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Polyacetylenic alcohol, panaxytriol, which was isolated from Panax ginseng C. A. Meyer, has antiproliferative activity against several kinds of tumor cells. In this paper, the effect of panaxytriol on the cytotoxicity of mitomycin C (MMC) against a human gastric carcinoma cell line, MK-1, was investigated. The combination of a subthreshold concentration of MMC and panaxytriol produced a significant cytotoxic effect, which indicates that the effects of panaxytriol and MMC are synergistic. A synergistic effect was observed when MK-1 cells were treated with the mixture of MMC and panaxytriol or treated with MMC followed by panaxytriol. In contrast, when MK-1 cells were exposed to panaxytriol and then to MMC, only an additive effect was induced. With the aim of finding a possible mechanism, the effect of panaxytriol on the accumulation of MMC into the MK-1 cells was examined. Cellular concentrations of MMC were measured by high-performance liquid chromatography (HPLC). When MK-1 cells were treated with a mixture of panaxytriol and MMC or first with MMC and then with panaxytriol, the cellular level of MMC was significantly higher than that in MK-1 cells treated with MMC alone, but no significantly increased accumulation was found when MK-1 cells were treated with panaxytriol followed by MMC. These results suggest that synergistic effects of panaxytriol and MMC may be induced by acceleration of the effect of MMC on cellular accumulation by panaxytriol. In addition, they suggest that the enhanced accumulation of MMC in MK-1 cells treated with panaxytriol can probably be attributed to the decreased fluidity of the cell membrane caused by panaxytriol. [ABSTRACT FROM AUTHOR]

A highly sensitive enzyme-linked immunosorbent assay (ELISA) for etoposide (EP) was developed, which is capable of accurately measuring as little as 40 pg EP/ml. Anti-EP sera were obtained by immunizing rabbits with EP conjugated with mercaptosuccinyl bovine serum albumin (MS.BSA) using N-[beta-(4-diazophenyl)ethyl]maleimide (DPEM) as a heterobifunctional coupling agent. An enzyme marker was similarly prepared by coupling EP with beta-D-galactosidase (beta-Gal; EC 3.2.23) via DPEM. This ELISA was specific for EP and showed a very slight cross-reactivity with its major metabolite, cis-hydroxy acid of EP (0.91%), but none with 4'-demethylepipodophyllotoxin and drugs commonly used with EP in combination chemotherapy for cancer treatment. The values for EP concentration detected by this assay were comparable with those detected by the high-performance liquid chromatography (HPLC) method. However, the ELISA was about 1,250 times more sensitive in detecting EP at lower concentrations. Using this assay, drug levels were easily determined in the blood and urine of rats for 7 h after i.v. administration of EP at a single dose of 3 mg/kg. Due to its sensitivity and specificity for EP, the ELISA should prove to be a valuable new tool for use in clinical pharmacological studies. [ABSTRACT FROM AUTHOR]

Abstract: We have carried out soft and hard X-ray spectroscopy of Sm-based filled skutterudites SmFe4 P12 and SmOs4 Sb12. Sm 3d–4f resonant photoemission and Sm 3d–4f X-ray absorption spectra suggest that SmOs4 Sb12 has much more strongly mixed Sm valence state than SmFe4 P12 in the bulk. On the other hand, the Sm trivalent state is dominant on the surface of SmOs4 Sb12. [Copyright &y& Elsevier]

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., Competing Interests: VThe authors declare no conflicts of interest., (2023 The Japan Society of Histochemistry and Cytochemistry.)

