Development of a Competitive Enzyme-Linked Immunosorbent Assay for the Determination of Sunitinib Unaffected by Light-Induced Isomerization

Hiroto Kataoka; Tetsuya Saita; Rintaro Sogawa; Yuta Yamamoto; Shunsuke Matsuo; Sakiko Kimura; Shinya Kimura; Masashi Shin

doi:10.1248/bpb.b21-00480

Abstract

Sunitinib is an oral multi-targeted tyrosine kinase inhibitor approved for treating metastatic renal cell carcinoma. This study reports a specific and sensitive competitive enzyme-linked immunosorbent assay (ELISA) for the pharmacokinetic evaluation of sunitinib. Anti-sunitinib serum was obtained from mice by using N-(2-(diethylamino)ethyl)-5-formyl-2,4-dimethyl-1H-pyrrole-3-carboxamide (DFPC) as a hapten, which has the same substructure as sunitinib, in order to avoid the effects of structural changes in the geometrical isomers of sunitinib. Enzyme labeling of sunitinib with horseradish peroxidase was similarly performed using DFPC. Serum sunitinib concentrations below the limit of quantification of 0.52 ng/mL were reproducibly measurable. This ELISA was speciﬁc for sunitinib (Z- and E-isomers) and showed very low cross-reactivity (0.094%) with its major metabolite, N-desethyl sunitinib. Its analytical applicability was demonstrated by a kinetic study with human liver microsomes. In addition, the levels of sunitinib measured by ELISA in a kinetic study with human liver microsomes were comparable with those measured by HPLC, and there was a strong correlation between the values determined by both methods (y = 1.065x − 51.2, R² = 0.9804). The developed ELISA provides for the specific and sensitive quantification of sunitinib without the influence of its major metabolite or light-induced geometric isomers. This ELISA will be a valuable tool in pharmacokinetic studies of sunitinib.

INTRODUCTION

Sunitinib is an epidermal growth factor receptor-tyrosine kinase inhibitor approved for the treatment of gastrointestinal stromal tumor, renal cell carcinoma, and pancreatic neuroendocrine tumor.^1,2) However, sunitinib administration can cause a variety of serious adverse effects, including thrombocytopenia, hypertension, neutropenia, fatigue, and stomatitis,³⁾ which may necessitate shortening the treatment duration, reducing the dose, or discontinuing treatment in many cases. For the safe and effective use of sunitinib in chemotherapy, therapeutic drug monitoring (TDM) is recommended because adjusting its trough level is an effective approach.^4,5)

As shown in Fig. 1, exposure of sunitinib to light while in solution causes geometric isomerization (Z-isomer → E-isomer, which is a cis–trans isomer of the double bond at the 3 position of the indole ring).⁶⁾ Due to this reaction, the effect of light must be considered when quantifying sunitinib levels. Several existing HPLC⁷⁾ and LC-tandem mass spectrometry methods^8,9) require that samples be shielded from light during all handling procedures in order to prevent E-isomer formation. This makes samples difficult to handle and poorly suited for TDM. The development of a quantification method that is not influenced by E-isomer formation would make it unnecessary to shield samples from light during collection and processing, which would be extremely useful when conducting TDM. We previously developed a simple enzyme-linked immunosorbent assay (ELISA) that uses a substructure of a molecularly targeted drug as a hapten in order to produce a specific antibody against the drug, which could be used in TDM for many molecularly targeted anti-cancer drugs.^10–13) As a result, we believed that if we used a substructure of sunitinib that does not undergo a structural change when exposed to light as a hapten, we might be able to produce an anti-sunitinib antibody that would not be affected by the structural changes caused by geometric isomerization.

Fig. 1. Chemical Structures of the E- and Z-Isomers of Sunitinib

In this paper, we successfully developed the first specific competitive ELISA for sunitinib that is not influenced by light-induced geometric isomerization using a polyclonal antibody against part of the structure of sunitinib and herein report the technique. The initial application of this assay for the measurement of sunitinib levels is demonstrated by a metabolic study in vitro with human liver microsomes.

