ATP-binding cassette transporter inhibitor potency and substrate drug affinity are critical determinants of successful drug delivery enhancement to the brain.

BACKGROUND: Pharmacotherapy for brain diseases is severely compromised by the blood-brain barrier (BBB). ABCB1 and ABCG2 are drug transporters that restrict drug entry into the brain and their inhibition can be used as a strategy to boost drug delivery and pharmacotherapy for brain diseases. METHODS: We employed elacridar and tariquidar in mice to explore the conditions for effective inhibition at the BBB. Abcg2;Abcb1a/b knockout (KO), Abcb1a/b KO, Abcg2 KO and wild-type (WT) mice received a 3 h i.p. infusion of a cocktail of 8 typical substrate drugs in combination with elacridar or tariquidar at a range of doses. Abcg2;Abcb1a/b KO mice were used as the reference for complete inhibition, while single KO mice were used to assess the potency to inhibit the remaining transporter. Brain and plasma drug levels were measured by LC-MS/MS. RESULTS: Complete inhibition of ABCB1 at the BBB is achieved when the elacridar plasma level reaches 1200 nM, whereas tariquidar requires at least 4000 nM. Inhibition of ABCG2 is more difficult. Elacridar inhibits ABCG2-mediated efflux of weak but not strong ABCG2 substrates. Strikingly, tariquidar does not enhance the brain uptake of any ABCG2-subtrate drug. Similarly, elacridar, but not tariquidar, was able to inhibit its own brain efflux in ABCG2-proficient mice. The plasma protein binding of elacridar and tariquidar was very high but similar in mouse and human plasma, facilitating the translation of mouse data to humans. CONCLUSIONS: This work shows that elacridar is an effective pharmacokinetic-enhancer for the brain delivery of ABCB1 and weaker ABCG2 substrate drugs when a plasma concentration of 1200 nM is exceeded.

Mutational analysis reveals the importance of residues of the access tunnel inhibitor site to human P-glycoprotein (ABCB1)-mediated transport.

Human P-glycoprotein (P-gp) utilizes energy from ATP hydrolysis for the efflux of chemically dissimilar amphipathic small molecules and plays an important role in the development of resistance to chemotherapeutic agents in most cancers. Efforts to overcome drug resistance have focused on inhibiting P-gp-mediated drug efflux. Understanding the features distinguishing P-gp inhibitors from substrates is critical. Cryo-electron microscopy has revealed distinct binding patterns, emphasizing the role of the L-site or access tunnel in inhibition. We substituted 5-9 residues of the L-site with alanine to investigate whether the binding of a second inhibitor molecule to the L-site is required for inhibiting drug efflux. We reveal, for the first time, that mutations in the L-site affect the drug efflux activity of P-gp, despite their distance from the substrate-binding pocket (SBP). Surprisingly, after the mutations were introduced, inhibitors such as tariquidar and zosuquidar still inhibited drug efflux by mutant P-gps. Communication between the transmembrane helices (TMHs) and nucleotide-binding domains (NBDs) was evaluated using the ATPase assay, revealing distinct modulation patterns by inhibitors for the mutants, with zosuquidar exhibiting substrate-like stimulation of ATPase. Furthermore, L-site mutations abolished ATP-dependent thermal stabilization. In silico molecular docking studies corroborated the altered inhibitor binding due to mutations in the L-site residues, shedding light on their critical role in substrate transport and inhibitor interactions with P-gp. These findings suggest that inhibitors bind either to the SBP alone, and/or to alternate site(s) when the L-site is disabled by mutagenesis.

The metabolic activation of and platelet response to vicagrel vary with P-glycoprotein deficiency, rather than P-glycoprotein inhibition, in mice.

This study aimed to determine changes in the hydrolysis of vicagrel, a substrate drug of arylacetamide deacetylase (Aadac) and carboxylesterase 2 (Ces2), in P-glycoprotein (P-gp)-deficient or P-gp-inhibited mice and to elucidate the mechanisms involved.Male wild-type (WT) and P-gp knock-out (KO) mice were used to investigate the systemic exposure of vicagrel thiol active metabolite H4 and platelet response to vicagrel, and the mRNA and protein expression levels of intestinal Aadac and Ces2. Moreover, WT mice were administered vicagrel alone or in combination with elacridar (a potent P-gp inhibitor) to determine drug-drug interactions.Compared with WT mice, P-gp KO mice exhibited significant increases in the systemic exposure of H4, the protein expression levels of intestinal Aadac and Ces2, and inhibition of ADP-induced platelet aggregation by vicagrel. Further, the H4 exposure was positively correlated with intestinal Aadac protein expression levels but did not vary with short-term inhibition of P-gp efflux activity by elacridar.P-gp-deficient mice, rather than elacridar-treated mice, exhibited significant upregulation of intestinal Aadac and Ces2 and thus, enhanced metabolic activation of and platelet response to vicagrel, suggesting that the metabolic activation of vicagrel may vary with P-gp deficiency, not P-gp inhibition, in mice.

