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Antimicrobial Agents and Chemotherapy, October 2001, p. 2925-2927, Vol. 45, No. 10
Center for Immunology and Microbial Diseases
and the Clinical Research Institute, Albany Medical College,
Albany, New York 12208,1 and
Departments of Antiinfectives2 and
Medicinal Chemistry,3 Pharmaceutical
Research Center, Bayer AG, D-42096 Wuppertal, Germany
Received 19 March 2001/Returned for modification 21 May
2001/Accepted 11 July 2001
BAY38-4766 and BAY43-9695 are nonnucleosidic compounds with
activities against human cytomegalovirus (HCMV). Two phenotypic assays
were used to determine the drug susceptibilities of 36 HCMV clinical
isolates to the BAY compounds and ganciclovir. Using either assay, both
BAY compounds at a concentration of approximately 1 µM inhibited the
replication of all 36 HCMV clinical isolates, including 11 ganciclovir-resistant clinical isolates, by 50%.
Human cytomegalovirus (HCMV) causes
considerable morbidity and mortality in immunocompromised hosts
(13). Organ transplant patients suffer from hepatitis and
pneumonia caused by HCMV infections, whereas AIDS patients suffer from
HCMV-induced retinitis and other complications (1). The
current Food and Drug Administration-approved therapies for retinitis
due to infection with HCMV include ganciclovir, foscarnet, cidofovir,
and fomivirsen (2, 11, 12, 14). These antiviral drugs are
active against infections caused by HCMV; however, they are not ideal
because of their toxicity and poor bioavailability. Furthermore,
long-term treatment with these antiviral agents often leads to the
selection of viral mutants that are resistant to one or more of these
drugs (3-5). Current research has led to the discovery of
several novel compounds with in vitro and in vivo activities against
HCMV (6, 7, 16). One such compound, BAY38-4766, is a
nonnucleosidic inhibitor of HCMV replication (17). Two
phenotypic drug susceptibility assays, a flow cytometric
fluorescence-activated cell sorter (FACS) assay (8-10) and a plaque reduction assay (PRA)
(15), were used to compare the effects of BAY38-4766, its
main metabolite, BAY43-9695, and ganciclovir on the in vitro
replication of ganciclovir-susceptible and ganciclovir-resistant HCMV
clinical isolates. BAY38-4766 and BAY43-9695 inhibited the replication
of ganciclovir-sensitive and ganciclovir-resistant HCMV clinical
isolates at concentrations less than or equal to 1 µM. These results
suggest that these compounds are potentially useful for treating
patients infected with ganciclovir-sensitive or ganciclovir-resistant HCMV.
The use of the FACS assay and the PRA for determining 50% inhibitory
concentrations (IC50s) for HCMV clinical isolates have been
described in detail previously (8-10). These two
phenotypic assays were used to determine the susceptibilities of the
AD169 laboratory strain of HCMV and 36 HCMV clinical isolates to
BAY38-4766, BAY43-9695, and ganciclovir. The FACS assay yielded average
IC50s of BAY38-4766, BAY43-9695, and ganciclovir for
the AD169 laboratory strain of 0.95 ± 0.17 (mean ± standard
deviation), 0.70 ± 0.30, and 3.05 ± 0.21 µM,
respectively. The PRA yielded average IC50s of these three
drugs for AD169 of 0.64 ± 0.14, 0.55 ± 0.06, and 3.50 ± 0.21 µM, respectively. The average IC50s of the two
BAY compounds and ganciclovir for 36 HCMV clinical isolates are
presented in Table 1. Both BAY compounds
inhibited the replication of all of the HCMV clinical isolates by 50%
at essentially the same concentrations. Of the 36 HCMV clinical
isolates, 25 were susceptible to ganciclovir (IC50s less
than 8 µM) and 11 were partially or completely resistant to
ganciclovir (IC50s between 9 and >96 µM). For the
ganciclovir-susceptible clinical isolates, the average
IC50s of BAY38-4766 and BAY43-9695 were approximately
one-third of the average IC50s of ganciclovir. Furthermore,
both BAY compounds inhibited the replication of ganciclovir-resistant HCMV clinical isolates by 50% at average concentrations near 1 µM.
