ELISA for the quantification of pilsicainide

Article abstract of DOI:10.1248/bpb.24.1113

A sensitive and specific ELISA for an antiarrhythmic drug, pilsicainide, was developed, which is capable of measuring as low as 1.6 ng/ml. Anti-pilsicainide antibody was obtained by immunizing rabbits with pilsicainide conjugated with bovine serum albumin

Full text of DOI:10.1248/bpb.24.1113

October 2001
Biol. Pharm. Bull. 24(10) 1113—1116 (2001)
1113
ELISA for the Quantification of Pilsicainide
Tetsuya SAITA,* Hiroshi FUJITO, and Masato MORI
Faculty of Hospital Pharmacy, Saga Medical School, 5–1–1 Nabeshima, Saga 849–8501, Japan.
Received May 28, 2001; accepted June 28, 2001
A sensitive and specific ELISA for an antiarrhythmic drug, pilsicainide, was developed, which is capable of
measuring as low as 1.6 ng/ml. Anti-pilsicainide antibody was obtained by immunizing rabbits with pilsicainide
conjugated with bovine serum albumin using diazotized N-(3-amino-2,6-dimethylphenyl)-8-pyrrolizidinylac-
etamide (3-aminopilsicainide). Enzyme labeling of pilsicainide with b-D-galactosidase was similarly performed
using a diazotized 3-aminopilsicainide. Cross-reactivity data showed that the antibody well recognizes both the
aromatic ring and the pyrrolizidine moieties, and thus specific enough to the structure of pilsicainide. The values
for the pilsicainide concentrations detected using this assay were comparable with those detected using HPLC.
There was a good correlation between the values determined by the two methods. Moreover, the ELISA was
about 30-fold more sensitive in detecting pilsicainide at lower concentrations. Using this assay, drug levels were
easily measured in the serum of rabbits after oral administration of pilsicainide at a single dose of 1 mg/kg. The
ELISA should be a valuable tool in therapeutic drug monitoring (TDM) and pharmacokinetic studies of pilsi-
cainide.
Key words pilsicainide; ELISA; antiarrhythmic drug
Since antiarrhythmic agents have a narrow range of effec- tachi 270-30 spectrometer (Tokyo, Japan). A proton nuclear
tive blood concentrations, and the therapeutic range and toxic magnetic resonance (1H-NMR) spectrum was taken with a
range are similar, therapeutic drug monitoring (TDM) is nec- JEOL-MY60FT spectrometer (Tokyo, Japan) at 60 MHz
essary.
using tetramethylsilane as an internal standard. Enzymic ac-
Pilsicainide is an antiarrhythmic agent developed in Japan tivity was measured with a fluorescence microplate reader
and classified as Ic in the Vaughan Williams classification.1,2) (Fluoroskan Ascent, Labsystems, Helsinki, Finland).
It has been reported to be effective for supraventricular and
Reagents Pilsicainide hydrochloride and 8-pyrrolizidine-
ventricular tachyarrhythmia.3—5) The therapeutic range of this acetic acid were supplied by Suntory Ltd. (Osaka, Japan).
drug was reported to be 0.2—0.9 mg/ml.6) This drug is elimi- b-D-Galactosidase (b-Gal; EC 3.2.1.23) from Escherichia
nated by renal excretion, and the elimination half-life is pro- coli and 4-methylumbelliferyl-b-D-galactopyranoside were
longed in patients with renal hypofunction.7) Therefore, it is obtained from Boehringer Mannheim (Mannheim, Germany).
necessary to adjust the administration interval and dosage ac-
Preparation of the Immunogen for Pilsicainide Sixty
cording to the renal function. Because overdose of this drug percent HNO3 (7 ml) was added dropwise to a mixture of pil-
results in adverse effects including circulatory disorders, sicainide hydrochloride (20 mg, 63 mmol) and concentrated
mental disorders, neuropathy, ventricular fibrillation, ventric- sulfuric acid (300 ml) at Ϫ20 °C, and the mixture was stirred
ular tachycardia, and syncope, pilsicainide also requires at Ϫ20 °C for 1.5 h. The reaction mixture was poured onto
TDM as other antiarrhythmic agents do.
