Short Communication Molecular Assessment of the Potential for Renal Drug Interactions Between Tenofovir and HIV Protease Inhibit

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Antiviral Therapy 12:267–272 Short communication Molecular assessment of the potential for renal drug interactions between tenofovir and HIV protease inhibitors Tomas Cihlar*, Adrian S Ray, Genevieve Laflamme, Jennifer E Vela, Leah Tong, Michael D Fuller, Anupma Roy † and Gerald R Rhodes

Gilead Sciences, Foster City, CA, USA †Present address: Telic, Inc., Palo Alto, CA, USA

*Corresponding author: Tel: +1 650 522 5637; Fax: +1 650 522 5890; E-mail: [email protected]

Background: Active renal secretion of tenofovir (TFV) across PIs with ritonavir (RTV) and lopinavir being the most proximal tubules occurs via uptake by human organic anion potent inhibitors of TFV transport (62% and 37% inhi-

transporters 1 and 3 (hOAT1 and hOAT3) coupled with bition, respectively, at their Cmax). In the absence of efflux by multidrug resistance protein 4 (MRP4). human serum, RTV at concentrations exceeding its ther-

Co-administration of some HIV protease inhibitors (PIs) apeutic Cmax also exhibited a minor effect on the cellular with tenofovir disoproxil fumarate (TDF), an oral prodrug of efflux of TFV by MRP4 (<30% inhibition at TFV, has been shown to increase systemic levels of TFV, 20 μM). However, no effects of PIs on hOAT1, hOAT3 or leading to a hypothesis that PIs may affect tubular MRP4 were detected in the presence of human serum secretion of TFV and potentially alter the renal safety of TDF. with the exception of RTV that inhibited hOAT3 by

Methods: The effect of PIs on the transport of TFV by approximately 35% at its Cmax. In addition, PIs did hOAT1, hOAT3 and MRP4 was assessed using in vitro not affect the cytotoxicity of TFV or TDF in MRP4- or cell-based transport models. MRP2-overexpressing cells. Results: At concentrations equal to their therapeutic Conclusion: These data indicate a low potential of PIs to

peak plasma levels (Cmax) all PIs showed <20% inhibition interfere with the active tubular secretion of TFV and to of TFV transport by hOAT1. hOAT3 was more sensitive to alter the clinical renal safety profile of TDF.

Introduction

Tenofovir disoproxil fumarate (TDF), an oral of TFV involves its tubular uptake by hOAT1 and prodrug of tenofovir (TFV), is widely used for the hOAT3 coupled with the tubular efflux via MRP4. treatment of HIV infection. TDF is rapidly hydrol- HIV protease inhibitors (PIs) atazanavir (ATV) and ysed in plasma, releasing parent TFV, which is elimi- lopinavir (LPV)/ritonavir (LPV/r; Kaletra®) increase the nated renally by a combination of glomerular plasma exposure of TFV by 25–35% [1,9]. As PIs filtration and active tubular secretion [1]. Prior interact with efflux transporters, such as Pgp and MRP2 studies have identified the human renal organic anion [10,11], it has been proposed that the drug interactions transporters 1 (hOAT1) and 3 (hOAT3) as the trans- between TFV and PIs may occur in the kidney as a result port systems that mediate the uptake of TFV into of TFV efflux inhibition by PIs [5,12]. It has also been proximal tubules [2,3]. In addition to basolateral hypothesized that this inhibition would increase the accu- uptake transporters, multiple apical efflux pumps are mulation of TFV in proximal tubules, changing its renal localized in renal proximal tubules – these include safety profile [5,6,12]. One preliminary study concluded P-glycoprotein (Pgp) and multidrug resistance that PIs may enhance the cytotoxicity of TFV in cells proteins, primarily MRP2 and MRP4 [4]. Although it overexpressing MRP2 [13,14]. To address the potential has been proposed that MRP2 mediates the tubular for these interactions, we studied the effects of PIs on the efflux of TFV [5,6], we and others have recently transport of TFV by hOAT1, hOAT3 and MRP4. In shown that MRP4 and not MRP2 is capable of trans- addition, we assessed whether PIs can enhance the cyto- porting TFV [7,8]. Hence, the active renal secretion toxicity of TFV in cells overexpressing MRP2 or MRP4.

