Darolutamide does not interfere with OATP‐mediated uptake of docetaxel

The addition of darolutamide, an androgen receptor signalling inhibitor, to therapy with docetaxel has recently been approved as a strategy to treat metastatic prostate cancer. OATP1B3 is an SLC transporter that is highly expressed in prostate cancer and is responsible for the accumulation of substrates, including docetaxel, into tumours. Given that darolutamide inhibits OATP1B3 in vitro, we sought to characterise the impact of darolutamide on docetaxel pharmacokinetics. We investigated the influence of darolutamide on OATP1B3 transport using in vitro and in vivo models. We assessed the impact of darolutamide on the tumour accumulation of docetaxel in a patient‐derived xenograft (PDX) model and on an OATP1B biomarker in patients. Darolutamide inhibited OATP1B3 in vitro at concentrations higher than the reported Cmax. Consistent with these findings, in vivo studies revealed that darolutamide does not influence the pharmacokinetics of Oatp1b substrates, including docetaxel. Docetaxel accumulation in PDX tumours was not decreased in the presence of darolutamide. Metastatic prostate cancer patients had similar levels of OATP1B biomarkers, regardless of treatment with darolutamide. Consistent with a low potential to inhibit OATP1B3‐mediated transport in vitro, darolutamide does not significantly impede the transport of Oatp1b substrates in vivo or in patients. Our findings support combined treatment with docetaxel and darolutamide, as no OATP1B3 transporter based drug–drug interaction was identified.

on OATP1B3 transport using in vitro and in vivo models.We assessed the impact of darolutamide on the tumour accumulation of docetaxel in a patient-derived xenograft (PDX) model and on an OATP1B biomarker in patients.Darolutamide inhibited OATP1B3 in vitro at concentrations higher than the reported C max .Consistent with these findings, in vivo studies revealed that darolutamide does not influence the pharmacokinetics of Oatp1b substrates, including docetaxel.Docetaxel accumulation in PDX tumours was not decreased in the presence of darolutamide.Metastatic prostate cancer patients had similar levels of OATP1B biomarkers, regardless of treatment with darolutamide.Consistent with a low potential to inhibit OATP1B3-mediated transport in vitro, darolutamide does not significantly impede the transport of Oatp1b substrates in vivo or in patients.Our findings support combined treatment with docetaxel and darolutamide, as no OATP1B3 transporter based drug-drug interaction was identified.
The combination of docetaxel and the androgen receptor inhibitor darolutamide can improve survival in metastatic hormone-sensitive prostate cancer patients.To enter tumor cells, docetaxel uses the drug transporter OATP1B3, which may be inhibited by darolutamide.Here, using in vitro and in vivo models and clinical samples, the authors investigated the impact of darolutamide on docetaxel pharmacokinetics.Darolutamide inhibited OATP1B3 but only above maximum serum concentrations.The drug had no impact on docetaxel accumulation in patient-derived xenograft tumors or OATP1B biomarkers in patient samples.An absence of drug trans-porter-related pharmacokinetic interactions supports the combined use of docetaxel and darolu-tamide.

| INTRODUCTION
A recent development in the treatment of metastatic prostate cancer is the addition of an androgen receptor signalling inhibitor (ARSi) to androgen deprivation therapy (ADT) and docetaxel chemotherapy.
The ARASENS phase III trial demonstrated increased overall survival in metastatic hormone-sensitive prostate cancer patients when the ARSi darolutamide was added to ADT and docetaxel, in comparison to placebo. 1 These data led to a second registration of darolutamide, following its previous approval for non-metastatic castration-resistant prostate cancer. 2Furthermore, a phase II trial (NCT05762536) is currently investigating the efficacy of darolutamide added to ADT and docetaxel or cabazitaxel in metastatic castration-resistant prostate cancer (mCRPC) patients after progression on an ARSi, in comparison to only ADT and docetaxel or cabazitaxel.
Despite the potential for increased efficacy with synergistic drug combinations, adding drugs to a chemotherapy regimen could have unintended consequences that impact patient outcomes.One of these consequences is potential drug-drug interactions (DDI) that impair the delivery of a substrate drug to the tumour.Membrane transport proteins are responsible for drug transport into cells.One of the largest families of drug transporters is the solute carrier (SLC) superfamily. 35][6][7] Previously, we identified docetaxel as a substrate of OATP1B3. 8,9Subsequently, we demonstrated in patient-derived xenograft (PDX) prostate cancer tumours that intratumoural docetaxel concentrations are directly correlated to OATP1B3 expression on the tumour cells. 10Thus, the cellular influx of docetaxeland consequent delivery to the tumourmay be obstructed by drug-induced inhibition of OATP function.Darolutamide and its major metabolite keto-darolutamide have been described to inhibit OATPs in vitro, but no in vivo studies have been performed to date. 11nsidering known discrepancies between in vitro and in vivo results 12 and the potential impact of this DDI on patient outcomes, we sought to assess this DDI using in vivo and clinical approaches.The aim of the current study was to assess the influence of darolutamide on OATP-mediated transport using in vitro and in vivo models and clinical samples.

