Preclinical evaluation of HBY 097, a new nonnucleoside reverse transcriptase inhibitor of human immunodeficiency virus type 1 replication

Article abstract of DOI:10.1128/AAC.39.10.2253

HBY 097 [(S)-4-isopropoxycarbonyl-6-methoxy-3-(methylthiomethyl)-3,4- dihydroquinoxaline-2(1H)-thione] was selected from a series of quinoxalines as a nonnucleoside inhibitor of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (NNRTI). HBY 097 was shown to be a highly potent inhibitor of HIV-1-induced cell killing and HIV-1 replication in a variety of human cell lines as well as in fresh human peripheral blood lymphocytes and macrophages. The compound was also active against a variety of clinical isolates of HIV-1 including different HIV-1 subtypes and viruses resistant to 3'-deoxy-3'-azidothymidine. Mutant reverse transcriptases which arise as a consequence of treatment with other nonnucleoside inhibitors of HIV-1 reverse transcriptase were still inhibited by HBY 097 at relatively low concentrations. An HIV-1(MN) variant resistant to inhibition by HBY 097 displayed in the reverse transcriptase gene a mutation causing a substitution at position 190 of a glutamic acid for a glycine residue (G190→E), which is characteristic for quinoxaline derivatives. The drug was demonstrated to possess a favorable toxicity profile and to show good oral bioavailability in both mice and dogs. As a consequence of its outstanding properties, HBY 097 was selected for further development and is at present undergoing clinical trials.

Full text of DOI:10.1128/AAC.39.10.2253

Preclinical evaluation of HBY 097, a new
nonnucleoside reverse transcriptase inhibitor
of human immunodeficiency virus type 1
replication.
J P Kleim, R Bender, R Kirsch, C Meichsner, A Paessens, M
Rösner, H Rübsamen-Waigmann, R Kaiser, M Wichers and K E
Schneweis
Antimicrob. Agents Chemother. 1995, 39(10):2253. DOI:
10.1128/AAC.39.10.2253.
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ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Oct. 1995, p. 2253–2257
0066-4804/95/$04.00ϩ0
Copyright ᭧ 1995, American Society for Microbiology
Vol. 39, No. 10
Preclinical Evaluation of HBY 097, a New Nonnucleoside Reverse
Transcriptase Inhibitor of Human Immunodeficiency
Virus Type 1 Replication
1
1
1
1
¨
JORG-PETER KLEIM, * RUDOLF BENDER, REINHARD KIRSCH, CHRISTOPH MEICHSNER,
2
1
2
¨
¨
ARNO PAESSENS, MANFRED ROSNER, HELGA RUBSAMEN-WAIGMANN,
ROLF KAISER,3 MARIA WICHERS,3 KARL EDUARD SCHNEWEIS,3
IRVIN WINKLER,1 AND GUNTHER RIESS
¨
1
General Pharma Research, Hoechst AG, D-65926 Frankfurt,1 Institute of Virology, Pharma Research Center,
Bayer AG, D-42096 Wuppertal,2 and Institute of Medical Microbiology,
University of Bonn, D-53105 Bonn,3 Germany
Received 26 May 1995/Returned for modification 6 July 1995/Accepted 31 July 1995
HBY 097 [(S)-4-isopropoxycarbonyl-6-methoxy-3-(methylthiomethyl)-3,4-dihydroquinoxaline-2(1H)-thione]
was selected from a series of quinoxalines as a nonnucleoside inhibitor of human immunodeficiency virus type
1 (HIV-1) reverse transcriptase (NNRTI). HBY 097 was shown to be a highly potent inhibitor of HIV-1-induced
cell killing and HIV-1 replication in a variety of human cell lines as well as in fresh human peripheral blood
lymphocytes and macrophages. The compound was also active against a variety of clinical isolates of HIV-1
including different HIV-1 subtypes and viruses resistant to 3؅-deoxy-3؅-azidothymidine. Mutant reverse tran-
scriptases which arise as a consequence of treatment with other nonnucleoside inhibitors of HIV-1 reverse
transcriptase were still inhibited by HBY 097 at relatively low concentrations. An HIV-1MN variant resistant
to inhibition by HBY 097 displayed in the reverse transcriptase gene a mutation causing a substitution at
position 190 of a glutamic acid for a glycine residue (G1903E), which is characteristic for quinoxaline
derivatives. The drug was demonstrated to possess a favorable toxicity profile and to show good oral bioavail-
ability in both mice and dogs. As a consequence of its outstanding properties, HBY 097 was selected for further
development and is at present undergoing clinical trials.
