Abstracts Vol. 1 [International Conference on AIDS (11th: 1996: Vancouver, Canada)]

Track A: Basic Science

Mo.A.1065 - Mo.A.1070

Mo.A. 1065
COMPLETE INHIBITION OF VIRAL BREAKTHROUGH BY COMBINATION OF
MKC-442 WITH AZT DURING A LONG-TERM CULTURE
Yuasa, Satoshi*,Yamada K*, Nakade K*, Okamoto M**, Makino M**, Baba M**. *Mitsubishi
Chemical Corp.,Yokohama, Japan; **Kagoshima University, Kagoshima, Japan
Objective:To investigate whether the combination of nonnucleoside reverse transcriptase
inhibitors (NNRTIs) and AZT is effective in inhibiting the emergence of HIV- I mutants in vitro.
Methods: 50% effective concentrations (ECS0) of AZT and NNRTIs, such as MKC-442 (6 -benzyl- I-ethoxymethyl-5-sopropyluracil), nevirapine, and loviride (o-APA) were measured
for their inhibitory effects on HIV- I (HE strain) replication in MT-4 cells on day 4 after virus
infection. Long-term culture of HIV- I infected cells was performed by subculturing with
fresh medium containing appropriate concentrations of NNRTIs and AZT to determine the
necessary concentrations for complete suppression of the viral breakthrough.
Results: EC50 of AZT, MKC-442, nevirapine, and loviride were 3.2, 9.4, 98, and 21 nM,
respectively After 48-day culture period, MKC-442, nevirapine, and loviride completely
inhibited viral breakthrough at a concentration of 1, 5, and I pM, respectively.These concentrations were 50- to 100 fold higher than their EC50 values.When the cells were treated
with either MKC-442 (0.04 and 0.2 pM), nevirapine (0.2 and I pM), or loviride (0.04 and
0.2 pM) in combination with AZ T (0.005 pM), the only combination of 0.2 pM MKC-442
and 0.005 pM AZT could inhibit completely the breakthrough of HIV- I after a 68-day culture period. Except for the combination of 0.04 pM MKC-442 and 0.005 pM AZT all of the
viruses isolated during combination treatments had various amino acid mutations in their
reverse transcriptase.
Conclusions: The combination treatment with a relatively high dose of MKC-442 and a low
dose of AZT may have potential to suppress the emergence of drug-resistance during a
long-term treatment in vivo and should be further pursued in HIV- I -infected patients.
Satoshi Yuasa, Mitsubishi Chemical Corp., Research Center, Pharmaceuticals Lab 2, I1000
Kamnroshida-cho, Aoba-Ku,Yokohama 227, Japan Telephone: [phone]
Fax: [phone]
Mo.A. 1066
ANALYSIS OF HIV- I VARIANTS RESISTANT TO AN IRREVERSIBLE PROTEASE
INHIBITORS, LB7I 1I48 AND LB71262.
Lee,Tae Gyu, Kwon,YD., Choi, H.I., Chang, H.J., Hwang, S.Y, Kim, S.C.,Yoo,YJ. LG Chemical
Ltd/ Research Park, DaeJeon, Korea
Objective:To investigate in vitro emergence of HIV- I variants resistant to the irreversible
protease inhibitors (LB7 11 48, LB7 I 262) containing cis-epoxide, and to analyze the effect of
the mutations on the enzyme activity as well as on the virus.
Methods: MT-2 cells infected with HIV- I NL-4-3 were cultured in the presence of increasing
dose of the protease inhibitor followed by the analysis of the clones by PCR and DNA
sequencing.The defined mutations were inserted into the protease gene of HIV- I NL-4-3.
Mutant virus was recovered after transfection into MT-2 cells and the susceptibility to the
inhibitor was measured.We also determined the kina/Ki value of the mutant enzyme
expressed in E. coli.
Results:Viral variants were selected in vitro in the presence of the irreversible protease
inhibitor; LB7 I 148. Molecular characterization of the variants revealed that isoleucine at
position 84 replaced by valine (184V) persisted temporarily at early stage. Under higher
concentration of the inhibitor a variant containing K451 and F53L became dominated, followed by an additional mutation ofV821.Variants selected in LB71262 contained 154M
change.The mutant virus containing these mutations displayed slightly reduced sensitivity to
the inhibitor: These mutants were not resistant to L-735524 and Reo31I-8959.The kina/Ki
value of the mutant enzyme decreased about 5 fold.
Discussion and Conclusions: Although viral mutants were isolated in the presence of the
irreversible inhibitor, IC50 and kinar/Ki value did not change significantly.This will be the next
generation of HIV I protease inhibitor:
Lee,Tae Gyu. LG Chemical Ltd/Research Park, 1 04-I MoonJi-dongYoosung-gu, DaeJeon,
305-380, Korea.lelephone [phone];Telefax [phone]
Mo.A. 1067
SIGNIFICANT DELAY OF HIV-I EMERGENCE AFTER PRETREATMENT WITH AN
IRREVERSIBLE PROTEASE INHIBITOR, LB71148.
KwonYoun Do, Lee,TG., Kim, S.C. LG Chremical Ltd/ Research Park, DaeJeon, Korea.
Objective: LB71148 containing cis-epoxide inhibits HIV- I protease irreversibly.We determined whether the irreversible inhibitor had an advantage over the conventional reversible
protease inhibitor:
Methods: MT-2 cells infected with HIV- I NL-4-3 were pretreated with the irreversible
inhibitor LB71148 or with the reversible inhibitor, L-735524. After removing the inhibitor,
virus in the media was detected by p24 assay as well as by TCID50 measurement. H9 cells
chronically infected with HIV- I B1118 were also treated as above.
Results: Removal of the reversible inhibitor resulted in sharp increase in the number of
virus in the media, probably due to the dissociation of the protease-inhibitor complexes
inside the cell. However emergence of virus in the media was delayed signifcantly when
cells were pretreated with the irreversible inhibiter
Discussion and conclusions: In order to block DlV- I growth, it is important to keep the
cencentration of the inhibitor in vivo. Since the irreversible inhibitor delays the emergence
of virus in the media longer than the reversible inhibitor in vitro, it will be the promising
candidate for HIV protease inhibitor
KwonYoung Do, LG Chemical Ltd/Research Park, 104-I Moonji-dong,Yoosung-gu, Daejeon,
305 380, Korea.Telephone [phone];Telefix [phone]
Mo.A. 1068
COMPARATIVE STUDY WITH TWO TREATMENT SCHEMES OF DDC PLUS AZT IN
HIV INFECTION.
Torres Roc/o.,Vrllarreal C., Robies M.,Terazas J. CanoC: Hospital de Infectologia, Centre
Mddico Nacisnal La Riza IMSS, Md~xrco D.F.
Objective: To compare efhcacy and tolerance between two different treatment schemes
with a double drug regimen (AZT plus DC) k patients with HIV infection. Group A
included DUC 0.375 nsg tid and three times a week, plus AZT 100 mg tid, three times a