| Cell lines and chemicals
Human embryonic kidney 293T (HEK 293T; RRID: CVCL_0063, ATCC, Manassas, VA, USA) cells stably expressing SLCO1B3 (coding for OATP1B3) or an empty vector were maintained as described previously. 13All human cell lines have been authenticated using STR profiling within the last 3 years.All experiments were performed with mycoplasma-free cells.Darolutamide was purchased from Selleckchem (Houston, TX, USA, cat.no.S7559) and keto-darolutamide was purchased from MedChemExpress (Monmouth Junction, NJ, USA, cat.

| Transport inhibition assays
Cells were seeded in 12-well plates, coated with poly-d-lysine, at a density of 2 Â 10 6 cells per well in phenol-red free DMEM supplemented with 10% FBS for 24 h prior to transport assays.Substrates and/or inhibitors were added in phenol-red free and serum-free DMEM for the desired time.Cells were lysed in 1 N NaOH for at least 4 h and neutralised in HCl before liquid scintillation counting using a Tri-Carb 4810TR (PerkinElmer, Waltham, MA, USA).Counts were normalised to total protein level, as assessed by a Pierce BCA Protein Assay (Thermo Fisher Scientific, Waltham, MA, USA, cat.no.23225).
According to the FDA Guidelines for In Vitro Metabolism and Transporter Mediated Drug-Drug Interaction Studies, a drug was considered a substrate for OATP1B3 if the uptake of the drug was ≥2-fold higher in OATP1B3 overexpressing cells, as compared to cells containing an empty vector. 14paration of cell membranes and intracellular fractions was completed as previously described. 15In short, HEK 293 T cells were seeded in poly-d-lysin coated 6-well plates at a density of 4 Â 10 6 cells per well and incubated as described above.Alternatively, cells were detached with TrypLE and processed using the ProteoExtract Native Membrane Protein Extraction Kit (EMD Millipore, Burlington, MA, USA, cat.no.444810), according to the manufacturer's protocol, before liquid scintillation counting.

| Cell viability assays
Cells were seeded in 96-well plates at a density of 5 Â 10 3 cells per well and incubated with drugs for the desired time in phenol-red free BUCK ET AL. and serum-free DMEM. 5 mg/mL 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide (MTT) was added for quantification of cell viability, which was normalised to vehicle treated cells.

| In vivo pharmacokinetic studies
Age and sex-matched Oatp1a/b(À/À) and wild-type (WT) mice on an FVB background (Taconic, Germantown, NY, USA) were housed in a temperature-and light-controlled environment.Mice within the same experiment were of similar age (±7 weeks) and weight (±6 g).Mice were randomly assigned to treatment groups (n = 4-5 per group 16 ).
Pravastatin was administered orally at a dose of 20 mg/kg in saline.Docetaxel and cabazitaxel were administered intravenously at a dose of 10 mg/kg in tween 80/EtOH/Saline (20:13:67, v/v/v).Darolutamide was administered orally at a dose of 200 mg/kg in PEG 400/propylene glycol/5% glucose (5:3:2, v/v/v).Drug dosages were previously established and found to be safe. 17When assessing darolutamide as a DDI perpetrator, darolutamide or vehicle was administered 15 min before pravastatin, docetaxel, or cabazitaxel.Blood samples for pharmacokinetic analysis were obtained according to a previously described sample strategy. 16r assessment of intratumoural docetaxel concentrations, 8 NMRI nu/nu male mice (Janvier, Le Genest-Saint-Isle, France) underwent subcutaneous implantation of human prostate cancer PC346C tumours. 18At a tumour volume of 300 mm 3 , 200 mg/kg darolutamide was administered daily for 7 days.Docetaxel (33 mg/kg i.v.) was administered once at day 5. Tumours and plasma were harvested at day 8.