This study sought to develop a specific and sensitive sandwich enzyme-linked immunosorbent assay (ELISA) for pharmacokinetic studies of dasatinib, a tyrosine kinase inhibitor. Anti-dasatinib antibodies were obtained from mice or rabbits by using two partial structures of dasatinib as haptens: 2-amino-N-(2-chloro-6-methylphenyl)-thiazole-5-carboxamide and 2-{4-(2-hydroxyethyl)-1-piperazinyl}-isonicotinic acid. The best combination of two antibodies for sandwich ELISA of dasatinib was determined using four anti-dasatinib antibodies derived from mice and rabbits. Using two dasatinib-specific rabbit antibodies, we successfully developed an ultra-specific and highly sensitive sandwich ELISA that is hardly affected by the main metabolite of dasatinib. The sandwich ELISA showed a linear detection range from 320 pg/mL to 1000 ng/mL. Serum dasatinib concentrations lower than 320 pg/mL were reproducibly measurable using the sandwich ELISA. The ELISA was specific to dasatinib and there were no cross-reactivities with the major metabolites 4'-hydroxy dasatinib and dasatinib carboxylic acid. The developed sandwich ELISA will be a valuable tool for pharmacokinetic studies of dasatinib. Furthermore, this study revealed that rabbit antibodies can sandwich drug molecules of a smaller size than mouse antibodies in sandwich ELISA., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Inc. All rights reserved.)

A monoclonal antibody (mAb) was produced against a fluvoxamine (FLV)-bovine serum albumin conjugate that was specific to both the conjugate and free form of FLV. The mAb enabled us to develop an immunohistochemistry (IHC) method for pharmacokinetic analysis of FLV at the cell and tissue levels. We demonstrated that IHC can be used to detect the localization of FLV in the small intestine, kidney, and liver 1 h after drug administration at the cell and tissue levels. Protease digestion is an important factor for obtaining appropriate IHC staining results for localization of drugs. In this study, precise FLV localization could be determined with only 1 h of protease digestion in the kidneys, but in the small intestine and liver, the staining results with two digestive conditions had to be merged. IHC provided new findings, such as (1) nerve cells are likely to uptake more FLV than other cells and tissues; (2) the ability of reabsorption and secretion in the kidney varies depending on the site, and the amount of FLV in the primary urine is regulated downstream of the proximal tubule S3 segment; and (3) some of the FLV is excreted in the bile., (© 2022. The Author(s) under exclusive licence to The Japanese Society for Clinical Molecular Morphology.)

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.

We investigated the pharmacokinetics of alogliptin (AG) at the cell and tissue level in healthy Wistar rats and a type 2 diabetic Goto-Kakizaki (GK) rat model. Immunohistochemistry of the renal tissue in these rats, post 1 hr of AG administration, showed that the signal was observed in the glomeruli, proximal tubule S3 segments, distal tubules, collecting ducts, and only in the brush border of the epithelial cells of the proximal tubule S1, S2 segments. After 6 hr of AG administration, the staining intensity of the regions other than the S3 segments was considerably reduced in Wistar rats, with no change observed in GK rats. At 24 hr, the staining intensity was considerably reduced, even in GK rats; however, the staining of the S3 segment remained unaltered in both. Hepatocytes in zone III of the hepatic lobule were more intensely stained than those in zone I in Wistar rats at 1 hr. However, almost no staining was observed in the hepatocytes of GK rats at 1 hr. Complete loss of signal was observed in the hepatocytes of the Wistar rats after 6 hr. This study revealed that the pharmacokinetics of AG in GK rats are different from those in Wistar rats., Competing Interests: VThe authors declare no conflicts of interest., (2022 The Japan Society of Histochemistry and Cytochemistry.)

O-Phosphoethanolamine (PEA) is an endogenous substance that is attracting interest as a biomarker for depression, and thus there is a need to develop a simple analytical method that specifically measures PEA. Therefore, this study aimed to develop a simple and specific enzyme-linked immunosorbent assay (ELISA) for PEA. Anti-PEA antibody was obtained by immunizing mice with an antigen conjugated with mercaptosuccinyl bovine serum albumin using m-maleimidobenzoyl-N-hydroxysulfosuccinimide ester (MBS). In this assay, the PEA to be quantified is chemically modified by benzoyl chloride that is allowed to compete with a PEA-MBS-HRP conjugate for binding to a limited amount of an anti-PEA antibody, which was used to coat the wells of a microtiter plate. This ELISA shows a linear range of detection of 0.11-27 μM, and a limit of quantification of 0.144 μM. The anti-PEA antibody showed high affinity for benzoyl PEA. No detectable cross-reactivity was found with benzoyl 2-aminoethanol, O-phospho-l-tyrosine or benzoyl sphingosine-1-phosphate. The values of plasma PEA levels measured by this ELISA were comparable to those measured by HPLC, and a strong correlation was observed between the values determined by the two methods. The developed ELISA should provide a valuable new tool for the quantification of PEA in human plasma., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Brigatinib and gilteritinib are oral tyrosine kinase inhibitors (TKIs). We aimed to develop a simple and sensitive indirect competitive enzyme-linked immunosorbent assay (ELISA) to quantify brigatinib and gilteritinib in various biological matrices. Antiserum against these TKIs was obtained from mice by using 3-methoxy-4-(-4-(4-methylpiperazin-1-yl) piperidin-1-yl) aniline as a hapten, which has a common substructure with these TKIs. The generated antibody was used to develop an indirect competitive ELISA for these TKIs in human serum. The lower limit of quantification of brigatinib and gilteritinib in human serum was 6.2 and 6.8 ng/mL, respectively. The developed ELISA was used to examine the pharmacokinetics of these TKIs after oral administration in mice and rats. This ELISA is expected to be a valuable tool in pharmacokinetic studies of these TKIs.

