Inhibition of Orbivirus Replication by Aurintricarboxylic Acid.

Bluetongue virus (BTV) and African horse sickness virus (AHSV) are vector-borne viruses belonging to the Orbivirus genus, which are transmitted between hosts primarily by biting midges of the genus Culicoides. With recent BTV and AHSV outbreaks causing epidemics and important economy losses, there is a pressing need for efficacious drugs to treat and control the spread of these infections. The polyanionic aromatic compound aurintricarboxylic acid (ATA) has been shown to have a broad-spectrum antiviral activity. Here, we evaluated ATA as a potential antiviral compound against Orbivirus infections in both mammalian and insect cells. Notably, ATA was able to prevent the replication of BTV and AHSV in both cell types in a time- and concentration-dependent manner. In addition, we evaluated the effect of ATA in vivo using a mouse model of infection. ATA did not protect mice against a lethal challenge with BTV or AHSV, most probably due to the in vivo effect of ATA on immune system regulation. Overall, these results demonstrate that ATA has inhibitory activity against Orbivirus replication in vitro, but further in vivo analysis will be required before considering it as a potential therapy for future clinical evaluation.

Enhanced transport of a novel anti-HIV agent--cosalane and its congeners across human intestinal epithelial (Caco-2) cell monolayers.

PURPOSE: Cosalane is a potent inhibitor of HIV replication with activity against a broad range of viral targets. However, oral bioavailability of this highly lipophilic compound is extremely poor (<1%). The purpose of this study is to screen a variety of permeation enhancers (cyclodextrin derivatives, cremophor EL, bile salts and mixed micelles) for their ability to enhance the transport of cosalane and its analogs/prodrugs across Caco-2 cell monolayers. METHODS: Cosalane and its different analogs/prodrugs were synthesized and their physicochemical properties were determined. Caco-2 cells were cultured at a density of 66,000 cells/cm(2) either on collagen coated clear polyester membranes or Transwell inserts. Side-bi-side diffusion cells and Transwell inserts were employed to study for the transport of cosalane and its analogs/prodrugs with various permeation enhancers across Caco-2 cell monolayers. RESULTS: Permeabilities of EH-3-39, EH-3-55 and EH-3-57 significantly improved compared to that of cosalane in the presence of bile salt, sodium desoxycholate. Among the various cyclodextrins studied, hydroxypropyl beta cyclodextrin (HP-beta-CD) and dimethyl beta cyclodextrin (DM-beta-CD) exhibited 22.3-fold and 19-fold permeability enhancement of cosalane respectively across Caco-2 cell monolayers. Sodium desoxycholate (10 mM) also showed a remarkable (105-fold) enhancement on the permeability of cosalane (P(app) 11.72+/-3.31 x 10(-6) cm/s) without causing any measurable cellular damage. Cremophor EL resulted in higher transport of 14C mannitol. The mechanism of enhancement effect can be mainly attributed to the alteration of membrane fluidity by cyclodextrin and opening of tight junctions by cremophor EL. CONCLUSIONS: Among the enhancers tested, 10 mM sodium desoxycholate and HP-beta-CD appear to be viable candidates for further development of an oral formulation of cosalane and its congeners.

Intestinal absorption and biodistribution of cosalane and its amino acid conjugates: novel anti-HIV agents.

Cosalane and its amino acid conjugates are potent inhibitors of HIV replication. The purpose of this study was to investigate: (1) the pharmacokinetic disposition of the diglycine (GC) and the diaspartic acid (ASPC) conjugates of cosalane in male Sprague-Dawley rats; (2) intestinal absorption of cosalane and its amino acid conjugates using in vitro (small intestinal segments), in situ (closed loop); and (3) biodistribution of GC and its absolute oral bioavailability in rat. Cosalane and its conjugates exhibited biexponential disposition with very long half-lives upon intravenous dosing. However, these compounds failed to permeate the small intestine unless sodium desoxycholate (5-20 mM) was used as an intestinal permeation enhancer. A rank order correlation in terms of permeation enhancement in a descending order is as follows: GC>Cosalane>ASPC. In situ studies revealed that although the bile salt enhanced the permeation of cosalane across the enterocyte, its hepatic uptake was extensive. However, 66% of the absorbed dose of GC escaped uptake by the reticuloendothelial system (RES) and its biodistribution studies showed that the uptake by the RES was significantly lower compared to the parent compound. GC had an absolute oral bioavailability of 5.10+/-1.51%. Therefore, GC appears to be a favorable candidate for further development.

Transport of cosalane-a highly lipophilic novel anti-HIV agent-across caco-2 cell monolayers.

