Intraspinal Grafting of Serotonergic Neurons Modifies Expression of Genes Important for Functional Recovery in Paraplegic Rats.

Serotonin (5-hydroxytryptamine; 5-HT) plays an important role in control of locomotion, partly through direct effects on motoneurons. Spinal cord complete transection (SCI) results in changes in 5-HT receptors on motoneurons that influence functional recovery. Activation of 5-HT2A and 5-HT7 receptors improves locomotor hindlimb movements in paraplegic rats. Here, we analyzed the mRNA of 5-HT2A and 5-HT7 receptors (encoded by Htr2a and Htr7 genes, resp.) in motoneurons innervating tibialis anterior (TA) and gastrocnemius lateralis (GM) hindlimb muscles and the tail extensor caudae medialis (ECM) muscle in intact as well as spinal rats. Moreover, the effect of intraspinal grafting of serotonergic neurons on Htr2a and Htr7 gene expression was examined to test the possibility that the graft origin 5-HT innervation in the spinal cord of paraplegic rats could reverse changes in gene expression induced by SCI. Our results indicate that SCI at the thoracic level leads to changes in Htr2a and Htr7 gene expression, whereas transplantation of embryonic serotonergic neurons modifies these changes in motoneurons innervating hindlimb muscles but not those innervating tail muscles. This suggests that the upregulation of genes critical for locomotor recovery, resulting in limb motoneuron plasticity, might account for the improved locomotion in grafted animals.

Drug-drug interaction potential of antitumor acridine agent C-1748: The substrate of UDP-glucuronosyltransferases 2B7, 2B17 and the inhibitor of 1A9 and 2B7.

BACKGROUND: The compound 9-(2'-hydroxyethylamino)-4-methyl-1-nitroacridine (C-1748), the promising antitumor agent developed in our laboratory was determined to undergo phase I metabolic pathways. The present studies aimed to know its biotransformation with phase II enzymes - UDP-glucuronosyltransferases (UGTs) and its potential to be engaged in drug-drug interactions arising from the modulation of UGT activity. METHODS: UGT-mediated transformations with rat liver (RLM), human liver (HLM), and human intestine (HIM) microsomes and with 10 recombinant human isoenzymes were investigated. Studies on the ability of C-1748 to inhibit UGT were performed with HLM, HT29 colorectal cancer cell homogenate and the selected recombinant UGT isoenzymes. The reactions were monitored using HPLC-UV/Vis method and the C-1748 metabolite structure was determined with ESI-TOF-MS/MS analysis. RESULTS: Pseudo-molecular ion (m/z 474.1554) and the experiment with ß-glucuronidase indicated that O-glucuronide of C-1748 was formed in the presence of microsomal fractions. This reaction was selectively catalyzed by UGT2B7 and 2B17. High inhibitory effect of C-1748 was shown towards isoenzyme UGT1A9 (IC50=39.7µM) and significant but low inhibitory potential was expressed in HT29 cell homogenate (IC50=84.5µM). The mixed-type inhibition mechanism (Ki=17.0µM;Ki'=81.0µM), induced by C-1748 was observed for recombinant UGT1A9 glucuronidation, whereas HT29 cell homogenate resulted in noncompetitive inhibition (Ki=94.6µM). CONCLUSIONS: The observed UGT-mediated metabolism of C-1748 and its ability to inhibit UGT activity should be considered as the potency for drug resistance and drug-drug interactions in the prospective multidrug therapy.

Modulation of UDP-glucuronidation by acridinone antitumor agents C-1305 and C-1311 in HepG2 and HT29 cell lines, despite slight impact in noncellular systems.

BACKGROUND: Among the studied antitumor acridinone derivatives developed in our laboratory, 5-dimethylaminopropylamino-8-hydroxytriazoloacridinone (C-1305) and 5-diethylaminoethylamino-8-hydroxyimidazoacridinone (C-1311) exhibited cytotoxic and antitumor properties against several cancer types and were selected to be evaluated in preclinical and early-phase clinical trials. In the present work, we investigated the impact of C-1305 and C-1311 on UDP-glucuronosyltransferase (UGT) activity. METHODS: Enzyme activity modulation was studied using HPLC by analyzing standard UGT substrate metabolism in the presence and absence of antitumor drugs. The investigations were performed in two model systems: (i) under noncellular conditions, including human liver microsomes (HLM) and recombinant UGT1A1, 1A9 and 1A10 isoenzymes and (ii) in tumor cells. RESULTS: There was observed a slight impact of studied drugs on enzyme activity. Only UGT1A1 action was altered by both compounds. The modulatory effects of UGT activity in cellular systems depended on the tumor cell type. In the case of HepG2, C-1305 and C-1311 strongly induced UGT activity, particularly for C-1311, at concentrations significantly lower than the EC50. This effect contradicted irinotecan mediated UGT inhibition. HT29 colon tumor cells were less sensitive than HepG2 to enzyme modulation in the presence of the studied compounds, particularly C-1305, where enzymatic inhibition similar to that of irinotecan was observed. CONCLUSIONS: The results demonstrated that UGT activity modulation should be expected in the case of antitumor therapy with C-1305 or/and C-1311. Analysis of the results indicated that these modulations would occur via cellular regulatory pathways not by direct drug-enzyme interactions.