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Materials and methods of MRP4 was verified by western blot and a cytotoxicity assay with adefovir [16]. Cells were incubated in the Drugs presence of 1 μM TDF or 100 μM TFV, and tested PIs TFV and TDF were synthesized at Gilead Sciences. for 1 h. Treated cells were isolated by spinning through [3H]TFV and [3H]TDF were purchased from Moravek the Niosyl M25 oil layer [17]. Cell pellets were Biochemicals (Brea, CA, USA). PIs were isolated from extracted with 70% methanol and analysed by ion-pair their therapeutic formulations by reverse phase high HPLC coupled with tandem mass spectrometry [17]. performance liquid chromatography (HPLC). Cytotoxicity assays Transport assays Madlin–Darby canine kidney (MDCK)II-MRP2 cells Chinese hamster ovary (CHO) cells stably transfected (kindly provided by Dr Piet Borst, Netherlands Cancer with hOAT1 (CHOhOAT1) have been described previ- Institute) [18] or CEM-R1 cells were treated in 96-well ously [15]. Baby hamster kidney (BHK)-21 cells stably plates with serially diluted TFV or TDF in the presence expressing hOAT3 (BHKhOAT3) were generated by a or absence of 10 μM PIs. After 3 days, concentrations

transfection of pIRESneo3 plasmid (Clontech, reducing cell viability by 50% (CC50) were determined Mountain View, CA, USA) containing a hOAT3 using CellTiter-Glo assay (Promega, Madison, WI, USA). coding sequence (GenBank: 042505) followed by the selection of a clone with the highest probenecid-sensitive Results uptake of TFV [3]. The transport assay with CHOhOAT1 and BHKhOAT3 cells was carried out in regular 24-well hOAT1 and hOAT3 plates and in BioCoatTM fibronectin-coated 12-well Six approved PIs were tested at their therapeutically plates (BD Biosciences, San Jose, CA, USA), respec- relevant concentrations for the inhibition of TFV tively. CHOhOAT1 and BHKhOAT3 cells were incubated transport by hOAT1 and hOAT3. At concentration with 1.2 μM [3H]TFV and various concentrations of corresponding to therapeutic peak plasma levels

PIs for 20 and 90 min, respectively, at 37˚C in (1×Cmax), nelfinavir (NFV) reduced the hOAT1 trans- Waymouth buffer [15] with or without 40% human port of TFV by >20%, whereas the other tested PIs

serum, and processed as described before [15]. Cell caused <15% reduction (Figure 1A). At 2×Cmax, all PIs lysates were counted for radioactivity and the except ATV and saquinavir (SQV) inhibited TFV trans- percentage of hOAT1- or hOAT3-specific uptake of port via hOAT1 by >20%. The effect of PIs on hOAT3 TFV relative to untreated control was calculated after was comparatively more pronounced (Figure 1B). At

the subtraction of background uptake in the presence 2×Cmax of RTV, LPV, NFV and LPV/r, transport of TFV

of 1 mM probenecid. was reduced by >50%, whereas 2×Cmax of amprenavir MRP4-mediated efflux of TFV was determined in and ATV showed a less pronounced effect. SQV did not HEK293T cells transiently transfected with pcDNA3.1 affect the activity of hOAT3. plasmids (Invitrogen, Carlsbad, CA, USA) containing PIs are known to be substantially bound by human the coding sequence of human MRP4 (GenBank: serum components [19], which is likely to affect the NM_005845.2) that had been PCR-amplified from a concentration of free drug available to interact with the cDNA clone (Origene #TC121947). Subconfluent cells transporter. Indeed, in the presence of 40% human were transiently transfected with pcDNA3.1 or serum, all PIs including LPV/r had no significant effects pcDNA–MRP4 using Lipofectamine 2000 and on TFV transport by hOAT1 (Figure 1A). Similarly, re-plated into 12-well poly-D-lysine-coated plates. Next ATV, NFV and SQV displayed no inhibition of TFV day, cells were preloaded with 1 μM [3H]TDF for 2 h transport by hOAT3 in the presence of serum. At

under ATP-depleting conditions (glucose-free DMEM 2×Cmax, only RTV and LPV/r had a significant impact,

medium supplemented with 10 mM NaN3 and 10 mM reducing the hOAT3 transport of TFV by 44% and

