Tadalafil administered once daily for lower urinary tract symptoms secondary to benign prostatic hyperplasia: a 1-year, open-label extension study.

OBJECTIVE: • To evaluate the 1-year safety of 5 mg of tadalafil once daily in men with lower urinary tract symptoms secondary to benign prostatic hyperplasia (BPH-LUTS); efficacy measures were included to evaluate the maintenance of efficacy after an additional year of treatment. PATIENTS AND METHODS: • In total, 427 men who completed a 12-week, placebo-controlled, dose- finding study assessing once-daily tadalafil (2.5, 5, 10 or 20 mg) or placebo elected to continue into the open-label extension period. Safety and efficacy parameters were assessed after 1 month and every 3 months. RESULTS: • In total, 299 patients (69.9%) completed the 1-year, open-label extension period. Treatment-emergent adverse events (TEAEs) were reported by 57.6% of patients, with most TEAEs being mild (44%) or moderate (45%) in severity; the most common TEAEs (≥ 2%) were dyspepsia, gastro-oesophageal reflux disease, back pain, headache, sinusitis, hypertension and cough. Twenty-two patients (5.2%) discontinued as a result of AEs. During the open-label extension period, mean prostate-specific antigen increased from 1.6 ± 1.3 ng/mL to 1.8 ± 1.4 ng/mL. • Mean post-void residual volume was 61.1 ± 60.4 mL at study entry and 42.2 ± 64.1 mL after the open-label extension period. Changes in the total International Prostate Symptom Score (IPSS), IPSS irritative and obstructive subscores, IPSS health-related quality of life and BPH Impact Index were maintained after 1 year. In sexually-active patients with erectile dysfunction, improvements in the International Index of Erectile Function-Erectile Function domain were maintained after 1 year. CONCLUSION: • In men with BPH-LUTS, 5 mg of tadalafil once daily during 1 year of treatment was well tolerated and efficacy changes were maintained.

Active site binding and sequence requirements for inhibition of HIV-1 reverse transcriptase by the RT1 family of single-stranded DNA aptamers.

Nucleic acid aptamers can potentially be developed as broad-spectrum antiviral agents. Single-stranded DNA (ssDNA) aptamer RT1t49 inhibits reverse transcriptases (RT) from HIV-1 and diverse lentiviral subtypes with low nanomolar values of Kd and IC50. To dissect the structural requirements for inhibition, RT-catalyzed DNA polymerization was measured in the presence of RT1t49 variants. Three structural domains were found to be essential for RT inhibition by RT1t49: a 5' stem (stem I), a connector and a 3' stem (stem II) capable of forming multiple secondary structures. Stem I tolerates considerable sequence plasticity, suggesting that it is recognized by RT more by structure than by sequence-specific contacts. Truncating five nucleotides from the 3' end prevents formation of the most stable stem II structure, yet has little effect on IC50 across diverse HIV-1, HIV-2 and SIV(CPZ) RT. When bound to wild-type RT or an RNase H active site mutant, site-specifically generated hydroxyl radicals cleave after nucleotide A32. Cleavage is eliminated by either of two polymerase (pol)-active site mutants, strongly suggesting that A32 lies within the RT pol-active site. These data suggest a model of ssDNA aptamer-RT interactions and provide an improved molecular understanding of a potent, broad-spectrum ssDNA aptamer.

Single-stranded DNA aptamer RT1t49 inhibits RT polymerase and RNase H functions of HIV type 1, HIV type 2, and SIVCPZ RTs.

Natural and selected resistance of HIV-1 to current anti-HIV drugs continues to pose serious problems to the development of HIV-1 antivirals. The viral reverse transcriptase (RT) is a proven therapeutic target. Single-stranded RNA and DNA (ssRNA and ssDNA) aptamers have been selected that specifically and potently inhibit RT function. In particular, the ssDNA aptamer RT1t49 was previously selected to recognize the RT from a subtype B strain of HIV-1 and binds with a reported K(d) of 4 nM. In the present work, we show that RT1t49 inhibits recombinant RT cloned from diverse branches of the primate lentiviral family. Aptamer concentrations required for half-maximal inhibition of all HIV-1, HIV-2, and SIV(CPZ) RTs assayed were in the low-to mid-nanomolar range for both polymerase and RNase H activities. Using pre-steady-state and order-of-addition kinetic analyses, we also established that this ssDNA aptamer competes with primer-template for access to RT, and that addition of a nucleoside analog RT inhibitor (NRTI) to the in vitro reaction enhanced the overall effectiveness of both drugs, while nonnucleoside analog RT inhibitors (NNRTIs) exhibited simple additivity. This is the first demonstration of universal inhibition of HIV and SIV(cpz) RTs by a nucleic acid aptamer and supports previous reports suggesting that resistance to RT1t49 may be exceptionally infrequent.

