[Development of a Simple and Sensitive Enzyme-linked Immunosorbent Assay for Sinomenine].

Sinomenine (SIN) is a major component contained in extracts of the Chinese medicinal herb Sinomenium acutum. SIN has various pharmacological properties, including cytoprotection, immunosuppression and anti-inflammation effects. Furthermore, recent studies have reported that SIN has anti-tumor and antidepressant effects, which has created a strong need for SIN kinetic studies. This paper reports a simple and sensitive competitive enzyme-linked immunosorbent assay (ELISA) for the pharmacokinetic evaluation of SIN. Anti-SIN serum was obtained by immunizing mice with an antigen conjugated with bovine serum albumin and carboxylic modified SIN using the N-succinimidyl ester method. Enzyme labeling of SIN with horseradish peroxidase was similarly performed using carboxylic modified SIN. Under optimized conditions, this ELISA shows a linear detection range from 40 to 5000 pg/mL, and a limit of detection of 12.1 pg/mL for 50-µL samples. This assay was specific for SIN and showed very slight cross-reactivity with dextromethorphan (0.45%), dimemorfan (0.22%) and codeine (0.01%), but no cross-reactivity with 2-methoxycyclohex-2-enone (<0.001%). Using this ELISA, SIN levels were easily determined in the blood of mice after oral administration of Kampo medicine, Boiogito. The ELISA may be a valuable tool for studies of the biological and pharmacological properties of SIN.

Localization of Sites of Osimertinib Action in Rat Intestine, Skin, and Lung by Immunohistochemistry.

Osimertinib is a third-generation, irreversible tyrosine kinase inhibitor (TKI) of epidermal growth factor receptor (EGFR) that selectively inhibits both EGFR-TKI-sensitizing and EGFR T790M resistance mutations and has shown efficacy in patients with non-small-cell lung cancer. In this study, we created osimertinib-specific antibodies and developed an immunohistochemistry (IHC) for locating the sites of osimertinib action. Moreover, we located osimertinib-protein conjugates in intestinal, dermal, and lung tissues of rats, thereby using our IHC to visualize the sites of the adverse effects of osimertinib, including diarrhea, skin disorder, and interstitial pneumonia. This report is the first to elucidate the localization of the sites of action of osimertinib in the rat intestine, skin, and lung and is expected to help clarify the mechanism of osimertinib-induced adverse effects.

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

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.

Development of a competitive enzyme-linked immunosorbent assay for therapeutic drug monitoring of afatinib.

Afatinib is an oral tyrosine kinase inhibitor (TKI) approved for treating advanced non-small cell lung cancer. It is necessary to develop a simple quantification method for TKIs in order to facilitate therapeutic drug monitoring (TDM) in clinical settings. This study sought to develop a simple and sensitive competitive enzyme-linked immunosorbent assay (ELISA) to quantify afatinib in plasma for routine pharmacokinetic applications. An anti-afatinib antibody was obtained using (S)-N-4-(3-chloro-4-fluorophenyl)-7-(tetrahydrofuran-3-yloxy)-quinazoline-4,6-diamine (CTQD), which has the same substructure as afatinib, as a hapten. Enzyme labeling of afatinib with horseradish peroxidase was similarly performed using CTQD. A simple competitive ELISA for afatinib was developed based on the principle of direct competition between afatinib and the enzyme marker for the anti-afatinib antibody, which had been immobilized on the plastic surface of a microtiter plate. Plasma afatinib concentrations below the limit of quantification of 30 pg/mL were reproducibly measurable. Also, the values of plasma afatinib levels measured from 20 patients were comparable with those measured by high-performance liquid chromatography, and there was a strong correlation between the values determined by both methods (Y = 0.976X - 0.207, r = 0.975). As indicated by its specificity and sensitivity, this newly developed ELISA for afatinib is an important tool for TDM and studies of the pharmacokinetics of afatinib.

Localization and Accumulation Studies of Dacomitinib in Rat Intestines and Skin by Immunohistochemistry.