We prepared a polyclonal antibody against a teicoplanin (TEIC)-bovine serum albumin conjugate that was specific to both conjugated and free forms of TEIC. We demonstrated that this antibody could be used to detect the time-dependent localization of TEIC in rat kidneys. Immunohistochemistry revealed immunoreactivity specifically in the microvilli and apical cytoplasm of epithelial cells in proximal tubule segments S1 and S2, 1 h after intravenous TEIC injection, with higher staining intensity in the S2 segments. The epithelial cells of S3 segments showed moderate immunostaining with a few cells exhibiting nuclear staining. Furthermore, we found that the distal tubules and collecting ducts contained both TEIC-positive and -negative cells. TEIC immunoreactivity decreased rapidly over time; only weak staining remained in the S3 segments, distal tubules, and collecting ducts 24 h after administration. No staining was detected 7 days after injection. These results were significantly different from those of our previous study obtained using vancomycin, which showed moderate staining in the proximal tubule segments S1 and S2, distal tubules, and the collecting ducts 8 days after administration. The lower TEIC accumulation in tissues may account for a lower risk of adverse events compared to that using vancomycin., (© 2021. The Japanese Society for Clinical Molecular Morphology.)

Background: This study evaluated the mechanism of decline in coronary pressure from the proximal to the distal part of the coronary arteries in the left anterior descending (LAD) versus the right coronary artery (RCA) from the insight of coronary hemodynamics using wave intensity analysis (WIA)., Methods: Twelve patients with angiographically normal LAD and RCA were prospectively enrolled. Distal coronary pressure, mean aortic pressure, and average peak velocity were measured at 4 different positions: 9, 6, 3, and 0 cm distal from each coronary ostium., Results: The distal-to-proximal coronary pressure ratio during maximum hyperemia gradually decreased in proportion to the distance from the ostium (0.92±0.03 and 0.98±0.03 at 9 cm distal to the LAD and RCA ostium). WIA showed the dominant forward-traveling compression wave gradually decreased and the backward-traveling suction wave gradually decreased in proportion to the decrease in coronary pressure through the length of the non-diseased LAD but not the RCA., Conclusions: The pushing wave and suction wave intensities on WIA were diminished in proportion to the distance from the ostium of the LAD despite the wave intensity not changing across the length of the RCA, which may lead to gradual intracoronary pressure drop in the angiographically normal LAD., Competing Interests: Declaration of Competing Interest The authors declare that there is no conflict of interest., (Copyright © 2021. Published by Elsevier Ltd.)

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, R 2  = 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.