MATERIALS AND METHODS

Chemicals and Reagents

Sunitinib malate was obtained from Cayman Chemical, Ann Arbor, MI, U.S.A. (PubChem ID, 6456015; Catalog No. 13159). N-Desethyl sunitinib was obtained from MedChemExpress Co., Ltd., Monmouth Junction, NJ, U.S.A. (PubChem ID, 10292573; Catalog No. HY-10873). N-(2-(Diethylamino)ethyl)-5-formyl-2,4-dimethyl-1H-pyrrole-3-carboxamide (DFPC) was obtained from FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan (PubChem ID, 20761785; Catalog No. AK-86805). Carboxymethoxyamine hemihydrochloride was obtained from Nacalai Tesque Inc., Kyoto, Japan (PubChem ID, 10129958; Catalog No. 07239-61). Horseradish peroxidase (HRP) was obtained from Sigma-Aldrich, St. Louis, MO, U.S.A. (Catalog No. P6782). 3,3′,5,5′-Tetramethylbenzidine was obtained from Sigma-Aldrich (PubChem ID, 41206; Catalog No. T2885). All other reagents and solvents were of the highest grade commercially available.

Preparation of the Sunitinib Immunogen

The sunitinib immunogen was prepared with part of the structure of sunitinib (DFPC) as shown in Fig. 2. DFPC (10 mg, 37.7 µmol) in 100 µL dimethylformamide (DMF) was dissolved in 250 µL of 0.1 M phosphate buffer (pH 5.5). The resulting solution was mixed with carboxymethoxyamine hemihydrochloride (4.12 mg, 37.7 µmol) in 50 µL of 0.1 M phosphate buffer (pH 5.5), followed by overnight incubation at 60 °C. The resulting mixture was extracted using ethyl acetate. The yield of DFPC–(O-carboxymethyl) oxime was tentatively estimated to be 97.6% according to HPLC measurements of the quantity of nonreacted DFPC. The resulting DFPC–(O-carboxymethyl) oxime was used without further purification for preparing the conjugates with bovine serum albumin (BSA) and HRP, respectively, as the sunitinib immunogen and the tracer in the ELISA.

Fig. 2. Preparation of the Immunogen for Sunitinib

The ethyl acetate solution of DFPC–(O-carboxymethyl) oxime was volatilized with nitrogen, the remaining reactant was dissolved in 95% DMF (0.5 mL), N-hydroxysuccinimide (8.7 mg, 75.4 µmol) and 1-ethyl-3-(3-dimethylaminopropyl)carbodimide hydrochloride (EDC) (28.9 mg, 150.7 µmol) were added, and the mixture was reacted at room temperature for 2 h. The reaction mixture was mixed with 10 mg BSA dissolved in 0.1 M phosphate buffer (1 mL, pH 7.0) containing 3 M urea and reacted at room temperature for 2 h. The obtained reaction product was dialyzed for 24 h against 1 mM phosphate buffer (pH 7.0) containing 3 M urea. The purified conjugate was stored frozen and used as an immunogen. The purified conjugate was examined spectrophotometrically and estimated to contain approximately 14.2 molecules of DFPC per BSA molecule, assuming the molar extinction coefficients of DFPC to be 10345 at 280 nm and 18568 at 310 nm, and that of BSA to be 43600 at 280 nm.

Preparation of Anti-sunitinib Immunoglobulin G (IgG)

Five-week-old female BALB/c mice (Kyudo Exp. Animals, Kumamoto, Japan) were injected intraperitoneally with 0.1 mg DFPC–BSA conjugate emulsiﬁed in incomplete Freund’s adjuvant. In total, the mice received three injections of the conjugate (0.05 mg) alone at 2-week intervals. At 7 d after the final injection, the mice were euthanized and serum was collected. The mixing serum from multiple mice (2 mL) were centrifuged at 1048 × g at 4 °C for 10 min and then heated at 55 °C for 30 min. IgG fractions were purified using a HiTrap Protein G column (GE Healthcare, Stockholm, Sweden) with 20 mM sodium phosphate (pH 7.0) as the binding buffer and 0.1 M glycine HCl (pH 2.7) as the elution buffer, according to the manufacturer’s protocol. The anti-DFPC IgG obtained was used directly as the anti-sunitinib antibody for ELISA. The experimental protocol was approved by the Ethics Review Committee for Animal Experimentation at Sojo University (2020-L-007).

Preparation of the DFPC–HRP Conjugate

DFPC was labeled by binding to HRP, essentially by the same method as that used for the preparation of the sunitinib immunogen. Ethyl acetate solution of DFPC–(O-carboxymethyl) oxime obtained by the same method as for the preparation of sunitinib immunogen was volatilized with nitrogen. The remaining reactant was dissolved in 95% DMF (0.5 mL), N-hydroxysuccinimide (8.7 mg, 75.4 µmol) and EDC (28.9 mg, 150.7 µmol) were added, and the mixture was reacted at room temperature for 2 h. A 50-µL aliquot of the reaction mixture was added directly to HRP (0.5 mg, 12.5 nmol) in 0.5 mL of 0.1 M phosphate buffer (pH 7.0), followed by incubation at room temperature for a further 2 h. The mixture was chromatographed on a Sephadex G-75 column (2.0 × 30.0 cm) using phosphate-buffered saline (PBS) containing 0.1% BSA to remove any remaining small molecules. Fractions (4 mL) were collected, and fractions 8 and 9, corresponding to the main peaks showing enzyme activity, were used as a label for ELISA.

