a Departments of Antiinfectives and b Medicinal Chemistry, Pharmaceutical Research Center, Bayer AG, Aprather Weg 18a, POB 101709, D-42096 Wuppertal, Germany; c Departments of Cancer Research and e Microbiology, Pharmaceutical Division, Bayer Corporation, 400 Morgan Lane, West Haven, CT 06516-4175, USA; d Pharmaceutical Research, Bayer S.p.A., Via Olgettina 58, 20132 Milano, Italy
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Abstract |
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Introduction |
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HCMV is widespread in the human population. In immunocompetent individuals, infection is inapparent or associated with mild symptoms. However, HCMV infection is the leading cause of neurological disease and hearing loss in congenitally infected newborns10 affecting some 8000 newborns per year in the USA. Furthermore, following the first 100 days after transplantation, HCMV-induced pneumonia develops in about 50% of heterologous bone marrow transplants with an 80% mortality rate if untreated.11 Approximately 1570% of kidney, liver, bone-marrow and heart/lung transplant recipients are affected by HCMV hepatitis and pneumonia resulting in decreased graft and patient survival.12 HCMV-positive graft donors and HCMV-negative graft recipients are the major risk factors in solid organ transplantation. HCMV was found in 90% of AIDS patients at autopsy. Before the advent of highly active antiretroviral therapy (HAART), HCMV retinitis occurred in about 1045% of patients with late-stage AIDS.13 In addition, some 710% of AIDS patients may have HCMV diseases of the gastrointestinal tract and the nervous system presenting with colitis, oesophagitis and wasting syndrome. While the use of HAART has diminished the impact of HCMV disease significantly,14 cessation of treatment in patients with virological and immunological failure under potent antiretroviral therapy led to recurrence of HCMV retinitis.15 Antiviral resistance emerges in 1437% of AIDS patients with HCMV retinitis treated for 9 months with ganciclovir, cidofovir or foscarnet.16,17
Currently, only inhibitors of herpesviral DNA polymerases are licensed for the prophylaxis and treatment of HCMV infections,18 but these anti-HCMV therapies do not eliminate virus or eradicate infection in any individual. Despite their antiviral potential all of these medications are associated with multiple side effects, such as dose-limiting bone marrow and kidney toxicity, as well as the emergence of single and double drug resistance.19,20 Even the recently developed phosphorothioate antisense oligonucleotide fomivirsen (ISIS 2922)21 for the treatment of HCMV retinitis in AIDS patients can only be applied intravitreally and is associated with increased intraocular pressure and ocular inflammation in 25% of treated patients.
We report on BAY 38-4766 {3-hydroxy-2,2-dimethyl-N-[4({[5-(dimethylamino)-1-naphthyl]sulfonyl}amino)-phenyl]propanamide} (Figure 1), a novel oral non-nucleosidic inhibitor of HCMV that targets virus-specific proteins known to be required for the cleavage and packaging of viral DNA by processing high molecular weight viral DNA concatemers to monomeric genome length. In particular, as this viral DNA processing machinery has no counterpart in human cells but is highly conserved among herpesviruses, and therefore very specific for these viruses, this approach facilitates the generation of novel drugs with excellent tolerability and also has the potential for broad-spectrum action against a number of human herpesviruses.
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Materials and methods |
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The laboratory strains HCMV-Davis [ATCC VR 807 of the American Type Culture Collection (ATCC), Rockville, MD, USA], HCMV-AD169 (ATCC VR 538), HCMV-Towne (ATCC VR 977), as well as the clinical isolates HCMV-He (kindly provided by Dr A. Eis, University of Bonn, Institute of Medical Microbiology, Germany), HCMV-UlmB, HCMV-B.K./19684, HCMV-R.R./17272, HCMV-M.F./16445 and HCMV-isolate 2 (obtained from Professor J. Mertens, University of Ulm, Germany), were propagated in human embryonic lung fibroblast (HELF) or normal human dermal fibroblast (NHDF; CellSystems, St Katharinen, Germany) cells at passages 1040 or 620, respectively. HELF and NHDF cells were cultivated in Eagle's minimal essential medium with Earl's salts (EMEM) supplemented with 10% (v/v) fetal calf serum (FCS), hereafter referred to as EMEM/10. Murine cytomegalovirus (MCMV) strain Smith (ATCC VR 194) was propagated in murine embryonic fibroblast (MEF) or NIH 3T3 cells (ATCC CRL 1658). MEF cells were prepared from embryos of late-stage pregnant BALB/c mice by trypsinEDTA disintegration and stored in liquid nitrogen at early passages, and were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% (v/v) FCS. NIH 3T3 cells were propagated in EMEM/10. All cell culture media and supplements were from Gibco-BRL, manufactured by Life Technologies Ltd (Paisley, UK). The following reference drugs were used: iv formulations of Cymevene (ganciclovir sodium; Syntex/Roche, Grenzach Wyhlen, Germany), Foscavir (foscarnet sodium; Astra Pharmaceuticals, Wedel, Germany) and Vistide (cidofovir; Pharmacia & Upjohn SA, Luxembourg, Luxembourg) as 50 mM solutions in 0.9% saline. BAY 38-4766 was used as a 50 mM solution in dimethylsulphoxide (DMSO).