2-deoxy-D-glucose), washed and supplemented with 22%, respectively. However, at 1×Cmax, only RTV complete DMEM with or without tested inhibitors. reduced the hOAT3-specific transport of TFV by Supernatants were harvested at various time points, >10% in the presence of serum (Figure 1B). cells were then washed and lysed with 0.4% Triton X-100. Supernatants and cell lysates were counted for MRP4 and MRP2 radioactivity and percentage of MRP4-specific efflux Transiently expressed MRP4 substantially acceler- was calculated relative to control without inhibitor. ated both the elimination of [3H]TFV from HEK293T cells preloaded with [3H]TDF and the Intracellular accumulation of TFV in CEM-R1 appearance of [3H]TFV in media (Figures 2A & 2B). CEM-R1 cells overexpress MRP4 as a result of resistance The efflux was sensitive to MRP inhibitor MK-571 selection with adefovir [16]. Continued overexpression and HPLC analysis confirmed that TFV and not TDF

Protease inhibitors and renal transport of tenofovir

Figure 1. Effect of protease inhibitors on TFV transport A

Tenofovir (TFV) transport by human renal organic anion transporters 1 (A) and 3 (B). Percentage of TFV uptake relative to corresponding untreated control has been

determined for each tested protease inhibitor under the following conditions: 1x and 2x therapeutic plasma peak level (Cmax; white bars and light gray bars, respectively); and 1x and 2x Cmax in the presence of 40% human serum (HS; dark gray bars and black bars, respectively). The results represent mean ±SD from at least three independent experiments performed in duplicate. Statistically significant differences (P<0.05) compared with corresponding untreated controls are highlighted with asterisks. Abbreviations with a corresponding 1x Cmax (μM) in parentheses: APV, amprenavir (15.0); ATV, atazanavir (4.5); NFV, nelfinavir (7.0); SQV, saquinavir (3.7); LPV, lopinavir (9.6); RTV, ritonavir (11.2); LPV/r, lopinavir + ritonavir (9.6/1.0); and PBC, probenecid (used at 50 μM as positive control).

is the substrate being effluxed by MRP4. At 20 μM approximately fourfold increase in the accumulation of concentration, only RTV, not LPV or ATV, inhibited TFV was observed relative to the incubation with TDF the MRP4-specific efflux of TFV into culture media alone (Figure 2D). By contrast, 20 μM RTV, LPV or ATV (Figure 2C). However, this effect was eliminated in the did not affect the intracellular accumulation of TFV in presence of 40% serum. Identical results were CEM-R1 cells (Figure 2D). In addition, no increase in obtained from the analysis of intracellular accumula- TFV accumulation in CEM-R1 cells was observed tion of TFV in the presence and absence of tested PIs following a 7-day treatment with 20 μM RTV (data not (data not shown). shown). Consistent with these results, 10 μM LPV, RTV The above observations were confirmed in CEM-R1 or ATV affected neither the cytotoxicity of TFV nor TDF cells stably overexpressing MRP4 [16]. Following the in CEM-R1 (Table 1). At concentrations above 10 μM, co-incubation of CEM-R1 cells with TDF and MK-571, PIs themselves affected CEM-R1 cell growth.

