The antiviral effects of RSV fusion inhibitor, MDT‐637, on clinical isolates, vs its achievable concentrations in the human respiratory tract and comparison to ribavirin

Background Respiratory syncytial virus (RSV) viral load and disease severity associate, and the timing of viral load and disease run in parallel. An antiviral must be broadly effective against the natural spectrum of RSV genotypes and must attain concentrations capable of inhibiting viral replication within the human respiratory tract. Objectives We evaluated a novel RSV fusion inhibitor, MDT‐637, and compared it with ribavirin for therapeutic effect in vitro to identify relative therapeutic doses achievable in humans. Method MDT‐637 and ribavirin were co‐incubated with RSV in HEp‐2 cells. Quantitative PCR assessed viral concentrations; 50% inhibitory concentrations (IC 50) were compared to achievable human MDT‐637 and ribavirin peak and trough concentrations. Results and conclusions The IC 50 for MDT‐637 and ribavirin (against RSV‐A Long) was 1.42 and 16 973 ng/mL, respectively. The ratio of achievable peak respiratory secretion concentration to IC 50 was 6041‐fold for MDT‐637 and 25‐fold for aerosolized ribavirin. The ratio of trough concentration to IC 50 was 1481‐fold for MDT‐637 and 3.29‐fold for aerosolized ribavirin. Maximal peak and trough levels of oral or intravenous ribavirin were significantly lower than their IC 50s. We also measured MDT‐637 IC 50s in 3 lab strains and 4 clinical strains. The IC 50s ranged from 0.36 to 3.4 ng/mL. Achievable human MDT‐637 concentrations in respiratory secretions exceed the IC 50s by factors from hundreds to thousands of times greater than does ribavirin. Furthermore, MDT‐637 has broad in vitro antiviral activity on clinical strains of different RSV genotypes and clades. Together, these data imply that MDT‐637 may produce a superior clinical effect compared to ribavirin on natural RSV infections.


| INTRODUCTION
Respiratory syncytial virus (RSV) causes acute bronchiolitis and pneumonia, which are important causes of childhood illness and death.
Every year ~3% of all US infants are hospitalized with RSV infection most of whom are previously healthy. 1 The burden of RSV in outpatients is even greater with ~39% of all outpatient visits under 2 years of age caused by this virus. 2 Treatment with nebulized beta-2 agonists, epinephrine, Heliox, or surfactant has been studied. But, the trials have either not shown convincing clinical benefit or offer very shortterm benefit, which does not alter the underlying disease process. [3][4][5][6][7] Corticosteroids have not shown benefit 8 and increase the concentration of RSV in the respiratory tract. 9 Nebulized hypertonic saline has also not shown benefit. 10 Ribavirin inhibits RSV replication and is the only FDA-approved specific treatment for RSV lower respiratory tract infection. Aerosolized ribavirin has been demonstrated to achieve around one log reduction in RSV-infected cotton rat lungs and reduces RSV measured from children's nasal secretions. 11,12 All randomized trials of aerosolized ribavirin therapy in children have shown either a statistically significant clinical benefit or a trend in this direction; however, the treatment and antiviral effects are small. 13 After 3 days of aerosolized ribavirin therapy, hospitalized children showed a reduction of 0.6 log pfu/mL compared to controls. 13 This ribavirin small antiviral effect is controversial because the method of RSV quantification employed in the studies utilized RSV quantitative culture, which is known to be affected ex vivo by concentrations of ribavirin within the secretions themselves. Therefore, the ribavirin data suggest that inhibiting RSV replication might reduce disease severity in children, especially when an antiviral is more potent than ribavirin. Ribavirin aerosol can only be delivered practically to inpatients because of its teratogenicity, the environmental contamination issues, and concern for healthcare worker exposure during treatment, necessitating the elaborate use of environmental protection devices. Additionally, ribavirin aerosol must dose either continuously or for three, dosing times each day, each lasting 2 hours. Intravenous and oral ribavirin have never been adequately studied for RSV therapy. No licensed vaccines currently exist for preventing RSV infections. Prophylactic administration of monoclonal antibodies has been effective in reducing, but not eliminating, severe RSV disease, and this antibody is only recommended for under 3% of the birth cohort in the United States. 14 Therapeutic uses of high potency RSV-monoclonal antibody have not shown clinical benefit. [15][16][17] The pediatric population needs an efficient and easily applied therapy for RSV.
More potent RSV antivirals are promising because higher viral loads and greater disease severity correlate in infants and more rapid natural clearance of RSV and shorter hospital stays correlate. 18,19 Furthermore, we have recently shown that the timing of RSV load and disease track together. 20 This implies that reducing viral load may translate into clinical benefit. Even at a time when viral replication is at its highest, application of antiviral agents early in the disease course might improve subsequent morbidity by significantly lowering viral load, reducing direct viral cytopathic effects, and aborting potential downstream immunopathology. 19,21,22 A major consideration for candidate experimental RSV antivirals is the requirement that the molecule is broadly active across the natural spectrum of viral genotypes. 23 The RSV G protein, which mediates viral attachment to target cells, is the most genetically and antigenically diverse RSV protein and forms the basis of genotyping. We have retained a series of primary low-passage isolates from a broad clade range including genotypes RSV-A and RSV-B (Fig. S1). 23 Antiviral activity of any new therapeutic must be preserved across this wide range of diverse RSV isolates.
MDT-637 (previously called VP14637, Teva Pharmaceuticals, Tel Aviv, Israel) is a substituted bis-tetrazole-benzhydryl phenol, which has been reported to reduce RSV replication by inhibition of the F-protein function. 24 This molecule has produced an antiviral effect in vivo (cotton rats) following delivery by small droplet aerosol. 25 It has been reformulated so as to be deliverable to humans. A phase-1 evaluation of single and multiple ascending doses of aerosolized MDT-637 to determine its safety and pharmacokinetics has been completed (ClinicalTrials.gov identifier NCT01475305).
We therefore evaluated the in vitro antiviral effects of MDT-637 (50% inhibitory concentration, IC 50 ) against a broad range of genetically diverse RSV strains and compared it to ribavirin. We also related the IC 50 of MDT-637 and ribavirin to the concentrations achieved within human respiratory secretions so as to evaluate the relative antiviral effect in humans infected with community-acquired RSV.

