Cobicistat boosts the intestinal absorption of transport substrates including HIV protease inhibitors and GS-7340 in vitro

Transcription

1 AAC Accepts, published online ahead of print on 30 July 2012 Antimicrob. Agents Chemother. doi: /aac Copyright 2012, American Society for Microbiology. All Rights Reserved Cobicistat boosts the intestinal absorption of transport substrates including HIV protease inhibitors and GS-7340 in vitro Eve-Irene Lepist, Truc K. Phan, Anupma Roy, Leah Tong, Kelly MacLennan, Bernard Murray, Adrian S. Ray* Gilead Sciences, Inc. Corresponding author: Adrian S. Ray Department of Drug Metabolism, Gilead Sciences, Inc., 333 Lakeside Dr., Foster City, CA Phone: (650) ;.Fax: (650) ; adrian.ray@gilead.com Running Title: Cobicistat inhibits intestinal transporters Key Words: P-glycoprotein, ATP-binding cassette sub-family B member 1, breast cancer resistance protein, ATP-binding cassette sub-family G member 2, caco-2, tenofovir, ritonavir, lopinavir, HIV protease inhibitor 1

2 The experimental pharmacoenhancer cobicistat (COBI), a potent mechanism based inhibitor of cytochrome P450 3A enzymes, was found to inhibit the intestinal efflux transporters P-glycoprotein and breast cancer resistance protein. Consistent with its transporter inhibition, COBI significantly increased the absorptive flux of potential candidates for clinical co-administration including the HIV protease inhibitors atazanavir and darunavir and the lymphoid cell- and tissue-targeted prodrug of the nucleotide analog tenofovir, GS-7340, through monolayers of caco-2 cells in vitro. Downloaded from on July 7, 2018 by guest 2

3 Cobicistat (COBI) is being developed as a pharamacoenhancer (booster) for coformulation with drugs that are metabolized by cytochrome P450 3A (CYP3A) enzymes. Similar to ritonavir (RTV; currently used as a pharmacoenhancer of protease inhibitors used to treat human immunodeficiency virus (HIV PIs)), COBI is a potent mechanismbased CYP3A inhibitor and its coadministration with CYP3A substrates can lead to desired boosting effects and unintended drug-drug interactions. COBI has been found to have a number of potentially differentiating attributes relative to RTV: (i) more selective CYP3A inhibition over other CYP enzymes, (ii) improved solubility and coformulatability, (iii) reduced induction potential mediated by the pregnane X receptor (PXR; also known as the nuclear receptor subfamily 1, group 1, member 2 (NR1I2)), and (iv) lower effects on adipocytes in vitro (21). COBI s lack of anti-hiv activity also eliminates the potential for selection of PI resistance mutations when boosting non-hiv PI drugs (21). Clinically COBI increases systemic levels of the CYP3A substrates midazolam or elvitegravir (EVG) to a similar extent as RTV (7, 13) and coadministration with COBI allows EVG to be administered once daily (16). A fixed dose, once a day, single tablet regimen including EVG/COBI and the nucleos(t)ide reverse transcriptase inhibitors tenofovir disoproxil fumarate and emtricitabine, also known as the QUAD, has completed registrational trials including meeting its primary endpoints in pivotal studies (2, 3, 20). Inhibition of efflux transporters expressed in the intestine can serve as a secondary mechanism for a pharmacoenhancer to increase systemic exposure to coadministered drugs by increasing their absorption. P-glycoprotein (Pgp; also known as multidrug resistance protein 1 (MDR1) or ATP-binding cassette sub-family B member 1 3