Competitive ELISA

Competitive ELISA is based on the principle of competition between enzyme-labeled and unlabeled drugs for an immobilized antibody, followed by measurement of the marker enzyme activity of the immunocomplex bound to the solid phase.^11,12,14) Briefly, following previously described methods,¹⁴⁾ microtiter plate wells (Nunc F Immunoplates I; Nunc, Roskilde, Denmark) were coated by adding 100 µL anti-sunitinib antibody (1 µg/mL in 10 mM Tris–HCl buffer (pH 8.5) containing 10 mM NaCl and 10 mM NaN₃, and allowed to stand for 1 h at 37 °C. The plates were washed twice with PBS containing 0.1% BSA and incubated with 100 µL of 10 mM Tris–HCl buffer (pH 8.5) containing 10 mM NaCl and 10 mM NaN₃ with 1% skim milk for 30 min at 37 °C to prevent nonspecific adsorption. The anti-sunitinib antibody-coated wells were filled with 50 µL of either sunitinib-treated samples or PBS containing 0.1% BSA as a control, followed immediately by the addition of 50 µL of the pooled DFPC–HRP conjugate (diluted 1 : 10000 in PBS containing 0.1% BSA for sunitinib). The wells were incubated for 3 h at 37 °C and washed carefully with PBS containing 0.1% BSA.

The activity of the enzyme conjugate that was bound to each well was measured by adding 100 µL of 0.42 mM 3,3′,5,5′-tetramethylbenzidine in 0.05 M acetate-citric acid buffer (pH 5.5) containing 3% dimethyl sulfoxide and 0.01% hydrogen peroxide, and incubating the wells at 37 °C for an appropriate duration. Next, 100 µL of 2.0 M H₂SO₄ was added to each well to stop the reaction, and the ensuing color intensity was measured using a spectrophotometer at 450 nm with an ELISA analyzer (ImmunoMini NJ-2300; Nalje Nunc Int. Co., Ltd., Tokyo, Japan). Concentrations were determined from the standard curve using semi-logarithmic graph paper.

Metabolism of Sunitinib by Human Liver Microsomes

Sunitinib metabolism was investigated using human liver microsomes. Briefly, the incubation mixture (1 mL total volume) contained 100 mM potassium phosphate buffer (pH 7.4), 100 mM nicotinamide adenine dinucleotide phosphate (NADPH), 2,000 ng/mL sunitinib, and 0.5 mg/mL human liver microsomes. The reaction was initiated by the addition of NADPH and incubated at 37 °C for various periods after preincubation for 30 min. The enzymatic reaction was stopped by the addition of a stop reagent (0.1 mL of 0.2% (v/v) formic acid/acetonitrile) at 0, 2, 5, 15, 30, 60, 90, and 120 min. Precipitated protein was removed by centrifugation (4800 × g for 2 min at 4 °C). The supernatant fractions were volatilized with nitrogen, and the residues were dissolved in 10 mM PBS (0.5 mL). The solution was analyzed directly by ELISA.

HPLC for Sunitinib

HPLC for sunitinib was performed according to a previous study.¹⁵⁾

Preparation and Measurement of the Z-Isomer of Sunitinib

As shown in Fig. 1, exposure of sunitinib to light while in solution causes geometric isomerization (Z-isomer → E-isomer). It has been reported that incubation of sunitinib solutions in a heated water bath for 5 min at 70 °C results in the quantitative (99%) reconversion of the E-isomer to the Z-isomer.⁶⁾ Therefore, samples of sunitinib solution were incubated in a heated water bath at 70 °C for 10 min, and the levels of Z-isomers were measured directly by ELISA under light-blocking conditions.

RESULTS AND DISCUSSION

Preparation of the Immunogen and Enzyme Conjugate for Sunitinib

The geometric isomer of sunitinib is a cis–trans isomer of the double bond at the 3 position of the indole ring (Fig. 1). Therefore, to obtain an anti-sunitinib antibody that would not be influenced by these structural changes, we created a sunitinib antigen using the DFPC substructure of sunitinib as a hapten (Fig. 2). DFPC has a formyl group; therefore, we chose to introduce a carboxymethyloxime group at the formyl group with carboxymethoxyamine. The DFPC–(O-carboxymethyl) oxime was coupled to BSA using the hydroxysuccinimide ester technique.¹⁶⁾ The resulting DFPC–BSA conjugate (sunitinib immunogen) induced the formation of specific antibodies in each of the five immunized mice. A DFPC–HRP conjugate (as a tracer) was also prepared by the same procedure (Fig. 2). The conjugate remained 103% and 98% of its enzyme and immunoreactivity enzyme activity respectively when stored in elution buffer (pH 7.0) at 4 °C for 6 months.