ice-water, neutralized with saturated NaHCO3, and extracted
The blood concentration of pilsicainide is currently mea- with AcOEt. The extract was washed with saturated NaCl
sured by HPLC.8,9) However, pretreatments such as extraction and then concentrated. The residue was purified by alu-
of samples is necessary and the procedure is complex. Thus, minium oxide gel 60 (70—230 mesh) column chromatogra-
the development of a simple quantification method of pilsi- phy with CHCl3–Et2O–EtOH (3 : 2 : 1, v/v). A total of 18 mg
cainide is needed. ELISA is generally very sensitive and spe- (90%) of N-(2,6-dimethyl-3-nitrophenyl)-8-pyrrolizidinylac-
cific, and has advantages in simplicity, cost, safety, and ca- etamide (3-nitropilsicainide) as a pale yellow oil was ob-
pacity of processing a large number of samples. If a kit and tained. 3-Nitropilsicainide was used for the next step without
automatic method are developed, it will be used in many fa- further purification. IR (CHCl3) cmϪ1: 2960, 1524, 1348. 1H-
cilities.
NMR (CDCl3) d: 1.25—3.07 (14H, m, CH2 of pyrrolidine
We present here the first report of ELISA for pilsicainide. and –CO–CH2–), 2.30 (3H, s, CH3), 2.39 (3H, s, CH3), 7.19
The present study reports the methodology for the antibody (1H, d, Jϭ9.15, 5-H of benzene), 7.70 (1H, d, Jϭ9.15, 4-H
production, the labeling of pilsicainide with b-D-galactosi- of benzene), 10.5—11.5 (H, br s, NH).
dase (b-Gal) to act as a tracer, the characterization of anti-
A solution of 18 mg of 3-nitropilsicainide was dissolved in
body specificity, and the technique developed for the mea- 3 ml of 60% ethanol, which was made basic by the addition
surement of pilsicainide by ELISA. The initial application of of 40 mg of MgO. To this mixture was added 80 mg of
the assay to the measurement of the drug levels in rabbits Na2S2O4 and vigorously shaken, and the solution was heated
demonstrates its usefulness for the assessment of basic phar- in a water-bath at 50 °C for 1 h. After cooling, ethanol was
macokinetic distributions.
evaporated off in vacuo, and the aqueous solution was ex-
tracted with AcOEt. The organic layer was separated and
evaporated down to give 3-aminopilsicainide (5 mg, 30%) as
a yellow solid.
MATERIALS AND METHODS
Equipment The IR spectrum was recorded using a Hi-
The resulting 3-aminopilsicainide was used without fur-
To whom correspondence should be addressed. e-mail: saita@post.saga-med.ac.jp
© 2001 Pharmaceutical Society of Japan

1114
Vol. 24, No. 10
ther purification for preparing the conjugates with bovine
serum albumin (BSA) and b-Gal, respectively, as the pilsi-
cainide immunogen and the tracer in the ELISA.
IgG (2.5 mg/ml) in 10 mM Tris–HCl buffer (pH 8.5) contain-
ing 10 mM NaCl and 10 mM NaN3 and allowed to stand for
1 h at 37 °C. After the plates had been washed twice with
60 mM phosphate buffer (pH 7.4) containing 10 mM ethylene-
diaminetetraacetate, 0.1% BSA and 0.1% NaN3 (buffer B),
they were incubated with 200 ml of 10 mM Tris–HCl buffer
(pH 8.5) containing 10 mM NaCl and 10 mM NaN3 containing
2% BSA for 20 min at 37 °C to prevent non-specific adsorp-
tion. The anti-pilsicainide IgG-coated wells were then filled
with 50 ml of either 10-fold diluted serum from drug-treated
rabbits, or standard normal rabbit serum samples diluted
1 : 10 with buffer B (containing various concentrations of pil-
sicainide), followed immediately by 50 ml of the pooled pilsi-
cainide-b-Gal conjugate (diluted 1 : 500 in buffer B for pilsi-
cainide). The wells were then incubated for 1 h at 37 °C and
once again washed thoroughly with buffer B.
