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. Author manuscript; available in PMC: 2019 Sep 1.
Published in final edited form as: Antiviral Res. 2018 Jul 21;157:128–133. doi: 10.1016/j.antiviral.2018.07.013

Antiviral activity of maribavir in combination with other drugs active against human cytomegalovirus

Sunwen Chou 1,2,*, Ronald J Ercolani 2, Katayoun Derakhchan 3
PMCID: PMC6097806  NIHMSID: NIHMS1501627  PMID: 30040968

Abstract

The human cytomegalovirus (CMV) UL97 kinase inhibitor maribavir is in Phase III clinical trials as antiviral therapy, including use for infections refractory or resistant to standard therapy. To assess its activity in combination with approved and experimental CMV antivirals, and with the mTor inhibitor rapamycin (sirolimus), drug effects were tested by in vitro checkerboard assays and the data were analyzed using a three dimensional model based on an independent effects definition of additive interactions. Baseline virus and representative drug-resistant mutants were tested. According to the volume of synergy at 95% confidence, maribavir showed additive interactions with foscarnet, cidofovir, letermovir and GW275175X when tested against wild type and mutant viruses, strong antagonism with ganciclovir, and strong synergy with rapamycin, the latter suggesting a potentially useful therapeutic combination.

Keywords: Cytomegalovirus, maribavir, ganciclovir, sirolimus, synergism, antagonism

1. Introduction

Effective antiviral prophylaxis and therapy for opportunistic human cytomegalovirus (CMV) infection in transplant recipients and other immunocompromised hosts has long relied mainly on ganciclovir and its oral prodrug valganciclovir, with foscarnet and cidofovir in secondary roles. Use of these viral DNA polymerase inhibitors has improved clinical outcomes, particularly when used as prophylaxis or preventive therapy, but have well-known limitations of toxicity as well as cross-resistance due to the same antiviral drug target (Kotton et al., 2018). Recently, the CMV terminase inhibitor letermovir was approved for prophylaxis in stem cell transplant recipients (Marty et al., 2017). The separate drug target avoids issues of cross-resistance, and opens the possibility of combination therapy targeting multiple viral gene products, a strategy proven successful for HIV and hepatitis C virus. Combination studies of letermovir with existing CMV DNA polymerase inhibitors show an additive effect (Wildum et al., 2015).

Maribavir is a potent and specific inhibitor of the CMV UL97 kinase (Biron et al., 2002) that has been investigated in vitro and in clinical trials over an extended period. The UL97 kinase has important biological functions during CMV replication, including cell cycle modulation and nuclear egress of nascent viral particles (Marschall et al., 2011; Prichard, 2009). Genetic inactivation of the UL97 kinase results in severe viral growth impairment, thus offering a separate antiviral drug target distinct from the polymerase and terminase gene products. After promising early phase clinical trials, maribavir was unsuccessful in Phase III trials of low doses as CMV prophylaxis in stem cell and liver transplant recipients (Marty et al., 2011; Winston et al., 2012), although the drug was well tolerated. Subsequent trials at higher doses showed indications of success in treatment of CMV infections refractory or resistant to standard therapy (clinicaltrials.gov NCT01611974), or as treatment of asymptomatic infection, and Phase III treatment trials are ongoing (NCT02927067 and NCT02931539).

