Transcript No Slide Title

Effects of Tenofovir on the In Vitro
Toxicity of Selected Antiretrovirals In
Human Renal Proximal Tubule Cells
F Vidal,1 JC Domingo,2 J Guallar,2 M Saumoy,1 B Cordobilla,2 M Giralt,2 F
Torres,3 F Villarroya,2 T Cihlar,4 and P Domingo3,5
1Hospital
Universitari Joan XXIII and Universitat Rovira I Virgili, Tarragona,
Spain;
de Barcelona, Barcelona, Spain; 3Universitat Autònoma
de Barcelona, Barcelona, Spain; 4Gilead Sciences, Inc, Foster City, USA;
5Hospital de la Santa Creu i Sant Pau, Barcelona, Spain
2Universitat
46th Interscience Conference on Antimicrobial Agents and Chemotherapy
September 27 - 30, 2006
San Francisco, California
Poster # H-1903
Introduction
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Tenofovir disoproxil fumarate (TDF) is an oral prodrug of tenofovir (TFV), a
nucleotide RT inhibitor that is widely used for the treatment of HIV-1 infection
Controlled clinical studies found TDF safe with the incidence of TDFa ssociated renal impairment being 1-3%1-3. Several reports described a subset
of patients with renal dysfunction attributed to TDF4-6
Common features of most TDF-related renal toxicity were the advanced stage
of HIV-1 infection, diabetes, extensive pre-treatment with antiretrovirals and/or
other potentially nephrotoxic drugs. The reported patients were receiving
advanced or salvage regimens, which frequently included lopinavir/ritonavir
(LPV/RTV) and/or didanosine (ddI) in addition to TDF4-6
The underlying mechanism of TFV-associated renal toxicity, particularly in
relation to its combinations with other antiretrovirals has yet to be established.
Prior studies have reported a low in vitro cytotoxic7 and mitotoxic8 potential of
TFV alone
To further understand the mechanisms of TFV-associated nephrotoxicity we
determined the in vitro cytotoxicity and mitochondrial toxicity of TFV alone and
in combination with selected antiretrovirals in human renal proximal tubule cells
(RPTECs)
VIdal, ICAAC 2006 September 27-30, 2006, San Francisco, CA. Poster #H-1903.
Methods
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Cells and drugs: Primary human RPTECs (Dominion Pharmakine, Spain) were
cultured in DMEM/F-12 (1:1) supplemented with 5 ng/ml selenium, 5 μg/ml insulin,
5 μg/ml transferrin, 40 ng/ml hydrocortisone, 10 ng/ml EGF and 4 pg/ml triiodothyronine.
AZT and ddI were provided by Glaxo (Herdtfordshire, UK) and Bristol-Myers-Squibb
(New Brunswick, NJ, USA), respectively, and TFV by Gilead Sciences, Inc. (Foster City,
CA, USA). RTV and LPV were provided by Abbott (Chicago, IL, USA)
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Cytotoxicity: Experiments were carried out in 96-well plates. Cell viability was
determined by the MTT-based assay and expressed as a percentage of the viability
determined for the untreated control. Cytotoxicity was evaluated at days 15 and 22 for
NRTIs and at day 12 for PIs. Data for every treatment were expressed as means ± SEM
of at least 6 independent experiments
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mtDNA quantification: mtDNA was quantified by real-time PCR using 20 ng of total
cellular DNA as template. A region of the mtDNA-encoded subunit II of cytochrome c
oxidase (COII) gene was used for the amplification (Assay-by-design, Applied
Biosystems). Abundance of nuclear DNA (nDNA) was determined by the amplification of
the intronless gene for CEBP-alpha (Assay-on-Demand, Hs 00269972-51, Applied
Biosystems). Four independent experiments were performed with each of them carried
out in duplicate. Results were calculated as the mean ratio of mtDNA to nDNA value
VIdal, ICAAC 2006 September 27-30, 2006, San Francisco, CA. Poster #H-1903.