Preparation of virus stocks
Confluent HELF cell cultures were infected with cellassociated or cell-free virus at a multiplicity of infection (moi) of 0.0010.005. After removal of the viral inoculum, the cells were incubated at 37°C/5% CO2 for a further 2 days after reaching 100% cytopathic effect (CPE). Culture supernatant containing cell-free virus was cleared by low speed centrifugation and stored at -140°C or in liquid nitrogen. Virus-infected cells were collected by trypsinization and distributed to confluent HELF tissue culture bottles. After reaching complete CPE, infected cells were collected in freezing medium (EMEM plus 20% FCS and 10% DMSO). Aliquots were carefully frozen and stored at -140°C or in liquid nitrogen. Confluent monolayers of MEF or NIH 3T3 cells were infected with MCMV strain Smith as described above. Reaching a complete CPE, infected cell cultures were harvested by three freezing and thawing cycles and subsequent sonification for 30 s. After low speed centrifugation, aliquots of cell-free virus were stored at -140°C.
Anti-HCMV and anti-MCMV cytopathogenicity assays
Test compounds were used as 50 mM DMSO stock solutions. Ganciclovir, foscarnet and cidofovir served as reference drugs. Addition of 50, 5, 0.5 and 0.05 mM DMSO-stock solutions to culture medium in duplicates was followed by serial two-fold dilutions in 96-well microtitre plates. Each well was supplemented with 1 x 104 to 3 x 104 cells of a suspension of infected and uninfected HELF or NHDF cells (moi = 0.0010.002). Wells without either drug and virus or wells without drug served as cell and virus controls, respectively. Final drug concentrations were between 250 and 0.0005 µM. Plates were incubated for 6 days at 37°C/5% CO2 until infected virus controls reached 100% CPE. A neutral red dye solution (3.33 g/L) was added to the wells (final concentration 56 µg/mL) and plates were stored at 37°C for 1 h followed by a 2 h staining in the dark at room temperature (RT). Neutral red-stained monolayers were fixed with 4% formaldehyde by incubation for 30 min at RT. After three washing cycles with deionized water, plates were dried at 56°C followed by visual evaluation using an overhead microscope (Plaque multiplier; Technomara, Zurich, Switzerland). The following data were drawn from the assay plates: EC50 (HCMV) = concentration of drug that inhibits the CPE by 50% compared with an untreated virus-infected control; CC50 (HELF) = highest concentration of drug with no visible cytostatic effects on cells compared with the untreated cell control; SI = selectivity index = CC50 (HELF)/EC50 (HCMV).
For anti-MCMV assays the same procedure was used, with some exceptions. A concentrated suspension of MEF or NIH 3T3 cells was mixed with a cell-free virus suspension (moi 0.050.1) and incubated for 15 min before being diluted to 1.3 x 105 cells/mL and added to the drug dilutions in 96-well plates. Incubation for 5 days was followed by fixation with 4% formaldehyde/Giemsa solution for 30 min. Plates were washed three times, dried and evaluated as described above.
