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

Mo.A. 1082 - Mo.A.1087

Monday, July 8, 1996

Mo.A. 1082
GEM 91,A gag-ANTISENSE PHOSPHOROTHIOATE: MECHANISMS OF INHIBITION
OF HIV REPLICATION AND ATTEMPTS TO GENERATE HIV RESISTANCE IN VITRO.
Yamaguchi, K.*, Papp B*, Zhang D*, Agrawal S**, Byrn, Randal A*. *Deaconess tiospia,
Harvard Medical School, Boston, and **Hybridon, Inc., Worcester MA USA.
Objective: To examine the mechanisms of the inhibitory activity of GEM 91 (Gene
Expression Modulator 9 I), a 25-mer antisense phosphorothioate designed to interact with
the conserved gag initiation site of HIV I, against HIV replication in vitro and to generate
and characterize GEM-9I resistant strains of HIV-1I.
Methods: In vitro experiments were designed to examine the inhibitory effects of GEM 91
on different stages of the HIV replicative cycle. Experiments focussed on the bindinr of viions to the cell surface, inhibition of virus entry and reverse transcription (HIV DNA production), inhibition of induction of HIV from latency inhibition of steady -state viral mRNA
levels, inhibition of virus production fr-om chronically infected cells, and inhibition of HIV
genornme packaging within virions. In vitro resistance experiments were performed by passaging HIV in the presence of increasing concentrations of GEM 91 or control inhibitors.
Results: We observed sequence dependent inhibition of virus entry/reverse transcriptioni
and reduction in steady state viral RNA levels and a dose dependent inhibition of induction
of HIV from latencyWe observed sequence-independent inhibition of virion binding to cells
and virus production by chronically infected cells.
The resistance-generating methodology was successful in generating 100-fold resistance to
control inhibitors yet we were unable to achieve more than 4.5 fold resistance to GEM 91
after repeated passages in vitro. No nucleotide sequence changes were observed in the
GEM-9I complementary region of passaged strains.
Conclusions: We observed sequence specific HIV inhibitory effects by GFM 9 I as well as
sequence nonspecific virus blocking effects.The inhibitory activity of GEM 91 ir vitro is due
to multiple mechanisms.While we were unable to generate significant resistance to GEM 91
i vitro only clin cal testing can determine if GEM 91 resistant isolates will be generated n
ivo.The results of this study support the ongoing Phase 1/II clinical evaluation of GEM 91 as
a therapeutic for HIV infection.
Randal A. Byrn, Deaconess Hospital, One Deaconess Road, Boston, MA 022 15 USA
Telephone: [phone] Fax: [phone] email: rbyrn(@nedhmail.nedh.harvard.edu
Mo.A. 1083
EFFECTS OF REVERSE TRANSCRIPTASE INHIBITOR THERAPY ON HIV- I VIRAL
BURDEN IN SEMEN
Gilliam, Bruce L, Dye r J, Cohen MS, Fiscus S, Eron, Jr. JJ. University of North Carolina at
Chapel Hill, Chapel Hill, NC
Previous studies have suggested that AZT may reduce the concentrati on of HIV in semen.
In order to more fully evaluate the effects of reverse transcriptase inhibitors on the HIV
concentration ir semen, we recently studied semen donated flom 12 me n participating in a
clinical trial with AZT or DDI +/- delavirdine, a non-nucleoside reverse transcriptase
inhibitor. CD4 count ranged from 80 to 391. Quanti tative seminal cell culture was positive
in half the subjects before therapy (range 3.1 to 2523.3 infectious units per ejaculate). HIV
RNA of seminal plasma was studied using the Organon-Technika nucleic acid sequence
based assay (NASBA). HIV-I RNA concentration range from 2,400 to 200,000 copies/mln
specimens obtained from untreated subjects. Follow-up cultures done at least 8 weeks after
initiation of therapy, had a lower number of infectious units per ejaculate from all patients
with initially positive cultures who have been evaluated to date. HIV I RNA was reduced in
all subjects examined with a median decrease of 1.2 logIl0 (16 fold) and a mean decrease
of 1.4 log10 (25 fold)(p0.0003 student T test).Two thirds of post-treatment samples had
HIV- I RNA levels below the limit of detection of the assay (i,000 copies/ml). Concomitant
evaluation of the ejaculate samples in subjects on no therapy or stable therapy is currently
being undertaken as an appropriate control. Preliminary analysis of this control data seems
to indicate that HIV- I RNA in seminal plasma is stable over an 8- 10 week period These
results suggest that treatment of HIV-I infected individuals with reverse transcriptase
inhibitors may decrease the concentration of HIV-I in the ejaculate and, therefore, the individual's infectiousness.
BL Gilliam, UNC Div. of Infect. Dis., CB#7030, 547 Burnett-Womack, Chapel Hill, NC 27599
phone: [phone], fax: [phone]; e mail bgilliam()med.unc.edu
Mo.A. 1084
APPLICATION OF A FLUORESCENT BASED PARTICLE CONCENTRATION HIV
PROTEASE ASSAY IN THE IDENTIFICATION OF THIRD-GENERATION NONPEPTIDIC DIHYDROPYRONE HIV PROTEASE INHIBITORS AS CLINICAL CANDIDATES
Tomich, Paul K,Thaisrivongs S, Aristoff P Romines K. Howe J, Watennaugh K, Chong K 1,
Kezdy F,Tomich C-S.Tomasselli ATarpley G. Upjohn Laboratories, Pharmacia & Upjohn Inc.,
Kalamazoo, MI 4900 1, USA
Objectives: Having previously introduced two generations of oraily bioavailable, nonpeptidic
mize orally bioaaaiable ithd-generator nionpetdic irhibitors wth good psUns7okinet
(PK) properties with signifcantly improved antiviral potencies.
Methods: Analogs were assayed for inhibitory activity against purified HIV protease. A novel
analysis for determination of K values fr om this end-point assay will be described The
dimeric, tandem and various mutant forms (V82A and V82F/184V) of HIV and the dimer c
form of HIV-2 enzymes were used to assess increased affinity and utility A series of
inhibitors were smultanenusly evalusted ir cirystsl stiructures complexed with the DIV protease that assisted the structure-based design effort.They were also assssed rn hIV- IIIB
infected H9 cells and DIV-IJRCSE infected PBMC antiviral assays.
Results: Us ng a novel end point kinetic analysis, Ki values were determined with a viety
of HIV enzyme structures.The dihydropyrone compound class proved especialy productive.
Within ornor, thie ir hibtor s tested hnd equivalent affinities with th variou for o DIV
I protease aend DIV proease. A linear correlation between Kr vlues and cnlated bird
sng energies based upon structures was observed with the DIV-2 enzyme.Third-genesrat on
nonpeptidic dihydropyrone compounds were identified as potent inhibitor s of DIV protease
(Ki value < 50 pM1) arnd demonstrated high ant visal activity (IC50 salon of 50 eM en DIV
11118B infected H9) cells; anrd IC50 value of 30 eM in DIV-IJRCSF infercted P8MG).

