*Article* **Acquired Drug Resistance Enhances Imidazoquinoline Efflux by P-Glycoprotein**

**Anunay J. Pulukuri <sup>1</sup> , Anthony J. Burt 1,2, Larissa K. Opp <sup>1</sup> , Colin M. McDowell <sup>3</sup> , Maryam Davaritouchaee 1,4,5 , Amy E. Nielsen <sup>1</sup> and Rock J. Mancini 1,5,\***


**Abstract:** Multidrug-Resistant (MDR) cancers attenuate chemotherapeutic efficacy through drug efflux, a process that transports drugs from within a cell to the extracellular space via ABC (ATP-Binding Cassette) transporters, including P-glycoprotein 1 (P-gp or ABCB1/MDR1). Conversely, Toll-Like Receptor (TLR) agonist immunotherapies modulate activity of tumor-infiltrating immune cells in local proximity to cancer cells and could, therefore, benefit from the enhanced drug efflux in MDR cancers. However, the effect of acquired drug resistance on TLR agonist efflux is largely unknown. We begin to address this by investigating P-gp mediated efflux of TLR 7/8 agonists. First, we used functionalized liposomes to determine that imidazoquinoline TLR agonists Imiquimod, Resiquimod, and Gardiquimod are substrates for P-gp. Interestingly, the least potent imidazoquinoline (Imiquimod) was the best P-gp substrate. Next, we compared imidazoquinoline efflux in MDR cancer cell lines with enhanced P-gp expression relative to parent cancer cell lines. Using P-gp competitive substrates and inhibitors, we observed that imidazoquinoline efflux occurs through P-gp and, for Imiquimod, is enhanced as a consequence of acquired drug resistance. This suggests that enhancing efflux susceptibility could be an important consideration in the rational design of next generation immunotherapies that modulate activity of tumor-infiltrating immune cells.

**Keywords:** Imiquimod; drug efflux; multidrug resistance; immunotherapy; Toll-Like Receptor

### **1. Introduction**

Multidrug-Resistant (MDR) cancers are implicated in over 90% of metastatic cancer deaths, including melanomas, breast cancer, and prostate cancer [1]. A general trend among MDR cancers is enhanced drug efflux, a term describing the expression of transport proteins that traffic drugs from within a cell to the extracellular space, thereby lowering intracellular drug concentration [2]. In MDR cancers, drug efflux is facilitated by the ABC (ATP-Binding cassette) superfamily which consists of at least 48 distinct transport proteins [3,4]. Of these, P-glycoprotein (P-gp or ABCB1/MDR1) was the first discovered [5,6] and is the most well-studied [7,8]. P-gp is particularly promiscuous, transporting structurally diverse compounds with minimal correlation other than a weak association with hydrophobicity [9–13]. Entire classes of chemotherapeutics, such as taxanes or anthracyclines, are substrates for P-gp-mediated drug efflux [14,15], which makes defeating this mechanism of chemoresistance particularly challenging [16]. However, this same promiscuity could be useful for enhancing extracellular concentrations of small molecule immunomodulator drugs that target bystander cells in the tumor microenvironment, such as tumor-infiltrating lymphocyte

**Citation:** Pulukuri, A.J.; Burt, A.J.; Opp, L.K.; McDowell, C.M.; Davaritouchaee, M.; Nielsen, A.E.; Mancini, R.J. Acquired Drug Resistance Enhances Imidazoquinoline Efflux by P-Glycoprotein. *Pharmaceuticals* **2021**, *14*, 1292. https://doi.org/10.3390/ ph14121292

Academic Editors: Mary J. Meegan, Niamh M O'Boyle and Jean Jacques Vanden Eynde

Received: 7 November 2021 Accepted: 7 December 2021 Published: 10 December 2021

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**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

(TIL) and tumor-associated macrophage (TAM) populations. As such, we were interested in examining the effect of MDR on the ability of small molecule immunomodulators to undergo drug efflux.