4 (ABCB1)) and the breast cancer resistance protein (BCRP; also known as ATP-binding cassette sub-family G member 2 (ABCG2)), both expressed at the apical side of the small intestine, have been highlighted by regulatory agencies and in the literature as key transporters affecting xenobiotic pharmacokinetics (5, 6). In addition to the role of CYP3A enzymes in RTV boosting, HIV PIs are known to be substrates for transporters including Pgp (11). Determination of the relative roles of transport and CYP3A inhibition in limiting HIV PI exposure is difficult due to their being substrates for both processes, but transporters clearly play a role in defining their pharmacokinetic profiles (15). RTV is known to increase the intestinal absorption of digoxin, a Pgp substrate that is not significantly metabolized by CYP3A, by inhibiting its transport by intestinal Pgp (4,14). In order for COBI to have similar boosting effects on HIV PIs to those observed with RTV, COBI will also likely have to cause similar inhibition of intestinal transporters. COBI has been reported to increase digoxin maximal plasma concentration and exposure by 40% and 20%, respectively, suggesting that it also has the potential to inhibit intestinal Pgp (8). The purpose of this study was to determine the mechanism for the clinically observed effects of COBI on digoxin pharmacokinetics and compare the potential for COBI to inhibit intestinal transport relative to that of RTV in vitro. Inhibition of Pgp- and BCRP-dependent accumulation of model substrates in transfected cell-lines. The inhibition of Pgp and BCRP by COBI relative to RTV and the known transport inhibitors cyclosporin A (CSA) and fumitremorgin C was studied by monitoring the effects of co-incubation on the Pgp- and BCRP-dependent accumulation of fluorescent probe substrates in Madin-Darby canine kidney (MDCKII) cells. Inhibition 4

5 of Pgp-mediated transport was assessed using the Pgp substrate calcein-am and MDCKII cells transfected with the human ABCB1 gene encoding Pgp (MDCKII-MDR1) as described previously (18). Similarly, inhibition of BCRP-mediated transport was studied using the BCRP substrate Hoechst and MDCKII cells transfected with the human ABCG2 gene encoding BCRP (MDCKII-BCRP). The 50% inhibition constants (IC 50 ) values, defined as the test article concentration needed to inhibit the maximal transporter specific transport by 50%, were calculated using non-linear curve fitting of inhibition versus concentration to a sigmoidal curve with a variable Hill Coefficient using GraphPad Prism 5 (GraphPad Software Inc., San Diego, CA). COBI inhibited Pgp and BCRP with IC 50 values of 36 ± 10 μm and 59 ± 28 μm, respectively. RTV, when incubated at its approximate solubility limit in assay buffers (20 μm) showed 35% inhibition of Pgp and 21% inhibition of BCRP. COBI showed similar Pgp and BCRP inhibition at the equimolar concentration of 20 μm but higher concentrations of COBI were achievable in assays (tested up to 90 μm) because of its increased solubility relative to RTV. Control compound CSA had an IC 50 of 1.8 ± 0.5 μm against Pgp and fumitremorgin C had an IC 50 of 0.4 ± 0.1 μm against BCRP under the same assay conditions. Reported inhibition constants are the mean ± standard deviation of at least 3 independent studies done in duplicate. Assessing the relevance of these inhibition constants in accordance with draft guidance issued by regulatory agencies (5, 6), both COBI and RTV have the potential to have a clinically relevant effect on intestinal Pgp and BCRP based on [I] 2 /IC 50 greater than 10 where [I] 2 is the dose divided by 250 ml and is approximately 600 µm for either RTV or COBI. Both compounds have limited potential to cause systemic transporter 5