HCMV and MCMV plaque assays
HELF and NHDF cells (1 x 105 to 2 x 105/well) or MEF and NIH 3T3 cells (1 x 105/well) were seeded in 24-well tissue culture plates. Confluent cell monolayers were infected with 4060 plaque-forming units (pfu) per well. After a 1 h adsorption period, a 0.5% methylcellulose (MC)EMEM/10 overlay medium and the appropriate drug solutions were added. HCMV-infected cell cultures were stained after 712 days with medium changes every 34 days and MCMV plaque assays after 5 days without overlay exchange, using neutral red dye or Giemsa's solution, respectively. Plaques were counted visually with the aid of an overhead microscope (Plaque viewer). The number of plaques in the treated wells was expressed as a percentage of untreated virus control and plotted against the logarithm of drug concentration. Drug concentrations producing 50% reduction in plaque formation (EC50) were determined graphically from the doseresponse curves. Assays were carried out two to four times in duplicate. For titration of crude virus stocks, confluent monolayers of cells were infected with a serial log dilution (10-110-6) of stock virus and processed accordingly.
Cytotoxicity and antiviral fluorescence assays
In order to evaluate drug toxicity, 96-well microtitre plates were prepared with 100 µL of EMEM/10 per well. After addition of 2 µL of 50 mM compound stock solutions in duplicate into 198 µL in row 2, serial two-fold dilutions were made with 100 µL up to row 12 and 100 µL of a HELF, NHDF or 3T3 cell suspension (5 x 103 cells/mL) were added per well. Row 1 served as an untreated cell control. After incubation for 6 days at 37°C and 5% CO2, the cells were washed once with phosphate-buffered saline (PBS), and 200 µL of a 10 µg/mL fluorescent dye solution in PBS, pH 7.2 (fluorescein diacetate) were dispensed per well. After 45 min, the fluorescence signal was measured with a Fluorskan Ascent fluorimeter (Labsystems, Finland) (excitation filter 485 ± 11 nm, emission filter 530 ± 15 nm). The relative fluorescence units (RFUs) of treated cells were expressed as percentages of untreated cell controls and CC50 values were determined graphically. The anti-HCMV and anti-MCMV cytopathogenicity assays followed the same procedure as described above, with the exception that the moi values were 0.03 and 0.2 and the incubation times 15 days and 7 days, respectively. Thereafter, medium was removed and the wells were washed with 200 µL of PBS, and 45 min after addition of 200 µL of the fluorescence dye solution, signals were measured as described. The RFUs of treated wells were expressed as percentages of the difference between RFUs of the untreated cell and virus controls and CC50 and EC50 values were estimated graphically.
Selection of HCMV- and MCMV-resistant strains
To select drug-resistant viral mutants, HCMV strain AD169 was serially passaged in HELF cells in the presence of BAY 35-5014, a structural analogue of BAY 38-4766. MCMV strain Smith was passaged on NIH 3T3 cells in the presence of BAY 38-4766. Selection of pre-existing mutants was started by infecting cultured cells with an moi of 0.0010.005 at a drug concentration EC50. Selection of the mutant virus generated was achieved by serial passage of progeny virus from the culture overlay medium in the presence of increasing compound concentrations (two-fold steps). The resultant mutant of HCMV strain AD169, as well as MCMV strain Smith, were growing at 50-fold and 500-fold above the EC50, respectively. Resistant progeny virus was plaque-purified by limiting dilution in the presence of the respective compounds. Stability of resistance was tested by serially passaging (1012 times) plaque-purified viruses without selective drug pressure.