As more CMV antiviral targets are developed, a detailed assessment is needed of how drugs of different classes act when used in combination. Early studies on antiviral combinations were conducted before full appreciation of the potential for cell culture conditions to affect maribavir inhibitory concentrations by as much as 100-fold (Chou et al., 2006), presumably because of variable host cell metabolic compensation for the biological functions of the UL97 kinase. Initial in vitro combination studies with maribavir variously reported additive or synergistic interactions with CMV DNA polymerase inhibitors including ganciclovir, and with older experimental terminase inhibitors (Evers et al., 2002; Selleseth et al., 2003). Since the UL97 kinase mediates the initial phosphorylation of ganciclovir that is required for its conversion to the active ganciclovir triphosphate inhibitor of the CMV DNA polymerase, it can be predicted mechanistically that maribavir should antagonize the action of ganciclovir by interfering with its phosphorylation. Unlike earlier reports, antagonism was experimentally documented between maribavir and ganciclovir starting in 2006 (Chou and Marousek, 2006; Drouot et al., 2016). Because the measured antiviral effect of maribavir is greater in less metabolically active cells (e.g. incubated at lower temperatures (Williams et al., 2003) or in the presence of cyclin dependent kinase or mTor inhibitors (Chou et al., 2006)), there is the possibility of useful synergy with host-acting drugs that are used in CMV treatment populations with an acceptable safety profile. In particular, the possibility of synergy with mTor inhibitors such as rapamycin (sirolimus) is attractive because these inhibitors have anti-CMV activity both in cell culture (Chou et al., 2006) and clinically (Pascual et al., 2016), though insufficient for complete viral suppression.

Given some discordance of the published experimental data, the purpose of this study was to revisit the antiviral effects of maribavir in combination with standard DNA polymerase inhibitors (foscarnet, cidofovir, and ganciclovir), terminase inhibitors (letermovir and an older benzimidazole compound GW275175X) or the mTor inhibitor sirolimus. This was done using a standardized cell culture system, readout of viral growth based on a reporter gene, replicates of checkerboard assays analyzed according to the same three dimensional model used for several previous CMV antiviral combination studies, and CMV strains representing wild type and mutant sequences of the corresponding antiviral targets. Mutants resistant to one or the other of the drugs tested in combination were assessed for their effect on synergy determinations.

2. Materials and Methods

2.1. Viral strains

The baseline bacterial artificial chromosome (BAC) clone BD1 (Chou, 2015) derived from standard laboratory strain AD169 was used to generate CMV strain T4175, with wild type sequence in all of the antiviral target genes: DNA polymerase UL54, terminase UL51, UL56, UL89 and kinase UL97. In addition, the BD series clones all contain a BAC vector, a secreted alkaline phosphatase (SEAP) reporter gene for viral growth quantitation located between US3 and US6, and a compensatory removal of the internal repeat of genes RL1 through RL13 to accommodate these inserted genetic elements (Chou, 2015). Mutant BAC clones containing known drug resistance mutations were generated from BD1 using previously described recombination techniques in genes UL54 (Chou, 2011), UL56 (Chou, 2015), UL89 (Chou, 2017a) and UL97 (Chou, 2010). Since the recombination method used a Frt-delimited upstream Kan selection marker and subsequent removal by Flp recombinase to leave a silent Frt motif, additional baseline strains containing the Frt motif upstream of UL54 (T4198), UL56 (T4190), UL97 (T4200) were generated and used as wild type controls as in previous studies.

2.2. Cell cultures

ARPEp cells were used at up to 45 passages from their original derivation and propagated in Dulbecco Minimal Essential Medium with 4.5 g/L glucose (DMEM) and 8% fetal bovine serum in the growth phase and 3% after viral inoculation (Chou et al., 2017). These are ARPE-19 cells made fully permissive for laboratory CMV strains by over-expression of the platelet derived growth factor receptor alpha receptor. Cells were trypsinized from confluent 75 cm2 monolayers, seeded into 96-well plates at ~15,000 cells/well and incubated at 37°C. Full confluency was reached within 3 days and the plates were used for antiviral assays 3 days after that. To mitigate uneven growth at the edges of plates, water-filled channels were designed around the margins of the plates. ARPEp cells in culture flasks were also used for routine propagation of viral strains and production of cell-free virus stock as used in the antiviral assays.

2.3. Antiviral compounds

Standard DNA polymerase inhibitors ganciclovir (Roche), foscarnet (Astra), cidofovir (Gilead), maribavir (Glaxo and Shire) and GW275175X (Underwood et al., 2004) (Shire) were obtained from their respective pharmaceutical sources. Letermovir was obtained from MedChemExpress (HY-15233). Rapamycin was obtained from LC Laboratories (RAP-5000). Ganciclovir and foscarnet were used as stock aqueous solutions of sodium salts of 125 to 200 mM. All other compounds were used as dimethyl sulfoxide (DMSO) stock solutions diluted into final working concentrations that included no more than 0.2% DMSO at the maximum drug concentration.