| Clinical samples
For analysis of the endogenous OATP1B biomarker glycochenodeoxycholate-3-sulfate (GCDCA-S), blood samples were collected from 18 patients with mCRPC, participating in a clinical trial (MEC-20-0394), treated with cabazitaxel, sequentially without and with darolutamide. 19Blood samples were collected before and during darolutamide treatment (600 mg bid for 6 weeks) simultaneously with cabazitaxel administrations until 24 h after the start of infusion.

| LC-MS/MS conditions
1][22][23] An LC-MS method was developed for the measure- revealed that the within-day precision and between-day precision of all QCs including lower limit of quantification (LLOQ) ranged from 1.9% to 6.8%, and the accuracy of all QCs ranged from 91.5% to 104%.The lower limit of quantification was 0.1 μg/mL in mouse plasma and the acceptable stability of pravastatin in matrix had already been fully evaluated by Sparidans et al. 24 A single one-step protein precipitation method was used to extract pravastatin from mouse plasma samples.Prior to analysis, frozen samples were thawed at room temperature, 5-μL aliquots of plasma were transferred into a 0.5-mL Eppendorf tube, followed by the addition of 5 μL  Vienna, Austria). 25A two-sided alpha of 0.05 was considered statistically significant.(D) concentrations were measured in plasma samples of male wild type and Oatp1a/1b(À/À) mice receiving darolutamide or vehicle at t = À0.25 h.Pravastatin, docetaxel and cabazitaxel were administered at t = 0 h.(E) Docetaxel concentrations were measured in tumour and plasma samples of male wild type mice (n = 4 per group) subcutaneously bearing a PC346C tumour, after treatment with darolutamide or vehicle for 7 days and docetaxel once at day 5.The ratio between tumour and plasma concentrations of docetaxel is depicted.

| Concentration-dependent inhibition of OATP1B3 by darolutamide in vitro
To determine whether darolutamide inhibits OATP1B3, cells overexpressing OATP1B3 or an empty vector were incubated with various concentrations of darolutamide or its major active metabolite keto-darolutamide, after which EβG, a prototypical OATP1B3 substrate, was added and its influx was quantified.Darolutamide and keto-darolutamide concentration-dependently inhibited EβG influx (IC 50 : 23.7 μM and 33.0 μM, respectively; Figure 1A,B).However, complete inhibition of EβG influx was only observed at concentrations beyond the reported C max in patients of 12.0 μM. 26This inhibition of OATP1B3 by darolutamide was noncompetitive, as determined by a Dixon plot (Figure S1A).In agreement with this, darolutamide was not a substrate of OATP1B3, as its uptake in OATP1B3 overexpressing cells was only 1.2 to 1.4-fold higher than in empty vector containing cells (Figure S1B).Furthermore, pre-incubation with darolutamide substantially augmented OATP1B3 inhibition (1.8 to 2.2-fold) in comparison to no pre-incubation (Figure S1C).
Next, we investigated the impact of darolutamide on OATP1B3-mediated uptake of docetaxel.Docetaxel transport was highly dependent on OATP1B3.OATP1B3 overexpressing cells transported more than twice the amount of docetaxel as compared to empty vector containing cells (113.8 vs. 55.3 pmol/mg protein; p < .001; Figure S2A).Similar to EβG, docetaxel influx was reduced by darolutamide and keto-darolutamide, but only at high concentrations (IC 50 : 51.6 μM and 21.1 μM, respectively; Figure 1C,D).The reliance of docetaxel on transport by OATP1B3 was further substantiated by viability assays.OATP1B3 overexpressing cells were significantly more sensitive to docetaxel than empty vector containing cells (IC 50 : 0.3 nM vs. 1.5 nM; p = .002;Figure 1E).Importantly, this discrepancy was reversible by adding the prototypical OATP inhibitor rifampin (IC 50 : 1.8 nM vs. 4.5 nM; p = .87for OATP1B3 overexpressing cells, compared to empty vector containing cells; Figure 1F).