The PRA yielded very similar ranges for and average IC50s of these three drugs for the HCMV clinical isolates. When the biases
and levels of precision of the IC50s of each of the three drugs for these HCMV clinical isolates, determined by the FACS assay
and the PRA, were compared, there was a <2-fold difference, suggesting
that the more rapid FACS assay is equivalent in efficacy to the PRA.
These results show that BAY38-4766 and BAY43-9695 inhibited replication
for a large number of HCMV clinical isolates, including some that were
partially or completely resistant to ganciclovir.
0066-4804/01/$04.00+0 DOI: 10.1128/AAC.45.10.2925-2927.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Susceptibilities of Human Cytomegalovirus Clinical
Isolates to BAY38-4766, BAY43-9695, and Ganciclovir
ABSTRACT
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TABLE 1.
IC50s of compounds for HCMV clinical isolates
To be therapeutically useful, antiviral compounds must be effective in
the presence of serum proteins. To determine the effect of serum
proteins on the ability of these BAY compounds to inhibit HCMV
replication, human foreskin fibroblasts were infected with the AD169
laboratory strain of HCMV in the presence of various concentrations of
these compounds in medium containing 4% human albumin and 1 mg of
-1 acid glycoprotein/ml and the cells were analyzed for the
percentage of IE antigen-positive cells by FACS analysis. In the
absence of any additives, the IC50s for AD169 were 0.65 µM for BAY38-4766 and 0.53 µM for BAY43-9695. In the presence of
serum proteins, the IC50s were 6.2 µM for BAY38-4766 and
8.42 µM for BAY43-9695. In contrast, the IC50s of
ganciclovir were 5.36 µM in the absence of serum proteins and 4.53 µM in the presence of serum proteins, confirming that ganciclovir
does not bind extensively to serum proteins. These results show that
the presence of plasma binding proteins decreases the virological activities of these compounds 10- to 16-fold and that ganciclovir was
unaffected by the presence of these binding proteins.
The preceding data show that both compounds have activities against
HCMV and that they bind serum proteins. To determine if combinations of
the two drugs are additive or synergistic, the AD169 laboratory strain
was grown in the presence of serum binding proteins and various
concentrations of the two compounds and the percentage of IE
antigen-positive cells was determined by FACS analysis. The results are
shown in Fig. 1. The value of the parameter was 0.216, and the 95% confidence interval was
0.074 to 0.358. The value of ± the 95% confidence interval
overlaps zero; therefore, the interaction is additive. These data
suggest that BAY43-9695, the main in vivo metabolite of BAY38-4766, may enhance the antiviral effect of the parent compound by establishing longer-lasting inhibitor levels after application.
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We have demonstrated that micromolar concentrations of BAY38-4766 and BAY43-9695 inhibited the replication of both ganciclovir-susceptible and ganciclovir-resistant HCMV clinical isolates. The antiviral activities of the two compounds are additive. However, protein binding studies demonstrated that approximately 90% of each of the two BAY compounds is bound to proteins. Both BAY compounds inhibit the replication of ganciclovir-resistant HCMV clinical isolates, suggesting that the BAY compounds have a different mode of action. These BAY compounds join other anti-HCMV compounds that have modes of action different from those of ganciclovir, foscarnet, and cidofovir (6, 7, 16). These novel compounds represent new classes of drugs that are potentially useful for controlling HCMV infections in immunocompromised patients infected with HCMV isolates that are resistant to the currently used antiviral drugs.
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ACKNOWLEDGMENTS |
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A 5 mM stock of ganciclovir was provided by the Virology Quality Assurance Program, Division of AIDS, NIAID. BAY38-4766 and its main metabolite, BAY43-9695, were provided by the Bayer Corporation, West Haven, Conn.
This work was supported in part by a grant from the Bayer Corporation and by grants AI45350 and AI45257 from the National Institutes of Health.
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FOOTNOTES |
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* Corresponding author. Mailing address: Center for Immunology and Microbial Diseases, Mail Code 151, Albany Medical College, 47 New Scotland Ave., Albany, NY 12208. Phone: (518) 262-5174. Fax: (518) 262-5748. E-mail: mcsharj{at}mail.amc.edu.
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Antimicrobial Agents and Chemotherapy, October 2001, p. 2925-2927, Vol. 45, No. 10
0066-4804/01/$04.00+0 DOI: 10.1128/AAC.45.10.2925-2927.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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