Preparation of viral DNA for sequencing and Southern blot analysis
Confluent monolayers of HELF cells (5 x 107) were infected with resistant HCMV AD169 at an moi of 0.25 0.5 and treated with various drug concentrations until untreated, infected control cells showed 100% CPE. Supernatant was harvested, cell debris removed by low-speed centrifugation and extracellular virions were collected by ultracentrifugation for 1 h, at 45 000g and 4°C. Pellets were resuspended using a PotterElvehjem homogenizer and further purified by a centrifugation step through a 15%/40% sucrose cushion for 1 h and 150 000g at 4°C. The Qiagen Blood & Cell Culture DNA kit (Qiagen, Hilden, Germany) was applied as instructed by the manufacturer. Construction of an overlapping cosmid library and sequence analyses were carried out by Qiagen. Resistant MCMV Smith was propagated using NIH 3T3 cells (moi = 0.005) and harvested as described above. Sequences homologous to mutant HCMV AD169 were amplified by PCR using a pfu polymerase and analysed by double-stranded sequencing. For Southern blot analyses, viral DNA was prepared using the Qiagen Blood & Cell Culture DNA kit as instructed by the manufacturer after disruption of collected cells by repeated freezing and thawing cycles. To quantify viral DNA, dot-blot hybridization was carried out using a DIG-labelled, randomly primed 300 bp PCR fragment (HCMV genome position 7031524). DNA (2.5 µg per lane) was digested overnight with 20 U KpnI, sizefractionated on a 0.7% agarose gel by electrophoresis and subjected to capillar transfer on positively charged nylon membranes. After UV-crosslinking, the blot was incubated four times for 3 min each time with Soak I (0.5 M NaOH; 1 M NaCl), twice with Soak II (3 M NaCl; 0.5 M TrisHCl, pH 7.4), baked at 120°C for 30 min, prehybridized in a standard hybridization buffer (5 x SSC, formamide, 50%; N-lauroylsarcosine, 0.1% w/v; SDS, 0.02%; blocking reagent, 2%; 20 mL/100 cm2 filter) for 2 h at 42°C and hybridized overnight in the presence of randomly DIG-labelled DNA probe (20 ng/mL). Detection was carried out by luminescence as instructed by the manufacturer (Roche, Germany).
MCMV pathogenesis model: in vivo passage of MCMV
Salivary glands of BALB/c mice were harvested 23 weeks post-infection and homogenized in EMEM/10 using an Ultra Turrax (IKA Labortechnik, Staufen, Germany). After freezing (-80°C) and thawing, the debris was removed by centrifugation at 2000g for 15 min at 4°C. Virus was concentrated by ultracentrifugation through a 15% sucrose cushion using a SW28 rotor at 100 000g for 90 min and titre was determined by plaque assay on MEF cells.
Infection and treatment of mice
Five- to 6-week-old male homozygous NOD/LtSz-SCID/j mice (Jackson Laboratory, Bar Harbor, ME, USA) were infected ip using 5 x 105 pfu in 0.2 mL. Approximately 20 h after infection, test compounds were administered as a suspension in 0.5% tylose to the animals twice daily for 8 consecutive days po using a gavage. Daily dosages of 3, 10, 30 and 100 mg/kg body weight (bw) per application in a total application volume of 10 mL/kg bw were given. Animals were killed 16 h after the last application and organs were removed for further analyses.
Isolation of nucleic acids and dot-blot analysis
The nucleic acids were extracted using the QIAamp Tissue Kit (Qiagen) as instructed by the manufacturer. After quantification of genomic DNA, dot-blot hybridization was carried out with a DIG-labelled, randomly primed 1300 bp PCR-amplified fragment from the HindIII J fragment of MCMV strain Smith. DNA (10 µg) was blotted on to a nylon membrane soaked four times for 3 min each time with 0.5 M NaOH/1 M NaCl, twice with 3 M NaCl/0.5 M TrisHCl, pH 7.4, baked at 120°C for 30 min, prehybridized in a standard hybridization buffer (5 x SSC, formamide, 50%; N-lauroylsarcosine, 0.1% w/v; SDS, 0.02%; blocking reagent, 2%; 20 mL/100 cm2) for 2 h at 42°C and hybridized overnight in the presence of the probe (25 ng/mL). After washing, immunological detection was carried out using the CDP-Star system (Roche) as instructed by the manufacturer. Intensity of chemiluminescence signals was quantified using the LumiImager system (Roche). The LumiImager signals were analysed by descriptive statistics and compared by Variance analysis with post hoc comparison of means (Statistica; StatSoft, Tulsa, OK, USA).