2.4. Cytotoxicity assay

Combinations of maribavir and other antiviral compounds were added to confluent uninfected ARPEp cell cultures in 96 wells at 2-fold increasing concentrations up to the maximum used for combination antiviral assays. One row of wells consisted of controls with no added drug. After 6 days of incubation in 100 μL of maintenance medium (DMEM with 3% fetal bovine serum), cytotoxicity was assessed by the MTT assay (Mosmann, 1983). Methylthiazolyldiphenyl- tetrazolium bromide (MTT, Sigma M5655) was added to each well as 10 μL of a 5 mg/mL solution in phosphate buffered saline, mixed and incubated for 2 hours at 37°C, followed by the addition of 100 μL of a 1:1 mixture of isopropyl alcohol and DMSO to solubilize the formazan produced by live cells. After mixing, the formazan was assayed by light absorbance at 570 nm with subtraction of the background absorbance at 690 nm. The absorbance of wells containing drug was compared with that of control wells.

2.5. Checkerboard combination antiviral assays

An 8 by 8 matrix of drug combinations was prepared in separate 96-well plates by loading one of the two drugs at one end of each axis at twice the maximum drug concentration and making serial two-fold dilutions in culture medium, leaving a final row or column without the drug. The resulting matrix had no drug in the left lower well, a single drug in rising 2-fold concentrations in the vertical and horizontal axes starting from that well, and the remaining wells with rising concentrations of drug mixtures reaching maximum concentrations of both drugs at the upper right well. After removal of culture media from 96-well cultures of ARPEp cells at 6 days after seeding, each well was inoculated with 50 μL of viral inoculum at a multiplicity of infection (MOI) of 0.02 and incubated for 90 minutes at 37°C. After removal of the viral inocula, the 8 by 8 matrix of drug concentrations was transferred to the ARPEp culture plate at 150 μL per well and the plates were incubated at 37°C under 6% CO2. Additionally, two separate columns of 8 inoculated wells were maintained under culture medium with no added drug, as controls for viral growth across a sample of wells. At day 1, 20 μL aliquots were collected from one column of 8 control wells and assayed for SEAP activity compatible with the intended MOI (Chou et al., 2005). At day 6, 10 μL aliquots were collected from each well of the 8 by 8 matrix and the second column of 8 control wells with no added drug. Supernatant SEAP activity for viral growth quantitation was assayed using a dioxetane chemiluminescent substrate (Chou et al., 2005). A minimum of 5 replicates of checkerboard assays were performed for each drug combination and viral strain, over at least 3 setup dates and batches of cells.

2.6. Data analysis

The drug concentration required to reduce viral growth by 50% (EC50) at 6 days as measured by culture supernatant SEAP activity (50% yield reduction) was calculated for each individual drug using the readings along the vertical and horizontal axis starting at the lower left well of each checkerboard assay, since those wells contained no more than one drug. The no drug control SEAP value (designated “VC” in MacSynergy II (Prichard and Shipman, 1990)) was the mean of those from the left lower well and 8 control wells without drug in a separate column of the 96- well plate. No checkerboard assays were accepted for analysis if the VC standard deviation was more than 15% of the mean value. EC50 values were calculated by fitting SEAP readings to an exponential growth curve as previously described (Chou et al., 2017).