Methods (cont’d)
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mtDNA-encoded COII mRNA expression: Quantification of COII mRNA was
performed by qPCR. Isolated total RNA was reverse-transcribed using random
primers. The amplification conditions were as for mtDNA assays. Cyclophilin mRNA was
used as a reference control for nuclear-encoded mRNA (Assay-on-Demand,
Hs99999904_m1, Applied Biosystems). Results were expressed as the mean of a ratio of
COII mRNA to cyclophilin mRNA signal from individual samples in duplicate
measurements. Four independent experiments were performed and results were
expressed as the ratio mean values
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Statistical analysis: Results were expressed as means and 95% CI, medians and IQRs.
ANOVA was performed to assess the cytotoxicity in RPTECs. The latest
measurements (22 days for NRTIs and 12 for PIs) were adjusted by the Bonferroni’s
method for pairwise comparisons; no multiplicity adjustments were applied to the rest of
comparisons since they were considered supportive. A non-parametric
ANOVA was used for mitochondrial analyses by applying a rank transformation on the
dependent variable. All analyses were performed using SAS version 9.1.3
software (SAS Institute Inc., Cary, NC, USA) and the level of significance was
established as 0.05 (two-sided)
VIdal, ICAAC 2006 September 27-30, 2006, San Francisco, CA. Poster #H-1903.
Cytotoxicity of TFV, ddI, AZT and Their
Combinations in RPTECs
140
Treatment Duration:
15 days
22 days
22 da ys with TFV for the last 5 days only
120
*
Viability [% control]
100
*
*
80
*
*
*
*
TFV 30
-
*
*
60
*
40
20
Viability [% control]
0
No
d ru g
3
30
TFV
300
-
TFV 3 0
ddI 3
-
T F V 30
ddI 40
-
T FV 3 0
dd I 200
-
TFV 3 0
AZT 3
-
AZT 40
TFV 3 0
AZ T 200
Concentrations in [µM]
Viability of RPTECs (expressed in % relative to untreated control) was determined following a treatment with NRTIs and their combinations.
Data represent means ± s.e.m. Groups marked with an asterisk (*) are statistically significantly different (Bonferroni adjusted) from
corresponding untreated (no drug) controls
VIdal, ICAAC 2006 September 27-30, 2006, San Francisco, CA. Poster #H-1903.
Cytotoxicity of LPV, RTV, and Their Combinations
with TFV in RPTECs
140
Viability [% control]
120
100
]lo
rtn
o
60 C
%
[
y
t
40 ili
b
a
iV
20
*
80
0
No
drug
TFV 30
RTV 10
-
RTV 10 TFV 30 RTV 10
TFV 30
LPV 20
-
RTV 10 TFV 30 RTV 10
TFV 30
LPV 40
Concentrations in [µM]
Viability of RPTECs (expressed in % relative to untreated control) was determined following a 12 day treatment with PIs in
the absence or presence of TFV. Data represent means ± s.e.m. Groups marked with an asterisk (*) are statistically
significantly different (Bonferroni adjusted) from untreated (no drug) control
VIdal, ICAAC 2006 September 27-30, 2006, San Francisco, CA. Poster #H-1903.
Effects of NRTIs on Mitochondrial
DNA Levels in RPTECs
200
% Untreated control
175
150
125
100
*
75
50
#
25
*
*
*
40
200
0
No
3
drug
30
300
TFV ( m M)
3
ddI ( m M)
3
#
#
*
*
40
200
3
ddI ( m M) +
TFV (30 m M)
40
200
AZT ( m M)
3
40
200
AZT ( m M) +
TFV (30 m M)
mtDNA content in RPTECs was determined following a 22 day treatment with tested NRTIs. Data are expressed as %
relative to mean control values. The line within each box shows the median and the upper and lower boundary indicate
the 75th and 25th percentile, respectively. Error bars above and below the box indicate 100 and 0 percentile,
respectively. Statistically significant differences from the control group (Bonferroni adjusted) marked with an asterisk
(*). Effects of TFV alone were not statistically significant (3µM, p = 0.15; 30µM, p = 0.20; 300µM p = 0.1; all Bonferroni
adjusted). Groups marked with (#) are significantly different from the corresponding groups without TFV (unadjusted
pairwise comparisons)
VIdal, ICAAC 2006 September 27-30, 2006, San Francisco, CA. Poster #H-1903.