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Figure 2. Effect of PIs on the MRP4-mediated efflux of TFV A B

(A and B) HEK293T cells were transiently transfected with an empty expression plasmid (control) or plasmid containing mutidrug resistance proteins 4 coding sequence and pre-incubated with 1 μM [3H] tenofovir disoproxil fumarate (TDF) prodrug for 2 h under ATP-depleting conditions. After switching to fresh media, intracellular (A) and extracellular (B) concentrations of [3H] tenofovir (TFV) in the absence and presence of MK571 were determined at 30 and 90 min. (C) Transfected cells were preloaded as above, washed and incubated in fresh media with or without 40% human serum (HS) in the presence of various concentrations of tested protease inhibitors for 90 min. Percentage of TFV efflux relative to no drug control was determined for each condition. Data are means ±S.D. from two independent experiments. An asterisk denotes a statistically significant change (P<0.05) in TFV efflux compared with untreated control. (D) CEM-R1 cells were incubated with 1 μM TDF in the presence of MK-571 or tested PIs. Cells were harvested after 1 h, extracted and analysed using high performance liquid chromatography combined with tandem mass spectrometry. An asterisk denotes statistically significant change (P<0.05) from untreated control. ATV, atazanavir; LPV, lopinavir; RTV, ritonavir.

Protease inhibitors and renal transport of tenofovir

Table 1. Effect of PIs on the cytotoxicity of TFV in cells it has been proposed that they may be a consequence overexpressing MRP4 or MRP2 of increased renal accumulation of TFV due to the

CC50, μM* inhibition of its tubular efflux via MRP2 [5,6,12]. PI treatment MRP4 cells† MRP2 cells† However, follow-up studies failed to provide any (10 mM) TFV TDF TFV evidence supporting the involvement of MRP2 in the efflux of TFV [7,8]. Furthermore, contrary to previous ‡ None 9,250 ± 100 ± 11,700 ± results [13,14], this study demonstrated the lack of 1,060 28 1,900 synergistic toxicity in MRP2-overexpressing cells ¶ Lopinavir 9,200 ± 110 ± 8,200 ± treated with the combination of TFV and various PIs. § 850 (0.99) 71 (1.10) 1,900 (0.70) Although recent studies identified MRP4 as tubular Ritonavir 9,000 ± 115 ± 11,300 ± efflux pathway for TFV [7,8], we demonstrated here 0 (0.97) 55 (1.15) 700 (0.96) the lack of any significant MRP4-mediated interac- Atazanavir 9,800 ± 100 ± 12,600 ± tions between PIs and TFV by showing that LPV, RTV, 400 (1.05) 60 (1.00) 1,900 (1.08) and ATV affected neither the accumulation nor the

*Mean ±SD from three independent experiments performed in triplicate. cytotoxicity of TFV in cells overexpressing MRP4. †CEM-R1 cells overexpressing mutidrug resistance protein 4 (MRP4) and Although some inhibition of MRP4-mediated efflux of Madlin–Darby canine kidney II (MDCKII) cells overexpressing MRP2 were used. ‡Tenofovir (TFV) and tenofovir disoproxil fumarate (TDF) showed 50% cytotoxi- TFV was detected in the presence of 20 μM RTV, this city dose (CC50) values of 5,750 and 30 μM, respectively, in control CEM-SS cells interaction is unlikely to bear any pharmacological that do not overexpress MRP4 [7]. TFV showed a CC50 value of 17,600 μM in control MDCKII cells that do not overexpress MRP2 [7]. §The numbers in paren- significance in vivo, because it was observed only in theses represent fold changes in CC50 values relative to a corresponding control the absence of serum and at concentrations exceeding without protease inhibitor (PI) treatment. ¶Lopinavir alone at 10 μM concentration showed approximately 30% reduction in the viability of MDCKII-MRP2 cells. the boosting RTV levels by 20-fold. With the excep- None of the other treatments with PIs alone at 10 μM reduced cell viability by tion of indinavir, PIs are not eliminated by the kidney >10% relative to untreated control. and the renal expression of Pgp and MRP2 is likely to minimize the concentrations of PIs inside proximal tubules. Together with conclusions that the pathways Notably, PIs also did not change the cytotoxicity of mediating the tubular secretion of TFV do not directly TFV in cells overexpressing MRP2 (Table 1). A minor overlap with renal transport systems interacting with shift in the cytotoxicity of TFV was found in the pres- PIs [7], these findings suggest that a significant compe- ence of 10 μM LPV; however, this was probably caused tition between TFV and PIs at the level of renal efflux by the toxicity of LPV alone in MRP2-overexpressing transport is unlikely to occur.

