Oseltamivir is the main medicine recommended by the World Health Organization in anticipation of next influenza pandemic. This anti-influenza viral agent is an ester prodrug, and the antiviral activity is achieved by its hydrolytic metabolite: oseltamivir carboxylate. In this study, we report that the hydrolytic activation is catalyzed by carboxylesterase human carboxylesterase (HCE) 1. Liver microsomes rapidly hydrolyzed oseltamivir, but no hydrolysis was detected with intestinal microsomes or plasma. The overall rate of the hydrolysis varied among individual liver samples and was correlated well with the level of HCE1. Recombinant HCE1 but not HCE2 hydrolyzed this prodrug and produced similar kinetic parameters as the liver microsomes. Several HCE1 natural variants differed from the wildtype enzyme on the hydrolysis of oseltamivir. In the presence of antiplatelet agent clopidogrel, the hydrolysis of oseltamivir was inhibited by as much as 90%

Existing anticoagulants effectively inhibit the activity of coagulation factors of the extrinsic and common pathway but have substantial limitations and can cause severe bleeding complications. Here we describe a novel therapeutic approach to thrombosis treatment. We have developed and characterized the efficacy and safety of selective second-generation antisense oligonucleotides (ASOs) targeting coagulation factor XI (FXI), a member of the intrinsic coagulation pathway. Systemic treatment of mice with FXI ASO led to a potent, specific, and dose-dependent reduction of FXI mRNA levels in the liver with corresponding reductions in plasma levels of FXI protein and activity. FXIASO treatment produced potent, dose-dependent antithrombotic activity in various venous and arterial thrombosis models, comparable with warfarin or enoxaparin. However, unlike warfarin or enoxaparin, FXI inhibition did not cause bleeding. Coadministration of FXI ASO with enoxaparin or the antiplatelet drug clopidogrel produced improved antithrombotic activity without increased bleeding. Finally, plasma-derived FXI concentrate was shown to effectively and rapidly reverse the anticoagulant effect of FXI antisense therapy. These results support the concept that inhibition of FXI through antisense therapy might serve as a new and effective strategy for the treatment and prevention of venous thromboembolism with improved specificity and safety.

Aspirin (acetylsalicylic acid) and clopidogrel are two major antithrombogenic agents that are widely used for the treatment and prevention of cerebro-and cardiovascular conditions such as stroke. Combined use produces enhanced therapeutic effect. Aspirin and clopidogrel both are esters, and hydrolysis leads to decreased or inactivated therapeutic activity. The aim of the study was to determine whether aspirin and clopidogrel are hydrolyzed by the same enzyme(s), thus reciprocally prolonging the antithrombogenic activity. To test this possibility, microsomes from the liver and intestine were assayed for the hydrolysis of aspirin and clopidogrel. In contrary to the hypothesis, aspirin and clopidogrel were hydrolyzed in a tissue-differential manner. Liver microsomes hydrolyzed both drugs, whereas intestinal microsomes hydrolyzed aspirin only. Consistent with the tissue distribution of two carboxylesterases human carboxylesterase (HCE) 1 and HCE2, recombinant HCE1 hydrolyzed clopidogrel, whereas recombinant HCE2 hydrolyzed aspirin. In addition, hydrolysis of clopidogrel among liver samples was correlated well with the level of HCE1, and hydrolysis of aspirin with HCE2. Certain natural variants differed from the wild-type enzymes on the hydrolysis of aspirin or clopidogrel. In the presence of ethyl alcohol, clopidogrel is converted to ethyl clopidogrel. Carboxylesterases are important pharmacological determinants for drugs containing ester linkages and exhibit a large interindividual variation. The isoform-specific hydrolysis of aspirin and clopidogrel suggests that these two antithrombogenic agents may have pharmacokinetic interactions with different sets of ester drugs, and the altered hydrolysis by polymorphic mutants provides a molecular explanation to the interindividual variation.

Edited by Tamas DalmayKeywords: Hepcarcin R RNA/MALAT-1 RNA processing Alternative splicing a b s t r a c t RNA processing is vital for the high fidelity and diversity of eukaryotic transcriptomes and the encoded proteomes. However, control of RNA processing is not fully established. R RNA is a class of conserved large non-coding RNAs (murine Hepcarcin; human MALAT-1) up-regulated in carcinomas. Using antisense technology, we identified that RNA post-transcriptional modification is the most significant global function of R RNA. Specifically, processing of the pre-mRNAs of genes including Tissue Factor and Endoglin was altered by hydrolysis of R RNA/MALAT-1. These results support the hypothesis that R RNA/MALAT-1 is a regulatory molecule exerting roles in RNA post-transcriptional modification.

