Discovery, Chemistry, and Preclinical Development of Pritelivir, a Novel Treatment Option for Acyclovir-Resistant Herpes Simplex Virus Infections.

When the nucleoside analogue acyclovir was introduced in the early 1980s, it presented a game-changing treatment modality for herpes simplex virus infections. Since then, work has been ongoing to improve the weaknesses that have now been identified: a narrow time window for therapeutic success, resistance in immunocompromised patients, little influence on frequency of recurrences, relatively fast elimination, and poor bioavailability. The present Drug Annotation focuses on the helicase-primase inhibitor pritelivir currently in development for the treatment of acyclovir-resistant HSV infections and describes how a change of the molecular target (from viral DNA polymerase to the HSV helicase-primase complex) afforded improvement of the shortcomings of nucleoside analogs. Details are presented for the discovery process leading to the final drug candidate, the pivotal preclinical studies on mechanism of action and efficacy, and on how ongoing clinical research has been able to translate preclinical promises into clinical use.

Different solid forms for optimizing route of administration of the herpes drug Pritelivir.

Pritelivir (AIC316, BAY 57-1293) was discovered as a highly potent drug against herpes simplex viruses with a novel mode of action, i.e. inhibition of the viral helicase-primase. A side by side comparison of the oral form against Valtrex™ in patients with genital herpes, showed superiority in phase II testing for Pritelivir. A number of different solid forms have been generated for additional, e.g. systemic, or topical applications.

Effect of Pritelivir Compared With Valacyclovir on Genital HSV-2 Shedding in Patients With Frequent Recurrences: A Randomized Clinical Trial.

Importance: Current therapy of herpes infections relies on nucleoside analogues. Pritelivir is a well-tolerated novel herpes simplex virus (HSV) helicase-primase inhibitor that reduced genital shedding and lesions. Objective: To compare the efficacy of pritelivir with valacyclovir for suppression of genital HSV-2 infection. Design, Setting, and Participants: A phase 2, randomized, double-blind, crossover clinical trial at clinical research centers in 4 US cities (October 2012-July 2013) compared daily oral doses of 100 mg of pritelivir with 500 mg of valacyclovir. The planned sample size was 98 adults, allowing for detection of a 50% reduction in viral shedding between the study treatments. Healthy adults with 4 to 9 annual genital HSV-2 recurrences were eligible. 45 participants were randomized to receive pritelivir [corrected] and 46 to receive valacyclovir first when the US Food and Drug Administration placed the trial on clinical hold based on findings in a concurrent nonclinical toxicity study, and the sponsor terminated the study. Interventions: Participants took the first drug for 28 days followed by 28 days of washout before taking the second drug for 28 days. Throughout treatment, the participants collected genital swabs 4 times daily for testing by HSV polymerase chain reaction assays. Main Outcomes and Measures: The primary end point was within-participant genital HSV shedding while receiving pritelivir compared with valacyclovir. Secondary end points included the quantity of HSV in positive swabs and the frequency of genital lesions and shedding episodes. Results: Of the 91 randomized participants (median age, 48 years; 57 women [63%]), 56 had completed both treatment periods at the time of the study's termination. In intent-to-treat analyses, HSV shedding was detected in 2.4% (173 of 7276 ) of swabs during pritelivir treatment compared with 5.3% (392 of 7453) during valacyclovir treatment (relative risk [RR], 0.42 [corrected]; 95% CI, 0.21 to 0.82; P = .01). In swabs with HSV, the mean quantity of HSV was 3.2 log10 copies/mL during pritelivir treatment vs 3.7 log10 copies/mL during valacyclovir treatment (difference, -0.1; 95% CI, -0.6 to 0.5; P = .83). Genital lesions were present on 1.9% of days in the pritelivir group vs 3.9% in the valacyclovir group (RR, 0.40; 95% CI, 0.17-0.96; P = .04). The frequency of shedding episodes did not differ by group, with 1.3 per person-month for pritelivir and 1.6 per person-month for valacyclovir (RR, 0.80; 95% CI, 0.52 to 1.22; P = .29). Treatment-emergent adverse events occurred in 62.3% of participants in the pritelivir group and 69.2% of participants in the valacyclovir group. Conclusions and Relevance: Among adults with frequently recurring genital HSV-2, the use of pritelivir compared with valacyclovir resulted in a lower percentage of swabs with HSV detection over 28 days. Further research is needed to assess longer-term efficacy and safety. Trial Registration: clinicaltrials.gov Identifier: NCT01658826.