Daptomycin (DAP) has a completely different mechanism of action compared with conventional drugs for methicillin-resistant Staphylococcus aureus (MRSA) and is widely used as the first-line drug for treatment of dermal soft tissue infection and sepsis caused by MRSA infection in clinical practice. However, DAP has serious side effects, including renal dysfunction and rhabdomyolysis, and thus therapeutic drug monitoring of DAP is recommended. The purpose of this study was to develop an enzyme-linked immunosorbent assay (ELISA) for DAP that is simpler and more sensitive compared with existing assay methods and can be used in pharmacokinetic studies. Anti-DAP antibody was obtained by immunizing mice with an antigen conjugated with mercaptosuccinyl bovine serum albumin using N-(4-maleimidobutyryloxy) succinimide as a heterobifunctional coupling agent. Enzyme labeling of DAP with horseradish peroxidase was performed using pyromellitic dianhydride. The generated antibody and enzyme conjugate were used to develop a highly sensitive and specific ELISA for DAP in human serum. This ELISA shows a linear range of detection from 0.3 to 72.9 ng/mL, and a limit of quantification of approximately 0.3 ng/mL. The developed ELISA should be a valuable tool for pharmacokinetic studies and therapeutic drug monitoring of DAP.

Alogliptin is one of a new class of therapeutic agents for type 2 diabetes called dipeptidyl peptidase-4 inhibitors. Here, we used immunohistochemistry to investigate the pharmacokinetics of alogliptin at the cell and tissue levels in the rat kidney and liver. One hour after alogliptin administration, the most noticeable immunoreactivity in the kidney was a moderate-to-strong staining in proximal tubule S3 segment epithelial cells. On the other hand, immunostaining was found only in the microvilli of S1 and S2 segment cells. Immunoreactivity was also observed in the glomerulus and distal tubules. Positive cells and almost negative cells coexisted in the collecting ducts. Twenty-four hours after administration, moderate immunostaining remained in the S3 segment but staining in other regions had almost disappeared. In the liver 1 hr after administration, hepatocyte staining differed in the hepatic lobule, with zone III being stronger than zone I. Immunostaining had almost disappeared 24 hr after administration. These findings suggest that alogliptin reabsorption at the kidney and uptake at the hepatocyte vary from region to region and that one or more types of transporter are involved in these processes. In addition, long-term alogliptin use may cause the drug to accumulate in S3 segment, leading to adverse events., Competing Interests: VThe authors declare no conflicts of interest., (2020 The Japan Society of Histochemistry and Cytochemistry.)

Among any drugs, no comparative pharmacological study on how prodrug and its active metabolite behave in animal bodies is available. Immunohistochemistry (IHCs) using newly prepared two monoclonal antibodies, AOS-96 and AOC-160, monospecific for oseltamivir (OS) and its metabolite oseltamivir carboxylate (OC) were developed, simultaneously detecting the uptake or excretion of OS and OC in the intestine, liver, and kidney of rats to which OS was orally administered. In the intestine, IHC for OS revealed OS highly distributed to the absorptive epithelia with heavily stained cytoplasmic small granules (CSGs). IHC for OC showed that OC also distributed highly in the epithelia, but without CSGs, suggesting that OS was partly converted to OC in the cells. In the liver, OS distributed in the hepatocytes and on their bile capillaries, as well as on the lumina from the bile capillaries to the interlobular bile ducts. OC distributed in the whole cell of the hepatocytes, but without CSGs nor on any lumina through the interlobular bile ducts. In the kidney, a few levels of OS distributed in the cytoplasm of almost all the renal tubule cells, but they contained numerous CSGs. In contrast, OC distributed highly in the proximal tubules, but very slightly in the lower renal tubules of the nephrons. Thus, it was concluded that the two drugs behave in completely different ways in rat bodies. This paper also discusses a possibility of the correlation of OS or OC levels in tissue cells with their known transporters., (© 2020 The Authors. Pharmacology Research & Perspectives published by John Wiley & Sons Ltd, British Pharmacological Society and American Society for Pharmacology and Experimental Therapeutics.)

Daptomycin (DAP) has a completely different mechanism of action compared to conventional methicillin-resistant Staphylococcus aureus (MRSA) drugs and is widely used clinically as the first-line drug for the treatment of skin soft tissue infection and sepsis caused by MRSA infection. However, the most serious side effects of DAP include renal dysfunction and rhabdomyolysis. Knowledge of the time sequence of localization of DAP in cells and tissues of animals may help in developing a better understanding of the actual overall pharmacokinetics of DAP. We prepared DAP-specific antibodies by immunizing mice with DAP-GMBS-BSA conjugate. The Anti-DAP antibody was specific for DAP, which enabled us to develop an immunocytochemical method for detecting the uptake of DAP in the rat kidneys. One hour after a single intravenous (i.v.) injection of DAP at 12 mg/kg, immunohistochemical observation showed a strong ring-like positive reaction in the cytoplasm immediately below the microvilli of proximal tubule epithelial cells. The distal tubules and collecting ducts contained DAP-positive and negative cells in the cross section of one tubule. Twenty-four hours after DAP administration, several strong positive reactions of different sizes were observed in the cytoplasm of epithelial cells at the proximal tubule. No staining was detected after 7 days. This study will be a useful tool for analyzing the pharmacokinetics of DAP.