Conclusions: C;:I
been opti,nzed wti!
saquinavit; i dinv,;
ty to the pepti-
compound. A tw,
studies and re
Paul K.Tomich,
Inc., 301 Hennet

Ible, third-generation, nonpeptdic HIV protease inhibitors have,.,,tiviral potency comparable to the peptide-derived inhibitors
Sr In preliminary studies iso ites with much reduced sensitiviiitors remain sensitive to th s class of unique. nonpeptidic, pyrone inhibitors hve been under extensive preclinicai safety
for clinical evaluation n the second half of 1996.
S& Biological Screenin,. Upjohn Liboraitones, Pharmacia & Upjohn,
ainazoo, MI 1t900 I. Phone: 6 16- -85 6491. Fax: [phone].

Mo.A. 1085
AN EFFICIENT ASYMMETRIC SYNTHESIS OF DIHYDROPYRONE HIV PROTEASE
INHIBITORS
Chrusciel Alan, orines, K.R. oris, J.K, Judge, FM., Lovasz, K, Tulinsk y.,VanderVelde,
S.L., Morris, J., Luke, (.. hrsciel, R.A., ustin, J.M.. Dolak,.A., SeeI, EPo Watt,W, Mizsak,
S.A., Gammill, R.B. Pharrnacia an nd Upohn, Inc., Kalamazoo, MI, UISA
Objectives: Several dihydropyrone structures containin cne or two chiral centers had
been identified as po tent HIV protease nhibito rs. Initially these compounds were synthe
sized as mixtu re ns twoenntome or four di rstereomers and sepd ted usin rc hiral
HPLC techniques to provide small qunties for testn.Th work demonstrated that the
stereochemistry of the chiral centers was intin tely related to biological activity Our goals
were to determine the absolute stereochemistry of the n ost potent stereoisomers and to
devise an efficient isyrnmetrcssynthesis of the desired dih dropyrore structures.
Methods: We first focus sed on theisynetiyhsmrcsynhesis of dihydropyrone protease
inhibitors with one chiral center Once this was accomplished, we modified the later steps
of the original synthesis to allow control of a second, remote chiral center: We used chira
HPLC techniques, x-ray crystillography and NMR experiments of key intermediates to elucidate the absolute stereochemistry of the biologically active stereoisomers.
Results: We developed a nline- step synthlesis e the dihy opyrone protee nhIbtors
which allows control of wo remote cfi al centers on kilor am scale.
Conclusions: Ehis asymmetric synthetic route facilitates development of dhydropyrone protease inhibitors by allowing efficient, tmely preparat Ion of the quantities required for preclinical and clinical eva ation of these candidates.
R. A. Chrusciel, 72,0 209 613, Pharmacia & Up ohn, Inc., K amnazoo, MI 4900! USA; Phone
616 385-5246; Fax [phone]; email rachrusc0opwinet upj.com
Mo.A. 1086
DRUG SAFETY EVALUATION OF DIHYDROPYRONES, NON-PEPTIDIC HIV
PROTEASE INHIBITORS, IN RATS AND DOGS
Kakuk Thonas.*, Cole SL, Z ya RM. Scwer de FJ, H oward lGM, Zhao Z', Koephnger
KA*,Zipp GL*, Ishii W'i1 tsu T". Phiarm ci & Upon, Inc, 30 Hen etta Sr.,
Kalamazoo, MI 49001 USA, " Pharmaci & Upjohn, Inc.,I.PL, 23 Wadai,-sukubl 5city,
Tsukuba, JAPAN
Objectives: To aid r por lizayn these dihydr opyrone co ouIds o a safety basis and to
provide safety data to support clinical developent in the second half of 1996.
Methods: Oral sine dy and multi -dose (up to 14 days) precl Inca studies for these dihydropyrones have either been completed or -reh n prores n the rat and dog. Dose levels