No Evidence of Pritelivir Resistance Among Herpes Simplex Virus Type 2 Isolates After 4 Weeks of Daily Therapy.

BACKGROUND: Pritelivir is a novel helicase-primase inhibitor in clinical development for treatment of herpes simplex virus type 2 (HSV-2) infections. In preclinical work, resistance-mediating mutations were identified in the HSV-2 genome at 3 loci in the UL5 gene and 1 locus in UL52. METHODS: To evaluate whether daily pritelivir treatment results in emergence of resistance-mediating mutations, we analyzed HSV-2 strains detected in genital swab specimens from trial participants who were randomly assigned to receive different dosages of pritelivir. We sequenced resistance regions from 87 participants' samples, the UL5 gene in 73 samples from 44 participants, and the UL52 gene in 71 samples from 43 participants. RESULTS: We found no evidence that pritelivir induced known resistance-mediating mutations or for amino acid variation at other loci. In one participant's HSV-2 isolate, we found a previously unidentified mutation close to the putative resistance-mediating region in UL5 and subsequently determined in vitro susceptibility to pritelivir. We characterized mutations from 32 cultivated HSV-2 isolates previously found to be susceptible to pritelivir in vitro and identified several novel mutations that most likely reflect preexisting variation in circulating HSV-2. CONCLUSIONS: This study demonstrates evidence of retained susceptibility of HSV-2 to pritelivir in immunocompetent persons following daily therapy for up to 28 days.

Acyldepsipeptide antibiotics kill mycobacteria by preventing the physiological functions of the ClpP1P2 protease.

The Clp protease complex in Mycobacterium tuberculosis is unusual in its composition, functional importance and activation mechanism. Whilst most bacterial species contain a single ClpP protein that is dispensable for normal growth, mycobacteria have two ClpPs, ClpP1 and ClpP2, which are essential for viability and together form the ClpP1P2 tetradecamer. Acyldepsipeptide antibiotics of the ADEP class inhibit the growth of Gram-positive firmicutes by activating ClpP and causing unregulated protein degradation. Here we show that, in contrast, mycobacteria are killed by ADEP through inhibition of ClpP function. Although ADEPs can stimulate purified M. tuberculosis ClpP1P2 to degrade larger peptides and unstructured proteins, this effect is weaker than for ClpP from other bacteria and depends on the presence of an additional activating factor (e.g. the dipeptide benzyloxycarbonyl-leucyl-leucine in vitro) to form the active ClpP1P2 tetradecamer. The cell division protein FtsZ, which is a particularly sensitive target for ADEP-activated ClpP in firmicutes, is not degraded in mycobacteria. Depletion of the ClpP1P2 level in a conditional Mycobacterium bovis BCG mutant enhanced killing by ADEP unlike in other bacteria. In summary, ADEPs kill mycobacteria by preventing interaction of ClpP1P2 with the regulatory ATPases, ClpX or ClpC1, thus inhibiting essential ATP-dependent protein degradation.

Mathematical modeling of herpes simplex virus-2 suppression with pritelivir predicts trial outcomes.