From the first empirical whole-organism Toll-Like Receptor (TLR) agonists in the late 1800s [17,18], a range of proinflammatory immunotherapeutics have emerged with mechanisms of action fundamentally orthogonal to P-gp-mediated drug resistance. Within this drug class, imidazoquinoline TLR 7/8 agonists promote TIL and TAM reprogramming along tumor-suppressing axes [19], resulting in a myriad of immune-mediated anti-cancer effects [20–25] in both mono [26] and combination [27] therapies. To achieve these effects, while also limiting systemic inflammatory toxicity, imidazoquinolines are typically administered locally, via topical application or intratumoral injection. This has led our group [28–30] and others [31–34] to develop prodrug delivery strategies that liberate imidazoquinolines, either within the tumor microenvironment, or within cancer cells themselves, by intracellular immunostimulant accumulation followed by drug efflux. Although these strategies have the potential to facilitate delivery of imidazoquinolines to TILs and TAMs, the results also point to drug efflux as a potential rate-limiting step in immune cell activation for imidazoquinolines liberated within cancer cells. This suggests that MDR cancer cells with enhanced transport protein expression could be more susceptible to small-molecule immunotherapeutics. However, the effect of acquired MDR on imidazoquinoline immunotherapeutic efflux has never been explored, apart from a recent study that exclusively examines Resiquimod trafficking [35]. As such, we hypothesized that identifying a pathway for imidazoquinoline efflux and establishing the effects of acquired MDR on efflux would inform the design of the next generation of small-molecule immunotherapeutic prodrugs.

Herein, we demonstrate that imidazoquinolines are highly variable as substrates for P-gp efflux, both in a membrane vesicle model, as well as in vitro across a range of MDR cancer cells (Figure 1). Overall, we find that efflux is significantly enhanced by the MDR phenotype depending on both the type of cancer and the substituent variation regarding N1 and C2 locations on the imidazoquinoline structure itself. *Pharmaceuticals* **2021**, *14*, x FOR PEER REVIEW 3 of 17

**Figure 1.** In this study we establish that Imiquimod (IMQ), Resiquimod (RSQ), and Gardiquimod (GDQ) are substrates of P-gp, and compare P-gp-mediated efflux between Multidrug-Resistant (MDR) cancer cell lines relative to parent cell lines. We also conclude that some efflux likely occurs through other transport proteins as well as passive transport into the extracellular space. **2. Results**  *2.1. Imiquimod, Resiquimod, and Gardiquimod Are Substrates of P-gp*  **Figure 1.** In this study we establish that Imiquimod (IMQ), Resiquimod (RSQ), and Gardiquimod (GDQ) are substrates of P-gp, and compare P-gp-mediated efflux between Multidrug-Resistant (MDR) cancer cell lines relative to parent cell lines. We also conclude that some efflux likely occurs through other transport proteins as well as passive transport into the extracellular space.

the highest concentrations tested. That said, each imidazoquinoline did liberate Pi in the activation test, confirming that all are P-gp substrates, albeit to varying degrees. Based upon this result, we calculated imidazoquinoline P-gp substrate affinity as (Table 1): IMQ (KD = 7.66 µM) > RSQ (KD = 24.37 µM) > GDQ (N.D.). These results were further confirmed using SwissADME, which predicted each imidazoquinoline to be a substrate for P-gp [36]. None of the three imidazoquinolines significantly inhibited maximal vanadate-sensitive ATPase activity when tested as inhibitors, an important point because P-gp-mediated

drug efflux occurs through multiple mechanisms (Figure S3) [37].

To determine if imidazoquinolines are substrates of P-gp, an ATPase assay using

### **2. Results**

*2.1. Imiquimod, Resiquimod, and Gardiquimod Are Substrates of P-gp*

To determine if imidazoquinolines are substrates of P-gp, an ATPase assay using purified membrane vesicles expressing P-gp was performed (Figure 2). All three imidazoquinolines were tested at the same concentrations, from 1.56 to 200 µM. Concentration dependent activation of P-gp ATP (quantified as liberated P<sup>i</sup> ) was detectable down to 6.25 µM for IMQ. RSQ also stimulated P-gp at higher concentrations, whereas GDQ was a poor substrate by comparison, only liberating P<sup>i</sup> above baseline at the highest concentrations tested. That said, each imidazoquinoline did liberate P<sup>i</sup> in the activation test, confirming that all are P-gp substrates, albeit to varying degrees. Based upon this result, we calculated imidazoquinoline P-gp substrate affinity as (Table 1): IMQ (K<sup>D</sup> = 7.66 µM) > RSQ (K<sup>D</sup> = 24.37 µM) > GDQ (N.D.). These results were further confirmed using SwissADME, which predicted each imidazoquinoline to be a substrate for P-gp [36]. None of the three imidazoquinolines significantly inhibited maximal vanadate-sensitive ATPase activity when tested as inhibitors, an important point because P-gp-mediated drug efflux occurs through multiple mechanisms (Figure S3) [37]. *Pharmaceuticals* **2021**, *14*, x FOR PEER REVIEW 4 of 17
