Paul A. Cossum

Intramolecular G-quartet Motifs Confer Nuclease Resistance to a Potent Anti-HIV Oligonucleotide

We have identified a potentially therapeutic anti-human immunodeficiency virus (HIV)-1 oligonucleotide composed entirely of deoxyguanosines and thymidines (T30177, also known as AR177: 5′-g*tggtgggtgggtggg*t-3′, where asterisk indicates phosphorothioate linkage). In acute assay systems using human T-cells, T30177 and its total phosphodiester homologue T30175 inhibited HIV-1-induced syncytium production by 50% at 0.15 and 0.3 μM, respectively. Under physiological conditions, the sequence and composition of the 17-mer favors the formation of a compact, intramolecularly folded structure dominated by two stacked guanine quartet motifs that are connected by three loops of TGs. The molecule is stabilized by the coordination of a potassium ion between the two stacked quartets. We now show that these guanine quartet-containing oligonucleotides are highly resistant to serum nucleases, with tof 5 h and >4 days for T30175 and T30177, respectively. Both oligonucleotides were internalized efficiently by cells, with intracellular concentrations reaching 5-10-fold above the extracellular levels after 24 h of incubation. In contrast, single-base mutated variants or random sequence control oligonucleotides that could not form the compactly folded structure had markedly reduced half-lives (tfrom 3 to 7 min), low cellular uptake, and no sequence-specific anti-HIV-1 activity. These data suggest that the tertiary structure of an oligonucleotide is a key determinant of its nuclease resistance, cellular uptake kinetics, and biological efficacy.

Differential Antifungal Activity of Isomeric Forms of Nystatin

ABSTRACT When nystatin is placed in RPMI and other biological fluids, there is loss of pure nystatin, with the development of two distinguishable chromatographic peaks, 1 and 2. Peak 1 appears identical to commercially prepared nystatin. By nuclear magnetic resonance (NMR) and mass spectral analysis, peak 2 appears to be an isomer of peak 1. The isomers are quantitatively and fully interconvertible. Formation of peak 2 is accelerated at a pH of >7.0 and ultimately reaches a near 55:45 (peak 1/peak 2 ratio) mixture. We sought to determine the relative activities of peaks 1 and 2 against Candidaspp. Peak 2 consistently showed higher MICs when it was the predominant form during the experiment. Time-kill analyses showed that peak 2 required ≥8× the concentration of peak 1 to produce a modest and delayed killing effect, which was never of the same magnitude as that produced by peak 1. In both types of assays, the activity of peak 2 corresponded with intra-assay formation of peak 1. Both MIC measurements and time-kill analysis suggest that peak 2 has considerably less activity, if any at all, againstCandida spp. Peak 2 may serve as a reservoir for peak 1.

The Nonclinical Safety Evaluation of the Anticancer Drug Atragen® (Liposomal All-Trans-Retinoic Acid)

ATRAGEN® is a liposome-encapsulated intravenous (IV) formulation of the anticancer drug all-trans-retinoic acid (tretinoin). Retinoids as a class of compounds produce a characteristic profile of toxicities collectively known as hypervitaminosis A. As part of the nonclinical regulatory submission, it was important to determine if liposome encapsulation of tretinoin would change the expected profile of toxicities. To this end, a single-dose study in rats and repeated-dose 28-day studies in rats and dogs were conducted. In the single-dose study, ATRAGEN was given as a single IV bolus via the tail vein at dosages of 5, 20, or 80 mg/kg. In the first repeated-dose studies in rats, ATRAGEN was given by tail vein infusion at dosages of 2.5, 15, or 25 mg/kg/day and in the second, at dosages of 1, 10 or 10 mg/kg/day. The second study in rats also included a group given free tretinoin at a dose of 10 mg/kg/day; lowered to 1 mg/kg/day. ATRAGEN was given to dogs as an IV infusion in the cephalic or saphenous vein at dosages of 2.5, 5, or 10 mg/kg/day. ATRAGEN was not acutely toxic in rats at doses of 5 or 20 mg/kg, whereas deaths were seen at 80 mg/kg. In contrast, free tretinoin at a dosage of 10 mg/kg caused the deaths of most male rats after the first dose in the repeated-dose study; consequently, the dose was lowered to 1 mg/kg/day for remaining males and all females in that study. In the 28-day repeated-dose studies, minimal toxicities were observed in either rats or dogs at ATRAGEN doses of 2.5 mg/kg/day or less. Both free tretinoin and ATRAGEN at 1 mg/kg/day were without signs of hypervitaminosis A in rats. Moderate to marked retinoid-associated hypervitaminosis A was observed in the 28-day rats studies in the dose range of 10 to 25 mg/kg/day. In dogs, repeated administration of ATRAGEN of 5 or 10 mg/kg/day also led to moderate to marked hypervitaminosis A. In both species, hypervitaminosis A was manifested primarily as bone and testicular toxicities. In bone, premature closure of epiphyseal growth plates and/or a decrease in the activity of cells in the growth plate were seen. Loss of the cartilaginous growth plate and replacement with less dense trabecular bone resulted in weakened bones, most evident in rats. Rats had increased levels of serum alkaline phosphatase (ALP) and bone fractures were common with ATRAGEN doses of 10 mg/kg/day and higher. In addition to effects on bone growth, endosteal fibroplasia, exostosis, and periosteal hemorrhages were observed in dogs. In both species, diffuse testicular atrophy, degenerative spermatic elements, and loss of seminiferous epithelium in the epididymis were observed microscopically. Hepatic enzyme levels were increased in rats, but no histopathological correlate was identified. Moderate to moderately severe nephrosis exemplified by a loss and/or degeneration of the kidney tubules was seen in dogs given 5 or 10 mg/kg/day. There was an increased weight of the spleens in rats receiving high dose volumes of liposomes; that is, control rats receiving empty liposomes and in rats receiving ATRAGEN in large dose volumes to provide tretinoin at dosages of 10 mg/kg/day and greater. Microscopically, there was an accumulation of macrophages with prominent vacuoles in the spleens of these rats. This effect on the spleen was not considered a pathological process but a clearance of liposomal material from the circulation by phagocytosis. No other toxicities were observed. Thus, these nonclinical safety studies of ATRAGEN conducted in rats or dogs found no unique toxicities from those observed previously in laboratory animals given tretinoin or other retinoids.