Pharmacokinetic and pharmacodynamic models estimate the potency of antiviral agents but do not capture viral and immunologic factors that drive the natural dynamics of infection. We designed a mathematical model that synthesizes pharmacokinetics, pharmacodynamics, and viral pathogenesis concepts to simulate the activity of pritelivir, a DNA helicase-primase inhibitor that targets herpes simplex virus. Our simulations recapitulate detailed viral kinetic shedding features in five dosage arms of a phase 2 clinical trial. We identify that in vitro estimates of median effective concentration (EC50) are lower than in vivo values for the drug. Nevertheless, pritelivir potently decreases shedding at appropriate doses based on its mode of action and long half-life. Although pritelivir directly inhibits replication in epithelial cells, our model indicates that pritelivir also indirectly limits downstream viral spread from neurons to genital keratinocytes, within genital ulcers, and from ulcer to new mucosal sites of infection. We validate our model based on its ability to predict outcomes in a subsequent trial with a higher dose. The model can therefore be used to optimize dose selection in clinical practice.

AIC649 Induces a Bi-Phasic Treatment Response in the Woodchuck Model of Chronic Hepatitis B.

AIC649 has been shown to directly address the antigen presenting cell arm of the host immune defense leading to a regulated cytokine release and activation of T cell responses. In the present study we analyzed the antiviral efficacy of AIC649 as well as its potential to induce functional cure in animal models for chronic hepatitis B. Hepatitis B virus transgenic mice and chronically woodchuck hepatitis virus (WHV) infected woodchucks were treated with AIC649, respectively. In the mouse system AIC649 decreased the hepatitis B virus titer as effective as the "gold standard", Tenofovir. Interestingly, AIC649-treated chronically WHV infected woodchucks displayed a bi-phasic pattern of response: The marker for functional cure--hepatitis surface antigen--first increased but subsequently decreased even after cessation of treatment to significantly reduced levels. We hypothesize that the observed bi-phasic response pattern to AIC649 treatment reflects a physiologically "concerted", reconstituted immune response against WHV and therefore may indicate a potential for inducing functional cure in HBV-infected patients.

Pharmacokinetics-pharmacodynamics of the helicase-primase inhibitor pritelivir following treatment of wild-type or pritelivir-resistant virus infection in a murine herpes simplex virus 1 infection model.

Herpes simplex virus (HSV) infections can cause considerable morbidity. Transmission of HSV-2 has become a major health concern, since it has been shown to promote transmission of other sexually transmitted diseases. Pritelivir (AIC316, BAY 57-1293) belongs to a new class of HSV antiviral compounds, the helicase-primase inhibitors, which have a mode of action that is distinct from that of antiviral nucleoside analogues currently in clinical use. Analysis of pharmacokinetic-pharmacodynamic parameters is a useful tool for the selection of appropriate doses in clinical trials, especially for compounds belonging to new classes for which no or only limited data on therapeutic profiles are available. For this purpose, the effective dose of pritelivir was determined in a comprehensive mouse model of HSV infection. Corresponding plasma concentrations were measured, and exposures were compared with efficacious concentrations derived from cell cultures. The administration of pritelivir at 10 mg/kg of body weight once daily for 4 days completely suppressed any signs of HSV infection in the animals. Associated plasma concentrations adjusted for protein binding stayed above the cell culture 90% effective concentration (EC90) for HSV-1 for almost the entire dosing interval. Interestingly, by increasing the dose 6-fold and prolonging the treatment duration to 8 days, it was possible to treat mice infected with an approximately 30-fold pritelivir-resistant but fully pathogenic HSV-1 virus. Corresponding plasma concentrations exceeded the EC90 of this mutant for <8 h, indicating that even suboptimal exposure to pritelivir is sufficient to achieve antiviral efficacy, possibly augmented by other factors such as the immune system.

Helicase-primase inhibitor pritelivir for HSV-2 infection.