Cosalane is a potent inhibitor of HIV replication with activity against a broad range of viral targets. However, the oral bioavailability of this highly lipophilic compound is extremely poor (<1%). Also, cosalane accumulates in high concentration in the liver after intravenous administration, with clear resistance to hepatic metabolism. In the present study, the transcellular permeability of cosalane was examined using Transwell(R) filter as well as plastic-grown confluent Caco-2 cell monolayers. A cell-culture-based biophysical model was adopted to understand the interactions of protein binding, membrane partitioning, and aqueous solubility of cosalane in limiting transcellular flux of cosalane across Caco-2 cell monolayers. The transcellular permeability (P(app)) of cosalane was extremely low (4.494 x 10(-8) cm/s) and the effect of p-glycoprotein on the efflux of cosalane was negligible. A characteristic disparity exists between the kinetics of cosalane uptake from apical (AP) donor solution and efflux into basolateral (BL) receiver side. The AP uptake of cosalane was rapid, exhibiting exponential kinetics, and reached equilibrium within 60 min, whereas the concomitant appearance of the compound into the BL receiver side was slow but linear over time. Furthermore, the uptake of cosalane was significantly reduced in the presence of bovine serum albumin (BSA). In unidirectional efflux studies, AP efflux of cosalane was limited in the absence of BSA. Also, no detectable metabolites were found in Caco-2 cell incubations. In conclusion, the present study demonstrates that diffusion of cosalane across Caco-2 cell monolayers is extremely limited and kinetically regulated essentially by the equilibrium between protein-bound and free drug partitioning into cell membrane.

Pharmacokinetics, biliary excretion, and tissue distribution of novel anti-HIV agents, cosalane and dihydrocosalane, in Sprague-Dawley rats.

Cosalane and dihydrocosalane are potent inhibitors of HIV replication with a broad range of activity. The purpose of this study was to investigate: 1) the pharmacokinetic disposition of both cosalane and dihydrocosalane in male Sprague-Dawley rats, and 2) biliary excretion, enterohepatic circulation, and tissue distribution of cosalane after i.v. and/or oral administration. Animals were administered i.v. (10 mg/kg) cosalane or dihydrocosalane through a jugular vein to obtain plasma profiles. Dose dependence of cosalane was studied over a dose range of 1.0 to 10 mg/kg. The extent of enterohepatic recycling, biliary excretion, and tissue distribution were studied after i.v. administration. Both cosalane and dihydrocosalane exhibited a biexponential disposition with very long half-lives of 749 +/- 216 and 1016 +/- 407 min, along with very large volumes of distribution 23.1 +/- 4.4 and 24.4 +/- 2. 5 liter/kg, respectively. Both cosalane (nondetectable) and dihydrocosalane (<1%) showed very poor oral bioavailability. The biliary and renal excretions of cosalane were found to be negligible with no detectable metabolites either in urine or bile. After oral administration, more than 87% of the cosalane dose was excreted in the feces as the parent compound. Also, cosalane was sequestered significantly in liver with quantifiable levels in all tissues tested, even 48 h after the dose was administered. Therefore it was concluded that the poor oral bioavailability of cosalane may be due to its poor enterocytic transport coupled with sequestration in liver parenchymal cell membrane layers.

Disposition of cosalane, a novel anti-HIV agent, in isolated perfused rat livers.

Cosalane is a potent inhibitor of HIV replication with a broad range of activity. In this study, the hepatic disposition of cosalane was investigated with a noncirculating isolated perfused rat liver technique. When 6 microM cosalane was infused into livers from untreated rats, the drug was highly extracted by the liver (only 2. 5% of influent cosalane concentration appeared in the effluent perfusate). Pretreatment of rats with various inducers of cytochrome P-450 before perfusion neither altered the effluent cosalane concentration nor resulted in the appearance of detectable metabolites in the effluent perfusate or liver homogenates. Hepatic uptake of cosalane was negligible when the drug was infused in the presence of BSA, and infusion of albumin after cosalane resulted in a significant displacement of the drug into the effluent perfusate. Furthermore, permeabilization of perfused livers with digitonin significantly diminished effluent cosalane concentration while enhancing cosalane uptake by the liver. Based on our data, it appears that a significant proportion of cosalane does not penetrate the hepatocyte membrane and may accumulate in the lipid bilayer of the cell membrane. This finding supports the proposed mechanism explaining the antiviral effect of cosalane which stipulates that this compound appears to imbed perpendicularly in the lipid bilayer of the cell membrane and the viral envelope. Also, cosalane does not seem to be metabolized by the liver as evidenced by the lack of detectable metabolites in the effluent perfusate, liver homogenates, and liver microsomal incubations.