ABSTRACT
This study aimed to estimate the absorption, distribution, metabolism and excretion (ADME) properties and safety of LDT5, a lead compound for oral treatment of benign prostatic hyperplasia that has previously been characterized as a multi-target antagonist of α1A-, α1D-adrenoceptors and 5-HT1A receptors. The preclinical characterization of this compound comprised the evaluation of its in vitro properties, including plasma, microsomal and hepatocytes stability, cytochrome P450 metabolism and inhibition, plasma protein binding, and permeability using MDCK-MDR1 cells. De-risking and preliminary safety pharmacology assays were performed through screening of 44 off-target receptors and in vivo tests in mice (rota-rod and single dose toxicity). LDT5 is stable in rat and human plasma, human liver microsomes and hepatocytes, but unstable in rat liver microsomes and hepatocytes (half-life of 11 min). LDT5 is highly permeable across the MDCK-MDR1 monolayer (Papp ∼32×10-6 cm/s), indicating good intestinal absorption and putative brain penetration. LDT5 is not extensively protein-bound and is a substrate of human CYP2D6 and CYP2C19 but not of CYP3A4 (half-life >60 min), and did not significantly influence the activities of any of the human cytochrome P450 isoforms screened. LDT5 was considered safe albeit new studies are necessary to rule out putative central adverse effects through D2, 5-HT1A and 5-HT2B receptors, after chronic use. This work highlights the drug-likeness properties of LDT5 and supports its further preclinical development.
Subject(s)
Humans , Animals , Male , Female , Mice , Rats , Drug Evaluation, Preclinical , Piperazines/pharmacology , Prostatic Hyperplasia/drug therapy , Drug Stability , Permeability , Piperazines/chemistry , Piperazines/metabolism , Time FactorsABSTRACT
p15INK4B, a cyclin-dependent kinase inhibitor, has been recognized as a tumor suppressor. Loss of or methylation of the p15INK4B gene in chronic myeloid leukemia (CML) cells enhances myeloid progenitor formation from common myeloid progenitors. Therefore, we examined the effects of overexpressed p15INK4B on proliferation and apoptosis of CML cells. Overexpression of p15INK4B inhibited the growth of K562 cells by downregulation of cyclin-dependent kinase 4 (CDK4) and cyclin D1 expression. Overexpression of p15INK4B also induced apoptosis of K562 cells by upregulating Bax expression and downregulating Bcl-2 expression. Overexpression of p15INK4B together with STI571 (imatinib) or BCR-ABL1 small interfering RNA (siRNA) also enhanced growth inhibition and apoptosis induction of K562 cells. The enhanced effect was also mediated by reduction of cyclin D1 and CDK4 and regulation of Bax and Bcl-2. In conclusion, our study may provide new insights into the role of p15INK4B in CML and a potential therapeutic target for overcoming tyrosine kinase inhibitor resistance in CML.
Subject(s)
Humans , Apoptosis/drug effects , Benzamides/pharmacology , Cell Proliferation/drug effects , /metabolism , Fusion Proteins, bcr-abl/metabolism , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/drug therapy , Piperazines/pharmacology , Pyrimidines/pharmacology , RNA, Small Interfering/pharmacology , Antineoplastic Agents/pharmacology , Benzamides/metabolism , Cyclin D1/drug effects , Cyclin D1/metabolism , /drug effects , /metabolism , /genetics , Drug Combinations , Drug Resistance, Neoplasm , Down-Regulation/drug effects , Fusion Proteins, bcr-abl/antagonists & inhibitors , Fusion Proteins, bcr-abl/genetics , Gene Expression/genetics , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/metabolism , Piperazines/metabolism , Protein Kinase Inhibitors/pharmacology , /drug effects , /metabolism , Pyrimidines/metabolism , /drug effectsABSTRACT
Sildenafil is a selective inhibitor of cyclic-guanosine monphosphat-specific phosphodiesterase type 5. It increases intracellular nitric oxide [NO] production in some cells. There are reports on its positive effect on uterine circulation, endometrial thickness, and infertility improvement. Endometrial epithelial cells [EEC] play an important role in embryo attachment and implantation. The present work investigates the effect of sildenafil on human EEC and their NO secretion in vitro. In this experimental in vitro study, endometrial biopsies [n=10] were washed in a phosphate buffered solution [PBS] and digested with collagenase I [2 mg/ml in DMEM/F12 medium] at 37°C for 90 minutes. Epithelial glands were collected by sequential filtration through nylon meshes [70 and 40 micro m pores], respectively. Epithelial glands were then treated with trypsin to obtain individual cells. The cells were counted and divided into four groups: control and 1, 10, and 20 micro M sildenafil concentrations. Cells were cultured for 15 days at 37°C and 5% CO[2]; the media were changed every 3 days, and their supernatants were collected for the NO assay. NO was measured by standard Greiss methods. Data were analyzed by one way ANOVA. There was no significant difference between groups in cell count and NO secretion, but the level of NO increased slightly in the experimental groups. The 10 micro M dose showed the highest cell count. EEC morphology changed into long spindle cells in the case groups. Sildenafil [1, 10, and 20 micro M] showed a mild proliferative effect on human EEC numbers, but no significant change was seen in NO production