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

Mo.A. 1049 - Mo.A. 1054

Monday, July 8, 1996

Conclusions: HIV -specificity is supposed to be conferred by the absence of SSDT in the
cells uninfected. t\t light of the results obtained, it is possible to hypothesize that the genes
for Exo VII, xseA arnd xseB, may be utilized in experiments of gene therapy which are now in
course.
Agostino Pughese Dip.Scierze Medico -Chirur-giche C.so Svizzera I164 -10149 Torino, ITALY
[phone]
Mo.A. 1049
HUMAN IN VITRO T-LYMPHOIESIS AS A MODEL FOR HIV GENE THERAPY
H, Zhu, A. Freedrn,, G Kurtzman, D Scadden. Massachusetts General Hospital, Harvard
Medical School. Boston, MA. and *Avigen, Alameda, CA.
Testing gene therapy strategies for AIDS requires the evaluation of transduced gene expression in cells differentiating along lines releivant to HIV infection.To further this end, we have
developed a method of inducing CD34+, CD2- human adult bone marrow cells to develop
functional and imunophenotypic characteristics of I cells. Cells cultured in the presence of
a human fetal thymic mronolayer interleukin- 12 and flt 3 ligand for 14 35 days acquired
expression of CD2, CD3, CD4 and CD8 in patterns resembling in vive T lymphopoiesis with
the intrathymic f(D2 CD3-, CD4+, CD8 subset predominating. In addition, RAG-2
expression became detectable by day 7 and a r-ange of TCRV( chains were present by day
21 by RT PCR while neither RAG 2 nor TCR were detectable in day 0 cells or in stroma.
The differentiated cells were capable of producing IL 2 upon mitogenic stimulation and
became susceptible to infection with a lymphotropic strain of HIV, HIV I iiiB.The ability of
adeno- associated virus (AAV) transduced genes driven by a modified CMV promoter to be
expressed in this differentiation system was evaluated. CD34+ CD2 cells were exposed to
recombinant AAV containing a CMV promotor beta galactosidase construct or control for
24 hours and cultured in the T lymphopoiesris system for 2 1 days. After 21 days, CD2+
sorted cells were found to have detectable beta-gal RNA by RT PCR, and to stain positively
with X gal in 71-79% of cells.This system may provide an in vitro method of analyzing gene
therapy construcs directed toward AIDS. [he AAV mediated gene transduction systems
evaluated appear to offer a method for delivering such constructs to precursor cells undergoing T cell differentiation.
David T. Scadden, M.D. Massachusetts General Hospital, CNY 7, Bldg 149 Charlestown,
MA 02 129
Mo.A. 1050
ANTI-HUMAN IMMUNODEFICIENCY VIRUS TYPE I (HIV- I) ACTIVITY OF Achyrocline
flaccida WEIN DC AND Gamochaeta simplicicaulis AQUEOUS EXTRACTS.
Salomon Horacio, Pampuro S, Libonatti O, Cavallaro L2, Garcia G2, Campos R2,
Coussio J I Departamento de Microbiologia, Facultad de Medicina, 2Citedra de Virologlria,
3Ctedra Farmacognosia, Faculdad de Farmnacia y Bioqulmica. Universidad de Buenos Aires.
Argentina.
Objective: To study anti-HIV I activities of South American plant extracts from both
Achyrocine flaccidcr Wein DC (AF) and rrochaeta simplicicaulis (GS) on infected lymphocytes of primary origin.
Methods:The plant extracts (aerial part) were extracted successively with solvents of
ncreasing polarity: hexane, dichloromethane, ethylacetate, methanol and hot waterThe final
aqueous plant extracts of AF and GS were yophilized. Screening and evaluation of plant
extracts for anti-HIV efficacy were analyzed using Peripheral Blood Mononuclear Cells
(PBMC) Infected with HIV I with 200 and 2000 fifty percent tissue culture infective dose
(TCID50) per I x 106 cells. After infection, cells were washed and dispensed in a 96 wells
plate in the presence of different concentrations of aqueous extracts. AZT treated cells
were evaluated as control. Cell free supernatant fluids were harvested at day 7, assayed for
p24 antigen and monitored by reverse transcriptase activity Dosis inhibiting 50 % of viral
production (IC50) and cellular growth (CCID50) were determined. Whether the plant
extracts were involved in the early step of cell infection and virucidal activity were studied.
Results: Selectivity Indexes (SI), defined as CCID50/IC50, of plant extracts in PBMC were
133 and 120 for AF and G5,respectively When we analized the effect ofTCID50 on the SI,
we found a 10 fold decrease of SI when the TCID50 was 10 times higher for infection of
PBMC.The high value of SI1 found and the direct correlation with the multiplicity of infection
reveal a high specificity of the antivr ral action of these extracts. Data obtained when the
extracts were present until the end of the experiment (7 days) or when the extracts were
removed after 4 hours suggested that HIV I infection was inhibited at an early step. In addition, the extracts were non virucdal.
Conclusions: The present work demonstrates the potent anti-HIV- I activity of AF and GS
extract on lymphocytes of primary origin. Natural plants products such as AF and GS that
might interfere with viral infection at early steps stresses the irmportance of further studying
the mechanisms of action of these plants.
SALOMON, ioracio. Paraguay 2 155 Piso II ( 12 I) Buenos Aires Argentina Phone: 54
1961 8124 -Fax: [phone]
Mo.A. 105 I
THIOCARBOXANILIDE DERIVATIVES: HIGHLY POTENT AND SELECTIVE HIV
INHIBITORS WITH A BROAD ACTIVITY SPECTRUM AGAINST MUTANT HIV- I
STRAINS RESISTANT TO OTHER NON-NUCLEOSIDE REVERSE TRANSCRIPTASE
INHIBITORS
J. Balzarini,WG. Brouwer2, D.C. Dee2, E.M. Osika2, A. Karisson3, [Dr Clercq-. Rega
Institute for Medical Research, Katholieke Universiteit Leuven, B-3000 Leuven, Belgium;
2Uniroyal Chemicail Company Ltd., Gueiph, Ontario, Canada N IE 5L7; 3Department of
Medical Biochemistry & Biophysics, Karolinska Institute, 5- 171 77 Stockholm, Sweden
A series of novel thiocarboxanilide derivatives have been synthesized and evaluated for
their acwtvry arainst human immunodebciency virus type I (HIV I) in cell culture.They are
highly specifcally inhibitory to HIV I strains and not active against other retroviruses including HIV 2 or other RNA or DNA viruses (i.e. herpesviruses).The prototype compound
(originally designated Uniroyal senior or UC-84) consists of an oxathiin group, linked
through a carboxamide moiety to chlorobenzoic acid that is esterifed through its carboxylic
acid function with in isopropyl group. Replacement of the carboxamide group by a thiocarboxamide moiety results in a marked increase in anti HIVI activityThe oxathiin moiety can
also be replaced ry a 2-methylfuranyl or 2 rethylthienyl ring resulting in a further increase