6 inhibition based on [I] 1 / IC 50 not greater than 0.1 where [I] 1 is the maximal plasma concentration not taking into account binding to plasma proteins and is approximately 1.5 µm for either RTV or COBI. The reported thermodynamic solubility of RTV and COBI also support the potential to reach concentrations in the gastrointestinal (GI) tract that significantly inhibited Pgp and BCRP in these assays (21). Taken together results indicate that COBI, like RTV, should inhibit Pgp and BCRP in the GI tract. Effect of COBI on the bidirectional permeability of model Pgp and BCRP substrates through caco-2 cell monolayers. In order to further assess the relative potential of COBI to inhibit intestinal transporters under more pharmacologically relevant conditions, studies were completed with caco-2 cells. Caco-2 cells are a relevant model system of GI absorption that supports the polarized expression of intestinal transporters including Pgp and BCRP (10). Permeability studies using monolayers of caco-2 cells were completed as described previously (18, 20). In studies done to determine transport inhibition, monolayers were pre-incubated for 60 minutes in the presence of assay buffer and inhibitor in order to saturate any transporter binding sites. Test compound concentrations in assay chambers were analyzed by liquid chromatography coupled to tandem mass spectrometry (LC/MS/MS). LC was done using 0.2% formate buffer, a linear gradient from 1% to 80% acetonitrile over 3.5 min, a 1.7 µm 50 x 2.1 mm BEH C 18 column and flow rate of 0.9 ml/min using an Acquity ultra-performance LC system (Waters Corporation, Milford, MA). MS/MS was done in positive ion mode tandem mass spectrometry using a Quattro Premier mass spectrometer (Waters Corporation). Transepithelial electrical resistance (TEER) and lucifer yellow permeability were determined to assure membrane integrity. Each individual experiment was done in 6

7 duplicate and the permeation of control compounds atenolol (low permeability), propranolol (high permeability) and vinblastine (efflux transport) was determined to meet acceptance criteria for each batch of assay plates. Digoxin and prazosin were chosen as model substrates for Pgp and BCRP, respectively, based on recommendations from the FDA and by the International Transporter Consortium (6, 9). COBI and RTV were incubated at their aqueous solubility limits at neutral ph of 90 and 20 µm, respectively. The previously reported higher thermodynamic solubility for COBI relative to RTV (21), coupled with a 50 mg higher clinical dose illustrate that COBI concentrations in the small intestine should exceed those achieved by RTV following oral administration. Similar to the known Pgp inhibitor CSA, COBI and RTV significantly increased the apical to basolateral (A-B) apparent permeability (P app ) of digoxin reflecting a decrease in the B-A P app and efflux ratio (B- A/A-B P app ; Fig. 1A). A similar increase in A-B P app of prazosin was observed in experiments studying the effect of COBI and RTV relative to the known BCRP inhibitor fumitremorgin C (Fig. 1B). The lack of a marked effect on the B-A P app of prazosin in the presence of BCRP inhibitors may reflect lower absolute levels of BCRP inhibition achieved by the inhibitors in the assay relative to that observed for digoxin and Pgp or rate-limiting permeability through the basolateral membrane masking the effects of BCRP inhibition at the apical membrane in the B-A direction. These data suggest similar inhibitory effect of COBI and RTV when incubated at their aqueous solubility limits, concentrations that should approximate pharmacologically relevant luminal concentrations in the small intestine, on the Pgp-mediated transport of digoxin and BCRP-mediated transport of prazosin. The effect of COBI or RTV on digoxin observed 7

8 in these studies is also consistent with increases in digoxin observed upon clinical coadministration (4, 8, 14). Effect of COBI on the bidirectional permeability of HIV PIs and GS-7340 through caco-2 cell monolayers. The effect of COBI and RTV incubated at their respective solubility limits in assay buffer on the bidirectional permeability of the HIV PIs atazanavir, darunavir, lopinavir and GS-8374, an experimental HIV PI (1), through caco-2 cell monolayers was assessed. Consistent with previous studies reporting HIV PIs to be Pgp substrates (11), significant efflux was observed for each of the HIV PIs. Coadministration of COBI and RTV comparably increased the A-B P app of HIV PIs reflecting a decrease in their efflux ratios (Fig. 2A-D). These results suggest that COBI has the potential to inhibit the intestinal efflux of co-administered PIs to a similar extent as RTV. In a separate experiment, the effect of COBI on GS-7340, a lymphoid cell- and tissue-targeted prodrug of tenofovir currently in clinical development (12), permeability across caco-2 monolayers was monitored over 2 hours. COBI increased the concentration of GS-7340 in the receiver well when tested in the A-B direction reflecting an increase in the A-B P app of GS-7340 from 0.74 x 10-6 cm/s to 3.1 x 10-6 cm/s while concomitantly reducing the B-A P app and efflux ratio (Fig. 2E-F). These results are consistent with the recently reported clinical finding that GS-7340 exposure is increased by approximately 2- fold upon coadministration with COBI (17). Similar to previously reported results showing RTV to inhibit the intestinal efflux transport of tenofovir disoproxil fumarate (TDF; 20), co-incubation with COBI was also found to increase the A-B permeability of TDF from 0.29 x 10-6 cm/s to 0.69 x 10-6 cm/s by decreasing its efflux ratio from 9.2 to 8

