Targeting drugs to specific organs, tissues, or cells is an attractive strategy for enhancing drug efficacy and reducing side effects. Drug carriers such as antibodies, natural and manmade polymers, and labeled liposomes are capable of targeting drugs to blood vessels of individual tissues but often fail to deliver drugs to extravascular sites. An alternative strategy is to use low molecular weight prodrugs that distribute throughout the body but cleave intracellularly to the active drug by an organ-specific enzyme. Here we show that a series of phosphate and phosphonate prodrugs, called HepDirect prodrugs, results in liver-targeted drug delivery following a cytochrome P450-catalyzed oxidative cleavage reaction inside hepatocytes. Liver targeting was demonstrated in rodents for, a HepDirect prodrug of the nucleotide analog adefovir (PMEA), and, a HepDirect prodrug of cytarabine (araC) 5Ј-monophosphate. Liver targeting led to higher levels of the biologically active form of PMEA and araC in the liver and to lower levels in the most toxicologically sensitive organs. Liver targeting also confined production of the prodrug byproduct, an aryl vinyl ketone, to hepatocytes. Glutathione within the hepatocytes rapidly reacted with the byproduct to form a glutathione conjugate. No byproduct-related toxicity was observed in hepatocytes or animals treated with HepDirect prodrugs. A 5-day safety study in mice demonstrated the toxicological benefits of liver targeting. These findings suggest that HepDirect prodrugs represent a potential strategy for targeting drugs to the liver and achieving more effective therapies against chronic liver diseases such as hepatitis B, hepatitis C, and hepatocellular carcinoma.Site-specific drug delivery is a concept that has the potential to increase local drug concentrations and thereby produce more effective medicines with fewer side effects (Tomlinson, 1987). Despite the obvious attractiveness of drug targeting and the substantial efforts made over the past 30 years, few drugs have reached the market that depend on a targeting mechanism. The most advanced strategies use sitespecific drug carriers such as antibodies (Payne, 2003), peptides (Arap et al., 1998), natural and man-made polymers (Meijer et al., 1990), and carbohydrate-or peptide-labeled nanoparticles (Akerman et al., 2002) and liposomes (Wu et al., 2002) capable of recognizing cell-and tissue-specific proteins expressed on the surface of the targeted cells. In many cases, drugs conjugated to the carrier molecules gain high tissue selectivity through the ability of the carrier molecule to recognize blood vessels of individual tissues via tissuespecific vascular markers expressed on the endothelium.Although impressive vascular specificity is achieved (Ruoslahti, 2002), drug exposure to extravascular sites is often severely compromised by limitations in drug-conjugate exchange across the endothelial barrier and the slow rate of drug-conjugate cleavage relative to the rate of drug removal from the vascular delivery site (Stell...

Spinal muscular atrophy (SMA) is a neurodegenerative disease characterized by progressive motor neuron loss and caused by mutations in SMN1 (Survival Motor Neuron 1). The disease severity inversely correlates with the copy number of SMN2, a duplicated gene that is nearly identical to SMN1. We have delineated a mechanism of transcriptional regulation in the SMN2 locus. A previously uncharacterized long noncoding RNA (lncRNA), SMN-antisense 1 (SMN-AS1), represses SMN2 expression by recruiting the Polycomb Repressive Complex 2 (PRC2) to its locus. Chemically modified oligonucleotides that disrupt the interaction between SMN-AS1 and PRC2 inhibit the recruitment of PRC2 and increase SMN2 expression in primary neuronal cultures. Our approach comprises a gene-up-regulation technology that leverages interactions between lncRNA and PRC2. Our data provide proof-of-concept that this technology can be used to treat disease caused by epigenetic silencing of specific loci.spinal muscular atrophy | lncRNA | PRC2 | SMN S pinal muscular atrophy is the leading genetic cause of infant mortality and is caused by deletions or mutation of Survival Motor Neuron 1 (SMN1) (1). Unique to humans, SMN1 is duplicated in the genome as SMN2, which is nearly identical in sequence. However, a C-to-T point mutation in exon 7 of SMN2 results in preferential skipping of this exon during pre-mRNA splicing and production of a truncated and unstable protein. A small fraction (10-20%) of pre-mRNA transcribed from SMN2 is spliced correctly to include exon 7 and produces a full-length SMN (SMN-FL, inclusive of exon 7) that is identical to the SMN1 gene product (2-4).Spinal motor neurons are highly sensitive to SMN1 deficiency, and their premature death causes motor function deficit in SMA patients (5, 6). The SMN2-derived SMN protein can extend spinal motor neuron survival, yet insufficient levels of SMN eventually lead to cell death. Overall, SMA patients with higher SMN2 genomic copy number have a less severe disease phenotype (7, 8). Type 0 or I patients, carrying one or two copies of SMN2, show onset of SMA within a few months of life with a life expectancy of less than 2. In contrast, type III and IV patients, carrying three or more copies, respectively, show juvenile or adult onset and slower disease progression (9). As further genetic evidence, SMA mouse models have been produced in which smn1 −/− mice, which would otherwise be embryonic lethal (10), can be rescued in the presence of high copy numbers of the human SMN transgene (11-13). Similar to the human disease spectrum, increased copy number of a human SMN transgene is inversely associated with decreased disease severity and mortality. We reasoned that increasing SMN2 transcription could phenocopy the beneficiary effect of SMN2 gene amplification and compensate for SMN1 deficiency. In addition, SMN1 heterozygotes are asymptomatic, whereas affected homozygotes have 10-20% of normal SMN levels. Therefore, we predict that modest SMN2 up-regulation will provide significant therapeutic benefit. H...

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Inhibition of platelet aggregation by acadesine was evaluated both in vitro and ex vivo in human whole blood using impedance aggregometry, as well as in vivo in a canine model of platelet-dependent cyclic coronary flow reductions. In vitro, incubation of acadesine in whole blood inhibited ADP-induced platelet aggregation by 50% at 240±60 ,IM.Inhibition of platelet aggregation was time dependent and was prevented by the adenosine kinase inhibitor, 5 '-deoxy 5-iodotubercidin, which blocked conversion of acadesine to its 5 '-monophosphate, ZMP, and by adenosine deaminase. Acadesine elevated platelet cAMP in whole blood, which was also prevented by adenosine deaminase. In contrast, acadesine had no effect on ADP-induced platelet aggregation or platelet cAMP levels in platelet-rich plasma, but inhibition of aggregation was restored when isolated erythrocytes were incubated with acadesine before reconstitution with platelet-rich plasma. Acadesine (100 mg/kg i.v.) administered to human subjects also inhibited platelet aggregation ex vivo in whole blood. In the canine Folts model of platelet thrombosis, acadesine (0.5 mg/kg per mm i.v.) abolished coronary flow reductions, and this activity was prevented by pretreatment with the adenosine receptor antagonist, 8-sulphophenyltheophylline. These results demonstrate that acadesine exhibits antiplatelet activity in vitro, ex vivo, and in vivo through an adenosine-dependent mechanism. Moreover, the in vitro studies indicate that inhibition of platelet aggregation requires the presence of erythrocytes and metabolism of acadesine to acadesine monophosphate (ZMP). (J. Clin. Invest. 1994. 94:1524-1532