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Results |
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The anti-HCMV activity and selectivity of BAY 38-4766 (Figure 1) and the reference drug ganciclovir were evaluated against different laboratory and human field strains in a plaque inhibition and an HCMV cytopathogenicity assay (6 day; neutral red dye staining) as well as an HCMV cytopathogenicity assay (15 day; fluorescence dye staining) using HELF or NHDF cells (Table 1
; Figures 1 and 2
). Results of plaque assays showed that all strains were sensitive to BAY 38-4766 with an averaged EC50 of 1.03 ± 0.57 µM. Most notably, the drug was also active against the ganciclovir-resistant clinical isolates, as well as the ganciclovir/foscarnet and ganciclovir/cidofovir double-resistant clinical isolates. Ganciclovir was about four times less active with an averaged EC50 of 4.32 ± 1.82 µM for the sensitive strains. Using two different HCMV pathogenicity assays antiviral activity data were confirmed for both drugs, although somewhat lower drug concentrations were needed to achieve 50% inhibition of cytopathogenicity of the laboratory strain HCMV Davis (EC50 = 0.3 and 1.9 µM for BAY 38-4766 and ganciclovir, respectively). No activity was found against HSV type 1 and type 2, varicella-zoster virus or human herpesvirus 6, nor against HIV, hepatitis B virus, adenovirus type 5 or measles virus. In contrast, BAY 38-4766 was found to be active against various monkey CMV strains (EC50 < 1 µM), but most pronounced inhibitory effects were found for various rodent CMV strains (data not shown) including the MCMV (see Figure 1
), offering the possibility of assessing the impact of the drug in a MCMV pathogenicity model in mice (see below). Compared with the HCMV Davis strain the impact of BAY 38-4766 on the replication of the MCMV strain Smith was nearly 10-fold stronger in both the plaque and cytopathogenicity assays (EC50 = 0.05 and 0.04 µM, respectively). Cytostatic activities of BAY 38-4766 in human NHDF and HELF cells as well as in murine 3T3 cells observed visually after staining from the respective HCMV or MCMV screening assays (Figure 1
) compared favourably with quantitative data of HCMV and MCMV screening assay procedures using a fluorescence dye signal evaluation (Figure 2
) as well as with data from 6 day cytotoxicity fluorescence assays (data not shown) using uninfected NHDF and 3T3 cells (CC50 = 93 and 63 µM, respectively). Based on these data, selectivity indices (SI = CC50/EC50) of nearly 300 (HCMV) and 1600 (MCMV) were calculated.
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The laboratory strain HCMV AD169 was made resistant to increasing concentrations of a BAY 38-4766-related compound over multiple in vitro passages (time >6 months) starting with the EC50. The resistant strain showed cross-resistance to BAY 38-4766 (Table 2a) and to various other BAY 38-4766-related structures (data not shown). As indicated by the resistance index (RI), no cross-resistance was shown for the nucleoside reference drugs, or the pyrophosphate analogue foscarnet, indicating a different mode of action. The same results were obtained with the resistant MCMV strain (Table 2b
) generated with increasing concentrations of BAY 38-4766 (within nearly 2 months). Interestingly, the MCMV-resistant strain showed much higher resistance compared with the resistant HCMV AD169 strain. The marketed reference drugs did not discriminate between the wild-type and resistant strains, again indicating a different target of drug interference.
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Due to its inhibitory potential against rodent CMV strains, BAY 38-4766 was tested in a murine pathogenicity model, in which immunodeficient mice were inoculated with MCMV. This viral infection is lethal. Spread of the virus into various organs occurs in this model as is also observed in human CMV infections under conditions of immunodeficiency. Survival of the mice after po treatment with BAY 38-4766 was compared with that of ganciclovir. Uninfected mice treated similarly with the test compounds did not develop weight loss. In acute toxicity studies the lethal dose, LD50, in mice and rats was >2000 mg/kg bw after oral administration of BAY 38-4766 (data not shown). Treatment of MCMV-infected animals for 8 days with increasing doses of ganciclovir and BAY 38-4766, respectively, showed a comparable effect of both drugs on survival, which was dose dependent (Figure 4). To clarify the in vivo drug activity in more detail, weight reduction and reduction of MCMV DNA in different target organs was determined for ganciclovir and BAY 38-4766. In this murine model, BAY 38-4766 decreased viral load in target organs comparably to ganciclovir. The clinical parameter (weight loss as a consequence of viral infection) showed a similar efficacy as well (see Tables 4a and b
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Discussion |
