Bridging the Gap: Conference Record [Abstract book, International Conference on AIDS (12th: 1998: Geneva, Switzerland)]

12th World AIDS Conference

Abstracts 42254-42262 825

508*/42258 Phosphorylation rate of 3'-azidothymidine in
maternal and fetal peripheral blood mononuclear
cells at term
Riad Agbaria. Ben-Gurion University, Department of Clinical Pharmacology,
Beer-Sheva 84105, Israel
Background: 3'-azidothymidine (AZT) therapy during pregnancy and of the newborn reduces the risk of maternal-infant transmission of HIV in HIV-seropositive
pregnant women. A prerequisite for AZT activity is intracellular phosphorylation to
AZT triphosphate (AZTTP), the active metabolite which inhibits the viral reverse
transcriptase. The purpose of this study was to determine and compare the phosphorylation rate of AZT in isolated maternal and fetal peripheral mononuclear
cells (PMNC) at term.
Methods: Maternal PMNC were isolated by ficoll from venous blood drawn from
HIV-seronegative pregnant women (36-40 weeks of gestation) 1-2 hr prior to
delivery. Immediately after delivery, cord blood was collected and the fetal PMNC
were isolated. The fresh isolated maternal and fetal PMNC were maintained in
RPMI medium and incubated with 10 /iM tritiated AZT, 10 jiCi/ml, for 6 and 12
hr. Cells were then harvested and methanolic cell extracts were prepared for AZT
phosphates determination by HPLC and radiochemical detection.
Results: There were no significant differences between AZT phosphate levels in maternal and fetal PMNC after 6 or 12 hr of incubation with AZT. AZT
monophosphate (AZTMP) was found to be the major metabolite (around 95%);
after 6 hr of incubation the maternal and fetal AZTMP levels won 1.244 ~ 1.264
(n = 5) and 1.300 ~ 0.149 pmoles/million cells (n = 6), respectively. Maternal and
fetal AZT diphosphate (AZTDP) levels were 0.044 ~ 0.015 and 0.034 ~ 0.005
pmoles/million cells, respectively. AZTTP levels in maternal and fetal PMNC were
0.052 4 0.015 and 0.045 ~ 0.007 pmols/million cells, respectively. Although the
ratio between AZT phosphates was the same in maternal and fetal PMNC, the
concentration of AZT phosphates decreased after 12 hr of incubation compared
to 6 hr.
Conclusion: These results showed that the fetal PMNC at term, like the maternal PMNC, are able to take up AZT effectively and generate AZT phosphates.
These results can be of major importance in understanding of both the efficacy
and toxicity of AZT in pregnant women and their fetuses.
510*/42259 The rate of decline of HIV-1 RNA in plasma
correlates with nelfinavir concentrations in plasma
Richard M.W. Hoetelmans1, M.H.E. Reijers2, G.F. Weberling3, S. Furriaans4,
F.M.A. Lange5. 1Slotervaart Hospital, Dept. of Pharmacy and Pharmacology
1066 EC Amsterdam; 20nze Lieve Vrouwe Gasthuis/NATEC; 3NATEC; 4HRL
AMC; 5NATEC/AMC, The Netherlands
Background: The rate of decline of HIV-1 RNA in plasma in antiretroviral drug
naive patients who are treated with a combination of d4T, 3TC, saquinavir (SQV)
and nelfinavir (NFV) shows large interindividual differences. Plasma concentrations of SQV and NFV were assayed to determine the influence of drug
concentrations on the rate of decline of HIV-1 RNA.
Methods: In 30 HIV-1 infected patients who are treated with quadruple combination therapy in the ADAM study, plasma HIV-1 RNA, SQV and NFV concentrations
in plasma were determined at weeks 0, 1, 2, 4, and 8. The decline of HIV-1 RNA
in plasma was fitted for each patient with an exponential function, which gives
an estimate of the slope of decline of HIV-1 RNA. All HIV RNA measurements
were used from the start of treatment to the first measurement -`50 copies/mL, or
until week 8. Plasma concentrations of SQV and NFV were determined by validated RP-HPLC assays. SQV and NFV concentrations were related to population
pharmacokinetic data, and were expressed as the ratio between determined and
population concentrations.
Results: Linear regression analysis showed a significant (p = 0.014) relationship between the maximum observed NFV concentration ratio and the slope of
the decline of HIV-1 RNA in plasma. No significant relationship (p = 0.16) was
observed when the SQV concentration ratio was used.
Conclusion: The rate of decline of plasma HIV-1 RNA in antiretroviral naive
patients, who are treated with a combination of d4T/3TC/SQV/NFV, is positively
correlated (p = 0.014) with NFV exposure in plasma. This outcome justifies further
evaluation of the effect of pharmacokinetic parameters on HIV dynamics.
509*/42260 Zidovudine (ZDV) phosphorylation determined at
intervals over 12 months in naive and antiretroviral
experienced HIV+ patients
David J. Back1, P.G. Hoggard1, S.E. Gibbons1, J. Lloyd1, M.G. Barry1,
S.H. Khoo2, C. Loveday3. Pharmacology University of Liverpool, Ashton Street,
Liverpool, L69 3GE; 2North Manchester Hospital, Manchester; 3Royal Free
Hospital, London, UK
Background: Nucleoside analogues (NA) require intracellular phosphorylation to
the active triphosphate before inhibiting HIV replication. Emergence of resistance