Effects of antiviral drugs in combination were assessed by MacSynergy II software (Prichard and Shipman, 1990) using a three dimensional model based on an independent effects definition of additive interactions, the same method as used in prior combination studies (Evers et al., 2002; Wildum et al., 2015). In this model, expected additive interactions of drug combination were calculated from the SEAP yield reduction readings from each drug alone, and the observed yield reductions under various drug combinations were compared with the expected (theoretical) yield reductions from an additive interaction by plotting values above and below a horizontal plane representing the expected values, indicating synergy and antagonism, respectively. The points defined by the various combinations of drug concentrations are integrated into a volume index of synergy/antagonism, with positive figures denoting synergy and negative values denoting antagonism. As a feature of the MacSynergy II software, the percentage SEAP yield reduction from each specific combination of drug concentrations and viral strain was averaged across multiple replicates of checkerboard assays and a standard deviation is calculated. From the mean and standard deviations of the percentage yield reductions, the volume index of synergy is calculated at confidence levels of 95%, 99% and 99.9% (the latter two with Bonferroni adjustment), enabling comparisons across drug combinations and studies (Prichard and Shipman, 1990). Conventionally, the volume index at 95% confidence is used to interpret synergy or antagonism, and we followed the published practice of interpreting indexes of ±50 as within the additive range, +100 or higher as strong synergy and −100 or lower as strong antagonism (Evers et al., 2002; Wildum et al., 2015). Because the SEAP yield reduction method used here gives percentage rather than logarithmic reductions in the viral quantitation signals, it was not necessary to modify the interpretation of volume indexes for logarithmic yield reductions as recently proposed (Smee and Prichard, 2017).

3. Results

3.1. Strains tested and single drug EC50 values

Table 1 summarizes the strains and drugs tested in combination with maribavir. A total of 106 checkerboard assays were performed to test the effect of maribavir in combination with each of 6 other drugs, against wild type virus and characteristic drug-resistant mutants. GW275175X is a benzimidazole pyranoside terminase inhibitor that was not advanced beyond Phase I clinical trial (Underwood et al., 2004), with UL89 D344E as its most common resistance mechanism (Chou, 2017a). For the recently approved terminase inhibitor letermovir, UL56 V236M has been observed twice in treated subjects (Marty et al., 2017). UL97 T409M and H411Y have been observed in maribavir-treated subjects (Schubert et al., 2013) and UL27 R233S was selected in vitro (Chou et al., 2012). The UL54 DNA polymerase mutant with in frame deletion of codons 981–2 was tested as it is resistant to all standard DNA polymerase inhibitors (Chou et al., 2005). The EC50 values for the individual antiviral drugs were compatible with published values for wild type and mutant viruses (Chou, 2017a, b; Chou et al., 2017; Chou et al., 2012; Chou et al., 2007). Specifically for maribavir, the baseline EC50 values determined here in ARPEp cells are comparable to values obtained in human embryonic lung (HEL) fibroblasts, and are much lower than those determined in human foreskin fibroblasts (Chou et al., 2006).

Table 1.

Checkerboard Combination Yield Reduction data

Drug A Maxa EC50b (Drug A) St Devc Drug B Maxa EC50b
(Drug B)		 St Devc CMV
Strain		 Genotype Nd Synergy Volume μM2% (95% confidence)
 Interpretatione
Synergism Antagonism
GW275175X 4 μM 0.67 0.10 Maribavir 0.6 μM 0.12 0.01 4190 Wild Type 6 0 −36 Additive
GW275175X 40 μM 6.0 0.81 Maribavir 0.6 μM 0.09 0.01 4274 UL89 D344E 6 0 −20 Additive
GW275175X 4 μM 0.89 0.22 Maribavir 48 μM 12 1.7 4352 UL97 T409M 5 3 −22 Additive
GW275175X 4 μM 0.70 0.16 Maribavir 8 μM 2.1 0.31 4353 UL97 H411Y 7 0 −16 Additive
Letermovir 12 nM 2.2 0.40 Maribavir 0.6 μM 0.12 0.02 4200 Wild Type 12 0 −44 Additive
Letermovir 400 nM 103 16 Maribavir 0.6 μM 0.12 0.03 4296 UL56 V236M 7 1 −19 Additive
Letermovir 12 nM 2.6 0.24 Maribavir 48 μM 12 2.0 4352 UL97 T409M 6 0 −28 Additive
Letermovir 12 nM 2.4 0.27 Maribavir 8 μM 2.2 0.33 4353 UL97 H411Y 5 0 −37 Additive
Ganciclovir 32 μM 2.0 0.05 Maribavir 1.0 μM 0.11 0.02 4175 Wild Type 5 0 −343 Antagonism
Rapamycin 8 nM 1.5 0.8 Maribavir 0.6 μM 0.11 0.03 4175 Wild Type 7 258 0 Synergism
Rapamycin 8 nM 1.3 0.39 Maribavir 1.2 μM 0.20 0.06 4347 UL27 R233S 7 152 0 Synergism
Rapamycin 8 nM 1.2 0.5 Maribavir 8 μM 1.8 0.7 4353 UL97 H411Y 9 153 0 Synergism
Foscarnet 200 μM 30 4.6 Maribavir 0.8 μM 0.09 0.01 4198 Wild Type 5 0 −41 Additive
Foscarnet 400 μM 93 16 Maribavir 0.6 μM 0.09 0.01 4376 UL54 981del2 6 0 −29 Additive
Cidofovir 1 μM 0.31 0.04 Maribavir 0.8 μM 0.10 0.03 4198 Wild Type 8 0 −28 Additive
Cidofovir 3 μM 1.3 0.18 Maribavir 0.6 μM 0.11 0.01 4376 UL54 981del2 5 3 −11 Additive
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Table Footnotes