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BAY 38-4766 is a representative of our newly discovered non-nucleosidic class of inhibitors of the human ß-herpesvirus, HCMV (Figure 1).2527 A large panel of laboratory HCMV strains and clinical isolates were shown to be several times more sensitive to BAY 38-4766 than to ganciclovir. Ganciclovir-resistant as well as ganciclovir/foscarnet and ganciclovir/cidofovir double-resistant clinical isolates were as susceptible to BAY 38-4766 as wild-type strains. These latter results indicate that BAY 38-4766 may act by a mode of action distinct from all these different DNA polymerase inhibitors. Calculated selectivity of anti-CMV activity in HELF or NHDF cells (reflecting three and four cell or virus generations) was nearly 300 in case of HCMV and nearly 1600 for MCMV in 3T3 cells (Figures 1 and 2
). In contrast to the lack of activity against the human
-herpesviruses (HSV type 1, type 2 and varicella-zoster virus), against a human
-herpesvirus, EBV, as well as the EBV-related mouse
-herpesvirus (MHV-68) and even against the human ß-herpesvirus 6 (HHV-6), BAY 38-4766 was found to be inhibitory to various monkey CMV strains but most particularly to rodent CMV strains.27
Sequence analyses of the genomes of two drug-resistant CMVs revealed several amino acid exchanges in UL89, encoding part of the putative viral terminase22,23 and UL104, a minor structural component of virions and capsids. Both proteins are essentially involved in the process of viral DNA concatemer cleavage and packaging of genomes into procapsids, as has been shown for respective homologues of the HSV or bacteriophage T4 maturation and packaging complex.2830 These data together with our DNA cleavage analysis indicate that both UL89 and UL104, alone or by interaction, most likely represent the molecular antiviral drug target. Therefore, maturation and spread of viral particles to uninfected host cells are inhibited by a new unique mechanism of action. Although it was proposed that inhibition of HCMV DNA maturation by the benzimidazole ribonucleoside BDCRB is mediated through the UL89 gene product and resistance to TCRB maps to the two ORFs UL89 and UL5622,23 we did not find cross-resistance of our HCMV AD169 sulphonamide-resistant strain to BDCRB.27 Unfortunately, because BDCRB is not active against MCMV, we could not confirm this result using our MCMV Smith BAY 38-4766-resistant strain. The fact that only two identified mutations of the highly resistant MCMV Smith map to UL89 exon II and UL104 indicates that in the case of HCMV, development of resistance is more complex compared with the murine virus, which was also reflected by a markedly prolonged generation time for drug-resistant HCMV. Thus, it can be expected that the requirement to accumulate multiple mutations to generate a resistant phenotype may translate into a relatively slow development of clinical HCMV resistance. However, future mutational analyses have to elucidate which amino acid exchanges confer resistance. In addition, the mechanism, which is distinct from those of the marketed drugs, will offer the possibility of treating patients who have acquired resistance to these agents.
Apart from offering a new highly specific approach to the inhibition of herpesviruses, this new mechanism of action could potentially also have beneficial immunological consequences. During treatment with BAY 38-4766, viral protein synthesis continues, but due to the lack of monomeric genomic length DNA, only empty particles (dense bodies) can be formed.31 It is conceivable that these non-infectious viral particles could aid the establishment of an antiviral immune response, leading to better control of the virus by the host. This mechanism appears possible in all cases where an immuno-incompetent host (re)gains immune competence (newborns, transplant recipients). However, proof of this theoretical benefit will have to await clinical studies.
In summary, by studying the mechanism of action of this novel drug class,32 which in vitro33 and in vivo34 selectively inhibits cytomegaloviruses, we have discovered an antiviral approach that may be of consequence for other members of the herpesvirus group as well, potentially allowing for the generation of broad-spectrum drugs. In addition, since a similar DNA maturation process does not occur in higher cells, this principle offers the potential for high selectivity, in contrast to many of the viral DNA polymerase inhibitors, which also interact with cellular enzymes and hence can have severe side effects. Furthermore, favourable pharmacokinetic data of BAY 38-476635 and its main active metabolite36 in mice, rats and dogs as well as excellent safety, tolerability and pharmacokinetic data after single oral doses in healthy male subjects37 with expected therapeutic benefits are encouraging markers of this new class of non-nucleosidic anti-HCMV inhibitors.
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Acknowledgements |
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Notes |
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Present address. Department of Cancer Research, Pharmaceutical Division, Bayer Corporation, 400 Morgan Lane, West Haven, CT 06516-4175, USA.
Present address. Research and Development, Mittenyi Biotec GmbH, Friedrich-Ebert-Strasse 68, D-51429, Bergisch Gladbach, Germany.
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Received 29 November 2000; returned 27 February 2001; revised 11 June 2001; accepted 13 July 2001