cells (CC50=20 μM). This observation is consistent with Co-administration of TDF with ATV or boosted LPV the inability of MRP2 to transport TFV [7,8]. increases the plasma levels of TFV by 25–35% [1,9]. Our results indicate that ATV did not affect the Discussion handling of TFV by hOAT1, hOAT3 or MRP4. Boosting should not lead to any substantial changes in This study evaluated the effects of PIs on hOAT1, the capacity of ATV to affect these transporters since it

hOAT3, and MRP4 renal transporters involved in the increases its Cmax by less than twofold. Similarly, we active tubular secretion of TFV. At therapeutically rele- found no physiologically relevant interactions of LPV or vant concentrations and in the presence of human RTV with hOAT1 and MRP4, but they caused a serum, all tested PIs displayed minimal inhibition of moderate inhibition of hOAT3, suggesting their poten- TFV transport by hOATs with the exception of LPV tial capacity to reduce the tubular uptake of TFV. and RTV, which caused 20% and 40% inhibition of However, even if it is clinically relevant, this mechanism

hOAT3, respectively, at 2× Cmax. However, a full dose would tend to decrease the accumulation of TFV in of RTV is now rarely used clinically because of its proximal tubules. In accordance, controlled clinical adverse effects. Furthermore, no significant inhibition studies showed a good renal safety of TDF when of hOAT3 was noted at boosting concentrations of co-administered with boosted PIs [20]. RTV. This interaction may be more relevant in a subset Our results suggest that other renal mechanisms for of patients with higher systemic exposure to PIs and/or the pharmacokinetic drug interactions between TFV lower levels of serum components binding PIs. and PIs should also be taken into consideration. PIs In contrast to the inhibition of hOAT1 and/or [10] and TDF [21] are substrates for Pgp, suggesting a hOAT3, drug interactions at the efflux level could possible interaction at the level of intestinal absorption increase the tubular accumulation of TFV, possibly that could lead to higher systemic exposure of TFV. having an adverse effect on the renal safety of TFV. In vitro studies have shown that PIs can increase the Cases of nephrotoxicity in patients treated with the permeability of TFV through intestinal cells exposed combination of TDF and PIs have been reported, and to TDF [22]. Alternatively, the interaction between