Nonnucleoside inhibitors of human immunodeficiency virus
type 1 (HIV-1) reverse transcriptase (RT), referred to as
NNRTIs, have been shown to be potent suppressors of in vitro
HIV-1 replication (see reference 7 for a review). Belonging to
different chemical classes, including tetrahydroimidazo (4,5,1-
jk)(1,4)-benzodiazepin-2(1H)-one and -thione (TIBO), dipyri-
dodiazepinone, pyridinone, bis(heteroaryl)piperazine, and ␣-
anilinophenylacetamide derivatives (10, 21, 24, 25, 32), all
NNRTIs are believed to bind to a lipophilic pocket within the
p66 subunit of the RT specified by HIV-1 (12, 17). Although
no data describing the structure of the corresponding enzyme
of HIV-2 are available, the lack of inhibitory potency of any
NNRTI against both in vitro HIV-2 RT activity and replica-
tion of HIV-2 in cell cultures may serve as proof of the absence
of a comparable tertiary structure of the protein in this mole-
cule.
The use of NNRTIs as therapeutic agents for the treatment
of HIV-1 infection has been limited so far by the rapid emer-
gence of drug-resistant viral variants in patients (30, 35, 39).
The most common RT amino acid change, which appeared
with most NNRTIs either as a result of the administration of
these drugs to HIV-1-infected individuals or during in vitro
selection experiments, is cysteine for a tyrosine at position 181
(Y1813C) (3, 18, 22, 28, 30, 35, 39).
arose from a screening program and which served as a lead
structure (14). S-2720 and other quinoxaline NNRTIs share
the favorable property of an improved resistance profile, se-
lecting for a characteristic G1903E mutant HIV-1 RT in vi-
tro, which possesses clearly reduced enzymatic activity (2, 14,
15).
Structure-activity studies led to (S)-4-isopropoxycarbonyl-6-
methoxy-3-(methylthiomethyl)-3,4-dihydro-quinoxaline-2
(1H)-thione (HBY 097) (23, 31), which was selected for further
development and is at present undergoing clinical trials. We
describe here the preclinical evaluation of HBY 097.
MATERIALS AND METHODS
Compounds. The quinoxaline derivative S-2720 was synthesized as described
previously (14). HBY 097 was obtained by refluxing 2-chloro-4-methoxynitro-
benzene with S-methylcysteine in 2-methoxyethanol by using 2 N NaOH as the
base. Reduction with Raney nickel in methanol-acetic acid was followed by
spontaneous cyclization to afford the quinoxaline ring system. Acylation at the 4
position is achieved with isopropyl chloroformate-pyridine in methylene chlo-
ride. Thiation was performed with phosphorous pentasulfide in toluene to yield
HBY 097. L-697661 and nevirapine were synthesized in-house by published
methods, and 3Ј-deoxy-3Ј-azidothymidine (AZT) was obtained from Burroughs
Wellcome (Research Triangle Park, N.C.).