Initial experiments demonstrated that overall influx of cabazitaxel
was not significantly increased in cells overexpressing OATP1B3 (85.0 vs. 88.0 pmol/mg protein, p = .37;Figure S3A).However, these data were confounded by extensive membrane binding by cabazitaxel (22.8 vs. 22.9 pmol/mg protein for membrane fractions; p = .95;Figure S3B).After correction by removal of membrane fractions, the intracellular fractions of OATP1B3 overexpressing cells were significantly more enriched with cabazitaxel than empty vector containing cells (15.0 vs. 11.6 pmol/mg protein, p < .01; Figure 1G).Nonetheless, this was only a 29% increase in the overexpressing cells.For T A B L E 1 Murine biomarker and pharmacokinetic parameters.docetaxel, on the other hand, intracellular fractions were marked by a substantial increase of 66% ( p .03; Figure S2B,C).Consistent with these darolutamide did not significantly cabazitaxel uptake between OATP1B3 overexpressing cells empty vector cells in both and intracellular fractions (182 vs. 173 protein, p = 16.7 vs. 22.4 pmol/mg protein, p = .57;Figure S3C).

| DISCUSSION
In an era of combination regimens for oncologic diseases, exclusion of DDIs is pivotal to achieve maximal therapeutic benefit. 28While such investigations in vitro are enforced by regulatory agencies, 14 in vivo investigations are not performed regularly before a combination of drugs is marketed. 29Thus, warnings are often presented in the label based exclusively on in vitro experiments, as is the case for darolutamide and OATP1B1/3.Darolutamide's label cautions against coadministration of substrates of OATP1B1 or OATP1B3, as their plasma concentrations may increase.However, prior to our study, this was not assessed in vivo or in patients.Beyond in vitro studies, the only additional evidence was an observed DDI between darolutamide and rosuvastatin, which could be due to transporters other than OATP1B1 or OATP1B3. 11The current study suggests that this warning may be inappropriate, given that we observed no interaction between darolutamide and OATP1B3 substrates in vivo and in patients.
Here, we show that darolutamide does not inhibit OATP1B3 at clinically-relevant concentrations in vitro.Nonetheless, given that in vitro experiments are not always predictive of in vivo observations 12 and especially considering our previous incongruent negative in vitro results 15 we pursued in vivo experiments.A major strength of the in vivo studies conducted here, is the investigation of transport of taxanes in an Oatp1a/1b knockout mouse model, in line with a previous study investigating transport of docetaxel. 30her previous studies on docetaxel and cabazitaxel transport by Oatps were predominantly performed in Oatp1b2 knockout mice, not accounting for the fact that members of the murine Oatp1a subfamily could maintain transport function and compensate for the loss of Oatp1b2. 8,31,32Maximum observed concentrations of darolutamide in mice receiving docetaxel were equal to nearly three times the IC 50 for inhibition of docetaxel uptake as established in vitro.Yet, no inhibition of Oatp1b was observed in vivo.Lack of inhibition may be the result of extensive protein binding by darolutamide and keto-darolutamide in vivo, which is 92% and 99% in human plasma, respectively, possibly hampering their inhibitory potential. 33Another explanation for the lack of inhibition could be that additional hepatic uptake transporters in vivo that are not inhibited by darolutamide, compensate for the transport of substrates.Regardless, our in vivo findings are consistent with our in vitro findings suggesting that there is no DDI between darolutamide and docetaxel.
We did not observe any influence of darolutamide on OATP1B biomarkers in patients.Our findings in vivo and in patients support combined treatment with docetaxel and darolutamide, as no SLC transporter based drug-drug interaction was identified.In accordance with this, recent pharmacokinetic analyses of the ARASENS phase III trial revealed no clinically relevant effect of darolutamide on docetaxel systemic exposure. 34In patients treated with darolutamide, docetaxel systemic exposure was only 6% higher when compared with patients receiving placebo.In contrast, concentrations of the prototypical Breast Cancer Resistance Protein (BCRP) and OATP substrate rosuvastatin were fivefold higher in healthy subjects when darolutamide was administered concomitantly. 11With the present knowledge, this is likely due to inhibition of BCRP by darolutamide, rather than an effect on OATP1B1 or OATP1B3.