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PIs and TDF may occur at the level of hepatic transport 10. Lee CG, Gottesman MM, Cardarelli CO, et al. HIV-1 protease inhibitors are substrates for the MDR1 multidrug and/or metabolism. Further evaluation of these alter- transporter. Biochemistry 1998; 37:3594–3601. native mechanisms is warranted. 11. Huisman MT, Smit JW, Crommentuyn KM, et al. Multidrug resistance protein 2 (MRP2) transports HIV protease inhibitors, and transport can be enhanced by other Acknowledgements drugs. AIDS 2002; 16:2295–2301. 12. Izzedine H, Launay-Vacher V, Deray G Antiviral drug- We would like to thank Piet Borst (Netherlands induced nephrotoxicity. Am J Kidney Dis 2005; 45:804–817. Cancer Institute, Amsterdam) for providing MDCKII- 13. Louie SG, Lam J, Neely M, Beringer P. Multidrug resistance protein-2 (MRP2) inhibition by ritonavir increases teno- MRP2 cells. fovir-associated renal epithelial cell cytotoxicity. 3rd International AIDS Society Conference on HIV Pathogenesis and Treatment; Rio de Janeiro, Brazil, 2005; References Abstract WePe3.3C09 14. Louie SG, Lam J, Neely M, Beringer P. Multidrug resistance 1. Kearney BP, Flaherty JF, Shah J. Tenofovir disoproxil protein-2 (MRP2) inhibition by ritonavir increases teno- fumarate: clinical pharmacology and pharmacokinetics. fovir-associated renal epithelial cell cytotoxicity. 6th Clin Pharmacokinet 2004; 43:595–612. International Workshop on Clinical Pharmacology of HIV 2. Cihlar T, Ho ES, Lin DC, Mulato AS. Human renal organic Therapy; Quebec, Canada; 2005. Abstract 2.16. anion transporter 1 (hOAT1) and its role in the nephrotox- 15. Ho ES, Lin DC, Mendel DB, Cihlar T. Cytotoxicity of icity of antiviral nucleotide analogs. Nucleosides antiviral nucleotides adefovir and cidofovir is induced by Nucleotides Nucleic Acids 2001; 20:641–648. the expression of human renal organic anion transporter 1. 3. Cihlar T, Bleasby K, Roy A, Pritchard J. Antiviral acyclic J Am Soc Nephrol 2000; 11:383–393. nucleotide analogs tenofovir and adefovir are substrates for 16. Schuetz JD, Connelly MC, Sun D, et al. MRP4: A previously kidney organic anion, but not cation transporters: unidentified factor in resistance to nucleoside-based Implications for renal drug interactions. 44th Interscience antiviral drugs. Nat Med 1999; 5:1048–1051. Conference on Antimicrobial Agents and Chemotherapy, 30 October – 2 November 2004, Washingtion DC, USA. 17. Ray AS, Myrick F, Vela JE, et al. Lack of a metabolic and Abstract A-443. antiviral drug interaction between tenofovir, abacavir and 4. Lee W, Kim R. Transporters and renal drug elimination. lamivudine. Antivir Ther 2005; 10:451–457. Annu Rev Pharmacol Toxicol 2004; 44:137–166. 18. Evers R, Kool M, Smith AJ, van Deemter L, de Haas M, 5. Rollot F, Nazal EM, Chauvelot-Moachon L, et al. Borst P. Inhibitory effect of the reversal agents V-104, Tenofovir-related Fanconi syndrome with nephrogenic GF120918 and Pluronic L61 on MDR1 Pgp-, MRP1- and diabetes insipidus in a patient with acquired immunodefi- MRP2-mediated transport. Br J Cancer 2000; ciency syndrome: the role of lopinavir-ritonavir-didanosine. 83:366–374. Clin Infect Dis 2003; 37:e174–e176. 19. Boffito M, Back D, Blaschke T, et al. Protein binding in 6. Zimmermann AE, Pizzoferrato T, Bedford J, Morris A, antiretroviral therapies. AIDS Res Hum Retroviruses 2003; Hoffman R, Braden G. Tenofovir-associated acute and 19:825–835. chronic kidney disease: a case of multiple drug interactions. 20. Johnson M, Grinsztejn B, Rodriguez C, et al. 96-week Clin Infect Dis 2006; 42:283–290. comparison of once-daily atazanavir/ritonavir and twice- 7. Ray AS, Cihlar T, Robinson KL, et al. Mechanism of active daily lopinavir/ritonavir in patients with multiple virologic renal tubular efflux of tenofovir. Antimicrobial Agents failures. AIDS 2006; 20:711–718. Chemother 2006; 50:3297–3304. 21. van Gelder J, Deferme S, Naesens L, et al. Intestinal 8. Imaoka T, Kusuhara H, Adachi M, Schuetz JD, Takeuchi K, absorption enhancement of the ester prodrug tenofovir SugiyamaY. Functional involvement of multidrug resistance- disoproxil fumarate through modulation of the biochemical association protein 4 (MRP4/ABCC4) in the renal barrier by defined ester mixtures. Drug Metab Dispos elimination of the antiviral drugs adefovir and tenofovir. 2002; 30:924–930. Mol Pharmacol 2007 71:619–627. 22. Ray AS, Tong L, Robinson KL, Kearney BP, Rhodes GR. 9. Kearney BP, Mathias A, Mittan A, Sayre J, Ebrahimi R, Role of intestinal absorption in increased tenofovir expo- Cheng AK. Pharmacokinetics and safety of tenofovir diso- sure when tenofovir disoproxil fumarate is co-adminstered proxil fumarate on coadministration with lopinavir/ with atazanavir or lopinavir/ritonavir. 7th International ritonavir. J Acquir Immune Defic Syndr. 2006; Workshop on Clinical Pharmacology of HIV Therapy, 43:278–283. 20–22 April 2006, Lisbon, Portugal. Abstract 49. Accepted for publication 13 October 2006