Cells and viruses. H9 cells (26, 27) were used to propagate HIV-1MN (9, 37),
and these reagents were obtained through the AIDS Research and Reference
Reagent Program, AIDS Program, National Institute of Allergy and Infectious
Diseases, Bethesda, Md. Peripheral blood lymphocytes (PBLs) were prepared to
grow HIV-1D34 (33), HIV-119/34-TI (14), HIV-1D 117 III, HIV-1MVP5180 (subtype
O) (11), HIV-1I757 (subtype C) (8), HIV-1TH93051 (subtype E), HIV-11420, HIV-
1D148/89 (34), and HIV-2ROD (5), as well as all primary clinical isolates. HIV-
1D148/89 is an AZT-resistant strain and displays RT amino acid substitutions
M413L, D673N, K703R, and T2153Y (13, 20). HIV-11420 harbors the same
genotypic AZT resistance pattern as HIV-1D148/89 and was obtained from a male
We have previously reported the synthesis and in vitro anti-
HIV-1 activities of 6-chloro-3,3-dimethyl-4-(isopropenyloxy-
carbonyl)-3,4-dihydroquinoxaline-2(1H)-thione (S-2720), which
* Corresponding author.
2253

2254
KLEIM ET AL.
ANTIMICROB. AGENTS CHEMOTHER.
patient with hemophilia A who underwent 26 months of AZT monotherapy at
the time of virus isolation. HIV-119/34-TI is a mutant virus generated via in vitro
selection for resistance against TIBO R82150, specifying A983G and L1003I
RT amino acid changes.
Cells were maintained in RPMI 1640 medium containing 10% fetal calf serum
and 40 IU of interleukin-2 per ml (for PBLs) in a humid 5% CO2 atmosphere.
For propagation of HIV-1D 117 III, fresh human mononuclear cells were prepared
from the blood of healthy donors by Ficoll gradient centrifugation. The cells were
suspended in RPMI 1640 medium containing 5% human type ABϩ serum and
were cultivated on hydrophobic membranes (Teflon bags; 3 ϫ 106 cells per ml).
After 24 h, the MNCs were infected by the addition of HIV-1D 117 III-infected
lymphocytes (5 ϫ 104 infected PBLs per ml of MNCs). Forty-eight hours after
infection, the cells were transferred to 24-well plates (Falcon), and the nonad-
hering cells were quantitatively washed off. The adherent cells were further
grown in a culture volume of 1.3 ml, and fresh medium was added every week
(38).
FIG. 1. Structural formulas of S-2720 and HBY 097.
during the experiment. The compounds were dissolved in a mixture of Epicholin
75–Cremophor EL–Glucofurol 75 (1:1:8.5) and were further diluted in water
(1:10). A total of 0.1 or 0.2 to 0.3 ml of these formulations was administered
either intravenously or orally, respectively. Blood samples obtained by heart
puncture after urethane narcosis were pooled (two animals per pool) for each
individual time point. Appropriate galenic formulations of S-2720 or HBY 097
were administered intravenously (only HBY 097) or orally to beagle dogs which
were fasted for 12 h with free access to water. Food was allowed again approx-
imately 5 h after drug administration. Blood samples were collected by venipunc-
ture and serum was obtained by coagulation for 30 to 60 min at 4ЊC. Within 1 h
the samples were centrifuged at 3,000 rpm for 10 min and 4ЊC. All samples were
stored at Ϫ20ЊC until further analysis. Serum samples were precipitated in 70%
(vol/vol) acetonitrile at 0ЊC. After centrifugation for 15 min at 16,000 ϫ g the
supernatant was analyzed by high-pressure liquid chromatography. A reversed-
phase column (C18, 4 by 250 mm) was eluted with acetonitrile-water-trifluoro-
acetic acid (60:40:0.1) at a flow rate of 1 ml/min at 25ЊC. The compounds were
detected by UV absorption at 335 nm. The detection limit was 10 ng/ml of serum
for both HBY 097 and S-2720. All analytical determinations were done in
duplicate. The pharmacokinetic parameters were calculated by fitting the drug
concentration in serum to a series of exponential equations by nonlinear least-
squares regression.