The activity of the enzyme conjugate bound to each well
was then measured by the addition of 125 ml of 0.1 mM 4-
methylumbelliferyl-b-D-galactopyranoside in buffer A, fol-
lowed by incubation of the wells at 37 °C for 60 min. The en-
zyme reaction was stopped by the addition of 75 ml of 0.5 M
glycine–NaOH buffer (pH 10.3) to each well, and the result-
ing 4-methylumbelliferone was measured by spectrofluorom-
etry at wavelengths of 355 nm for excitation and 460 nm for
emission using a fluorescence microplate reader.
HPLC Method The HPLC system consisted of a
Shimadzu Model LC-10AT liquid chromatograph equipped
with a spectrophotometric detector SPD-10AV and a 5ϫ
100 mm Radial-pak cartridge 5CN (10 mm) column (Waters,
Milford, MA, U.S.A.). The column was eluted with acetoni-
trile and 10 mM ammonium acetate (70 : 30, v/v) at a flow
rate of 2.5 ml/min, and the eluate was monitored at 210 nm.
The retention time and peak height were measured with a
Shimadzu C-R7A chromatopac.
One milliliter of 0.1 N NaOH was added to 0.5 ml of serum
in a 15ϫ10 mm glass test tube. After the addition of 100 ml
of mexiletine (10 mg/ml) as the internal standard, the mixture
was extracted using 5 ml of chloroform and shaken for
10 min. After centrifugation for 5 min at 2270ϫg, the or-
ganic layer (4 ml) was transferred to another glass tube. The
extracted solvent was evaporated to dryness at 40 °C under
N2 gas. The residue was reconstituted in 50 ml of acetonitrile,
and 40 ml was injected.
3-Aminopilsicainide (approximately 3 mg, 10.1 mmol) in
40 ml DMF was acidified by the addition of 100 ml of 1 N
acetic acid and then diazotized with sodium nitrite (1.5 mg,
21.7 mmol) in 50 ml distilled water at 0 °C for 10 min. The so-
lution (diazotized 3-aminopilsicainide) was immediately
mixed with BSA (10 mg) in 1 ml of 0.5 M boric acid buffer
(pH 9.5), followed by 1 h incubation at room temperature.
The mixture was chromatographed on a Sephadex G-100
column (2.8ϫ42 cm) with 0.1 M phosphate buffer (pH 7.0)
containing 3 M urea. The degree of coupling of diazotized
pilsicainide with BSA was determined by amino acid analy-
sis after hydrolysis with 6 N HCl at 110 °C for 24 h.10) Fifteen
moles of the hapten was found to be coupled with one mole
of BSA on the basis of the decrease in moles of histidine and
tyrosine. The amount of protein was determined by Lowry’s
method.11)
Preparation of Pilsicainide Antibody An aliquot of a
solution containing 1 mg pilsicainide-BSA conjugate (1.5 ml)
was emulsified with an equal volume of Freund’s complete
adjuvant. Two white female rabbits were each given multiple
subcutaneous injections over sites along both sides of their
backs. Booster injections were then given three times at bi-
weekly intervals, using one-half the amount of the dose of
the first immunization. The rabbits were bled from an ear
vein 10 weeks after immunization began. The sera (10 ml)
were separated by centrifugation and heated at 55 °C for
30 min. Fractions of IgG were extracted from the sera with
50% saturated ammonium sulfate and chromatographed on a
column of DEAE-Sephacel (2.1ϫ23 cm) using 17.5 mM
phosphate buffer (pH 6.8) as an eluant.12) The fraction passed
through the column was lyophilized and used as anti-pilsi-
cainide IgG for ELISA.