week in alternating days. Group B included DDC 0.750 mg tid, three times a week plus
AZT 500 mg per day divided in three doses three times a week in alternating days.
Methods: Patients of both sex, older than I 8 years old, HIV positive by ELISA and WesternBlot, without a history of clinical disorders were included, with a CD4 cell count < 500
mm3 and > 200 mm3 and non pregnant women. An aleatory number was assigned, and
treatment was given in concordance with the aleatory number.The following tests hematic
biometry CD4, CD8 lymphocytes, liver function tests, serum amylase, were done basal,
every two weeks the first month, afterward every two months.
Results: Group A (GA) 26 cases 22 males and 4 women; group B (GB) 31 cases 29 male
and 2 women: GA: average age 32.4 ~ 6 years old (range 20-50), GB: 30 ~ I I years old
(range 2 I-5 I); GA: weigth 67.2 ~ 9 kg (mean 56-93), GB: 64 + 9 kg (range 56-95);
Karnofsky index both groups 100%. GA: Hemoglobin level of 14.4 ~ 1.6 gr/dL (range 10 -17), GB: hemoglobin level of 14.6 ~ I1.6 gr/dL (range I I-17); GA: hematocrit of 44.5 ~ 4.9%
(range 32-52), GB: hematocrit of 45. I ~ 4. I (range 34-52). Leucocytes GA: 4895 mm3 ~
I249 mm3 (range 3400-8500), GB: 5277 ~ I1206 (range 3700-6700); neutrophils GA: 2758
~ 1377 mm (range 1204-7000), GB: 496 1 ~ 1320 mm3; lymphocytes GA: 2107 ~ 556
mm3 (range 1100-3575), GB: 2221 ~ 686 (rane Ill 10-3680); CD4 lymphocytes GA 353 ~
99 mm3 (range 200-494), GB: 323 ~ 105 mm (range 200-484); CD8 lymphocytes GA:
1316 ~ 50 mm3 (range 55 1 -2327), GB: I 347 ~ 574 mm3 (range 616-2948). After a follow
up of II months we did not found a significative difference independ t-test in the hematologic data until the 13 months we found a significative difference ( p.0001), with appared ttest, we found a significative difference in CD4 basal to 13 months (p 0.035 GB) the GA
there were 8 opportunistic infectious episodes versus I I in GB.There was no toxicity
event in either group.
Conclusions: we consider that at present both schemes have the same efficacy and tolerance, but it necessary to continue the follow up.
RocioTorres I.Tepalcatzin #36 Col. Ajusco Del Coyoacan CP.4300 Mexico D.F.
Mo.A. 1069
NEGATIVELY CHARGED HUMAN SERUM ALBUMINS AS INHIBITORS OF HIV- I
REPLICATION: MECHANISM OF ACTION, IN VITRO ACTIVITY AGAINST DISTINCT
HIV- I ISOLATES AND IN VIVO EFFICACY IN MICE
M.E. Kui ers I, PJ. Swart I, M. Schutten2, J.G. Huisman3, H. Schuitemaker3, A.D.M.E.
Osterhaus, D.K.F. Meijer1.I University Centre for Pharmacy Groningen,The Netherlands;2
Erasmus University Rotterdam,The Netherlands; 3 Central Laboratory of the Netherlands
Red Cross Blood Transfusion Service, Amsterdam,The Netherlands
Objective: Negatively charged albumins (NCAs), with the prototypes Suc-HSA and AceHSA, are polyanionic proteins with a potent antiviral activity on HIV- I laboratory strains. In
the present study the mechanism of their antiviral action was studied. In addition we determined the in vitro antiviral activity of these NCAs on phenotypically distinctive patient HIV- I
isolates and evaluated the in vivo efficacy of these polyanionic proteins in a mouse chimeric
model for HIV- I infection,
Methods: Formation of syncytia and determination of products in the transcription cycle
were used to clarify the potential level of interference of the NCAs in the replication cycle.
This was also studied by the interaction of a series of peptides of gpl20 of distinct HIV- I
isolates, as well as by binding studies using (PHA-prestimulated) human PBLs. For the in vitro