Determination of IC50s and 50% cytotoxic concentrations. A total of 105 cells
were infected with virus stocks at 0.01 PFU per cell, seeded onto 24-well plates,
and incubated for 72 to 96 hours in the presence of compounds (initially dis-
solved in dimethyl sulfoxide [DMSO] and diluted in cell culture medium). For
experiments with macrophages, compounds were added either at the beginning
of the experiment (day 0) or 2 h prior to infection (day 1). Virus replication was
evaluated either by light microscopy for syncytium formation or by testing 1:1 to
1:1,000 dilutions of the culture supernatants by a p24 antigen capture assay
(Innotest HIV-1/HIV-2 antigen; Medipro, Ketsch, Germany). The concentra-
tions of compounds that inhibited virus replication by 50% (IC50) were calcu-
lated by using data from the antigen assay. In general, the presence of syncytium
formation was examined by light microscopy, and syncytium formation was used
to confirm the results of the p24 antigen assay.
For determination of the 50% cytotoxic concentrations, media containing
dilutions of compounds dissolved in DMSO were added to wells containing 2.5
ϫ 105 cells and the mixtures were incubated. The number of cells was deter-
mined after 96 h of cultivation by using an automated cell counter (Sysmex
CC-130) to determine the drug concentration causing a 50% decrease in cell
proliferation.
Inhibitory effects of drugs on the oxidative metabolism of cells. Test com-
pounds were dissolved in DMSO and were further diluted in medium. The
human T-lymphocyte cell lines H9 and J.Jhan were incubated with the test
compounds for 40 h. After the addition of MTT (Promega 96), the cells were
incubated for another 4 h and were finally solubilized for 1 h. The A540 was
determined.
RESULTS
Selection of an HIV-1 variant resistant to inhibition by HBY 097 and geno-
typic analysis of the RT gene. HIV-1MN was passaged on H9 cells in the presence
of increasing amounts of HBY 097 through a total of 11 passages (0.5, 1, 2, 4, 8,
16, 64, 128, and 500 nM and 2 and 10 ␮M HBY 097, respectively) to obtain
HIV-1097/1. For determination of proviral HIV-1 pol gene sequences, purified
cellular DNA was used in a nested PCR protocol. The first reaction mixture
contained 0.5 ␮g of DNA and 10 pmol of primers JA99 and RTRIT137 per 50
␮l (1). An aliquot of the first PCR mixture was subjected to a second amplifi-
cation with primers JA100 (1) and RTRIT138 (5Ј-biotin-CTGTCTTTTTCTG
GTAGCACTATAGG). The biotinylated DNA strand was separated from the
unlabeled strand by using streptavidin-coated magnetic beads (Dynal) and served
as a template for dideoxy chain termination sequencing with primers RT1SEQ2F
(5Ј-fluorescein-CAATGGCCATTGACAGAAG) and RT8KF (5Ј-fluorescein-
CTGCATTTACCATACCTAG). Analysis of the reaction products on an A.L.F.
automated DNA sequencer (Pharmacia) allowed determination of the sequences
corresponding to HIV-1 RT amino acids 35 to 242.
For specific detection of the G1903E mutation by selective PCR (19, 29),
lysates of HIV-1MN-infected cells were obtained at different drug concentrations
(passages). The 1.7-kbp RT gene was amplified with primers RT101 (5Ј-ACTT
TAAATTTTCCCATTAG) and RT102 (5ЈGGGCCTTATCTATTCCATC). A
1-␮l aliquot of the first reaction mixture was used for either wt 190 PCR with
oligonucleotides RT101 and RTGLUG (5ЈCCTATTTCTAAGTCAGATC) or
for 190E PCR with primers RT101 and RTGLUA (5ЈCCTATTTCTAAGTC
AGATT). Products of the heminested amplifications of 599 bp were detected on
a 2% agarose gel.