Preparation of the Pilsicainide-b-Gal Conjugate Pilsi-
cainide was labeled by binding to b-Gal, essentially by the
same principle used for the previous preparation of pilsi-
cainide immunogen, using 3-aminopilsicainide. 3-Aminopil-
sicainide (approximately 1 mg, 3.4 mmol) in 20 ml DMF was
acidified by the addition of 100 ml of 1 N acetic acid and then
diazotized with sodium nitrite (0.5 mg, 7.2 mmol) in 50 ml
distilled water at 0 °C for 10 min. Next, 70 ml portions of the
above reaction mixture containing diazotized 3-aminopilsi-
cainide (ca. 1.4 mmol) was added directly to b-Gal (156 mg,
0.28 nmol) in 0.5 ml of 0.5 M phosphate buffer (pH 7.5), fol-
lowed by 1 h incubation at room temperature. The mixture
was chromatographed on a Sepharose 6B column (2.0ϫ
40 cm) using 20 mM phosphate buffer (pH 7.0) containing
0.1 M NaCl, 1 mM MgCl2, 0.1% BSA and 0.1% NaN3 (buffer
A) to remove any remaining small molecules. Four-milliliter
fractions were collected, and fractions 14 to 16, representing
the main peak showing enzyme activity, were combined and
used as a label in the ELISA.
Pharmacokinetic Evaluation White female rabbits in
the weight range of 3.5 to 4 kg were used in this study. Pilsi-
cainide was orally administered at a dose of 1 mg/kg to the
rabbits. The drug was dissolved in isotonic sodium chloride
at concentrations of 1 mg/ml. Blood samples were collected
at 0.25, 0.5, 1, 2, 4, 6 and 8 h post-administration, and the
serum was stored at Ϫ20 °C until assayed for pilsicainide
concentration. The serum was diluted 10-fold with buffer B
to obtain a pilsicainide concentration appropriate for a mea-
surement by ELISA.
ELISA for Determination of Pilsicainide ELISA is
based on the principle of competition between enzyme-la-
beled and unlabeled drugs for an immobilized antibody, fol-
lowed by measurement of the marker enzyme activity of the
immunocomplex bound to the solid phase. Briefly, the wells
in microtiter plates (Nunc F Immunoplates I; Nunc, Reskilde,
Denmark) were coated by loading 150 ml of anti-pilsicainide
RESULTS AND DISCUSSION
Pilsicainide has no suitable reactive structure for making
immunogens such as the pilsicainide-BSA conjugate. There-
fore, we chose to introduce an amino group at the pilsi-
cainide molecule via nitration of the aromatic ring (Fig. 1).
1
From the H-NMR spectral data, we confirmed this nitration

October 2001
1115
Fig. 2. Standard Curves for Pilsicainide in the Buffer and Serum
The curve shows the amount (%) of bound enzyme activity for various doses of pilsi-
cainide (B) as a ratio of that bound using pilsicainide-b-Gal alone (B0). ᭺, buffer; ᭹,
serum. Each point represents the meanϮS.D. of 3 replicates.
Table 1. Precision and Recovery (%) of ELISA for Pilsicainide in Serum
Added (ng/ml) Estimated (ng/ml) Recovery (%) C.V. (%)
Intra-assay
1.6
8.0
40.0
200.0
1.6
8.0
40.0
200.0
1.61Ϯ0.17
7.86Ϯ0.47
38.0Ϯ1.58
208.0Ϯ16.43
1.63Ϯ0.16
7.94Ϯ0.42
38.8Ϯ1.64
196.6Ϯ14.42
100.6
98.3
95.0
104.0
101.9
99.3
10.6
6.0
4.2
7.9
9.8
5.3
4.2
7.3
Fig. 1. Preparation of the Immunogen for Pilsicainide
Inter-assay
occurred at the C-3 position of the aromatic ring. The nitro
group was reduced to an amino group which was then cou-
pled by diazotization to tyrosine and histidine residues on
the carrier protein. The pilsicainide-BSA conjugate, with
15.0 mol of pilsicainide per mol of BSA, induced the forma-
tion of a specific antibodies in each of the two rabbits immu-
nized. Pilsicainide-b-Gal conjugate was also prepared by es-
sentially the same procedure. The conjugate thus obtained
was stable in eluted buffer (pH 7.0) at 4 °C for more than 6
months during which no loss of the enzyme activity or im-
munoreactivity was seen.