An all-in-one nanoparticle for overcoming drug resistance: doxorubicin and elacridar co-loaded folate receptor targeted PLGA/MSN hybrid nanoparticles.

Overexpression of permeability-glycoprotein (P-gp) transporter leads to multidrug resistance (MDR) through cellular exclusion of chemotherapeutics. Co-administration of P-gp inhibitors and chemotherapeutics is a promising approach for improving the efficacy of therapy. Nevertheless, problems in pharmacokinetics, toxicity and solubility limit the application of P-gp inhibitors. Herein, we developed a novel all-in-one hybrid nanoparticle system to overcome MDR in doxorubicin (DOX)-resistant breast cancer. First, folic acid-modified DOX-loaded mesoporous silica nanoparticles (MSNs) were prepared and then loaded into PEGylated poly(lactic-co-glycolic acid) (PLGA) nanoparticles along with a P-gp inhibitor, elacridar. This hybrid nanoparticle system had high drug loading capacity, enabled both passive and active targeting of tumour tissues, and exhibited sequential and pH-triggered release of drugs. In vitro and in vivo studies in DOX-resistant breast cancer demonstrated the ability of the hybrid nanoparticles to reverse P-gp-mediated drug resistance. The nanoparticles were efficiently taken up by the breast cancer cells and delivered elacridar, in vitro. Biodistribution studies demonstrated substantial accumulation of the folate receptor-targeted PLGA/MSN hybrid nanoparticles in tumour-bearing mice. Moreover, deceleration of the tumour growth was remarkable in the animals administered with the DOX and elacridar co-loaded hybrid nanoparticles when compared to those treated with the marketed liposomal DOX (Caelyx®) or its combination with elacridar.

Cryo-EM structure of P-glycoprotein bound to triple elacridar inhibitor molecules.

P-glycoprotein (P-gp) is an ATP-binding cassette transporter known for its roles in expelling xenobiotic compounds from cells and contributing to cellular drug resistance through multidrug efflux. This mechanism is particularly problematic in cancer cells, where it diminishes the therapeutic efficacy of anticancer drugs. P-gp inhibitors, such as elacridar, have been developed to circumvent the decrease in drug efficacy due to P-gp efflux. An earlier study reported the cryo-EM structure of human P-gp-Fab (MRK-16) complex bound by two elacridar molecules, at a resolution of 3.6 Å. In this study, we have obtained a higher resolution (2.5 Å) structure of the P-gp- Fab (UIC2) complex bound by three elacridar molecules. This finding, which exposes a larger space for compound-binding sites than previously acknowledged, has significant implications for the development of more selective inhibitors and enhances our understanding of the compound recognition mechanism of P-gp.

Coadministration of ABCB1/P-glycoprotein inhibitor elacridar improves tissue distribution of ritonavir-boosted oral cabazitaxel in mice.

Developing an oral formulation for the chemotherapeutic cabazitaxel might improve its patient-friendliness, costs, and potentially exposure profile. Cabazitaxel oral availability is restricted by CYP3A-mediated first-pass metabolism, but can be substantially boosted with the CYP3A inhibitor ritonavir. We here tested whether adding the ABCB1/P-glycoprotein inhibitor elacridar to ritonavir-boosted oral cabazitaxel could further improve its tissue exposure using wild-type, CYP3A4-humanized and Abcb1a/b-/- mice. The plasma AUC0-2h of cabazitaxel was increased 2.3- and 1.9-fold in the ritonavir- and ritonavir-plus-elacridar groups of wild-type, and 10.5- and 8.8-fold in CYP3A4-humanized mice. Elacridar coadministration did not influence cabazitaxel plasma exposure. The brain-to-plasma ratio of cabazitaxel was not increased in the ritonavir group, 7.3-fold in the elacridar group and 13.4-fold in the combined booster group in wild-type mice. This was 0.4-, 4.6- and 3.6-fold in CYP3A4-humanized mice, illustrating that Abcb1 limited cabazitaxel brain exposure also during ritonavir boosting. Ritonavir itself was also a potent substrate for the Abcb1 efflux transporter, limiting its oral availability (3.3-fold) and brain penetration (10.6-fold). Both processes were fully reversed by elacridar. The tissue disposition of ritonavir-boosted oral cabazitaxel could thus be markedly enhanced by elacridar coadministration without affecting the plasma exposure. This approach should be verified in selected patient populations.