a.

Maximum concentration and six two-fold dilutions of this concentration were tested in checkerboard assays

b.

Mean EC50 for the individual drug, concentration units same as in Max column

c.

Standard deviation of EC50 values

d.

Number of replicates of checkerboard testing (over at least 3 setup dates)

e.

Additive = synergy volume in ±50 range, Synergism = synergy volume 100 or more, Antagonism = synergy volume −100 or less

Across all the checkerboard assays, the maribavir EC50 against baseline CMV strains was 0.11 μM (SD 0.02, 42 replicates), against the UL97 mutant H411Y 2.0 μM (SD 0.51, 21 replicates), against UL97 mutant T409M 12.3 μM (SD 1.7, 11 replicates), and against UL27 mutant R233S 0.20 μM (SD 0.06, 7 replicates). Comparable published values as assayed in HEL cells are 0.11 μM, 1.2 μM, 11 μM and 0.2 μM respectively (Chou et al., 2012; Chou and Marousek, 2008; Chou et al., 2007). Single-drug EC50 values in Table 1 also confirm the expected lack of cross resistance of terminase-resistant mutants UL56 v236M and UL89 D344E to maribavir.

3.2. Antiviral activities of maribavir in combination with other drugs

Five to 12 replicates of checkerboard combination assays were performed on at least 3 separate dates and batches of cells to assess antiviral effects in combination. The ranges of tested drug concentrations were centered on the EC50 values of the individual drugs. All drug concentrations were below those documented to be cytotoxic in published literature (Williams et al., 2003), as confirmed in MTT cytotoxicity assays for the ARPEp cells used in this study. Pairwise combinations of 8 μM maribavir with each of ganciclovir (32 μM), cidofovir (4 μM) and foscarnet (400 μM), and 48 μM maribavir with each of GW275175X (40 μM), letermovir (200 nM) and rapamycin (20 nM) were tested in ARPEp cells. In three replicates of the MTT assay, there was no reduction from control values of the formazan-generated light absorbance after exposure to any of the above drug combinations or six of their 2-fold dilutions, indicating no measurable cytotoxicity at the drug concentrations used in the checkerboard assays. Quality control criteria for the checkerboard assays included a measurable EC50 endpoint for each drug alone, relative standard deviations of the mean percentage SEAP yield reductions at each drug combination of less than 16%, and no negative SEAP yield reductions averaging less than −2% from the no-drug control value. The averaged results across replicates as calculated using the MacSynergy II worksheet (Prichard and Shipman, 1990) yielded the volume of synergy indices at 95% confidence as shown in Table 1, which can be compared with similar data from previous studies (Evers et al., 2002; Wildum et al., 2015) calculated using the same technical approach.