is a central problem for the success of antiviral therapy and while attention has
focused on virological mechanisms of resistance it is important to consider that
cellular/pharmacological mechanisms (i.e. decreased intracellular phosphorylation of NA with time) may play a key role. The aim of this study was to determine
intracellular concentrations of ZDV and its mono-, di-and tri- phosphates in PBMCs
at intervals over 12 months in HIV+ patients receiving combination therapy of
ZDV/ddl or ZDV/3TC.

Methods: 24 subjects (both antiretroviral naive and ZDV experienced) were
recruited and blood samples collected at 0, 1 & 2 h at commencement of study
(Day 0), 2 weeks, 1, 2, 3, 6, 9 and 12 months. Intracellular nucleotides were
measured by a validated combined HPLC-RIA method. Viral load was measured
throughout using the NASBA assay.
Results: 10 patients completed the trial to 12 months (6 experienced, 4 naive);
a further 4 to 6 months, and 3 to 3 months. The remainder were less than
3 months. Reasons for discontinuing the study included the intensity of blood
sampling and switches in drug combinations. For the 10 patients who completed
to 1 year there was no significant difference from baseline for either intracellular
total ZDV phosphates or the active ZDV triphosphate (0.08 ~ 0.05, 0.10 ~ 0.07,
0.09 ~ 0.08, 0.13 ~ 0.10, 0.09 I 0.07, 0.12 ~ 0.07, 0.06 A 0.05, 0.10 ~ 0.07
pmoles/10 6 cells.h; mean ~ s.d. at day 0, 2 weeks, 1,, 2, 3, 6, 9 & 12 months).
Comparable data were obtained to 3 or 6 months on the larger cohort. Virological
markers were assessed in relation to individual phosphorylation profiles and data
will be presented.
Conclusions: There is no evidence of i) a systematic decrease in intracellular
phosphorylation of ZDV with time or ii) a difference between naive and experienced patients, suggesting that altered phosphorylation is not the key factor
governing the emergence of resistance. However, there is marked inter-individual
variability.
S511*/42261 Plasma concentrations of saquinavir (SQV)
determine HIV-1 RNA response over a 48-week
period
Richard M.W. Hoetelmans, R.P.G. Heeswijk, Van, P.L. Meenhorst, F.W. Mulder,
W.A. Scheele, F.H. Beijnen. Slotervaart Hospital, Dept. of Pharmacy and
Pharmacology 1066 EC Amsterdam, The Netherlands
Background: The usefulness of therapeutic drug monitoring of SQV concentrations in plasma was investigated. SQV concentrations of all HIV-1 infected
outpatients from the Slotervaart Hospital, Amsterdam, who were treated with
SQV (Invirase) 1200 mg tid plus two RT inhibitors, were routinely monitored.
Observed SQV concentrations were related to the decline in plasma HIV-1 RNA
load after 12, 24, 36, and 48 weeks of therapy.
Methods: SQV plasma concentrations of 130 patients were routinely monitored
from July 1996 until January 1998. During this period 505 plasma samples were
assayed for SQV with a validated HPLC assay. Observed SQV concentrations
were related to population pharmacokinetic data (from 20 patients), and SQV