Pharmacokinetics of the KRASG12C inhibitor adagrasib is limited by CYP3A and ABCB1, and influenced by binding to mouse plasma carboxylesterase 1c.

Adagrasib (Krazati™) is the second FDA-approved specific KRASG12C inhibitor for non-small cell lung cancer (NSCLC) patients harboring this mutation. The impact of the drug efflux transporters ABCB1 and ABCG2, and the drug-metabolizing enzymes CYP3A and carboxylesterase 1 (CES1) on the pharmacokinetics of oral adagrasib were studied using genetically modified mouse models. Adagrasib was potently transported by human ABCB1 and modestly by mouse Abcg2 in vitro. In Abcb1a/b-/- and Abcb1a/b;Abcg2-/- mice, the brain-to-plasma ratios were enhanced by 33- and 55-fold, respectively, compared to wild-type mice, whereas ratios in Abcg2-/- mice remained unchanged. The influence of ABC transporters was completely reversed by coadministration of the dual ABCB1/ABCG2 inhibitor elacridar, increasing the brain penetration in wild-type mice by 41-fold while no signs of acute CNS toxicity were observed. Tumor ABCB1 overexpression may thus confer adagrasib resistance. Whereas the ABC transporters did not affect adagrasib plasma exposure, CYP3A and Ces1 strongly impacted its apparent oral availability. The plasma AUC0-8 h was significantly enhanced by 2.3-fold in Cyp3a-/- compared to wild-type mice, and subsequently 4.3-fold reduced in transgenic CYP3A4 mice, indicating substantial CYP3A-mediated metabolism. Adagrasib plasma exposure was strongly reduced in Ces1-/- compared to wild-type mice, but tissue exposure was slightly increased, suggesting that adagrasib binds to plasma Ces1c in mice and is perhaps metabolized by Ces1. This binding could complicate interpretation of mouse studies, especially since humans lack circulating CES1 enzyme(s). Our results may be useful to further optimize the clinical safety and efficacy of adagrasib, and give more insight into potential drug-drug interactions risks.

Nano-gold micelles loaded Dox and Elacridar for reversing drug resistance of breast cancer.

The aim of this study was to provide a new effective carrier for rescuing the sensitivity of drug-resistant in breast cancer cells. Nano-gold micelles loaded with Dox and Elacridar (FP-ssD@A-E) were chemically synthesised. With the increase in the amount of Dox and Elacridar, the encapsulation rate of FP-ssD@A-E gradually increased, and the drug loading rate gradually decreased. FP-ss@A-E had a sustained-release effect. Dox, Elacridar, FP-ss@AuNPs, and FP-ssD@A-E significantly improved cell apoptosis, in which, FP-ssD@A-E was the most significant. FP-ssD@A-E significantly decreased the cell viability and improved the Dox uptake. The levels of VEGFR-1, P-gp, IL-6, and i-NOS were significantly decreased after Dox, Dox + Elacridar, FP-ss@AuNPs, and FP-ssD@A-E treatment. It was worth noting that FP-ssD@A-E had the most significant effects. The prepared FP-ssD@A-E micelles, which were spherical in shape, uniform in particle size distribution, and had good drug loading performance and encapsulation efficiency.

Investigations with Drugs and Pesticides Revealed New Species- and Substrate-Dependent Inhibition by Elacridar and Imazalil in Human and Mouse Organic Cation Transporter OCT2.

Multiple drugs are used to treat various indications as well as pesticides that are ingested unintentionally and enter the bloodstream. The residence time or bioavailability of these substances in circulation depends on several mechanisms, such as drug−drug interaction (DDI), drug−pesticide interaction, metabolizing enzymes and the hepatic and renal transport systems, involved in the elimination of the compounds from the body. One of these transporters is the Organic Cation Transporter 2 (OCT2) member of the solute carrier (SLC22) transporter family. OCT2 is highly expressed in the proximal tubule epithelial cells in human and mouse kidney, where it mediates the uptake of endogenous organic cations as well as numerous drugs and xenobiotics, and contributes to the first step of renal clearance. In this study, we examined OCT2 on two subjects: First, the transferability of data from mouse to human, since mice are initially examined in the development of new drugs to assess the renal excretion of organic cations. Second, to what extent the choice of substrate affects the properties of an inhibitor. For this purpose, the functional properties of hOCT2 and mOct2 were validated under the same experimental conditions with the known substrates metformin and 1-Methyl-4-phenylpyridinium iodide (MPP). While hOCT2 and mOct2 showed very low affinities for metformin with Km values of 3.9 mM and 3.5 mM, the affinity of hOCT2 and mOct2 for MPP (62 and 40 µM) was 64- and 89-fold higher, respectively. For our positive control inhibitor decynium22, we determined the following IC50 values for hOCT2 and mOct2: 2.2 and 2.6 µM for metformin uptake, and 16 and 6.9 µM for MPP uptake. A correlation analysis of the inhibitory effects of 13 drugs and 9 pesticides on hOCT2- and mOct2-mediated transport of metformin showed a correlation coefficient R2 of 0.88, indicating good interspecies correlation. Nevertheless, the bioenhancer elacridar and the fungicide imazalil showed species-dependent inhibitory potentials. Concentration-dependent inhibition of hOCT2- and mOct2-mediated metformin uptake by elacridar showed IC50 values of 20 µM and 1.9 µM and by imazalil 4.7 µM and 0.58 µM, respectively. In conclusion, although our data show comparable species-independent interactions for most compounds, there can be large species−specific differences in the interactions of individual compounds, which should be considered when extrapolating data from mice to humans. Furthermore, a comparison of the inhibitory potential of elacridar and imazalil on metformin uptake with that on MPP uptake reveals substrate-dependent differences in hOCT2 and mOct2 for both inhibitors. Therefore, it might be useful to test two different substrates in inhibition studies.