BACKGROUND: Pritelivir, an inhibitor of the viral helicase-primase complex, exhibits antiviral activity in vitro and in animal models of herpes simplex virus (HSV) infection. We tested the efficacy and safety of pritelivir in otherwise healthy persons with genital HSV-2 infection. METHODS: We randomly assigned 156 HSV-2-positive persons with a history of genital herpes to receive one of four doses of oral pritelivir (5, 25, or 75 mg daily, or 400 mg weekly) or placebo for 28 days. Participants obtained daily swabs from the genital area for HSV-2 testing, which was performed with a polymerase-chain-reaction assay. Participants also maintained a diary of genital signs and symptoms. The primary end point was the rate of genital HSV shedding. RESULTS: HSV shedding among placebo recipients was detected on 16.6% of days; shedding among pritelivir recipients was detected on 18.2% of days among those receiving 5 mg daily, 9.3% of days among those receiving 25 mg daily, 2.1% of days among those receiving 75 mg daily, and 5.3% of days among those receiving 400 mg weekly. The relative risk of viral shedding with pritelivir, as compared with placebo, was 1.11 (95% confidence interval [CI], 0.65 to 1.87) with the 5-mg daily dose, 0.57 (95% CI, 0.31 to 1.03) with the 25-mg daily dose, 0.13 (95% CI, 0.04 to 0.38) with the 75-mg daily dose, and 0.32 (95% CI, 0.17 to 0.59) with the 400-mg weekly dose. The percentage of days with genital lesions was also significantly reduced, from 9.0% in the placebo group to 1.2% in both the group receiving 75 mg of pritelivir daily (relative risk, 0.13; 95% CI, 0.02 to 0.70) and the group receiving 400 mg weekly (relative risk, 0.13; 95% CI, 0.03 to 0.52). The rate of adverse events was similar in all groups. CONCLUSIONS: Pritelivir reduced the rates of genital HSV shedding and days with lesions in a dose-dependent manner in otherwise healthy men and women with genital herpes. (Funded by AiCuris; ClinicalTrials.gov number, NCT01047540.).

Geno- and phenotypic characterization of human cytomegalovirus mutants selected in vitro after letermovir (AIC246) exposure.

Letermovir is a novel antiviral compound currently in clinical development for the prevention of human cytomegalovirus (HCMV) infections. In contrast to all currently approved anti-HCMV drugs that target the viral DNA polymerase, letermovir acts via a distinct mode of action involving the viral terminase subunit pUL56. To extend our understanding of potential letermovir resistance mechanisms, we used marker transfer to characterize mutations identified in letermovir-resistant HCMV variants that were selected in cell culture.

Inactivated ORF virus shows antifibrotic activity and inhibits human hepatitis B virus (HBV) and hepatitis C virus (HCV) replication in preclinical models.

Inactivated orf virus (iORFV), strain D1701, is a potent immune modulator in various animal species. We recently demonstrated that iORFV induces strong antiviral activity in animal models of acute and chronic viral infections. In addition, we found D1701-mediated antifibrotic effects in different rat models of liver fibrosis. In the present study, we compare iORFV derived from two different strains of ORFV, D1701 and NZ2, respectively, with respect to their antifibrotic potential as well as their potential to induce an antiviral response controlling infections with the hepatotropic pathogens hepatitis C virus (HCV) and hepatitis B virus (HBV). Both strains of ORFV showed anti-viral activity against HCV in vitro and against HBV in a transgenic mouse model without signs of necro-inflammation in vivo. Our experiments suggest that the absence of liver damage is potentially mediated by iORFV-induced downregulation of antigen cross-presentation in liver sinus endothelial cells. Furthermore, both strains showed significant anti-fibrotic activity in rat models of liver fibrosis. iORFV strain NZ2 appeared more potent compared to strain D1701 with respect to both its antiviral and antifibrotic activity on the basis of dosages estimated by titration of active virus. These results show a potential therapeutic approach against two important human liver pathogens HBV and HCV that independently addresses concomitant liver fibrosis. Further studies are required to characterize the details of the mechanisms involved in this novel therapeutic principle.

In vitro and in vivo activities of AIC292, a novel HIV-1 nonnucleoside reverse transcriptase inhibitor.