For the combination of maribavir with the standard polymerase inhibitors cidofovir and foscarnet (Table 1), additive interactions were found against both wild type and mutant drug- resistant CMV strains. This differs from a previous report of synergy (Evers et al., 2002) and is more consistent with other reports of additive effects for the same drug pairs (Chou and Marousek, 2006; Selleseth et al., 2003). For the terminase inhibitors GW275175X and letermovir, additive effects were also found with maribavir. This contrasts with the strong synergy observed between maribavir and a related benzimidazole terminase inhibitor BDCRB (Evers et al., 2002). Letermovir was reported to have additive effects in combination with standard CMV DNA polymerase inhibitors (Wildum et al., 2015). The predominantly additive effects with a slight bias toward an antagonistic volume score were observed with both wild type strains and representative drug-resistant mutants. The volume of synergy 3-dimensional surface plot (MacSynergy II), at 99.9% confidence level for the data points, is shown for selected drug combinations in Figure 1. Figure 1A shows a typical additive result from the maribavir-letermovir combination against wild type virus. For most of the drug concentrations studied, there was no perceptible deviation from the predicted additive effect, but for a few of the higher drug concentrations in combination, there was a less than additive effect contributing to the mildly negative overall volume of synergy. Another way of stating this effect is that a virus yield reduction of 83% achieved by 12 nM letermovir was not further reduced with added maribavir at its maximal tested concentration of 0.6 μM, which on its own gave a yield reduction of 69%.

Figure 1. 3-Dimensional synergy plots for antiviral drug combinations with maribavir.

Figure 1

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The plots were generated from the results of 5 to 12 checkerboard assays of selected maribavir (MBV) drug combinations against wild type virus (Table 1), using MacSynergy II software. The difference between the observed viral SEAP growth inhibition and the amount expected from an additive effect of the drug concentrations was calculated (at 99.9% confidence level for these plots). The green-shaded plane positioned at the 0% inhibition % change represents the expected (theoretical) additive effect of the drug concentrations. Deviations from this plane at various drug concentrations are shown along their respective axes, with positive indicating synergy and negative indicating antagonism. A: Letermovir (LMV) combination, showing an additive effect at most concentrations and a limited zone of less than expected combination effects at the highest drug concentrations. B: Ganciclovir (GCV) combination, showing a strongly antagonistic effect. C: Rapamycin (RAPA) combination, showing a strongly synergistic effect.

The strongly antagonistic interaction of maribavir and ganciclovir is confirmed by a volume of synergy score of −343 (Table 1), or −247 at 99.9% confidence level, and by the deeply negative volume of synergy plot in Figure 1B. The yield reductions of 9–14% less than expected (99.9% confidence level) for additive effects encompass maribavir and ganciclovir concentrations close to or slightly higher than their respective single drug EC50 values.

A strongly synergistic interaction is shown for maribavir and rapamycin for wild type virus and mutants with low-grade (UL27 R233S ~2-fold) and moderate (UL97 H411Y ~20-fold) increases in maribavir EC50 (Table 1). The volume of synergy score for wild type virus is higher than that for two maribavir-resistant mutants, but a synergistic score is maintained for all 3 tested strains even when calculated at the 99.9% confidence level (scores of 180, 90 and 62, respectively). Figure 1C shows the volume of synergy plot for wild type virus, indicating that concentrations close to or higher than the respective single drug EC50 values combined to give yield reductions that are 5–12% higher than additive at 99.9% confidence. A yield reduction of 57% at 8 nM rapamycin or 65% at 0.6 μM maribavir became a yield reduction of 93% with a combination of 8 nM rapamycin and 0.6 μM maribavir.

4. Discussion

This study aimed to resolve some uncertainties left by conflicting older literature regarding the antiviral action of maribavir in combination with standard and experimental antivirals. The present findings show a predominantly additive antiviral effect of maribavir in combination with cidofovir, foscarnet, or the terminase inhibitors letermovir and GW275175X. The strong antagonism of maribavir and ganciclovir is confirmed. Conversely, a strong synergism is demonstrated between maribavir and rapamycin (sirolimus) at clinically meaningful concentrations.