Preparation of recombinant RTs and in vitro RT assays. Total DNA was
prepared from cells infected with HIV-1 strains. RT-coding regions were ampli-
fied by PCR with primers RT1 and RT2B for HIV-1 and primers HIV2RT1 and
HIV2RT2 for HIV-2 (14, 15). RT mutants were generated by site-specific mu-
tagenesis as described previously (14, 15). PCR products of 1.7 kbp were inserted
into the BamHI-XbaI cloning sites (the SacI-HindIII cloning sites for the HIV-2
RT gene) of the Escherichia coli expression vector pMalcRI. The Ptac promoter
was induced with 0.3 mM isopropyl-␤-D-thiogalactopyranoside to produce re-
combinant proteins in bacteria transformed with plasmid DNA encoding RT
genes. Extraction of enzymes fused to the E. coli maltose binding protein and
purification of enzymes were performed as described previously (14, 15). The
IC50s of the test compounds were obtained by using a heteropolymeric primer-
template and scintillation proximity assay reagents essentially as described pre-
viously (14, 15).
Inhibition of virus replication by HBY 097. HBY 097 is a
derivative of the quinoxaline lead compound S-2720 (Fig. 1).
We first determined the activity of HBY 097 against different
strains of HIV-1 commonly used in our laboratories for the in
vitro evaluation of anti-HIV compounds, including viruses re-
sistant to inhibition by AZT and TIBO R82150 (Table 1).
Antiviral activity at very low concentrations was seen against
all of the strains with the exception of HIV-1097/1. However,
like other NNRTIs, HBY 097 did not prevent the replication of
HIV-2. When compared with S-2720, HBY 097 was only
slightly more active against the viruses tested, but it proved to
be at least 10 times more potent than two other NNRTIs
(L-697661 and nevirapine) which were evaluated. The phos-
phorylation-independent inhibitory activity of HBY 097 was
seen against HIV-1D 117 III in mononuclear cell cultures, as
expected with a NNRTI.
The compound was also demonstrated to be highly active
against a series of fresh clinical HIV-1 isolates. Forty-one
HIV-1 isolates were tested, including 17 viruses from which the
RT gene sequences were determined: the median IC50s and
IC90s of HBY 097 for all strains were 2.0 and 7.1 nM, respec-
tively. AZT resistance and the different cytopathogenicities of
the isolates investigated did not affect their susceptibilities to
the quinoxaline derivative (Table 2).
No antiviral activity was found against HIV-related animal
retroviruses such as the simian immunodeficiency virus or visna
virus in vitro. The compound was also devoid of activity against
adenovirus type 5, herpes simplex virus types 1 and 2, influenza
virus type A2 (A/Aichi), and rhinovirus type 2 in vitro (data not
shown).
Determination of possible cytotoxic properties of HBY 097.
A 50% cytotoxic concentration of 25 ␮M was found with HBY
Pharmacokinetics. Female NMRI mice weighing 22 to 25 g were used for the
experiments. The animals had free access to food and drinking water before and

VOL. 39, 1995
Virus
EVALUATION OF HBY 097 AGAINST HIV-1 REPLICATION
2255
TABLE 1. Antiviral activities of HBY 097 and different RT inhibitors against laboratory strains of HIV
IC50 (␮M)a
HBY 097
S-2720
L-697661
Nevirapine
AZT
HIV-1MN
0.006 Ϯ 0.0005
0.002 Ϯ 0.0004
0.001 Ϯ 0.0007
Ͻ0.010
0.05 Ϯ 0.01
Ͼ10
0.006 Ϯ 0.001
0.012 Ϯ 0.003
0.001 Ϯ 0.0002
0.001 Ϯ 0.0002
Ͼ10
0.007 Ϯ 0.001
0.003 Ϯ 0.0008
0.003 Ϯ 0.001
0.067 Ϯ 0.013
0.030 Ϯ 0.007
0.046 Ϯ 0.011
0.208 Ϯ 0.030
0.030 Ϯ 0.004
0.032 Ϯ 0.009
0.021 Ϯ 0.006
0.007 Ϯ 0.0009
Ͼ30
HIV-1D34
HIV-11420
HIV-1D148/89
HIV-119/34-TI
HIV-1097/1
HIV-1D117III
HIV-1MVP5180
HIV-1I757
Ͼ1.00
Ͼ10
0.008 Ϯ 0.002
Ͼ10
Ͼ10
0.025 Ϯ 0.007
0.150 Ϯ 0.020
HIV-1TH93051
HIV-2ROD
Ͼ10
Ͼ10
Ͼ10
a Values are means Ϯ standard deviations.
