1, v/v). A total of 80 zotized AMMB (ca. 3.3 mmol) was added directly to b-Gal
mg (40%) of 3-methoxy-4-[3-(morpholin-4-yl)propoxy] ni- (156 mg, 0.28 nmol) in 0.5 ml of 0.5 M phosphate buffer (pH
trobenzene (MMNB) as a pale yellow oil was obtained. IR 7.5), followed by 1-h incubation at room temperature. The
1
(CCl4) cmꢀ1: 2956, 1512, 1338, 1274. H-NMR (CDCl3) d: mixture was chromatographed on a column of Sepharose 6B
2.05 (2H, t, Jꢁ6.8 Hz, –CH2CH2CH2–), 2.39—2.67 [6H, m, (2.0ꢂ40 cm) using 20 mM phosphate buffer (pH 7.0) contain-
N(CH2)3], 3.72 [4H, t, Jꢁ4.7 Hz, O(CH2)2], 3.95 (3H, s, ing 0.1 M NaCl, 1 mM MgCl2, 0.1% BSA, and 0.1% NaN3
CH3O), 4.2 (2H, t, Jꢁ6.4Hz, CH2OAr), 6.87—7.81 (3H, (buffer A) to remove the remaining small molecules. Four-
m, ArH).
milliliter fractions were collected, and fractions 15 to 17, rep-
Coupling Reaction for Hapten A solution of 10 mg of resenting the main peaks showing enzyme activity, were
MMNB was dissolved in 1 ml of 60% ethanol and alkaline combined and used as a label in the ELISA.
with 40 mg of MgO. To this mixture was added 80 mg of
ELISA for Gefitinib ELISA is based on the principle of
Na2S2O3 which was vigorously shaken, and the solution competition between enzyme-labeled and unlabeled drugs
was heated in a water bath at 50 °C for 2 h. After cooling, for an immobilized antibody, followed by measurement of
ethanol was distilled by evaporation in vacuo, and the aque- the marker enzyme activity of the immunocomplex bound to
ous solution was washed with AcOEt to remove unreacted the solid phase. Briefly, the wells in microtiter plates (Nunc F
MMNB. The aqueous layer was evaporated, and the residue Immunoplates I; Nunc, Reskilde, Denmark) were coated by
was dissolved in ethanol. The resulting suspension was loading 150 ml of anti-gefitinib IgG (1.0 mg/ml) in 10 mM
filtered and the filtrate evaporated in vacuo to give 4-amino- Tris–HCl buffer (pH 8.5) containing 10 mM NaCl and 10 mM
2-methoxy-[3-(morpholin-4-yl)propoxy]benzene (AMMB) NaN3 and allowed to stand for 1 h at 37 °C. After the plates
(5 mg, 50%) as a yellow solid. The resulting AMMB was had been washed twice with 60 mM phosphate buffer (pH
conjugated with bovine serum albumin (BSA) and b-Gal, re- 7.4) containing 10 mM ethylenediaminetetraacetate, 0.1%
spectively, as a gefitinib immunogen and a tracer.
BSA and 0.1% NaN3 (buffer B), they were incubated with
AMMB (approximately 5 mg, 20 mmol) in 20 ml dimethyl- 200 ml of 10 mM Tris–HCl buffer (pH 8.5) containing 10 mM
formamide was acidified by the addition of 100 ml of 1 M NaCl and 10 mM NaN3 containing 2% BSA for 20 min at
acetic acid and then diazotized with sodium nitrite (2.8 mg, 37 °C to prevent nonspecific adsorption. The anti-gefitinib
40.5 mmol) in 50 ml of distilled water at 0 °C for 10 min. The IgG-coated wells were then filled with 50 ml of either gefi-
solution was immediately mixed with BSA (10 mg) in 1 ml tinib-treated samples, or buffer B as a control, followed im-
of 0.5 M borate buffer (pH 9.5), followed by 1-h incubation at mediately by 50 ml of the pooled gefitinib-b-Gal conjugate
room temperature. The mixture was chromatographed on a (diluted 1 : 300 in buffer B for gefitinib). The wells were then
Sephadex G-100 column (2.8ꢂ42 cm) with 0.1 M phosphate incubated overnight at room temperature and once again
buffer (pH 7.0) containing 3 M urea. The degree of coupling washed thoroughly with buffer B.