The optimal quantities and optimal incubation time for
each reaction were established. The dose-response standard
curves of pilsicainide obtained in the buffer and serum are
shown in Fig. 2. The range of pilsicainide detection by
ELISA were between 320 pg and 1 mg/ml of pilsicainide. For
practical purposes, the working range was arbitrarily set be-
tween 1.6 and 200 ng/ml based on the precision and accuracy
findings for the ELISA in serum (Table 1), which showed
this ELISA to be a reproducible technique. Recoveries of
four different pilsicainide levels ranging from 1.6 to 200
ng/ml were satisfactory (95.0 to 104.0%, nϭ5). The coeffi-
cients of variation for intra- and interassays at four different
pilsicainide levels between 1.6 to 200 ng/ml were 4.2 to
10.6% and 4.2 to 9.8% (each nϭ5), respectively. The ELISA
for pilsicainide detected as little as 1.6 ng/ml of the drug and
was reproducible, as indicated by the overall mean coefficient
of variation of 6.9% intra-assays and inter-assays at four dif-
ferent levels of pilsicainide. The standard curve in the serum
system was essentially the same as that in the buffer system
(Fig. 2). The therapeutic range of pilsicainide was reported to
97.0
98.3
Values represent the meanϮS.D. of 5 experiments.
be 0.2—0.9 mg/ml.6) Therefore, this ELISA may be sensitive
enough to quantify pilsicainide for TDM.
The antibody specificity was determined by the cross-reac-
tivity with two kinds of similar compounds, which were de-
fined as the ratio of each compound to pilsicainide in the
concentrations required for 50% inhibition of bound b-Gal
activity. The anti-pilsicainide antibody showed 0.04% cross-
reaction with 8-pyrrolizidineacetic acid. No detectable cross-
reaction, however, was found with lidocaine (Table 2). These
findings suggest that the antibody well recognizes both the
aromatic ring and the pyrrolizidine moieties, and thus spe-
cific enough to the structure of pilsicainide.
The ELISA method was compared with an HPLC method
for measuring human serum samples containing known
amounts of standard pilsicainide. The HPLC technique ana-
lyzed the 10 samples of various pilsicainide concentrations
ranging from 0.25 to 10 mg/ml, showing a linear relationship
between the peak height and the added pilsicainide dose.
ELISA determination was carried out using these pilsicainide
samples, properly diluted to adjust the drug-concentration in
the measurable range of the ELISA. Figure 3 shows that
there was a linear correlation between the values determined
by the two methods, providing the equation Yϭ0.96Xϩ0.14,
where Y is the concentration value determined by HPLC
analysis and X is that determined by ELISA; the correlation

1116
Vol. 24, No. 10
Table 2. Specificity of Anti-pilsicainide IgG
Compounds
% cross-reaction (50%)
100.0
Pilsicainide
Lidocaine
Ͻ0.025
8-Pyrrolizidineacetic acid
0.04
Fig. 4. Serum Pilsicainide Levels in Rabbits after a Single Oral Adminis-
tration of Pilsicainide
Three rabbits, each weighing 3.5 to 4.0 kg, were injected with 1 mg/kg pilsicainide.
At each interval, blood was collected and the serum pilsicainide was measured by
ELISA. Each point represents the meanϮS.D. of the three rabbits.
metabolite.
In conclusion, the ELISA procedure for pilsicainide re-
ported here is sensitive, specific, reproducible, simple and
adaptable for analyses of a large number of samples. This
ELISA will be a valuable tool in TDM and pharmacokinetic
studies.
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Fig. 3. Correlation of Pilsicainide Quantification by the Present ELISA
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