097 on PBLs. In addition, we measured the toxicities of HBY
097 and S-2720 in cell culture using the MTT assay. Neither
S-2720 nor HBY 097 affected the oxidative pathway in H9
cells, as measured by formazan formation (50% effective con-
centrations, Ͼ290 ␮M). Likewise, HBY 097 and S-2720 dis-
played low cytotoxic potentials when incubated with J.Jhan
cells (50% effective concentrations, 200 and Ͼ290 ␮M, respec-
tively).
Characterization of HBY 097-resistant HIV-1 and in vitro
activity of HBY 097 against drug-resistant HIV-1 RT mu-
tants. HIV-1MN was passaged on H9 cells in the presence
of increasing concentrations of HBY 097. At passage 11 (10
␮M HBY 097, HIV-1097/1) the proviral RT gene sequence
was analyzed: a point mutation in codon 190 of the RT
gene was detected (GGA3GAA), leading to a G1903E RT
mutant. No mutations other than the latter transition were
found. By using a selective PCR method, it was demonstrat-
ed that the proportion of G1903E RT-specifying genomes
increased during the course of the selection experiment
(Fig. 2). The gradual rise of RT G1903E-mutated genomes
was confirmed by sequence analysis of PCR products ob-
tained from different passages. Selection for the resistance of
HIV-1MN against the pyridinone L-697661 resulted in the
appearance of the RT Y1813C amino acid change, as ex-
pected.
(TSAO)-specific E3138K RT substitution (4) either did not
affect the inhibitor’s potency or mediated only moderate
decreases in enzyme susceptibility to HBY 097 activity. Mul-
tiple RT mutants carrying the G1903E substitution and a
triple mutant with L1003I, V1083I, and Y1813I amino acid
changes were highly resistant to inhibition by HBY 097 (Table
3).
Pharmacokinetic properties of HBY 097 upon oral and in-
travenous administration to mice and dogs. HBY 097 was
rapidly absorbed in mice after administration of an oral dose
(100 mg/kg of body weight), achieving maximum levels in
plasma approximately 30 min after dosing (3 ␮g/ml). In con-
trast, only low drug levels were achieved in plasma with S-2720
(0.14 ␮g/ml). Mean elimination half-life values for intravenous
doses were comparable for both drugs (35 min for S-2720 and
24 min for HBY 097) (Table 4). Assuming dose proportion-
alities, the oral bioavailabilities for HBY 097 and S-2720 were
calculated from the area under the data values for intravenous
and oral administration. When compared with S-2720, HBY
Using recombinant RTs expressed in E. coli, we showed
that the G1903E RT is the only single mutant causing high-
level resistance to inhibition by HBY 097. Other mutations
known to confer viral resistance against NNRTIs and the
[1-[2Ј,5Ј-bis-O-(tert-butyldimethylsilyl)-␤-D-ribofuranosyl]py-
rimidine]-3Ј-spiro-5Љ-(4Љ-amino-1Љ,2Љ-oxathiole-2Љ,2Љ-dioxide)
TABLE 2. Inhibition of in vitro replication of primary
clinical isolates of HIV-1 by HBY 097
HIV-1 strain
Mean (range)
IC50 (nM)
Mean (range)
IC90 (nM)
characterizationa
AZT resistant (n ϭ 14)
AZT susceptible (n ϭ 17)
SI (n ϭ 38)
3.3 (0.1–11)
1.7 (0.1–3.9)
2.0 (0.1–16)
1.8 (0.6–4.0)
2.0 (0.1–16)
5.1 (0.9–21)
6.2 (0.4–19)
7.4 (0.4–57)
3.3 (2.0–4.0)
7.1 (0.4–57)
NSI (n ϭ 3)
Total (n ϭ 41)
a AZT-resistant strains either had at least one of the five known resistance-
conferring mutations (13, 20) in the RT gene or the IC90 of AZT for the strains
was Ͼ1.5 ␮M. Differentiation of SI and NSI viruses was done as described
previously (36).