In vivo experiments excluded inhibition of hepatic Oatps by darolutamide, as well as inhibition of tumour uptake of docetaxel in an OATP1B3 expressing PDX tumour.Likewise, darolutamide did not influence hepatic OATP1B function in patients, as assessed by a validated endogenous biomarker. 27Based on these results, there is no clinically relevant DDI between darolutamide and OATP1B3 substrates including docetaxel.Our findings support the use of combination treatment of metastatic prostate cancer with docetaxel and darolutamide in its current mode, as no drug transporter-related pharmacokinetic interaction was identified in vivo and drug delivery to the tumour was not compromised.
ment of pravastatin concentrations.In brief, a Vanquish UHPLC coupled with a Altis Plus triple quadrupole mass spectrometer from Thermo Fisher Scientific was used for LC-MS analysis.An Accucore aQ column (50 Â 2.1 mm, dp = 2.6 μm, Thermo Fisher Scientific) was protected by a C18 AQUASIL guard cartridge (2.1 mm Â 10 mm, dp = 3 μm, Thermo Fisher Scientific).The temperature of the autosampler rack was 4 C, and the temperature of the column was maintained at 40 C. The mobile phase was composed of solvent A (0.1% formic acid in H 2 O) and solvent B (0.1% formic acid in acetonitrilemethanol, 1:3 v:v).The total run time was 5.0 min.The gradient conditions were as follows: 0-0.5 min, 5% B; 0.5-4.0min, 5 to 95%, 4.0-4.5 min, 95% B; 4.5-5.0min, 5% B with a flow rate of 0.4 mL/min.The MS assay setting with the negative voltage applied to the ESI capillary was set at 3000 V, and the capillary temperatures was 350 C with a vaporiser temperature of 380 C. Argon was used as the collision gas at a pressure of 1.5 mTorr.Precursor molecular ions and product ions were recorded for confirmation and detection of pravastatin (423.383> 321.217), using [ 2 H 3 ]-pravastatin as an internal standard (426.467> 321.217).Results from assay validation studies of internal standard working solution at 20 μg/mL and 90 μL of neat methanol.The samples were vortex-mixed for 30 s and centrifuged at 13,000 rpm for 10 min at 4 C. Next, 75-μL aliquots of the supernatant were added to autosampler vials (Agilent Technologies, Palo Alto, CA), and a 5-μL volume was injected into the LC-MS/MS system.Pharmacokinetic parameters were estimated using noncompartmental analysis in Phoenix WinNonLin version 8.3 (Certara, Princeton, NJ, USA).

2. 7 |
Statistical analysisAll data are presented as mean ± standard deviation (SD), except for Area Under the Curve (AUC) values and maximum observed plasma concentrations (C max ), which are presented as geometric mean with coefficient of variation (CV) and plasma concentration time curves, which are presented as geometric mean ± 95% confidence interval.Differences between groups were tested for statistical significance by an unpaired Student's t-test or a two-way ANOVA in case of multiple categorical variables.For AUC and C max data, a log-transformation was performed first, as these data are assumed to follow a log-normal distribution.Differences between samples collected from the same patient were tested by a paired Student's t-test.Dose-response curves were interpolated by loglogistic regression.IC 50 values were compared using the function EDcomp of the package drc in R version 4.1.1(R Core Team,

3
While we did not observe an OATP-mediated DDI between darolutamide and docetaxel in our murine model nor in our patient-derived Darolutamide does not influence GCDCA-S concentrations in patients.GCDCA-S concentrations were measured in plasma samples of patients with mCRPC (n = 18) before and during treatment with darolutamide.T A B L E 2 Clinical biomarker and pharmacokinetic parameters.