9 when TDF was incubated at 50 µm (values are the average of 4 wells in each directions from 2 independent experiments done in duplicate). COBI as a substrate for Pgp and BCRP in transfected cells. To further characterize COBI s interaction with Pgp and BCRP and understand the mechanism for the observed inhibition, bidirectional permeability assays were completed in MDCKII cells transfected with the genes for the human transport proteins to determine if COBI is a substrate for these efflux transporters (Fig. 3). MDCKII cells were grown to confluence over 4 to 6-days on 24-well PET transwell plates (BD Biosciences). Experiments were conducted as described above for caco-2 transepithelial transport studies and samples analyzed by LC/MS/MS. Similar quality control and acceptance criteria were used as those described above for caco-2 studies. TEER values and the permeability of lucifer yellow, atenolol and propranolol were determined to meet acceptance criteria for each batch of assay plates. Efflux ratios were determined to be at least 3-fold higher in transfected versus nontransfected monolayers for the model Pgp substrate vinblastine and BCRP substrate prazosin. The overexpression of Pgp or BCRP in MDCKII cells increased the efflux ratios of COBI reflecting a decrease in A-B P app and an increase in B-A P app. Consistent with Pgp- and BCRP-dependent transport, COBI efflux was decreased in the presence of the Pgp inhibitor CSA and the BCRP inhibitor Ko134. These results illustrate that COBI is a substrate for both Pgp and BCRP suggesting that the observed inhibition may be due to competition for the binding sites of the respective transporters. In conclusion, we have shown that the experimental pharmacoenhancer COBI has a similar potential to inhibit intestinal transporters to the currently used 9

10 pharmacoenhancer RTV. COBI is a substrate of Pgp and BCRP and likely has a competitive mode of inhibition with co-administered agents. COBI affects these transporters during intestinal absorption, facilitated by its high solubility and resulting high concentrations in the GI tract. Combined, these results suggest that COBI can inhibit intestinal transporters and increase the absorption of co-administered substrates, including HIV PIs and GS Downloaded from on July 7, 2018 by guest 10

11 Acknowledgements Parental MDCKII cells and MDCKII cells stably transfected with the human MDR1 or ABCG2 gene (encoding Pgp and BCRP, respectively; MDCKII-MDR1 and MDCKII- BCRP cells) were kindly provided by Piet Borst of The Netherlands Cancer Institute

12 References 1. Callebaut C, Stray K, Tsai L, Williams M, Yang ZY, Cannizzaro C, Leavitt SA, Liu X, Wang K, Murray BP, Mulato A, Hatada M, Priskich T, Parkin N, Swaminathan S, Lee W, He GX, Xu L, Cihlar T In vitro characterization of GS-8374, a novel phosphonate-containing inhibitor of HIV-1 protease with a favorable resistance profile. Antimicrob. Agents Chemother. 55: Cohen C, Elion R, Ruane P, Shamblaw D, DeJesus E, Rashbaum B, Chuck SL, Yale K, Liu HC, Warren DR, Ramanathan S, Kearney BP Randomized, phase 2 evaluation of two single-tablet regimens elvitegravir/cobicistat/emtricitabine/tenofovir disoproxil fumarate versus efavirenz/emtricitabine/tenofovir disoproxil fumarate for the initial treatment of HIV infection. AIDS. 25: DeJesus E, Rockstroh J, Henry K, Molina JM, Gathe J, Ramanathan S, Wei X, Szwarcberg J, Jandourek A, Cheng A Week 48 Results of an Ongoing Global Phase 3 Study Comparing Elvitegravir/Cobicistat/Emtricitabine/Tenofovir (Quad) with Atazanavir/ritonavir plus Emtricitabine/Tenofovir in Treatment-naïve HIV-1+ Subjects Showing Efficacy, Safety, and Pharmacokinetics, abstr Abstr. 19th Conference on Retroviruses and Opportunistic Infections Ding R, Tayrouz Y, Riedel KD, Burhenne J, Weiss J, Mikus G, Haefeli WE Substantial pharmacokinetic interaction between digoxin and ritonavir in healthy volunteers. Clin. Pharmacol. Ther. 76:

13 European Medicines Agency. Guidelines on the investigation of drug interactions ent_detail.jsp?webcontentid=wc &murl=menus/document_library/docum ent_library.jsp&mid=0b01ac058009a3dc 6. Food and Drug Administration. Guidance for Industry: Drug Interaction Studies - study design, data analysis, implications for dosing and labeling recommendations uidances/ucm pdf 7. German P, Warren D, West S, Hui J, Kearney BP Pharmacokinetics and bioavailability of an integrase and novel pharmacoenhancer-containing single-tablet fixed-dose combination regimen for the treatment of HIV. J. Acquir. Immune Defic. Syndr. 55: German P, Mathias A, Wei L, Murray B, Warren D, Kearney BP The Effect of Cobicistat and Cytochrome P450 2D6, 2B6 and P-glycoprotein Using Phenotypic Probes. Abstr. 12 th Int. Workshop Clin. Pharmacol. HIV Ther. 9. Giacomini KM, Huang SM, Tweedie DJ, Benet LZ, Brouwer KL, Chu X, Dahlin A, Evers R, Fischer V, Hillgren KM, Hoffmaster KA, Ishikawa T, Keppler D, Kim RB, Lee CA, Niemi M, Polli JW, Sugiyama Y, Swaan PW, Ware JA, Wright SH, Yee SW, Zamek-Gliszczynski MJ Membrane transporters in drug development. Nat. Rev. Drug Discov. 9:

14 Hidalgo IJ, Raub TJ, Borchardt RT Characterization of the human colon carcinoma cell line (Caco-2) as a model system for intestinal epithelial permeability. Gastroenterology. 96: Kim RB, Fromm MF, Wandel C, Leake B, Wood AJ, Roden DM, Wilkinson GR The drug transporter P-glycoprotein limits oral absorption and brain entry of HIV-1 protease inhibitors. J. Clin. Invest. 101: Lee WA, He GX, Eisenberg E, Cihlar T, Swaminathan S, Mulato A, Cundy KC Selective intracellular activation of a novel prodrug of the human immunodeficiency virus reverse transcriptase inhibitor tenofovir leads to preferential distribution and accumulation in lymphatic tissue. Antimicrob. Agents Chemother. 49: Mathias AA, German P, Murray BP, Wei L, Jain A, West S, Warren D, Hui J, Kearney BP Pharmacokinetics and pharmacodynamics of GS-9350: a novel pharmacokinetic enhancer without anti-hiv activity. Clin. Pharmacol. Ther. 87: Penzak SR, Shen JM, Alfaro RM, Remaley AT, Natarajan V, Falloon J Ritonavir decreases the nonrenal clearance of digoxin in healthy volunteers with known MDR1 genotypes. Ther. Drug. Monit. 26: Polli JW, Jarrett JL, Studenberg SD, Humphreys JE, Dennis SW, Brouwer KR, Woolley JL Role of P-glycoprotein on the CNS disposition of amprenavir (141W94), an HIV protease inhibitor. Pharm. Res. 16:

