for their technical assistance throughout this study. VIII.?. 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. strong class=”kwd-title” Keywords: alogliptin, immunohistochemistry, localization, intestine, rat I.?Introduction Globally, the number of diabetic patients, which was 108 million in 1980, increased to 422 million in 2014 , ~4 times increase in 40 years. Diabetes is classified as type 1 diabetes when little or no insulin is produced and type 2 diabetes when insulin secretion and insulin action is insufficient. Majority of people are affected by type 2 diabetes . Therapeutic agents for type 2 diabetes include sulfonylureas (stimulate insulin secretion from pancreatic -cells), biguanides CB-6644 (reduce insulin resistance), -glucosidase inhibitors, and incretin-related agents. Recently, incretin-related agents such as dipeptidyl peptidase-4 (DPP-4) inhibitors and glucagon-like peptide (GLP)-1 receptor agonists are being widely used in the treatment of type 2 diabetes patients. The DPP-4 inhibitors augment the glucose-dependent insulin secretion through enhancement of the action of endogenous incretins, such as GLP-1 and glucose-dependent insulinotropic polypeptide (GIP) by inhibiting DPP-4, a degrading enzyme of incretin . Compared to the use of conventional drugs, such as sulfonylureas, the incretin-based therapies are considered to have a lower risk of hypoglycemia and weight gain, acute pancreatitis and pancreatic cancer [5, 6, 31]. However, there are reports that saxagliptin, a DPP-4 inhibitor, induced recurrent acute pancreatitis . The DPP-4 inhibitors induced morphological abnormalities in the pancreas treated with incretin therapy . Also there appears to be a statistical association between DPP-4 inhibitor use and pancreatic carcinoma . Although DPP-4 circulates in blood as a soluble enzyme [21, 24], the major fraction of the total bodys DPP-4 is not localized in plasma, but is present in peripheral tissues in a membrane-bound form [15, 16, 18, 21]. Thus, knowledge of the time sequence of the localization of DPP-4 inhibitors in cells and tissues of animals would be useful in developing a better understanding of the mechanisms behind the action and/or adverse effects of the drugs and their appropriate usage. However, only a few reports about the cell and tissue localization of the DPP-4 inhibitors have been obtained by autoradiography using radio-labeled drugs [16, 20, 28]. For over 10 years, we have successfully developed immunohistochemical procedures for detecting cell and tissue localization of some drugs, such as daunomycin [11, 32], gentamicin , amoxicillin , and vancomycin . We now report on the preparation and characterization of a specific monoclonal antibody to alogliptin (AG), one of the DPP-4 inhibitors, and the development of an IHC method for the localization of AG in the intestine of rats orally administered with the drug. II.?Materials and Methods Preparation of CB-6644 immunogen (AG-GMBS-BSA conjugate) The immunogen was prepared according to our previous method for anti-daunomycin serum using a heterobifunctional agent em N /em -(-maleimidobutyryloxy)succinimide (GMBS; Dojindo Laboratories, Kumamoto, Japan) [9, 11]. Briefly, AG (2 mg, 5.9 mol; Takeda Pharmaceutical Co. Ltd., Osaka, Japan) in 2.0 ml of 0.1 M phosphate buffer, pH 7.0; and 1.6 mg (5.7 mol) GMBS in CB-6644 0.5 ml tetrahydrofuran were mixed, constantly stirred, and incubated at room temperature for 60 min, thus yielding a GMBS-acylated AG solution. The sample was centrifuged for 10 min at 2,000 rpm, and the supernatant was collected. Acetylmercaptosuccinyl BSA (AMS-BSA, 15 mg, approximately 0.1 mol) was dissolved in 200 l of 0.1 M phosphate buffer, pH 7.0, and incubated with 50 l of 0.5 M hydroxylamine, pH 7.4, at room temperature for 10 min to remove the acetyl group. The resulting mercaptosuccinyl BSA (MS.BSA) was diluted with 1 ml of 0.1 M phosphate buffer, pH 7.0, and added immediately to GMBS-acylated AG supernatant and incubated for 60 min with slow stirring. The conjugate was applied to a 2.5 cm by 45 cm Sephadex G-75 column equilibrated with 10 mM phosphate buffer (pH 7.0) and eluted with the same buffer. The eluate, monitored at 280 nm, was collected in 3 ml fractions and the concentration of the conjugate was determined by Modified Lowery Protein Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA). The peak fraction was used for immunization. Preparation of anti-AG mAbs Three five-week-old female BALB/c Rabbit Polyclonal to CARD11 mice were injected intraperitoneally.