Nonnucleoside reverse transcriptase inhibitors (NNRTIs) are important and frequently used elements of highly active antiretroviral therapy (HAART) for the treatment of human immunodeficiency virus type 1 (HIV-1) infection. However, the development of drug resistance, as well as the side effects of existing drugs, defines a medical need for novel NNRTIs with excellent tolerability, improved activity against NNRTI-resistant viruses, and a low barrier to resistance. Within the chemical class of diarylpyrazole-[imidazolidinone]-carboxamides, AIC292 was identified as a promising novel HIV-1 NNRTI and has successfully completed single-dose clinical phase I studies. Here, we report on the antiviral activity of AIC292, evaluated in vitro against wild-type and NNRTI-resistant HIV-1 isolates and in vivo using an engineered mouse xenograft model. AIC292 inhibited wild-type HIV-1 laboratory strains at low nanomolar concentrations, was well tolerated in different cell lines, and showed excellent selectivity in a lead profiling screen. In addition, activity of AIC292 could be demonstrated against a broad panel of wild-type HIV-1 group M and group O clinical isolates. AIC292 also retained activity against viruses harboring NNRTI resistance-associated mutations (RAMs), including the most prevalent variants, K103N, Y181C, and G190A. Interestingly, viruses bearing the L100I RAM were hypersusceptible to AIC292. Two-drug combination assays showed no antagonistic interactions between AIC292 and representative marketed HIV drugs with regard to antiviral activity. Furthermore, AIC292 displayed potent antiviral in vivo efficacy in a mouse xenograft model when applied once daily. Taken together, these data show that AIC292 represents a molecule with the antiviral properties of a novel NNRTI for the treatment of HIV-1 infection.

The ClpP peptidase of Wolbachia endobacteria is a novel target for drug development against filarial infections.

BACKGROUND: Filarial infections causing lymphatic filariasis or onchocerciasis (river blindness) can be treated with antibiotics (e.g. doxycycline) targeting the essential endosymbiotic Wolbachia bacteria. The depletion of Wolbachia inhibits worm development and causes worm death. Available antibiotics have restrictions for use in children and pregnant or breastfeeding women. Therefore, alternative antibiotics are needed that can be given to all members of the population and that are active with a shorter therapy time. Antibiotics of the acyldepsipeptide class have been shown to inhibit the growth of bacteria by overactivating the peptidase ClpP. The novel mode of action of this class of antibiotics could lead to faster killing of intracellular bacteria. OBJECTIVES: To characterize acyldepsipeptide activity against the Wolbachia ClpP. METHODS: The activity of acyldepsipeptides was investigated against Wolbachia in vitro in insect cells and also against worms in culture. In addition, structural effects were investigated by fluorescence microscopy and electron microscopy. The activity of ClpP was also investigated in vitro. RESULTS: We show that acyldepsipeptides are active against recombinant Wolbachia ClpP and endobacteria resident within insect cells in vitro, and some derivatives were also active against filarial worms in culture. As a consequence of treatment, the worms became immotile and died, the latter confirmed by a viability assay. CONCLUSIONS: The mode of action of the acyldepsipeptides in Wolbachia is the dysregulation of ClpP, causing the uncontrolled degradation of proteins, including the cell division protein FtsZ. Our results demonstrate that wolbachial ClpP is a target for further antifilarial antibiotic discovery.

Discovery of substituted N-phenylbenzenesulphonamides as a novel class of non-nucleoside hepatitis C virus polymerase inhibitors.