n Sprague-Dawley rats w er e 0, 63, 125, 250, 500 and 1000 mg/ks F/day, and n dBeagle do gs
were 0,75, 50, and 300 rng/kg/darlyThe d4ily dos es were divided and given s two equal
doses, AM and PM, wish about 8 hours between doses.Typi ai tov cologic p arameters were
evaluated as elas to icokinetics and CytIochro e P4IS0 nduction in hepatic mrosomes.
Results: An ex ample of th e results are iven for one dihydopyrone compound i the rat.
s/ith this compoundlIred tioe fomtn he gcIyand m4 Ind n ty or studies indicated exposure (AUC an d Crnax values) was consi stently deater in firnale rats i than in males,
increased with doses of up to500 k/dynd decreased after repeated dosing reaching
steady-state b y day 8. he nobserved-oI dverse effect ev 1 (NOA eL) was 500mg/kg/day.
At 1000 m/k/day the ormpound ws severe toxicrss- of 6 female rts died: this was
associated wit r a d plsr level o 76 4 vues anied fon 465 pM to 655 Mn
survivors); both sexes sIowed clinical signs of ciscomfo t. Ihe ier was the targetetssueof
toxicity as noted by increases i clotting time,r-eweiht, Cytofhroe siP450 Isoform 3A
activity, and hepatocelluar hypertrophy.
Conclusions:The toxicoloic Isofile of tile dihydropyrone cla eo compounds willbe presented.To date, thle toxicologc infri ao 0o ione rio slpcundrin the rat suggests a similar
target organ to U 96988 ausdf 10301 / (1 st und 2rh eneratoro non peptidDc HV prote se inhibitor clinic al ardi, tes).
DrThomas J. Kakuk, Ph.rmaci & Upohn, Inc. (1)27 -i00 226),. 301 Henrietta Street,
Kalamazoo,M Ml49001,T-elephone: 616-385 798- Fax: 616 <85 7629
Mo.A. 1087
THE PHARMACOKINETICS AND ORAL BIOAVAILABILITY OF THREE DIHYDROPYRONE NON-PEPTIDIC HIV PROTEASE INHIBITORS IN RATS AND DOGS.
Francis j. Schwende, suM IlM.tHoward, Karen F. Wilkinson, (race J.Wlson, Bob D. Rush,Kong
T Chong, Gail L. Zpp, feggy L.Possert, Lilua Siou. Pt,irsmac & Upjohn Compan,
Kalamazoo, MI, USA.
Objective: To characteize the pharnaokinetics and ora l bn oav, illties (%F) of three dihydropyrone, non peptidic HIV protease nhibitors n rats and dogs.
Methods: Rats were surgically implanted with superior versa Caw vcannula to failitate etravenous dosing and serial blood samping.Test compounds were administered as solutions in
dlute aqueous sodin hydroxide at doses of 5 m/kg (Intravenous) and 10 mg/kg (ormal),
Solid forms of the Iree acid and dsiodun salts were arn stered to dous in gelatine capsules. Resultant blood 10evels of tet npounds were determined by specfic and quantita
tve high peformance quid chro atoraph nethods isn non compartmental pharmacokinetic psirsmetes swere nilculseed.
Results: Snocted pha racokrotc parameters re tabulated below along with IC90 values
(eterined mm DIVIIB dnted 19 cell. Acceptble ona bIonvalb te an- total plasna
clearances more observed for ill Ihree assalogues. Maxirmum plnains concenrtioers aftes
oral dosig exceeded piedireed offcacs rpa i orensrations bnsed o e free na
bsntion of dounm st e rnm s poef oid npsrhalys i eui slotishobut
nbor ption Iof-iopous3nd irtnlsed 5' mhe Ioh free aid Iwuas mted by Ilow dissolution.

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