Dacomitinib, a second-generation tyrosine kinase inhibitor, was irreversible inhibitor forming covalent bonds with the kinase domains of EGFR and other ErbB family receptors. Dacomitinib has been approved for the treatment of locally advanced or metastatic non-small cell lung cancer. In this study, we aimed to develop an immunohistochemistry to detect dacomitinib-ErbB family receptor conjugates. Immunostaining was performed in rat intestine and skin tissues after oral administration of dacomitinib. Following a single oral dose of dacomitinib, strong staining was observed after 24 hr in the ileum and colon, with only slight staining in the duodenum and jejunum. In the skin, strong staining was observed in the epidermis, hair follicles, and sebaceous glands. Moreover, significant amounts of dacomitinib remained for up to 72 hr post-administration in the ileum, colon, and skin. This report is the first to elucidate the localization and accumulation of dacomitinib in the rat intestine and skin and should be valuable during efforts to clarify the mechanism dacomitinib-induced diarrhea or skin toxicities.

Quantification of Sorafenib in Human Serum by Competitive Enzyme-Linked Immunosorbent Assay.

The multikinase inhibitor sorafenib has been used in the treatment of hepatocellular carcinoma, renal cell carcinoma, and differentiated thyroid carcinoma. Here we have demonstrated the production of the first specific antibody against sorafenib. Anti-sorafenib serum was obtained by immunizing mice with an antigen conjugated with bovine serum albumin and carboxylic modified 4-(4-aminophenoxy)-N-methyl-2-pyridinecarboxamide (AMPC) using the N-succinimidyl ester method. Enzyme labeling of sorafenib with horseradish peroxidase was similarly performed using carboxylic modified AMPC. A simple competitive enzyme-linked immunosorbent assay (ELISA) for sorafenib was developed using the principle of direct competition between sorafenib and the enzyme marker for anti-sorafenib antibody, which had been adsorbed by the plastic surface of a microtiter plate. Serum sorafenib concentrations lower than 0.04 µg/mL were reproducibly measurable using the ELISA. This ELISA was specific to sorafenib and showed very slight cross-reactivity (2.5%) with a major metabolite, sorafenib N-oxide. The values of serum sorafenib levels from 32 patients measured by this ELISA were comparable with those measured by HPLC, and there was a strong correlation between the values determined by the two methods (Y=1.016X-0.137, r=0.979). The specificity and sensitivity of the ELISA for sorafenib should provide a valuable new tool for use in therapeutic drug monitoring and pharmacokinetic studies of sorafenib.

Development of a specific and sensitive enzyme-linked immunosorbent assay for the quantification of imatinib.

Imatinib is an oral tyrosine kinase inhibitor used for first-line treatment of chronic myeloid leukemia. Therapeutic drug monitoring targeting trough plasma levels of about 1000 ng/mL may help to optimize imantinib's therapeutic effect. This paper reports a specific and sensitive enzyme-linked immunosorbent assay (ELISA) for a pharmacokinetic evaluation of imatinib. Anti-imatinib antibody was obtained by immunizing rabbits with an antigen conjugated with bovine serum albumin and succinimidyl 4-{(4-methyl-1-piperazinyl)methyl}-benzoate. Enzyme labeling of imatinib with horseradish peroxidase was similarly performed using succinimidyl 4-{(4-methyl-1-piperazinyl)methyl}-benzoate. A simple ELISA for imatinib was developed using the principle of direct competition between imatinib and the enzyme marker for anti-imatinib antibody which had been adsorbed by the plastic surface of a microtiter plate. Serum imatinib concentrations lower than 40 pg/mL were reproducibly measurable using the ELISA. This ELISA was specific to imatinib and showed very slight cross-reactivity (1.2%) with a major metabolite, N-desmethyl imatinib. Using this assay, drug levels were easily measured in the blood of mice after their oral administration of imatinib at a single dose of 50 mg/kg. The specificity and sensitivity of the ELISA for imatinib should provide a valuable new tool for use in therapeutic drug monitoring and pharmacokinetic studies of imatinib.

[Development of a specific and sensitive enzyme-linked immunosorbent assay for vindesine].