The RNA-dependent RNA polymerase NS5B of the hepatitis C virus (HCV) has emerged as one of the key targets for antiviral drug discovery. Here we describe a novel non-nucleoside inhibitor (NNI) chemotype identified by screening: The substituted N-phenylbenzenesulphonamides (SPBS) which showed reversible inhibition of NS5B from HCV genotype 1b with IC(50) values up to 40 nM. Based on the decreased inhibitory activity against a recombinant NS5B protein carrying the mutation L419M or M423T we assumed that the SPBS inhibitors bind to the thumb site II which has already been described as the allosteric binding site for the NNI carboxy thiophene. The postulated binding site was consequently confirmed by solving two co-crystal structures of NS5B in complex with SPBS analogues at 2.3 and 2.2Å resolutions. The inhibitors are hydrogen-bonded to the main chain Ser476 and Tyr477 and to the side chain of Arg501. In addition, the inhibitors displayed van der Waals interactions with several residues of the hydrophobic binding pocket Leu419, Ile482, Leu497, Met423 and Trp528. Notably, the two SPBS analogues reported here revealed significant differences in addressing the NH-group of the main chain Tyr477 by hydrogen-bonds, water-mediated or directly, which provoked a shift of the carboxyphenyl group of the inhibitors towards the His475 position for the water-mediated binding mode. Interestingly, the differences observed in the binding mode led to a different cross resistance profile at positions M423 and I482. Using a panel of 38 individual NS5B proteins derived from different HCV genotypes, we could demonstrate inhibitory activity of the SPBS against polymerases from HCV genotypes 1a and 1b whereas the inhibitor class failed to inhibit any of the non-genotype 1 polymerases efficiently. Furthermore we demonstrated initial antiviral activity for SPBS against the subgenomic replicons of HCV genotypes 1a and 1b, respectively, and no considerable cytotoxic potential against a panel of ten different cell types.

In vitro evaluation of the activities of the novel anticytomegalovirus compound AIC246 (letermovir) against herpesviruses and other human pathogenic viruses.

AIC246 (letermovir) is a potent anticytomegalovirus drug in clinical development. Here, we report a consistent antiviral efficacy of AIC246 against human cytomegalovirus laboratory strains, clinical isolates, and virus variants resistant to approved drugs. Furthermore, we describe a remarkable selectivity of AIC246 for human cytomegaloviruses compared to that of other alpha-, beta-, or gammaherpesviruses or nonrelated pathogenic viruses, including adeno-, hepadna-, retro-, orthomyxo-, and flaviviruses. Our data confirm and support an excellent and selective anticytomegaloviral activity of AIC246.

The novel anticytomegalovirus compound AIC246 (Letermovir) inhibits human cytomegalovirus replication through a specific antiviral mechanism that involves the viral terminase.

Human cytomegalovirus (HCMV) remains the leading viral cause of birth defects and life-threatening disease in transplant recipients. All approved antiviral drugs target the viral DNA polymerase and are associated with severe toxicity issues and the emergence of drug resistance. Attempts to discover improved anti-HCMV drugs led to the identification of the small-molecular-weight compound AIC246 (Letermovir). AIC246 exhibits outstanding anti-HCMV activity in vitro and in vivo and currently is undergoing a clinical phase IIb trial. The initial mode-of-action studies suggested that the drug acts late in the HCMV replication cycle via a mechanism distinct from that of polymerase inhibitors. Here, we extend our mode-of-action analyses and report that AIC246 blocks viral replication without inhibiting the synthesis of progeny HCMV DNA or viral proteins. The genotyping of mutant viruses that escaped AIC246 inhibition uncovered distinct point mutations in the UL56 subunit of the viral terminase complex. Marker transfer analyses confirmed that these mutations were sufficient to mediate AIC246 resistance. The mapping of drug resistance to open reading frame UL56 suggests that viral DNA processing and/or packaging is targeted by AIC246. In line with this, we demonstrate that AIC246 affects the formation of proper unit-length genomes from viral DNA concatemers and interferes with virion maturation. However, since AIC246-resistant viruses do not exhibit cross-resistance to previously published terminase inhibitors, our data suggest that AIC246 interferes with HCMV DNA cleavage/packaging via a molecular mechanism that is distinct from that of other compound classes known to target the viral terminase.

RNA-dependent RNA polymerases from different hepatitis C virus genotypes reveal distinct biochemical properties and drug susceptibilities.

