Effects of Tenofovir on the In Vitro Toxicity of Selected Antiretrovirals in Human Renal Proximal Tubule Cells

1. 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; 2Universitat 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 46th Interscience Conference on Antimicrobial Agents and Chemotherapy September 27 - 30, 2006 San Francisco, California Poster # H-1903

2. Introduction • 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)

3. Methods • 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) • 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 • 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

4. Methods (cont’d) • 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 • 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 9.1.3software (SAS Institute Inc., Cary, NC, USA) and the level of significance wasestablished as 0.05 (two-sided)

5. Cytotoxicity of TFV, ddI, AZT and Their Combinations in RPTECs Viability [% control] 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

6. 1 4 0 1 2 0 1 0 0 * 8 0 Viability [% control] 6 0 4 0 2 0 0 N o - T F V 3 0 - R T V 1 0 T F V 3 0 R T V 1 0 - R T V 1 0 T F V 3 0 R T V 1 0 d r u g T F V 3 0 T F V 3 0 R T V 1 0 L P V 2 0 L P V 4 0 Concentrations in [µM] Cytotoxicity of LPV, RTV, and Their Combinations with TFV in RPTECs ] l o r t n o C % [ y t ili b a i V 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

7. Effects of NRTIs on Mitochondrial DNA Levels in RPTECs 200 175 150 125 100 % Untreated control * 75 # 50 # * # * * 25 * * 0 No drug 3 30 300 3 40 200 3 40 200 3 40 200 3 40 200 m m m m m TFV ( M) ddI ( M) ddI ( M) + AZT ( M) AZT ( M) + m m TFV (30 M) TFV (30 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)

8. Effects of NRTIs on Mitochondrial COII mRNA in RPTECs 200 175 150 125 100 % Untreated control 75 # * 50 * 25 * * * 0 No drug 3 30 300 3 40 200 3 40 200 3 40 200 3 40 200 m m m m m TFV ( ddI ( ddI ( M) + AZT ( M) AZT ( M) + M) M) m m TFV (30 M) TFV (30 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)

9. Conclusions • 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 • 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 • 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 • The results suggest that TFV is unlikely to be the unique offending drug in the reported cases of renal dysfunction in TDF-treated patients • 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

10. References • Schooley RT, et al., Study 902 Team. AIDS 2002; 16:1257-1263. • Gallant JE, et al,. 903 Study Group. JAMA 2004; 292:191-201. • Gallant JE, et al, for the Study 934 Group. N Engl J Med 2006; 354:251-260. • Karras A, et al. Clin Infect Dis 2003; 36:1070-1073. • 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. • Molina JM, et al. Once-Daily vs.Twice-Daily Lopinavir/ritonavir in Antiretroviral-Naïve Patients: 96-Week Results. IAS 2005, Rio de Janeiro, Brazil, July 2005.

11. Acknowledgements • This work was supported by grants from: • 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.