of the antiviral potency of the compounds. Replacement of the metabolically unstable ester
function by a metabolically highly stable oxime t-butyl ether function, as n UC I0, or by a
branched pentenyloxy ether entity as in UC-781 (ithe furanyl thiocarboxanilide derivative)
or UC-82 (the thienyl thiocarboxanilide derivative) resulted in the generation of novel thiocarboxanilide compounds with an anti-HIV-I potency in the range of I to 5 ng/ml and an
antiviral selectivity exceeding 50,000.The thiocarboxanilides UC 78 1 and UC-82 show a
highly favorable inhibition spectrum against a series of mutant virus strains that contain the
100 Leu Ile, 103 Lys Asn, 106 Val Ala, 138 Gu Lys and 181 Tyr Cys amino acid mutation in
their reverse transcriptase (50% effective concentration in the range of 2 to 20 ng/ml).
These compounds proved to be fully stable in human serurm at 37~C for at least 24 h.

E. De Clercq, Rega Institute for Medical Research, Katholieke Universiteit Leuven,
Minderbroedersstraat 10, B-3000 Leuven, Belgium.Tel. [phone]. Fax 32- 16 -
3.73.40.

Mo.A. 1052
DESIGN, SYNTHESIS AND BIOLOGICAL ACTIVITY OF AMINOGLYCOSIDE-LIKE
INHIBITORS OF THE REV/RRE INTERACTION
Brown William*, Cedergren R**, Charron M', Duschene J", Ellington A* *, Falardeau G-,
Gilard JW*, Hamel M, Leclerc F*, Mansour T', Mounir 5,rinivasan J "Yuen L*.
*BioChem Therapeutic Inc., Laval, QC, Canada; I"University of Montreal, Montreal, QC,
Canada; ***University of Indiana, Bloomington, Indiana, USA.
Objective: To identify inhibitors of HIV which function by disrupting the Rev/RRE(Rev
responsive element) interaction, which were designed by using a proprietary molecular
modelling docking program.
Methods: Illustration of the use of a proprietary molecular modelling docking program of
the RBE(Rev binding element) to design inhibitors of the Rev/RRE interaction.The predict
ed molecules were then synthesized and evaluated in a Rev binding assay
Results: The known potent inhibitors of the Rev/RRE interaction, Neomycin B and
Tobramycin were docked into a computer generated 3D model of the RBE. Based on the
information obtained, novel aminoglycoside like molecules were designed and synthesized.
These molecules were later evaluated in a Rev binding assay to validate these predictions.
Conclusions: The molecular modelling docking program has proved to be a powerful tool
in the prediction of molecular inhibitors of the Rev/RRE interaction.
W Brown, 275 Armand Frappier, Laval, QC, Canada H7V 41\7 Telephone: [phone]
Fax: [phone]
Mo.A. 1053
EFFICACY OF F-5~ GEL AGAINST HIV-I AND HIV-2 IN INFECTED HUMAN
PERIPHERAL BLOOD MONONUCLEAR CELLS (PBMC)
Wetherall N.T*, Hodges-Savola C.*, Werness, L.*, Gosselin L.F.* Saunol C.*, Colin P.
Viromed Laboratories Inc., Minneapolis, Minnesota; * Axcan Ltd., Mont-St Hilaire, Quebec,
Canada
Objective:To determine the in-vitro cytotoxicity and antiviral efficacy of F 5~ Gel (used in
Protectaid~ sponge), on HIV I and HIV 2 infected PBMCs, which are involved in the heterosexual transmission of HIV
Methods: HIV I strain HTLV IIIB and HIV-2 strain CBL-20 were used in this study PBMCs