Mice deficient for the cellular prion protein (PrPC) do not develop prion disease; accordingly, gene-based strategies to diminish PrPC expression are of interest. We synthesized a series of chemically modified antisense oligonucleotides (ASOs) targeted against mouse Prnp messenger RNA (mRNA) and identified those that were most effective in decreasing PrPC expression. Those ASOs were also evaluated in scrapie-infected cultured cells (ScN2a) for their efficacy in diminishing the levels of the disease-causing prion protein (PrPSc). When the optimal ASO was infused intracerebrally into FVB mice over a 14-day period beginning 1 day after infection with the Rocky Mountain Laboratory (RML) strain of mouse prions, a prolongation of the incubation period of almost 2 months was observed. Whether ASOs can be used to develop an effective therapy for patients dying of Creutzfeldt–Jakob disease remains to be established.

We have systematically investigated the therapeutic potenine acetyltransferase (ChAT) activity in cultures following tial of cationic liposome-mediated neurotrophic gene transcalcium-dependent depolarization injury. In in vivo studies, fer for treatment of CNS injury. Following determination of following intraventricular injections of NGF cDNA comoptimal transfection conditions, we examined the effects of plexed with DC-Chol liposomes, ELISA detected nine-to dimethylaminoethane-carbamoyl-cholesterol (DC-Chol) 12-fold increases of NGF in rat CSF. Further studies liposome-mediated NGF cDNA transfection in injured and showed that liposome/NGF cDNA complexes could attenuuninjured primary septo-hippocampal cell cultures and rat ate the loss of cholinergic neuronal immunostaining in the brains. In in vitro studies, we detected an increase of NGF rat septum after traumatic brain injury (TBI). Since deficits mRNA in cultures 1 day after transfection. Subsequent in cholinergic neurotransmission are a major consequence ELISA and PC12 cell biological assays confirmed that culof TBI, our studies demonstrate for the first time that DCtured cells secreted soluble active NGF into the media from Chol liposome-mediated NGF gene transfection may have day 2 after gene transfection. Further experiments showed therapeutic potential for treatment of brain injury. that such NGF gene transfection reduced the loss of chol-

To identify chemistries and strategies to improve the potency of MOE second generation ASOs, we have evaluated gapmer antisense oligonucleotides containing BNAs having N-O bonds. These modifications include N-MeO-amino BNA, N-Me-aminooxy BNA, 2',4'-BNA(NC)[NMe], and 2',4'-BNA(NC) bridged nucleoside analogues. These modifications provided increased thermal stability and improved in vitro activity compared to the corresponding ASO containing the MOE modification. Additionally, ASOs containing N-MeO-amino BNA, N-Me-aminooxy BNA, and 2',4'-BNA(NC)[NMe] modifications showed improved in vivo activity (>5-fold) compared to MOE ASO. Importantly, toxicity parameters, such as AST, ALT, liver, kidney, and body weights, were found to be normal for N-MeO-amino BNA, N-Me-aminooxy BNA, and 2',4'-BNA(NC)[NMe] ASO treated animals. The data generated in these experiments suggest that N-MeO-amino BNA, N-Me-aminooxy BNA, and 2',4'-BNA(NC)[NMe] are useful modifications for applications in both antisense and other oligonucleotide based drug discovery efforts.

Aldehyde oxidase (AO) is a cytosolic enzyme that contributes to the Phase I metabolism of xenobiotics in human and preclinical species. We compared AO activity in cytosol and cryopreserved hepatocytes from human, monkey, rat and mouse livers to assess species differences. We also evaluated possible species differences in drug interactions using seven drugs known to inhibit human cytosolic AO i.e. raloxifene, perphenazine, menadione, maprotiline, ketoconazole, erythromycin, and estradiol. AO activity was measured using the formation of vanillic acid from vanillin. The rate of vanillic acid formation was 2 +/- 0.2 nmol/min/mg in human liver cytosol and 0.79 +/- 0.45 nmol/min/million cells in cryopreserved human hepatocytes. AO activity (V(max,app)) was highest in monkey and lowest in rat. Mouse liver cytosol had the lowest K(m,app) (1.44 +/- 0.16 microM) and highest intrinsic clearance (8.97 ml/min/mg) and rat liver cytosol the highest K(m,app) (10.9 +/- 1.2 microM) and lowest intrinsic clearance (0.47 ml/min/mg). There was a 4.25-fold difference in AO activity between the 5 human hepatocyte preparations. Drug interaction studies with the seven marketed drugs revealed marked species-specific inhibition. Our data indicates major differences in the rate of AO metabolism, and inhibition of AO across species, indicating that results from animal studies cannot be safely extrapolated to humans. Cryopreserved hepatocytes and cytosolic fractions from animals and humans provide qualitatively similar data within the species.