Combination therapy directed at multiple viral drug targets remains insufficiently tested for CMV and has only recently become feasible with the introduction of newer antiviral options. Taken together, the available in vitro data show that maribavir can be used in combination with other licensed CMV antiviral drugs with an expectation of additive but not synergistic effects, except that maribavir strongly antagonizes the action of ganciclovir, an effect predicted from inhibition of pUL97-mediated ganciclovir phosphorylation.

A therapeutic opportunity may arise from the observed strong synergism between maribavir and rapamycin in vitro. The latter compound is the same as sirolimus, an mTor inhibitor used in transplant recipients and associated with significantly decreased CMV infection as compared with those treated with calcineurin inhibitors (Bowman et al., 2018). The maribavir-rapamycin synergy was suggested in a study of cell culture conditions affecting maribavir, but the effect was not quantitated (Chou et al., 2006). The range of rapamycin concentrations used here to show synergy are clinically relevant, as the usual therapeutic whole blood rapamycin concentrations are in the 4–12 nM range (Stenton et al., 2005). Mechanistically, the combination of maribavir and rapamycin is appealing because neither drug is able to shut off CMV replication completely by itself (Kudchodkar et al., 2004; Prichard, 2009). Cellular metabolic activity enables a reduced level of CMV replication even in the absence of UL97 kinase activity that is being inhibited by maribavir, with the level of growth impairment dependent on host cell physiology (Chou et al., 2006; Prichard, 2009; Webel et al., 2014). Rapamycin is shown here to inhibit CMV replication up to ~60% at low nanomolar concentrations under the prevailing cell culture conditions, but the much greater effect (>90% reduction) in synergy with maribavir suggests that the metabolic pathways being inhibited by rapamycin affect the cellular compensation for absence of UL97 kinase activity during CMV replication.

Clinical use of maribavir and sirolimus in combination requires consideration of drug interactions. The CYP3A4 pathway is involved in the metabolism of both maribavir and sirolimus, and the co-administration of maribavir and sirolimus is expected to increase sirolimus drug exposure, although the amount of change has not been well-documented (Pescovitz et al., 2009). A lung transplant recipient treated with maribavir as salvage therapy for ganciclovir- resistant CMV infection (case #2 of 6) experienced a rapid and sustained control of viral load but required dose adjustment for elevated sirolimus levels after starting maribavir (Avery et al.,2010). This case along with the supportive in vitro data suggests that further study of the maribavir-sirolimus combination in CMV therapy is warranted. Antiviral efficacy analyses of maribavir clinical trials should be stratified by the status of co-administration of sirolimus.

Highlights.

  • Maribavir was tested for anti-CMV effect in vitro in combination with other antivirals or with rapamycin (sirolimus)

  • Multiple checkerboard assays were analyzed using a 3-dimensional independent effects model

  • Maribavir showed additive interactions with foscarnet, cidofovir, letermovir and GW275175X

  • The combination of maribavir and ganciclovir is confirmed to be strongly antagonistic

  • The strongly synergistic combination of maribavir and sirolimus may be therapeutically useful

Acknowledgements

The authors thank Dr. Galen Carey (Shire, Cambridge, MA) and Dr. David Ehmann (Shire, Cambridge, MA) for their review and comments. This work was supported by a Shire-U.S. Department of Veterans Affairs (VA) Cooperative Research and Development Agreement (CRADA) and National Institutes of Health (NIH grant AI116635). VA provided research resources and facilities. KD is an employee of Shire. SC is principal investigator on an unrelated Merck-VA CRADA. Role of funding sources: All CRADA payments are to the institution for research costs; no portion is income for the principal investigator. Study design was a Shire-VA collaboration, except that maribavir-rapamycin combination assays were solely NIH-funded research. Collection, analysis and interpretation of data were performed by VA staff. The manuscript was written by the first author with review and comment by other authors and Shire staff. The contents are not a statement of views of any agency of the United States Government.

Footnotes

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