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., Competing Interests: VThe authors declare no conflicts of interest., (2019 The Japan Society of Histochemistry and Cytochemistry.)

Afatinib, a second-generation tyrosine kinase inhibitor, was designed to bind covalently to and irreversibly inhibit active ErbB family receptors. The major metabolites of afatinib in human plasma are adducts of afatinib covalently bound to plasma proteins via. the Michael addition reaction. These findings suggest that afatinib may form covalent bonds with proteins in tissue and be localized in tissue. However, there is no method for the specific detection of afatinib-protein conjugates localized in tissue. In this paper, we aimed to develop an immunohistochemical protocol to detect afatinib-protein conjugates. Immunostainings were performed with male rat intestinal tract and skin at 24 h after an oral administration of afatinib. In the intestinal tract, strong staining was observed in the ileum and colon, but only slight staining was observed in the duodenum and jejunum. In the skin, strong staining was observed in the epidermis, sebaceous glands and hair follicles. Immunohistochemistry for afatinib-protein conjugates could be a useful tool to detect the localization of such conjugates. This study is the first to elucidate the localization of afatinib-protein conjugates in the rat intestinal tract and skin and is expected to be of great use in efforts to clarify the mechanism underlying afatinib-induced diarrhoea or skin toxicities., (Copyright © 2019 Elsevier GmbH. All rights reserved.)

The tyrosine kinase inhibitor ponatinib is extensively metabolized in the body, and consequently the development of specific immunoassays for pharmacokinetic studies and therapeutic drug monitoring of ponatinib is challenging. If two antibodies simultaneously recognize the entire structure of ponatinib, they could be utilized to establish an ultra-specific sandwich immunoassay for ponatinib, free of any interference from ponatinib metabolites. In this study, we created two types of anti-ponatinib polyclonal antibodies that recognize two different ponatinib epitopes, and sandwiched almost all structural components of ponatinib in these two antibodies in order to develop an enzyme-linked immunosorbent assay (ELISA) technique not affected by any ponatinib metabolites. After optimization, this sandwich ELISA showed a linear detection range of 640 pg/mL to 2000 ng/mL and a limit of quantification of 640 pg/mL. This sandwich ELISA was specific to ponatinib and showed no cross-reactivity with the major metabolite M14. Comparison between the sandwich ELISA and HPLC, using serum samples from 15 rats orally administered a single dose of 15 mg/kg ponatinib, showed a linear regression (y = 0.9662x + 3.5354, r = 0.9683). Thus, in this study, we successfully developed the first ultra-specific sandwich ELISA for ponatinib in serum., (Copyright © 2019 Elsevier Inc. All rights reserved.)

This study evaluated the impact of optical coherence tomography (OCT)-derived low-backscattered tissue on mid-term coronary endothelial function after drug-eluting stent (DES) implantation. Although OCT enables detailed in vivo evaluation of neointimal tissue characterization after DES implantation, its association with physiological vascular healing response is unclear. Thirty-three stable angina pectoris patients underwent OCT examination and endothelial function testing with intracoronary infusion of incremental doses of acetylcholine 8-month after DES implantation in a single lesion of the left anterior descending artery. Neointimal tissue was classified into two patterns based on the predominant OCT light backscatter: high backscatter and low backscatter. Although the presence of uncovered or malapposed stent strut was not associated with the degree of vasoconstriction, the degree of vasoconstriction was significantly greater in the DES with low-backscattered neointima than in the DES without low-backscattered neointima (- 32.1 ± 25.7 vs. - 4.1 ± 20.1%, p = 0.003). Moreover, there was an inverse linear relationship between low backscatter tissue index and degree of vasoconstriction after acetylcholine infusion (r = 0.50 and p = 0.003). The endothelium-dependent vasomotor response after 8-month of DES was impaired in patients with low neointimal tissue backscatter on OCT imaging. OCT assessment of low-backscattered tissue may be used as surrogate markers for impairment of endothelial function after DES.