The RNA-dependent RNA polymerase of the hepatitis C virus (HCV) is the key enzyme for viral replication, recognized as one of the promising targets for antiviral intervention. Several of the known non-nucleoside HCV polymerase inhibitors (NNIs) identified by screening approaches show limitations in the coverage of all six major HCV genotypes (GTs). Genotypic profiling therefore has to be implemented early in the screening cascade to discover new broadly active NNIs. This implies knowledge of the specific individual biochemical properties of polymerases from all GTs which is to date limited to GT 1 only. This work gives a comprehensive overview of the biochemical properties of HCV polymerases derived from all major GTs 1-6. Biochemical analysis of polymerases from 38 individual sequences revealed that the optima for monovalent cations, pH and temperature were similar between the GTs, whereas significant differences concerning concentration of the preferred cofactor Mg(2+) were identified. Implementing the optimal requirements for the polymerases from each individual GT led to significant improvements in their enzymatic activities. However, the specific activity was distributed unequally across the GTs and could be ranked in the following descending order: 1b, 6a>2a, 3a, 4a, 5a>1a. Furthermore, the optimized assay conditions for genotypic profiling were confirmed by testing the inhibitory activity of 4 known prototype NNIs addressing the NNI binding sites 1 to 4.

Effects of therapy using a helicase-primase inhibitor (HPI) in mice infected with deliberate mixtures of wild-type HSV-1 and an HPI-resistant UL5 mutant.

Point mutations in the HSV-1 UL5 (helicase) gene confer resistance to helicase-primase inhibitors (HPIs), e.g. BAY 57-1293. Such mutations normally occur at a frequency of < or =10(-6)PFU. However, individual HSV-1 laboratory strains and some clinical isolates contained resistance mutations (e.g. UL5: Lys356Asn) at 10(-4)PFU. To address the possibility that pre-existing mutants at high frequency might have an impact on therapy using HPIs, deliberate mixtures were prepared to contain the SC16 UL5: Lys356Asn mutant in SC16 wild-type in the proportion of 1/500 or 1/50PFU. Mice were infected in the neck-skin with 5x10(4)PFU/mouse of wt alone, mutant alone, or the respective mixture. The mutant could not be detected in infectious virus yields from mice inoculated with the 1/500 mixture. However, resistant mutant was recovered from some treated mice inoculated with the 1/50 mixture. All mice inoculated with mixtures remained responsive to BAY 57-1293-therapy with no increase in clinical signs compared to treatment of wt-infected mice.

In vitro and in vivo activities of the novel anticytomegalovirus compound AIC246.

Human cytomegalovirus (HCMV) remains a serious threat for immunocompromised individuals, including transplant recipients and newborns. To date, all drugs licensed for the treatment of HCMV infection and disease target the viral DNA polymerase. Although these drugs are effective, several drawbacks are associated with their use, including toxicity and emergence of drug resistance. Hence, new and improved antivirals with novel molecular targets are urgently needed. Here we report on the antiviral properties of AIC246, a representative of a novel class of low-molecular-weight compounds that is currently undergoing clinical phase II studies. The anti-HCMV activity of AIC246 was evaluated in vitro and in vivo using various cell culture assays and an engineered mouse xenograft model. In addition, antiviral properties of the drug were characterized in comparison to the current gold standard ganciclovir. We demonstrate that AIC246 exhibits excellent in vitro inhibitory activity against HCMV laboratory strains and clinical isolates, retains activity against ganciclovir-resistant viruses, is well tolerated in different cell types (median selectivity index, 18,000), and exerts a potent in vivo efficacy in a mouse xenograft model. Moreover, we show that the antiviral block induced by AIC246 is reversible and the efficacy of the drug is not significantly affected by cell culture variations such as cell type or multiplicity of infection. Finally, initial mode-of-action analyses reveal that AIC246 targets a process in the viral replication cycle that occurs later than DNA synthesis. Thus, AIC246 acts via a mode of action that differs from that of polymerase inhibitors like ganciclovir.