Frank J. Dutko

Self-emulsifying drug delivery systems: formulation and biopharmaceutic evaluation of an investigational lipophilic compound.

Self-emulsifying drug delivery systems (SEDDSs) represent a possible alternative to traditional oral formulations of lipophilic compounds. In the present study, a lipophilic compound, WIN 54954, was formulated in a medium chain triglyceride oil/nonionic surfactant mixture which exhibited self-emulsification under conditions of gentle agitation in an aqueous medium. The efficiency of emulsifi-cation was studied using a laser diffraction sizer to determine particle size distributions of the resultant emulsions. An optimized formulation which consisted of 25% (w/w) surfactant, 40% (w/w) oil, and 35% (w/w) WIN 54954 emulsified rapidly with gentle agitation in 0.1 N HCl (37°C), producing dispersions with mean droplet diameters of less than 3 µm. The self-emulsifying preparation was compared to a polyethylene glycol 600 (PEG 600) solution formulation by administering each as prefilled soft gelatin capsules to fasted beagle dogs in a parallel crossover study. Pharmacokinetic parameters were determined and the absolute bioavailability of the drug was calculated by comparison to an i.v. injection. The SEDDS improved the reproducibility of the plasma profile in terms of the maximum plasma concentration (Cmax) and the time to reach the maximum concentration (tmax). There was no significant difference in the absolute bioavailability of WIN 54954 from either the SEDDS or the PEG formulations.

The structure of coxsackievirus B3 at 3.5 A resolution.

BACKGROUND Group B coxsackieviruses (CVBs) are etiologic agents of a number of human diseases that range in severity from asymptomatic to lethal infections. They are small, single-stranded RNA icosahedral viruses that belong to the enterovirus genus of the picornavirus family. Structural studies were initiated in light of the information available on the cellular receptors for this virus and to assist in the design of antiviral capsid-binding compounds for the CVBs. RESULTS The structure of coxsackievirus B3 (CVB3) has been solved to a resolution of 3.5 A. The beta-sandwich structure of the viral capsid proteins VP1, VP2 and VP3 is conserved between CVB3 and other picornaviruses. Structural differences between CVB3 and other enteroviruses and rhinoviruses are located primarily on the viral surface. The hydrophobic pocket of the VP1 beta-sandwich is occupied by a pocket factor, modeled as a C16 fatty acid. An additional study has shown that the pocket factor can be displaced by an antiviral compound. Myristate was observed covalently linked to the N terminus of VP4. Density consistent with the presence of ions was observed on the icosahedral threefold and fivefold axes. CONCLUSIONS The canyon and twofold depression, major surface depressions, are predicted to be the primary and secondary receptor-binding sites on CVB3, respectively. Neutralizing immunogenic sites are predicted to lie on the extreme surfaces of the capsid at sites that lack amino acid sequence conservation among the CVBs. The ions located on the icosahedral threefold and fivefold axes together with the pocket factor may contribute to the pH stability of the coxsackieviruses.

In vitro and in vivo activities of WIN 54954, a new broad-spectrum antipicornavirus drug.

WIN 54954 (5-[5-[2,6-dichloro-4-(4,5-dihydro-2-oxazolyl)phenoxy]pentyl]-3- methylisoxazole) is a new member of the class of broad-spectrum antipicornavirus compounds known to bind in a hydrophobic pocket within virion capsid protein VP1. In plaque reduction assays, WIN 54954 reduced plaque formation of 50 of 52 rhinovirus serotypes (MICs ranged from 0.007 to 2.2 micrograms/ml). A concentration of 0.28 microgram/ml was effective in inhibiting 80% of the 52 serotypes tested (EC80). WIN 54954 was also effective in inhibiting 15 commonly isolated enteroviruses, with an EC80 of 0.06 microgram/ml. Furthermore, WIN 54954 was effective in reducing the yield of two selected enteroviruses in cell culture by 90% at concentrations approximately equal to their MICs. The therapeutic efficacy of intragastrically administered WIN 54954 was assessed in suckling mice infected with coxsackievirus A-9 or echovirus type 9 (Barty) 2.5 days prior to initiation of therapy. Single daily doses of 2 and 100 mg/kg protected 50% of the mice from developing paralysis (PD50) following infection with coxsackievirus A-9 and echovirus-9, respectively. At the PD50 doses for these two viruses, levels of WIN 54954 in serum were maintained above the in vitro MICs for a significant portion of the dosing interval. The dose-dependent reduction in viral titers observed in coxsackievirus A-9-infected mice correlated well with the therapeutic dose response. The potency and spectrum of WIN 54954 make it a potentially useful compound for the treatment of human enterovirus and rhinovirus infections.

Three-dimensional structures of drug-resistant mutants of human rhinovirus 14.

Mutants of human rhinovirus 14 were isolated and characterized by searching for resistance to compounds that inhibit viral uncoating. The portions of the RNA that code for amino acids that surround the antiviral compound binding site were sequenced. X-ray analysis of two of these mutants, 1188 Val----Leu and 1199 Cys----Tyr, shows that these were single-site substitutions which would sterically hinder drug binding. Differences in the resistance of mutant viruses to various antiviral compounds may be rationalized in terms of the three-dimensional structures of these mutants. Predictions of the structures of mutant rhinovirus 14 with the substitutions 1188 Val----Leu, 1199 Cys----Tyr and 1199 Cys----Trp in VP1 were made using a molecular dynamics technique. The predicted structure of the 1199 Cys----Tyr mutant was consistent with the electron density map, while the 1188 Val----Leu prediction was not. Large (up to 1.4 A) conformational differences between native rhinovirus 14 and the 1199 Cys----Tyr mutant occurred in main-chain atoms near the mutation site. These changes, as well as the orientation of the 1199 tyrosine side-chain, were correctly predicted by the molecular dynamics calculation. The structure of the predicted 1199 Cys----Trp mutation is consistent with the drug-resistant properties of this virus.