Validation of the Sunitinib ELISA

A calibration curve model, limit of detection, limit of quantification, precision (intra-assay and inter-assay), and spike-in recovery for the sunitinib ELISA were determined for human serum. The parameter requirements used in the following sections comply with the current bioanalytical guidelines of the US Food and Drug Administration.¹⁷⁾

Figure 3 shows the calibration curve of sunitinib as obtained using human serum. The calibrator range was 0.2–145.8 ng/mL sunitinib. The best curve fitting was obtained by applying the four-parameter logistics regression algorithm. The mean correlation coefficient from these seven calibration curves was 0.99922 ± 0.0004. The lower limit of detection was determined to be 0.38 ng/mL by interpolation at 3 standard deviation (S.D.) above the mean background signal. The lower limit of quantification was determined to be 0.52 ng/mL by interpolation at 10 S.D. above the mean background signal. Intra-assay precision in the low, mid, and high assay range (six levels, n = 6 each) resulted in coefficients of variability between 1.7 and 9.2% (Table 1). Inter-assay precision in the low, mid, and high assay range (six levels, over five independent runs) resulted in coefficients of variability between 3.3 and 9.8% (Table 1). Spike-in recovery in the low, mid, and high assay range (six levels) was between 98.4 and 108.6% (Table 1). Dilutional linearity of a spiked sample showed a recovery between 99.4 and 99.5% over the working range when diluted 10–100-fold, respectively. The developed ELISA fulfilled the acceptance criteria for all addressed validation parameters.¹⁷⁾ In addition, the calibration curve of sunitinib in the serum system was similar to that in the buffer system.

Fig. 3. Standard Curve of the Developed ELISA for Sunitinib in Human Serum

The curve shows the bound enzyme activity (%) for various doses of sunitinib (B) as a ratio to that bound using DFPC-HRP alone (B₀). Each point represents the mean ± standard deviation (S.D.) (n = 6).

Table 1. Recoveries of Sunitinib from Human Serum and Precision of ELISA for Sunitinib

Assay	Added (ng/mL)	Estimated (ng/mL)	Recovery (%)	CV (%)
Intra-assay	0.6	0.65 ± 0.06	108.6	9.2
	1.8	1.84 ± 0.14	102.0	7.8
	5.4	5.64 ± 0.31	104.5	5.4
	16.2	16.0 ± 0.41	99.1	2.5
	48.6	47.8 ± 1.75	98.4	1.7
	145.8	149.5 ± 2.56	102.6	1.7
Inter-assay	0.6	0.65 ± 0.05	108.3	9.8
	1.8	1.62 ± 0.12	104.6	7.4
	5.4	4.72 ± 0.28	100.7	6.5
	16.2	13.8 ± 0.52	98.9	4.5
	48.6	41.7 ± 1.88	98.8	5.2
	145.8	133.5 ± 4.36	105.2	3.3

Values represent the mean ± S.D. (n = 5).

The lower limit of quantification of sunitinib was 0.52 ng/mL. The therapeutic range of concentrations for sunitinib is between 50–100 ng/mL (sunitinib plus N-desethyl sunitinib).^18,19) During long-term therapy, the plasma level of N-desethyl sunitinib is comparable with that of sunitinib.^18,19) Therefore, this ELISA seems to have adequate sensitivity to quantify sunitinib in TDM and pharmacokinetic studies.

Specificity of the Sunitinib ELISA

Antibody cross-reactivity was determined by the displacement of bound DFPC-HRP with comparable compounds.¹⁴⁾ The cross-reactivity values were set as the ratio of each compound to sunitinib at the concentration needed for 50% inhibition of DFPC-HRP binding to the antibody. The anti-sunitinib antibody showed 100% cross-reactivity with DFPC as the hapten antigen, 100% with sunitinib (the ratio of E- to Z-isomer is approximately 1 : 1.2), 100% with the Z-isomer of sunitinib, and 0.094% with N-desethyl sunitinib (Table 2). All ELISA analyses of the Z-isomer of sunitinib were performed under light-blocking conditions to avoid light-induced geometric isomerization. These experimental results suggest that this ELISA is not affected by the major metabolite N-desethyl sunitinib or light-induced geometric isomers in the determination of the serum levels of sunitinib. Therefore, this ELISA may have sufficient specificity to enable the quantification of sunitinib for TDM and pharmacokinetic studies.