of diazotized AMMB with BSA was determined based on
The activity of the enzyme conjugate bound to each well
amino acid analysis after hydrolysis with 6 M HCl at 110 °C was then measured by the addition of 125 ml of 0.1 mM 4-
for 24 h.14) Seven moles of the hapten were found to be cou- methylumbelliferyl-b-D-galactopyranoside in buffer A, fol-
pled with one mole of BSA on the basis of the decrease in lowed by incubation of the wells at 37 °C for 60 min. The en-
moles of histidine and tyrosine. The amount of protein was zyme reaction was stopped by the addition of 75 ml of 0.5 M
determined using Lowry et al.’s method.15)
glycine–NaOH buffer (pH 10.3) to each well, and the result-

October 2005
1835
ing 4-methylumbelliferone concentration was determined by
spectrofluorometry at wavelengths of 355 nm for excitation
and 460 nm for emission using a fluorescence microplate
reader. Concentrations were calculated from the standard
curve using semilogarithmic graph paper.
Pharmacokinetic Evaluation Three white female rab-
bits in the weight range of 3.5 to 4.0 kg were used in this
study. Gefitinib was orally administered at a dose of 5 mg/kg
to the rabbits. The drug was suspended in a carrier composed
of 10% DMSO and isotonic sodium chloride at concentra-
tions of 5 mg/ml. Blood samples were collected at 0.5, 1, 2,
3, 4, 5, 6, 8, and 24 h postadministration, and the serum was
stored at ꢀ30 °C until the assay of gefitinib concentration.
The serum was diluted 40-fold with buffer B to obtain a gefi-
tinib concentration appropriate for measurement using
ELISA.
Fig. 1. Chemical Structures of Gefitinib and Its Major Metabolite
RESULTS AND DISCUSSION
The major metabolite of gefitinib in humans is O-
desmethylgefitinib (Fig. 1). During long-term therapy, the
plasma level of O-desmethylgefitinib is comparable with that
of gefitinib. Therefore, to develop an ELISA for gefitinib
which can be applied to the pharmacokinetic studies of gefi-
tinib, it is necessary to produce an anti-gefitinib antibody that
does not show a cross-reaction with the major metabolite O-
desmethylgefitinib. In general, the antibody specificity
on hapten appears to be toward the group farthest away from
the region of conjugation to the carrier protein in the im-
munogen structure.16,17) Thus, to produce an antibody spe-
cific for both the 3-morpholinopropoxy and the methoxy
moieties of gefitinib, gefitinib immunogen was prepared
using a partial structure of gefitinib (AMMB) (Fig. 2). The
AMMB was coupled by diazotization to tyrosine and histi-
dine residues on the carrier protein. The AMMB-BSA conju-
gate, with 7.0 mol of AMMB per mol of BSA, induced the
formation of specific antibodies in each of two the rabbits
immunized. AMMB-b-Gal conjugate was also prepared fol-
lowing essentially the same procedure. The conjugate thus
obtained was stable for more than 6 months in eluted buffer
(pH 7.0) at 4 °C without any loss of the enzyme and im-
munoreactive enzyme activity.
Fig. 2. Preparation of the Immunogen for Gefitinib
Using anti-gefitinib antibody and gefitinib-b-Gal as a
tracer, an ELISA for the quantification of gefitinib was devel-
oped. The dose–response standard curve of gefitinib obtained
in the human serum is shown in Fig. 3. The curve was essen-
tially linear on a semilogarithmic plot between 0.8 and
100 ng/ml. For practical purposes, the working range was ar-
bitrarily set between 0.8 and 100 ng/ml based on the preci-
sion and accuracy findings for the ELISA in serum (Table 1),
which showed this ELISA to be a reproducible technique.
Recoveries of four different gefitinib levels ranging from 0.8
to 100 ng/ml were satisfactory (98.8 to 107.5%, nꢁ5). The
coefficients of variation for intra- and interassays at four dif-
ferent gefitinib levels between 0.8 to 100 ng/ml was 6.3 to
9.5% and 5.1 to 9.0% (each nꢁ5), respectively. The detec-
tion limit of gefitinib in the ELISA was 800 pg/ml (Student’s
t-test, nꢁ3, pꢃ0.001 compared with the B0 value). The
plasma concentration range in the usual clinical dose of gefi-
tinib is between about 10 and 200 ng/ml (data on file at As-
Fig. 3. Standard Curve for Gefitinib in Human Serum
The curve shows the amount (%) of bound enzyme activity for various doses of gefi-
tinib (B) as a ratio of that bound using gefitinib-b-Gal alone (B0). Each point represents
traZeneca). Therefore this ELISA may be sufficiently sensi- the meanꢄS.D. of 5 replicates.