exposure in each patient was expressed as the ratio between observed and
population concentrations.
Results: Linear regression analysis showed significant positive relationships
between the minimum observed SQV concentration ratio and the decline in HIV-1
RNA after weeks 12 (p = 0.012, n = 107), 24 (p = 0.004, n = 101), 36 (p = 0.024,
n = 82), and 48 (p = 0.004, n = 63). Patients with an estimated SQV trough
concentration >50 ng/mL had a significant higher likelihood to have a sustained
decline in HIV-1 RNA - 2 log units below baseline after 48 weeks of therapy (p =
0.008).
Conclusions: SQV plasma concentrations are significantly related to the decline in plasma HIV-1 RNA after 12, 24, 36, and 48 weeks of therapy. Therapeutic
drug monitoring of SQV plasma concentrations is indicated to optimise antiretroviral therapy. A trough concentration -50 ng/mL should be reached for sustained
inhibition of viral replication.
42262 Enhanced activation of nucleoside analogues in the
presence of hydroxyurea in vitro
David J. Back, Stephen Kewn, P.G. Hoggard, J.S. Henry-Mowatt, M.G. Barry.
Pharmacology University of Liverpool, Ashton Street, Liverpool, L69 3GE, UK
Objectives: To investigate the effects of hydroxyurea (HU) on the activation of
the nucleoside analogues lamivudine (3TC), zidovudine (ZDV), and didanosine
(ddl) in a promonocytic cell line (U937).
Methods: U937 cells (4 x 106 cells/plate) were incubated with either [3H]3TC
(0.65 /iCi; 0.013 1iM), [3H]ZDV (0.65 /iCi; 0.02 /iM), or [3H]ddl (1.5 /ICi; 0.01
1iM), and HU (0, 1, 10, or 100 I M), for 24 h. Cells were extracted in 60%
methanol and phosphates were detected by radiometric HPLC. Additional cell
extracts (subjected to identical conditions but minus radiolabelled drug) were also
taken and deoxycytidine triphosphate (dCTP) levels determined using a DNA
polymerase assay similar to those previously described (Sherman & Fyfe, Anal.
Biochem., 1989, 180; 222-226). This enabled the calculation of 3TCTP/dCTP
ratios.
Results: HU significantly increased the total phosphorylation of 3TC at 10 and
100 p M concentrations (eg. at HU 100 /iM, 111% of control total 3TC phosphates;
P -. 0.001). 3TCTP levels were also increased by HU (100 pM) (116% of control
3TCTP production), dCTP levels were reduced significantly by HU (100 pM)
(67% of control dCTP levels; P - 0.001). The resulting 3TCTP/dCTP ratio was
enhanced by a factor of 2. ZDV phosphorylation was also increased by HU at
10 and 100,tM (eg. at HU 10 iM, 150% of control total ZDV phosphates; P

0.01), with a significant increase in ZDVTP levels noted at HU 100 /M (180% of
control ZDVTP production; P < 0.05). dTTP levels have been shown to decrease
in the presence of HU (Gao et al., Mol. Pharmacol., 1994. 46; 767-772), thus an
enhanced ZDVTP/dTTP ratio would be expected. The metabolism of ddl to its
active triphosphate, dideoxyadenosine-5'-triphosphate (ddATP), was unaffected
by HU at any of the concentrations studied.