Table 2. Percent Cross-Reactivity of Metabolite and Analogs Measured by ELISA

Compounds	Cross-reactivity (%)
DFPC	100.0
Sunitinib (Z + E)^a)	100.0
Sunitinib (Z)	100.0
N-Desethyl sunitinib	0.094

a) The ratio of E- to Z-isomer is approximately 1 : 1.2.

Kinetics of Sunitinib in Human Liver Microsomes

This ELISA was applied to a kinetic study of sunitinib in human liver microsomes. As shown in Fig. 4, immediately after the start of the reaction, a rapid decrease in sunitinib was observed. From 30 min after the initiation of the reaction, the reaction rate was lowered. These results suggest that sunitinib was metabolized by human liver microsomes. Previous metabolic studies using human biological samples have shown that sunitinib is metabolized primarily into N-desethyl sunitinib by CYP3A4; N-desethyl sunitinib is further metabolized into an inactive N-desethyl metabolite by CYP3A4.²⁰⁾ In vitro studies have also identified other trace metabolites, including an N-oxide metabolite.²⁰⁾ These findings suggest that our ELISA specifically quantifies sunitinib levels without being influenced by these metabolites. Furthermore, the ELISA was shown to be applicable to the study of the metabolic kinetics of sunitinib. Therefore, this ELISA may be sufficiently specific to quantify sunitinib for pharmacokinetic studies in animals and humans.

Fig. 4. Kinetics of Sunitinib in Human Liver Microsomes

Human liver microsomes (0.5 mg/mL) were incubated at 37 °C in a total volume of 1 mL of 100 mM potassium phosphate buffer (pH 7.4) containing 100 mM NADPH and 2000 ng/mL sunitinib. Each point represents the mean ± S.D. of three replicates.

N-Desethyl sunitinib shows similar biochemical activity and potency and reaches similar plasma concentrations as the parent compound.²¹⁾ Therefore, the sum of the plasma concentrations of sunitinib and its metabolite have to be considered to calculate the target plasma concentration for TDM analysis. For this reason, it is also necessary to develop an ELISA against N-desethyl sunitinib. If the same process underlying our method for creating an anti-sunitinib antibody was applied to create an anti-N-desethyl sunitinib antibody through the use of a substructure of N-desethyl sunitinib, e.g., N-[2-(ethylamino)ethyl]-5-formyl-2,4-dimethyl-1H-pyrrole-3-carboxamide, as a hapten, this antibody could theoretically be used to develop a specific ELISA targeting N-desethyl sunitinib that would not be influenced by the parent compound or by light-induced geometric isomerization of N-desethyl sunitinib.

Correlation between Sunitinib Concentrations Determined by the Developed ELISA and HPLC

The developed ELISA was compared with an existing HPLC method for sunitinib quantification. Samples from the metabolic kinetic study of sunitinib in human liver microsomes were used to verify the suitability of the ELISA for TDM and pharmacokinetics studies of sunitinib (Fig. 5). A good correlation was found between the values of both methods. The equation y = 1.065x − 51.2 was derived, with y as the concentration determined by HPLC analysis and x as the concentration determined by ELISA. A correlation coefficient of 0.9804 was obtained (n = 14). These experimental results strongly suggest that the ELISA is not affected by sunitinib metabolites or light-induced geometric isomers in the determination of sunitinib in biological materials. Therefore, this ELISA may have adequate sensitivity and specificity to quantify sunitinib for TDM and pharmacokinetic studies in animals and humans.

Fig. 5. Correlation between Sunitinib Concentrations in Human Liver Microsome-Treated Samples Determined by ELISA and HPLC

The HPLC values is the sum of E- and Z-isomer of sunitinib.

CONCLUSION

By using a substructure of sunitinib that does not change with light-induced geometric isomerization as a hapten, we were able to produce an anti-sunitinib antibody with equal cross-reactivity to the Z- and E-isomers of sunitinib. We used this anti-sunitinib antibody to create a specific and highly sensitive ELISA that is not influenced by the primary metabolite N-desethyl sunitinib or by light-induced geometric isomerization of sunitinib. This ELISA will be a valuable tool in pharmacokinetic studies of sunitinib.

Acknowledgments

We are grateful to Dr. Yutaro Yamamoto for technical assistance.

Conflict of Interest

The authors declare no conflict of interest.

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