FIG. 2. Increasing resistance of HIV-1MN against HBY 097 as monitored by
selective PCR. Lanes 1 to 5: 2 nM, 16 nM, 64 nM, 128 nM, and 10 ␮M HBY 097,
respectively. (A) PCR for G1903E (GAA) mutant codon; (B) PCR for G190
(GGA) wild-type codon.

2256
KLEIM ET AL.
ANTIMICROB. AGENTS CHEMOTHER.
TABLE 3. In vitro inhibition of mutant recombinant
RTs by HBY 097
be highly potent and specific inhibitors of both in vitro HIV-1
replication and HIV-1 RT activity (2, 14, 23, 31). Structure-
activity relationship studies resulted in the lead compound
S-2720 (14). Since all of the known RT inhibitors cause the
appearance of resistant viruses both in vitro (cell culture) and
in vivo (patients), S-2720 was used to investigate its resistance
profile in tissue cultures infected with HIV-1. It was found,
that, unlike other NNRTIs, S-2720 selected for a novel RT
G1903E substitution, which caused high-level viral resistance
to the drug and contemporarily reduced the enzymatic activity
of the resistant RT.
These observations prompted us to select a quinoxaline de-
rivative for further development. HBY 097 fulfilled a number
of essential preconditions. The most important difference be-
tween HBY 097 and S-2720 is the favorable bioavailability
of HBY 097 in both mice and dogs (Table 4). Furthermore,
the cytotoxic potential of the new drug was demonstrated
to be relatively low, resulting in a selectivity index in the range
of 104. The antiviral activity of the compound was studied
intensively and was shown to be outstanding against different
subtypes of HIV-1 and also against a panel of 41 clinical iso-
lates (Tables 1 and 2). The IC50s of HBY 097 against a number
of NNRTI-resistant recombinant RTs demonstrate the domi-
nant role of the G1903E substitution. This result was con-
firmed by in vitro selection for HBY 097-resistant HIV-1 vari-
ants (16; this study). Assuming that drug-resistant viruses
specific for any RT inhibitor will occur, the appearance of a
viral variant with decreased RT activity and delayed growth
properties should indicate that a new compound has favorable
attributes (6). However, it still must be demonstrated that the
in vitro selection for an attenuating mutation reflects what
might happen in HIV-1-infected patients treated with HBY
097.
Fold increase in IC50
compared with that
for wild type
Amino acid
substitution
IC50 of HBY
Enzyme
097 (␮M)a
RT-1 MN
RT-1 E
Wild type
G1903E
G1903A
G1903S
Y1813I
Y1813C
Y1883L
L1003I
V1063A
M2303L
P2363L
E1383K
0.08 Ϯ 0.0025
Ͼ29
1.0
Ͼ363
8.9
12.5
49
7.0
25
7.4
7.3
12.5
1.4
2.8
RT-1 A
RT-1 S
RT-1 I3
RT-1 C
0.71 Ϯ 0.0068
1.0 Ϯ 0.031
3.9 Ϯ 0.049
0.56 Ϯ 0.069
2.0 Ϯ 0.043
0.59 Ϯ 0.01
0.58 Ϯ 0.01
1.0 Ϯ 0.031
0.11 Ϯ 0.0043
0.22 Ϯ 0.0063
0.08 Ϯ 0.0029
Ͼ29
RT-1 L3
RT-1 I2
RT-1 A2
RT-1 L4
RT-1 L2
RT-1 K
RT-1 R2
RT-1 EC
RT-1 EI3
RT-1 II3
K1043R
1
Y1813C, G1903E
Y1813I, G1903E
L1003I, V1083I,
Y1813I
Ͼ363
Ͼ363
Ͼ363
Ͼ29
Ͼ29
RT-1
K1033R, V1063A,
Y1813I, G1903E
M1843V, G1903E
G1903E, T2153Y
Ͼ29
Ͼ363
IERA
RT-1 VE
RT-1 EY
Ͼ29
Ͼ29
Ͼ363
Ͼ363
a Values are means Ϯ standard deviations.