Binding affinities of structurally related human rhinovirus capsid-binding compounds are related to their activities against human rhinovirus type 14.

The binding affinities (Kds) and the rates of association and dissociation of members of a chemical class of antiviral compounds at their active sites in human rhinovirus type 14 (HRV-14) were determined. On the basis of analysis by LIGAND, a nonlinear curve-fitting program, of saturation binding experiments with HRV-14, the Kds for Win 52084, Win 56590, disoxaril (Win 51711), and Win 54954 were found to be 0.02, 0.02, 0.08, and 0.22 microM, respectively. The independently determined kinetic rates of association and dissociation resulted in calculated Kd values which were in agreement with the Kd values determined in saturation binding experiments. Scatchard plots of each of four compounds for the binding data indicated that approximately 40 to 60 molecules were bound per HRV-14 virion. Hill plots showed no evidence of cooperativity in binding. Furthermore, the antiviral activities (MICs in plaque reduction assays with HRV-14) for this limited series of compounds (n = 4) correlated well (r = 0.997) with the observed Kds. Likewise, the absence of detectable binding of Win 54954 to the drug-resistant mutant HRV-14 (Leu-1188) corresponded to a lack of antiviral activity. The positive relationship between the antiviral activities and the Kds that were determined may have implications for the molecular design of capsid-binding antirhinovirus drugs.

Structures of four methyltetrazole-containing antiviral compounds in human rhinovirus serotype 14.

Four novel antiviral WIN compounds, that contain a methyl tetrazole ring as well as isoxazole, pyridazine or acetylfuran rings, have had their structures determined in human rhinovirus serotype 14 at 2.9 A resolution. These compounds bind in the VP1 hydrophobic pocket, but are shifted significantly towards the pocket pore when compared to previously examined WIN compounds. A putative water network at the pocket pore is positioned to hydrogen bond with these four WIN compounds, and this network can account for potency differences seen in structurally similar WIN compounds.

3-Pyridines as replacements for an isoxazole ring: The antirhinoviral activity of pyridine analogues related to disoxaril

Abstract In a series of rhinovirus inhibitors related to Disoxaril, replacement of 3-methylisoxazole with all 3 isomers of pyridine showed the 3-pyridyl isomer most similar to 3-methylisoxazole in spectrum of activity over 15 human rhinovirus serotypes.

Crystallographic and Pharmacological Studies of Antiviral Agents Against Human Rhinovirus

Picornaviruses are small RNA containing viruses that cause diseases in mammals such as polio, foot-and-mouth disease, and the common cold. For reasons not yet fully understood, some members of this family have a small number of serotypes while others have many (e.g. poliovirus has three serotypes while rhinovirus has 100 serotypes). One serotype is immunologically distinct from another such that an animal can become immune to one serotype but is still susceptible to infection by another. For this reason, vaccines have been developed to prevent poliovirus infection, but the great number of rhinovirus serotypes has thwarted the development of a rhinovirus vaccine. Therefore, in the case of rhinovirus, the only hope for a “cure” seems to lie in a pharmaceutical approach.

Antiviral Compounds Bind to a Specific Site Within Human Rhinovirus

The approximately 100 serotypes of human rhinovirus (HRV) [1], the most commonly isolated virus from those suffering from an upper respiratory infection (common cold), represent a formidable therapeutic challenge [2]. A number of structurally unrelated compounds (Figure 39.1) have been shown to inhibit HRV uncoating (i.e., the release of viral RNA into the cytosol) as a result of site-specific binding to virions. Recently, X-ray crystallographic analysis of disoxaril, [5-[7-[4-(4,5-dihydro-2-oxazolyl)phenoxy]heptyl]-3methylisoxazole]=HRV(type 14) complexes has identified the specific binding site in the viral capsid [3,4]. In this chapter the molecular details and biological consequences of the binding of several antiviral compounds to HRV-14 are discussed.

Drugs as Molecular Tools

Chemotherapeutic agents such as acyclovir and zidovudine have been extremely useful in treating patients with herpes simplex virus infections or AIDS, respectively. However, there is another scientific use for drugs or compounds which is to use them as molecular tools in the research laboratory in order to dissect the replication of viruses and to discover new facts about viruses. For example, alpha-amanitin, an inhibitor of the cellular DNA-dependent RNA polymerase II (Pol II), is used to define whether a particular virus RNA species is synthesized with the involvement of Pol II or solely by viral polymerases. With herpesviruses, cycloheximide, an inhibitor of protein synthesis, is used to define the “immediate early” class of viral genes. If a viral RNA is synthesized in the presence of cycloheximide, then the synthesis of that viral RNA is not dependent on the synthesis of any viral protein and that viral gene can be classified as immediate early. Arabinosyl cytosine (AraC) is an anticancer/antiherpesvirus drug which inhibits DNA synthesis in cell culture systems but allows RNA and protein synthesis to proceed. One can ask whether the replication of an RNA virus is dependent on cellular DNA synthesis by determining if virus replication is sensitive to AraC. These examples demonstrate how chemotherapeutic agents can be used as molecular tools in the research laboratory.