| Compounds
MDT-637 (MicroDose Therapeutx Inc., NJ, USA) was solubilized in 5 mL of 100% dimethylsulfoxide. Consecutive 10-fold serial dilutions subsequently were prepared with DMEM until the first experimental drug concentration (0.1 μmol/L) was reached. Half-log dilutions were then performed with DMEM from 0.1 μmol/L to 31.6 pmol/L. Ribavirin (Calbiochem, CA, USA) was diluted using the same serial dilution as described for MDT-637.

| Exposure of RSV to Drug
The laboratory and clinical isolates of RSV (Multiplicity of Infection [MOI]; 0.001) and various concentrations (ranging from 0.1 μmol/L to 31.6 pmol/L) of MDT-637 (or ribavirin) were added to the HEp-2 cell monolayers at the same time. After 72 hours of incubation at 37°C, the cells and supernatants were harvested. An aliquot was immediately serially diluted and used for plaque assay as described in section "Quantification by Culture." The remainder cell-supernatant mixture was frozen at −80°C and subsequently quantified by qPCR.

| Quantification by culture (qCx)
Plaque assays were performed as previously described 26 using the human RSV-A Long strain (for A strains) and RSV-B, 9320 (for B strains) as a quantitative standard.

| Quantitative real time-PCR (qPCR)
Viral RNA was extracted from the samples frozen at −80°C using  26 . PCR was performed as previously described. 27

| Statistics
All statistical analyses were performed using Graphpad prISM, Version 5.0d.

| Comparison of the in vitro antiviral effect of MDT-637 to ribavirin
The IC 50 s for MDT-637 and ribavirin against RSV-A Long were 1.42 ng/mL and 16 973 ng/mL by qPCR, respectively. By qCulture, the IC 50 s were 1.83 ng/mL for MDT-637 and 20 509 ng/mL for ribavirin. These represent over 11 000-fold greater potency of MDT-637 (Table 1 and Figure 1).

| In vitro antiviral effect of MDT-637 against broad range of RSV isolates
MDT-637 IC 50 s were measured with three laboratory strains (RSV-A Long, RSV-A2, and RSV-9320) and three clinical strains (RSV-Memphis-37, RSV-LAP0824, and RSV-HAN1135; Fig. S1). The IC 50 s ranged from 0.36 to 3.4 ng/mL measured by qPCR (Table 2). IC 50 s were measured by qCx for RSV strains, A Long, A2, and Memphis-37, and correlated with the corresponding IC 50 s measured by qPCR validating the fidelity of the two measurement techniques (Table S1). For subsequent strains, antiviral effect was measured only by the qPCR. Peak extrapolated MDT-637 concentrations were over 6,000-fold greater than the IC 50 s (Table 3).

| Ratios of achievable concentrations of RSV antivirals to their corresponding the IC 50 s
Using RSV-A Long, the ratio of achievable peak respiratory secretion concentration to IC 50 was 6,041-fold for MDT-637 and 25-fold for ribavirin. The ratio of trough concentration to IC 50 was 1,481-fold for MDT-637 and 3.29-fold for ribavirin (Table 4).  commonly used, that is, high dose, short course, three times a day dosing. 35 Using RSV-A Long, the ratio of achievable peak respiratory secretion concentration to IC 50 was 6041-fold for MDT-637 and 25fold for aerosolized ribavirin. The ratio of trough concentration to IC 50 was 1481-fold for MDT-637 and 3.29-fold for aerosolized ribavirin (Table 4). Under the working condition, achievable MDT-637 concentrations in respiratory secretions exceed the IC 50 s by factors from several hundred-to thousand-fold greater than does aerosolized ribavirin concentrations in human respiratory secretions. The commonly reported clinical practice of administering ribavirin by the oral and intravenous route for severe RSV infection was also evaluated. 36,37 Our results predict the futility of such oral or intravenous ribavirin dosing for this infection.

| CONCLUSIONS
This study looking at the antiviral effects of the RSV F-protein inhibitor MDT-637 shows that MDT-637 appears to have antiviral activity against laboratory and clinical strains representing broad RSV genetic diversity. It is from several hundred-to thousand-fold more potent in vitro compared to ribavirin. MDT-637 concentrations achievable in human nasal washes are from hundred-to thousand-fold higher than the IC 50 s, suggesting that an antiviral effect may be achievable in human. Comparing IC 50 s to achievable human drug concentrations predicts that MDT-637 may produce a superior clinical effect compared to ribavirin on natural human RSV infections.