Tokyo, Japan) at 50 ml/min for 2 min at RT, and then with a freshly prepared answer of 2% GA in PBS for 6 min. may impact 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 [35], ~4 occasions increase in 40 years. Diabetes is usually classified as type 1 diabetes when little or no insulin is usually produced and type 2 diabetes when insulin secretion and insulin action is usually insufficient. Majority of people are affected by type 2 diabetes [35]. Therapeutic brokers for type 2 diabetes include sulfonylureas (stimulate insulin secretion from pancreatic -cells), biguanides (reduce insulin resistance), -glucosidase inhibitors, and incretin-related brokers. Recently, incretin-related brokers 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 [29]. Compared to the use of standard drugs, such as sulfonylureas, the incretin-based therapies are considered to have a lower risk of hypoglycemia and weight gain, acute pancreatitis and pancreatic malignancy [5, 6, 31]. However, there are reports that saxagliptin, a DPP-4 inhibitor, induced recurrent acute pancreatitis [23]. The DPP-4 inhibitors induced morphological abnormalities in the pancreas treated with incretin therapy [19]. Also there appears to be a statistical association between DPP-4 inhibitor use and pancreatic carcinoma [27]. 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 Astragaloside III 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 Astragaloside III 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 [12], amoxicillin [13], and vancomycin [14]. We now report around the preparation and characterization of a specific monoclonal antibody to alogliptin (AG), one of the DPP-4 inhibitors, and the development of an IHC Astragaloside III method for the localization of AG DNM2 in the intestine of rats orally administered with the drug. II.?Materials and Methods Preparation of 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 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 heat for 10 min to remove the acetyl group. The producing 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.