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

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.

Development of immunohistochemistry for detecting fluvoxamine in rat tissues using newly prepared monoclonal antibody: its precise localization in small intestine, kidney, and liver of rats.

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.

Development of an enzyme-linked immunosorbent assay for the quantification of O-Phosphoethanolamine in human plasma.

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.

An Indirect Competitive Enzyme-Linked Immunosorbent Assay for the Determination of Brigatinib and Gilteritinib Using a Specific Polyclonal Antibody.

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.

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.

Immunohistochemical localization and pharmacokinetics of the anti-MRSA drug teicoplanin in rat kidney using a newly developed specific antibody.

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.

Development of a sandwich enzyme-linked immunosorbent assay for the quantification of ponatinib in serum.

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.

Preparation of Antibodies and Development of an Enzyme-Linked Immunosorbent Assay for the Tyrosine Kinase Inhibitors Lapatinib and Nilotinib.

In this paper, we describe the production of the first specific antibodies against the tyrosine kinase inhibitors lapatinib and nilotinib. Anti-lapatinib antibody was obtained by immunizing rabbits with an antigen conjugated with bovine serum albumin using 3-chloro-4-((3-fluorobenzyl)oxy)aniline. Anti-nilotinib antibody was produced by immunizing mice with an antigen conjugated with bovine serum albumin using 2-(5-amino-2-methylanilino)-4-(3-pyridyl)pyrimidine. The generated antibodies were used to develop highly sensitive and specific enzyme-linked immunosorbent assays (ELISAs) for lapatinib and nilotinib in human serum. The assays were capable of detecting lapatinib and nilotinib at serum concentrations as low as 40 and 8 ng/mL, respectively. Using the two ELISAs, drugs levels were easily measured in the serum of rats after a single dose oral administration of lapatinib or nilotinib. The assays are therefore expected be valuable tools for therapeutic drug monitoring in the clinical setting and pharmacokinetic studies of lapatinib and nilotinib.

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 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.

Immunocytochemistry for vancomycin using a monoclonal antibody that reveals accumulation of the drug in rat kidney and liver.

We prepared monoclonal antibodies against N-(γ-maleimidobutyryloxy)succinimide-conjugated vancomycin (VM). The monoclonal antibody was specific for conjugated or free VM. The monoclonal antibody enabled us to develop an immunocytochemical method for detecting the uptake of VM in the rat kidney and liver. Three hours after a single intravenous (i.v.) injection of VM at the therapeutic dose, the immunocytochemistry revealed that VM accumulated in large amounts in both the S1 and S2 segments and in much smaller amounts in the S3 segment of the proximal tubules as well as in the distal tubules and collecting ducts. The drug was detected in the cytoplasm, cytoplasmic irregular granules, nuclei, and microvilli of the proximal tubule cells. The distal tubules and collecting ducts contained scattered swollen cells in which both the nuclei and cytoplasm were heavily immunostained. Twenty-four hours after injection, most of the swollen cells returned back to normal size and had somewhat decreased immunostaining. Also, significant amounts of VM remained accumulated for as long as 8 days postadministration. In the liver, similar drug accumulation was observed in the Kupffer cells and the endothelial cells of the hepatic sinusoids but not in the hepatocytes, suggesting that vancomycin cannot be eliminated via the liver. Immunoelectron microscopic studies demonstrated that in the collecting ducts, uptake of VM occurred exclusively in the lysosomes and cytoplasm of the principal cells and scarcely in the intercalated cells. Furthermore, double fluorescence staining using rats simultaneously administered with VM and gentamicin strongly suggests that both drugs colocalized in lysosomes in the proximal tubule cells of kidneys.

Immunohistochemical Localization of Alogliptin, a DPP-4 Inhibitor, in Tissues of Normal and Type 2 Diabetes Model Rat.

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.

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.

[Development of a Sensitive Enzyme-linked Immunosorbent Assay for Daptomycin].

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.

[Localization and Accumulation Studies of Daptomycin in Rats Kidney Using Immunohistochemistry].

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.

Metabolism and disposition of oseltamivir (OS) in rats, determined by immunohistochemistry with monospecific antibody for OS or its active metabolite oseltamivir carboxylate (OC): A possibility of transporters dividing the drugs' excretion into the bile and kidney.

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.

Immunohistochemical Pharmacokinetics of the Anti-diabetes Drug Alogliptin in Rat Kidney and Liver.

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.

Light-microscopic immunocytochemistry for gentamicin and its use for studying uptake of the drug in kidney.

Gentamicin (GM) is a widely used antibiotic but shows renal toxicity. We produced a serum against GM (anti-GM) conjugated to bovine serum albumin with N-(gamma-maleimidobutyryloxy)succinimide. The antiserum was monospecific for GM and did not cross-react with the analog streptomycin, tobramycin, kanamycin, or amikacin. The antiserum also detected glutaraldehyde-fixed GM, and this enabled us to develop an immunocytochemical method for detecting the uptake of GM in rat kidney. Twelve hours after a single intravenous administration of GM, immunocytochemistry revealed that GM accumulated in the S1, S2, and S3 segments of the proximal tubules, as well as in the distal tubules and collecting ducts. By 12 h after injection, the drug was detected in cytoplasmic granules of the proximal tubule cells. However, early (1 h) after injection, drug accumulation was detected in the microvilli of these cells. The distal tubules and collecting ducts contained scattered swollen cells, reminiscent of necrotic cells, in which both the nuclei and the cytoplasm reacted strongly with GM. No staining occurred in the kidneys of saline-injected control rats. These results agree with previous studies showing that GM is endocytosed in the proximal tubules and accumulates in lysosomes. Additionally, our results show that GM also accumulates in the distal tubules and collecting ducts. This was achieved by systematically varying the pretreatment conditions-an approach necessary for detecting GM in different subcellular compartments. This approach should be useful for accurately detecting the uptake and toxicity of the antibiotic in different tissues.