Cross-clade inhibition of recombinant human immunodeficiency virus type 1 (HIV-1), HIV-2, and simian immunodeficiency virus SIVcpz reverse transcriptases by RNA pseudoknot aptamers.

Reverse transcriptase (RT) remains a primary target in therapies directed at human immunodeficiency virus type 1 (HIV-1). RNA aptamers that bind RT from HIV-1 subtype B have been shown to protect human cells from infection and to reduce viral infectivity, but little is known about the sensitivity of the inhibition to amino sequence variations of the RT target. Therefore, we assembled a panel of 10 recombinant RTs from phylogenetically diverse lentiviral isolates (including strains of HIV-1, simian immunodeficiency virus SIVcpz, and HIV-2). After validating the panel by measuring enzymatic activities and inhibition by small-molecule drugs, dose-response curves for each enzyme were established for four pseudoknot RNA aptamers representing two structural subfamilies. All four aptamers potently inhibited RTs from multiple HIV-1 subtypes. For aptamers carrying family 1 pseudoknots, natural resistance was essentially all-or-none and correlated with the identity of the amino acid at position 277. In contrast, natural resistance to aptamers carrying the family 2 pseudoknots was much more heterogeneous, both in degree (gradation of 50% inhibitory concentrations) and in distribution across clades. Site-directed and subunit-specific mutagenesis identified a common R/K polymorphism within the p66 subunit as a primary determinant of resistance to family 1, but not family 2, pseudoknot aptamers. RNA structural diversity therefore translates into a nonoverlapping spectrum of mutations that confer resistance, likely due to differences in atomic-level contacts with RT.

Differential susceptibility of HIV-1 reverse transcriptase to inhibition by RNA aptamers in enzymatic reactions monitoring specific steps during genome replication.

Nucleic acid aptamers to HIV-1 reverse transcriptase (RT) are potent inhibitors of DNA polymerase function in vitro, and they have been shown to inhibit viral replication when expressed in cultured T-lymphoid lines. We monitored RT inhibition by five RNA pseudoknot RNA aptamers in a series of biochemical assays designed to mimic discrete steps of viral reverse transcription. Our results demonstrate potent aptamer inhibition (IC50 values in the low nanomolar range) of all RT functions assayed, including RNA- and DNA-primed DNA polymerization, strand displacement synthesis, and polymerase-independent RNase H activity. Additionally, we observe differences in the time dependence of aptamer inhibition. Polymerase-independent RNase H activity is the most resistant to long term aptamer suppression, and RNA-dependent DNA polymerization is the most susceptible. Finally, when DNA polymerization was monitored in the presence of an RNA aptamer in combination with each of four different small molecule inhibitors, significant synergy was observed between the aptamer and the two nucleoside analog RT inhibitors (azidothymidine triphosphate or ddCTP), whereas two non-nucleoside analog RT inhibitors showed either weak synergy (efavirenz) or antagonism (nevirapine). Together, these results support a model wherein aptamers suppress viral replication by cumulative inhibition of RT at every stage of genome replication.

HIV-1 inactivation by nucleic acid aptamers.

Although developments in small-molecule therapeutics for HIV-1 have been dramatic in recent years, the rapid selection of drug-resistant viral strains and the adverse side effects associated with long-term exposure to current treatments propel continued exploration of alternative anti-HIV-1 agents. Non-coding nucleic acids have emerged as potent inhibitors that dramatically suppress viral function both in vitro and in cell culture. In particular, RNA and DNA aptamers inhibit HIV-1 function by directly interfering with essential proteins at critical stages in the viral replication cycle (Figure 1). Their antiviral efficacy is expected to be a function, in part, of the biochemical properties of the aptamer-target interaction. Accordingly, we present an overview of biochemical and cell culture analyses of the expanding list of aptamers targeting HIV-1. Our discussion focuses on the inhibition of viral enzymes (reverse transcription, proteolytic processing, and chromosomal integration), viral expression (Rev/RRE and Tat/TAR), viral packaging (p55Gag, matrix and nucleocapsid), and viral entry (gp120) (Table 1). Additional nucleic acid-based strategies for inactivation of HIV-1 function (including RNAi, antisense, and ribozymes) have also demonstrated their utility. These approaches are reviewed in other chapters of this volume and elsewhere.