Effects of NRTIs on Mitochondrial
COII mRNA in RPTECs
200
% Untreated control
175
150
125
100
75
#
50
*
*
25
*
*
0
No
3
drug
30
300
TFV ( m M)
3
40
200
ddI ( m M)
3
40
*
200
ddI ( m M) +
TFV (30 m M)
3
40
200
AZT ( m M)
3
40
200
AZT ( m M) +
TFV (30 m M)
Expression of mtDNA-encoded mRNA for COII mRNA in RPTECs treated with NRTIs was determined after 22 days.
Data are expressed as percentage relative to the mean control values. The line within each box shows the median and
the upper and lower boundary indicate the 75th and 25th percentile, respectively. Bars above and below the box
indicate 100 and 0 percentile, respectively. Statistically significant differences from the control group (Bonferroni
adjusted) marked with an asterisk (*). Effects of TFV alone were not statistically significant (3µM, p = 0.15; 30µM,
p = 0.20; 300 µM; p = 0.1; all Bonferroni adjusted). Groups marked with (#) are significantly different from the
corresponding groups without TFV (unadjusted pairwise comparisons)
VIdal, ICAAC 2006 September 27-30, 2006, San Francisco, CA. Poster #H-1903.
Conclusions
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TFV does not show any significant in vitro cytotoxicity or mitochondrial toxicity in
primary human RPTECs whereas AZT exhibits minor and ddI more profound effects
both on the cell viability and the status of mitochondria
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RTV and LPV are significantly cytotoxic in RPTECs only when combined together
at concentrations substantially exceeding their therapeutic levels and TFV does not
enhance the cytotoxicity of LPV/RTV. This finding supports the favorable clinical
safety profile of TDF in combination with LPV/r9
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Importantly, combining TFV with either NRTIs or PIs does not further enhance
cytotoxic effects with the exception of a slight increase of ddI-induced mtDNA
depletion when TFV is present at supratherapeutic concentrations
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The results suggest that TFV is unlikely to be the unique offending drug in the
reported cases of renal dysfunction in TDF-treated patients
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Multiple effects including the combinations with other therapeutics and/or
compromised baseline renal functions may be necessary to induce nephrotoxicity
in a limited subset of individuals treated with TDF
VIdal, ICAAC 2006 September 27-30, 2006, San Francisco, CA. Poster #H-1903.
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Peyrière H, et al. J Acquir Immune Defic Syndr 2004; 35:269-273.
Malik A, et al. J Infect 2005; 51:e61-e65.
Cihlar T, et al. Antivir Res 2002; 54:37-45.
Birkus G, et al. Antimicrob Agents Chemother 2002; 46:716-723.
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2005, Rio de Janeiro, Brazil, July 2005.
VIdal, ICAAC 2006 September 27-30, 2006, San Francisco, CA. Poster #H-1903.
Acknowledgements
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This work was supported by grants from:
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–
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Marato de TV3 (02/1830 and 02/0631), Fondo de Investigación Sanitaria (FIS 02/1282, 05/1501,
05/1591).
FIPSE 3161/00.
Red Temática Cooperativa de Investigación en Sida (RIS G03/173), ISCIII.
Unrestricted grant from Gilead Sciences, Inc.
M. Saumoy was the recipient of a research grant from the Instituto de Salud Carlos III.
VIdal, ICAAC 2006 September 27-30, 2006, San Francisco, CA. Poster #H-1903.