Knowledge of time sequence of localization of drugs in cells and tissues of animals may help in developing a better understanding of the actual overall pharmacokinetics of the drugs. We produced monoclonal antibody (mAb) against alogliptin (AG), a dipeptidyl peptidase-4 (DPP-4) inhibitor, conjugated to BSA with N -(γ-maleimidobutyryloxy)-succinimide. The mAb was specific for AG and did not cross-react with sitagliptin, vancomycin or amoxicillin. The mAb enabled us to develop an immunohistochemical method for detecting the localization of AG in the rat small intestine. One hour after a single oral administration of AG, immunohistochemistry revealed that the immunoreactivity of AG was observed in almost all of cells and tissues of the duodenum. The microvilli of the absorptive epithelial cells were moderately stained. The staining pattern of AG at jejunum and ilium was almost the same as that of duodenum, but the staining intensity, especially at absorptive epithelial cells and intestinal gland epithelial cells, became stronger towards the distal part of the small intestine. These results suggested that AG may be more actively absorbed from the lower part of the small intestine than in the upper part. It may affect the function of cells with membrane-bound DPP-4 because it was reported that membrane-bound form of DPP-4 exists in the microvilli of the absorptive epithelial cells., Competing Interests: VThe authors declare no conflicts of interest.

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.

Background: Coronary angioscopy (CAS) is used to comprehensively evaluate vascular responses after drug-eluting stent (DES) implantation. This study sought to evaluate the capability of CAS for evaluating DES strut coverage grade and color grade of the intima compared with histological images in coronary autopsy specimens. Methods and Results: A total of 23 DES extracted from 11 autopsy hearts were imaged by CAS. All stent segments were graded as white or yellow according to the luminal surface color, and thrombus was evaluated according to a previous report. Neointimal coverage over the DES was graded as 0 (stent struts fully visible) to grade 3 (stent struts fully embedded and invisible). Of 76 segments, neointimal coverage was graded as 0 in 35 (46%), 1 in 22 (29%), 2 in 8 (11%), and 3 in 11 (14%). The neointimal thickness increased significantly with increasing neointimal coverage grade on angioscopy. Neointimal color was graded as white in 40 (53%) and yellow in 36 segments (47%). Histological analysis revealed that yellow neointima contained fibroatheroma, foam cells accumulation or superficial calcium deposition. A thrombus was identified in 13 segments. Thrombi adherent around the stent strut were partly intimal erythrocyte accumulation around the strut., Conclusions: In-stent yellow segment had atherosclerotic components. CAS could evaluate vascular status comprehensively after DES implantation.

A selective and sensitive competitive enzyme-linked immunosorbent assay (ELISA) method was developed and validated for the quantification of erlotinib in 50 µL of samples of human serum. Anti-erlotinib serum was obtained by immunizing mice with an antigen conjugated with bovine serum albumin and 3,4-bis(2-methoxyethoxy)benzoic acid using the N -succinimidyl ester method. Enzyme labeling of erlotinib with horseradish peroxidase was similarly performed using 3,4-bis(2-methoxyethoxy)benzoic acid. A simple competitive ELISA for erlotinib was developed using the principle of direct competition between erlotinib and the enzyme marker for anti-erlotinib antibody, which had been immobilized on the plastic surface of a microtiter plate. Serum erlotinib concentrations lower than 40 ng/mL were reproducibly measurable using the ELISA. This ELISA was specific to erlotinib and showed very slight cross-reactivity (6.7%) with a major metabolite, O -desmethyl erlotinib. Using this assay, drug levels were easily measured in the blood of mice after oral administration of erlotinib at a single dose of 30 mg/kg. ELISA should be used as a valuable tool for therapeutic drug monitoring and in pharmacokinetic studies of erlotinib.