This paper reports a specific and sensitive enzyme-linked immunosorbent assay (ELISA) for pharmacokinetic studies of vindesine (VDS). Anti-VDS antibody was obtained by immunizing rabbits with VDS conjugated with bovine serum albumin using N-[ß-(4-diazophenyl) ethyl] maleimide as a heterobifunctional coupling agent. An enzyme marker was similarly prepared by coupling VDS with horseradish peroxidase using N-(4-diazophenyl) maleimide. The detection limit of VDS by ELISA was approximately 24 pg/mL with 50-mL samples. This assay was specific for VDS and showed very slight cross-reactivity with other vinca alkaloids, vincristine (0.18%) and vinblastine (0.11%). The values for the VDS 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, ELISA was about 50-fold more sensitive in detecting VDS at lower concentrations. The sensitivity and specificity of ELISA should provide a useful tool for pharmacokinetic studies of VDS.

Immunocytochemistry for bestatin and its application to drug accumulation studies in rat intestine and kidney.

The in vivo role of transporters in drug disposition, in the context of other transporters, and metabolism has not been established. We prepared an anti-bestatin serum against bestatin conjugated to albumin with glutaraldehyde (GA). The antiserum was specific for GA-conjugated bestatin and weakly reacted with free bestatin, but no reaction occurred with structurally unrelated compounds according to both the inhibition and binding ELISAs. The antiserum allowed us to develop an immunocytochemical (ICC) method for detecting the uptake of bestatin in the rat intestine and kidney. Three hours after a single oral administration of bestatin, the ICC method revealed that the drug distributed in the microvilli, cytoplasm and nuclei of the absorptive epithelial cells at much larger amounts than in all other cell types in the small intestine. However, no drug was detected in the mucin goblets in the epithelium. In the kidney, the drug distributed to a greater extent in the S3 segment than in the S1 and S2 segments of the proximal tubule, and also was detected in the microvilli of the proximal tubule cells (S1, S2 and S3). These findings that bestatin accumulated in large amounts, especially in the cells and/or at the sites where the transporters PEPT1 and PEPT2 occur, corresponded well to those observed with ß-lactam amoxicillin in the previous ICC studies. Thus, this may indicate a possibility that both the transporters might be involved, at least in part, in the distribution of bestatin in the small intestine and kidney under the conditions examined.

Distribution study of peplomycin in rat kidney revealed by immunocytochemistry using monoclonal antibodies.

Peplomycin (PEP), an anti-tumor antibiotic related structurally to bleomycin, is widely used, especially for squamous cell carcinoma but shows renal toxicity. We prepared monoclonal antibodies (mAbs) against N-(γ-maleimidobutyryloxy)succinimide-conjugated PEP. The mAbs were monospecific for PEP, but did not react with bleomycin and other anticancer antibiotics. The mAbs enabled us to develop an immunocytochemical (ICC) method for detecting the uptake of PEP in the rat kidney. Two hours after a single i.v. administration of PEP, ICC revealed immunostaining for PEP in irregularly shaped cytoplasmic granules of the proximal tubules in which the microvilli were also stained. Also, staining occurred in the distal tubules and collecting ducts, in both of which we observed scattered swollen cells, reminiscent of necrotic cells, in which both the nuclei and cytoplasm reacted strongly with the antibody. Twenty-four hours after injection, PEP in the proximal tubules completely vanished, but yet significant amounts of PEP remained in both the distal tubules and collecting ducts. Distribution patterns of PEP in cells of the kidneys resembled, in some ways, those of our recent ICC studies for an organic cation aminoglycoside antibiotic gentamicin. This ICC suggests that PEP taken up in the proximal tubule cells is localized in the lysosomes, and organic cation transporters and bleomycin hydrolase might be involved in entrance and/or disappearance of PEP in this cell type. Furthermore, the distal tubules and collecting ducts may be the sites readily affected by some chemotherapeutic agents.

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

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 diazotized 4-amino-2-methoxy-1-[3-(morpholin-4-yl)propoxy]benzene. Enzyme labeling of gefitinib with beta-D-galactosidase 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 measured 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.