antiviral activity of the NCAs patient HIV- I isolates (NSI/ SI variants) were used, either cultivated in PBMC or in primary macrophages.The efficacy study was performed with
immunosuppressed mice which were i.p. grafted with human PBMC. I.p. injections of 0.3 to
300 mg/kg Suc-HSA were given within 15 to 30 minutes before the mice were challenged
with HIV- I.When Graft versus Host disease was observed in these mice, the human cells in
the peritoneal cavity were isolated and tested for the presence of p24 antigen.
Results: Formation of syncytia was inhibited by the NCAs. Early products of reverse transcription, in cells inoculated with HIV- I in the presence of NCAs, were also absent.
Interaction of NCAs and HIV- I peptides was only observed for the positively charged V3
loop and the C-terminal part of gp 20, predominantly caused by electrostatic interactions as
indicated with competition studies with heparin and dextran sulfate, although hydrophobic
interactions were also involved. Furthermore proteolytic cleavage of the tip of the V3 loop
(GPGRAF sequence) caused a complete loss of binding affinity of the NCAs for the V3 loop.
In addition, it was observed that the NCAs bind to uninfected human lymphocytes and that
prestimulation of these lymphocytes with PHA markedly increased the binding of the NCAs
to the cells.The IC50 values of the NCAs against the primary HIV- I isolates were all in the
nanomolar range. Furthermore, the NCAs showed equipotent inhibition of replication of a
macrophage-tropic HIV- I variant in either PBMC or primary macrophages. I.p. injections of 3
and 300 mg/kg Suc-HSA were sufficient to completely protect the mice against infection
with HIV-I, while even an i.p. injection of 0.3 mg/kg partially protected the grafts.
Conclusions: These results indicate that the NCAs interfere at an early level in the virus
replication cycle and suggest that the antiviral activity of the NCAs is based on shielding of
the positively charged V3 loop from a functional interaction with the target cell surface,
both by binding to the HIV- I particles and by binding to inducable receptors on the target
cells. Preliminary results indicate that NCAs also prevent proteolytic cleavage of the V3 loop
that is supposed to precede the initiation of the actual fusion process.These three aspects
may explain the preferential infuence of the NCAs on virus/cell fusion instead of
gplI20/CD4 binding.This combined with the potent irn vitro anti-HIV- I activity in various
lV-I infected cell types and the antiviral offiracy in the HlV-I mouse model, render these
conjugates promising candidates for the testing of their anti-HPV activity in humans.
M. E. Kuipers, University Centre for Pharmacy Ant. Deusingln 2, 97 I 3 AW GroningenThe
Netherlands.Tel: #31-S50-3633276; Fax: #[phone] I; email: [email protected]
Mo.A. 1070
ANTIVIRAL EFFECTS OF MILK PROTEINS:ACYLATION RESULTS IN POLYANIONIC
PROTEINS WITH POTENT ACTIVITY AGAINST HIV TYPE I AND II IN VITRO
Huisman Han/, Kuipers, M.E2., Smit, C2., De Clercq, E3., Meije, D.K.F2., Swart, PJ2. I Central
Lab. of the Neth. Red Cross Blood Transf Service, Plesmanlaan I25, I066 CX Amsterdam,
NL. 2 University Centre of Pharmacy A. Deusinglaan 2, 9713 AW Groningen, NL. 3Rega
Inst. for Med. Res. 8-3000 Leuven, Belgium.
Objective: A number of sative and modified milk proteins from bovine or human sources
were analyzed for" their anti-DIV and anti-HIV-2 effect in vitro.

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