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Vol. 28, No. 10
Table 1. Precision and Recovery of ELISA for Gefitinib in Human Serum
Added (ng/ml) Estimated (ng/ml) Recovery (%) C.V. (%)
Intraassay
0.8
4.0
20.0
100.0
0.8
4.0
20.0
100.0
0.86ꢄ0.06
4.14ꢄ0.3
20.8ꢄ1.3
104.8ꢄ10
0.81ꢄ0.07
3.95ꢄ0.2
19.9ꢄ1.7
100.8ꢄ9.1
107.5
103.5
104.0
104.8
101.3
98.8
7.0
7.2
6.3
9.5
8.6
5.1
8.5
9.0
Interassay
99.5
100.8
Values represent the meanꢄS.D. of 5 experiments.
Table 2. Specificity of Anti-gefitinib IgG
Compounds
Fig. 4. Serum Gefitinib Levels in Rabbits after a Single Oral Administra-
tion of Gefitinib
% Cross-reaction (50%)
Three rabbits, each weighing 3.5 to 4.0 kg, were injected with 5 mg/kg gefitinib. At
each interval, blood was collected and the serum gefitinib level was measured by
ELISA. Each point represents the meanꢄS.D. of three rabbits.
anti-gefitinib antibody. In addition, the other three morpholin
open-ring metabolites of gefitinib would not show the cross-
reaction. On the other hand, another metabolite (desfluoroge-
fitinib) would show a cross-reaction similar to that of gefi-
tinib. However, the maximal concentration of this metabolite
detected in human plasma was relatively low (data on file at
AstraZeneca). Therefore this ELISA may be sufficiently spe-
cific to quantify gefitinib for pharmacokinetic studies in hu-
mans.
Gefitinib
MMNB
100.0
100.0
MPP
3.8
4-(3-Hydroxypropyl)morpholine
0.18
In conclusion, the ELISA procedure for gefitinib reported
here is sensitive, specific, reproducible, simple, and adapt-
able for the analyses of numerous samples. This ELISA will
be a valuable tool in pharmacokinetic and toxicity studies of
gefitinib.
4-Nitroguaiacol
ꢃ0.023
Acknowledgment This study was supported in part by a
Grant-in-Aid for Scientific Research (No. 16922068) from
the Japan Society for the Promotion of Science.
tive to quantify gefitinib in pharmacokinetic studies.
An antibody specificity was determined based on the dis-
placement of bound gefitinib-b-Gal by similar compounds.
Values of the cross-reactivity were defined as the ratio of
each compound to gefitinib in the concentrations required for
50% inhibition of gefitinib-b-Gal binding to the antibody.
The anti-gefitinib antibody showed 100.0% cross-reactivity
with MMNB used as a hapten antigen, 3.8% with MPP, and
0.18% with 4-(3-hydroxypropyl)morpholine. No detectable
cross-reaction, however, was found with 4-nitroguaiacol
(Table 2). These findings suggest that the antibody well rec-
ognizes both the 3-morpholinopropoxy and methoxy moi-
eties of gefitinib well, and thus is sufficiently specific for the
structure of gefitinib.
As a demonstration of the potential of the ELISA, gefitinib
was orally administered at a dose of 5 mg/kg to the rabbits,
and serum concentration kinetics were monitored (Fig. 4).
Gefitinib was rapidly absorbed, reached a peak concentration
in the serum of 926.7ꢄ174.7 ng/ml (meanꢄS.D.) at 60 min
after dosing, and then slowly decreased. The ELISA determi-
nations, however, may quantify the total amounts of gefitinib
immunoreactivity including gefitinib metabolites with cross-
reactivity with anti-gefitinib antibody. Five metabolites of
gefitinib have already been identified in human plasma (data
on file at AstraZeneca). The cross-reactivity of these metabo-
lites has not yet been confirmed. However, it is estimated that
the O-desmethylgefitinib, the major metabolite, shows a sim-
ilar cross-reaction to MPP judging from the specificity of the
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