097 achieved a considerably higher degree of oral absorption
(48 versus 10%).
The excellent oral bioavailability of HBY 097 compared with
that of S-2720 was also confirmed in dogs. Oral administration
of HBY 097 resulted in maximum levels in serum of more than
2 ␮g of drug per ml with both 34- and 83-mg/kg doses, whereas
with a dose as high as 100 mg of S-2720 per kg, the amount of
drug in serum was found to be less than 0.1 ␮g/ml. With the
administration of 5.3-mg/kg intravenous doses, maximum lev-
els of about 6 ␮g of HBY 097 per ml were achieved. The mean
elimination half-life of HBY 097 for the intravenous adminis-
tration route was 1.6 h. The oral bioavailability of HBY 097 in
dogs was calculated to be 54%, assuming dose proportionality
(Table 4).
We conclude that the properties of HBY 097 summarized
above may help to address the main problem seen with other
NNRTIs: the prevention of a more prolonged clinical benefit
of treated patients caused by the rapid selection of drug-resis-
tant viruses (30, 35, 39).
Since AZT is still the only first-line treatment for patients
infected with HIV-1, it is of particular interest how the muta-
tions selected for by AZT and the mutants found with qui-
noxalines might interact. However, a viral variant displaying
the RT G1903E amino acid change together with the AZT-
specific substitutions has so far not been able to be generated
by in vitro selection procedures.
Preliminary results from a phase I single-dose study in
healthy volunteers have confirmed the good bioavailability and
good tolerability of HBY 097 in humans (data not shown).
HBY 097 is undergoing further clinical trials in patients in-
fected with HIV-1.
DISCUSSION
We have described here the preclinical investigation of HBY
097, a member of the quinoxaline class of NNRTIs. These
specifically substituted quinoxalines were previously shown to
TABLE 4. Pharmacokinetic properties of HBY 097 and S-2720 in mice and dogsa
Dose Route of AUD t1/2 Cmax Tmax
CL
(ml/min)
Bioavailability
(%)
Species
Compound
(mg/kg) administration (␮g ⅐ min/ml) (min) (␮g/ml) (min)
Mouse
Mouse
Mouse
Mouse
Dog
S-2720
10
i.v.
p.o.
i.v.
p.o.
i.v.
i.v.
16.7
17.1
34.7
24.1
0.7
0.1
1.6
3.0
1.2
5.8
2.1
2.5
5
30
5
20
5
5
4.3
5.0
S-2720
100
10
48
HBY 097
HBY 097
HBY 097
HBY 097
HBY 097
HBY 097
7.4
100
2
5.3
33.8
82.5
35.7
229.9
50.7
292.8
873.6
1,434.4
99.0
95.2
597.8
317.4
732.8
888.6
Dog
Dog
Dog
p.o.
p.o.
120
90
54
a i.v., intravenous; p.o., oral; AUD, area under the data curve; t1/2, half-life; Cmax, maximum concentration of drug in serum; Tmax, time to maximum concentration
of drug in serum; CL, clearance.

VOL. 39, 1995
EVALUATION OF HBY 097 AGAINST HIV-1 REPLICATION
2257
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