15 Ramanathan S, Mathias AA, German P, Kearney BP Clinical pharmacokinetic and pharmacodynamic profile of the HIV integrase inhibitor elvitegravir. Clin. Pharmacokinet. 50: Ramanathan S, Wei X, Custodio J, Wang H, Dave A, Cheng A, Kearney BP Pharmacokinetics of a novel EVG/COBI/FTC/GS-7340 single tablet regimen, abstr.o-13. Abstr. 13 th Int. Workshop Clin. Pharmacol. HIV Ther. 18. Ray AS, Cihlar T, Robinson KL, Tong L, Vela JE, Fuller MD, Wieman LM, Eisenberg EJ, Rhodes GR Mechanism of active renal tubular efflux of tenofovir. Antimicrob. Agents Chemother. 50: Sax P, DeJesus E, Mills A, Zolopa A, Cohen C, Wohl D, Gallant J, Liu H, Quirk E, Kearney BP Elvitegravir/Cobicistat/Emtricitabine/Tenofovir (Quad) Has Non-inferior Efficacy and Favorable Safety Compared to Efavirenz/Emtricitabine/Tenofovir in Treatment-naïve HIV-1+ Subjects, abstr Abstr. 19th Conference on Retroviruses and Opportunistic Infections Tong L, Phan TK, Robinson KL, Babusis D, Strab R, Bhoopathy S, Hidalgo IJ, Rhodes GR, Ray AS Effects of human immunodeficiency virus protease inhibitors on the intestinal absorption of tenofovir disoproxil fumarate in vitro. Antimicrob. Agents Chemother. 51: Xu L, Liu H, Murray BP, Callebaut C, Lee MS, Hong A, Strickley RG, Tsai LK, Stray KM, Wang Y, Rhodes, GR, Desai MC Cobicistat (GS-9350): A Potent and Selective Inhibitor of Human CYP3A as a Novel Pharmacoenhancer. ACS Med. Chem. Lett. 1:

16 Legends to Figures FIG 1. Effect of RTV (20 µm) and COBI (90 µm) on the bi-directional permeability through monolayers of caco-2 cells of 10 µm of the Pgp substrate digoxin (panel A) and BCRP substrate prazosin (panel B). The known Pgp inhibitor CSA (10 µm) and BCRP inhibitor fumitremorgin C (2 µm) were used as positive controls. The black bars show apical to basolateral (A-B) and the open bars basolateral to apical (B-A) permeability and efflux ratios are indicated above graphs for each experimental condition. Results are the mean ± standard deviation of at least 4 independent experiments done in duplicate and statistical significance was assessed by comparing results to no co-treatment wells using paired two-tailed Student s t tests (*, P <0.05; **, P<0.01). FIG 2. Effect of RTV (20 µm) and COBI (90 µm) on the bi-directional permeability through monolayers of caco-2 cells of 10 µm of the HIV PIs atazanavir (panel A), darunavir (panel B), lopinavir (panel C) and GS-8374 (panel D). The black bars show apical to basolateral (A-B) and the open bars basolateral to apical (B-A) permeability and efflux ratios are indicated above graphs for each experimental condition. Results are the mean ± standard deviation of at least 4 independent experiments done in duplicate and statistical significance was assessed comparing directional results to no co-treatment wells by using paired two-tailed Student s t tests (*, P <0.05; **, P<0.01; ***,P<0.001). Effect of COBI (90 µm) on the bi-direction permeability of GS-7340 (10 µm) through caco-2 monolayers over a 2 hour time course in the A-B (panel E) and B-A (panel F) directions. Open symbols depict presence and solid symbols depict absence of COBI. 16

17 Results are the mean ± standard deviation of duplicate measurements from two independent experiments FIG 3. Bi-directional permeability of COBI (10 µm) in MDCKII-WT, MDCKII-MDR1 (panel A) and MDCKII-BCRP cells (panel B). The black bars show apical to basolateral (A-B) and the open bars basolateral to apical (B-A) permeability and efflux ratios are indicated above graphs for each experimental condition. CSA (10 µm) and Ko134 (10 µm) were used as known inhibitors of Pgp and BCRP, respectively. Results are the average of duplicate wells from a representative side by side experiment done comparing wild type MDCKII (MDCKII-WT) to MDCKII-MDR1 or MDCKII-BCRP cells in the presence or absence of respective inhibitors. 17

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