were obtained from an HIV-negative donor and cultured at 36-38'C in a humidified atmosphere of 5-7% CO2. F-5~ Gel was diluted 1/100, 1/500, 1/1000, 1/5000 and I/ 0000 in
the culture media. Cells were exposed to HIV I or HIV-2 at a MOI of 0.01. P 24 or P 27
antigen as well as reverse transcriptase (RT), produced by PBMCs infected by either HIV-I
or HIV-2 were assayed after an incubation per-iod of 6 days for HIV I and 8 days for HIV 2.
Cytotoxicity was assessed at the end of these periods using an inverted microscope. Cell
culture supernatants were collected to determine the level of viral infectivity via a P-24 or
P-27 enzyme immune assay On the same day aliquots of supernata nts were also taken for
reverse transcriptase activity
Results: On day 6 (HIV I) or 8 (HIV 2), evidence of severe Cytotoxicity was observed in
non infected PBMCs treated with F-5R Gel at dilution of I/100 to I/1000, while slight
cytotoxicity was observed at 1/5000. Cells were unaffected by a I/10000 dilution. Similar
findings were observed for cellular proliferation as determined by a Microculture
Tetrazolium AssayThe percent viral inhibition in HIV I infected cells was less than or equal
to 17.4% for all dilutions of test substance, but greater than 87.7% for I/100 -I/I1000 dilution
for HIV-2. EC90 was 1:650 for HIV 2. Finally when compared to enzyme levels in virus controls, RT activity was greatly reduced in preparation exposed to HIV- I or HIV 2 at 1/100 -I/1000 F-5~ Gel dilutions.
Conclusion:The anti HIV I/HIV-2 effects of F 5~ Gel observed appear to be due mainly to
cytotoxicity against PBMCs at dilutions I/ 100 1/1000 Since the undiluted gel has been
shown to be non-irritant to the cervico-vaginal mucosa, these effects could be clinically
important since PBMCs are involved in the heterosexual transmission of HIV
L.F. Gosselin, 597 Laurier Blvd., Mont-St-Hilaire, Quebec, Canada J3H 4X8 Telephone (5 14)
467-5138 Fax: [phone]
Mo.A. 1054
ENTRAPMENT OF FOSCARNET IN LIPOSOMES:A STRATEGIC APPROACH FOR
THE TREATMENT OF HIV AND CMV INFECTIONS.
Dusserre Nathalie, Omanr R., Desormeaux, A.,Tremblay M., Beauchamip, D., Pouln, L.,
Bergeron, M.G. Centre de Recherche en Infectiologie, Centre Hospitalier de I'Universite
Laval, Ste-Foy Quebec, Canada.
Objective: To evaluate the potential therapeutic applications of liposome-encapsulated foscarnet for the treatment of HIV and CMV infections.
Methods: In vitro experiments have been performed to evaluate the accumulation and anti
HIV efficacy of free and liposome-encapsulated foscarnet in different cell ines.The pharmacokinetics and tissue distribution of free and liposomal foscarnet have been evaluated in
female Sprague-Dawley rats following the administration of a single intravenous dose (10
mg foscarnet/kg).The protective role of liposomes on serum calcium abnormalities associated to foscarnet therapy has been also evaluated.
Results: Results clearly showed that the incorporation of foscarnet into Iiposomes greatly
enhanced the drug uptake in murine monocyte smacrophage RAW 264.7 cells and in
human premonocytoid U937 cells. Comparable or even greater anti-HIV efficacy was
observed for the liposomal formulation in U937 cells infected with HIV IIIB and in cno

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