Co-administration of MDR1 and BCRP or EGFR/PI3K inhibitors overcomes lenvatinib resistance in hepatocellular carcinoma.

Lenvatinib is the first-line treatment for hepatocellular carcinoma (HCC), the most common type of primary liver cancer; however, some patients become refractory to lenvatinib. The underlying mechanism of lenvatinib resistance (LR) in patients with advanced HCC remains unclear. We focused on exploring the potential mechanism of LR and novel treatments of lenvatinib-resistant HCC. In particular, we established a Huh7 LR cell line and performed in vitro, bioinformatic, and biochemical assays. Additionally, we used a Huh7-LR cell-derived xenograft mouse model to confirm the results in vivo. Following LR induction, multidrug resistance protein 1 (MDR1) and breast cancer resistance protein (BCRP) transporters were markedly upregulated, and the epidermal growth factor receptor (EGFR), MEK/ERK, and PI3K/AKT pathways were activated. In vitro, the co-administration of elacridar, a dual MDR1 and BCRP inhibitor, with lenvatinib inhibited proliferation and induced apoptosis of LR cells. These effects might be due to inhibiting cancer stem-like cells (CSCs) properties, by decreasing colony formation and downregulating CD133, EpCAM, SOX-9, and c-Myc expression. Moreover, the co-administration of gefitinib, an EGFR inhibitor, with lenvatinib retarded proliferation and induced apoptosis of LR cells. These similar effects might be caused by the inhibition of EGFR-mediated MEK/ERK and PI3K/AKT pathway activation. In vivo, co-administration of lenvatinib with elacridar or gefitinib suppressed tumour growth and angiogenesis. Therefore, inhibiting MDR1 and BCRP transporters or targeting the EGFR/PI3K pathway might overcome LR in HCC. Notably, lenvatinib should be used to treat HCC after LR induction owing to its role in inhibiting tumour proliferation and angiogenesis. Our findings could help develop novel and effective treatment strategies for HCC.

P-Glycoprotein (MDR1/ABCB1) Restricts Brain Accumulation of the Novel EGFR Inhibitor EAI045 and Oral Elacridar Coadministration Enhances Its Brain Accumulation and Oral Exposure.

EAI045 is a fourth-generation allosteric tyrosine kinase inhibitor (TKI) of the epidermal growth factor receptor (EGFR). It targets T790M and C797S EGFR mutants in the treatment of non-small cell lung cancer (NSCLC). EAI045 and cetuximab combined induce tumor regression in mouse models of EGFR-mutant lung cancer. We investigated the pharmacokinetic roles of the multidrug efflux and uptake transporters ABCB1 (P-gp), ABCG2 (BCRP), and OATP1A/1B, and of the drug-metabolizing enzyme CYP3A in plasma and tissue distribution of EAI045 and its metabolites, using genetically modified mouse models. In vitro, EAI045 was a good transport substrate of human ABCB1. In vivo, oral EAI045 (20 mg/kg) was rapidly absorbed. Relative to wild-type mice, EAI045 brain-to-plasma ratios were increased 3.9-fold in Abcb1a/1b-/- and 4.8-fold in Abcb1a/1b;Abcg2-/- mice. However, in single Abcg2-/- mice they were unchanged. EAI045 oral availability was not markedly altered. Oral coadministration of elacridar, an ABCB1/ABCG2 inhibitor, increased the plasma AUC0-30min and brain-to-plasma ratios of EAI045 by 4.0-fold and 5.4-fold, respectively, in wild-type mice. EAI045 glucuronide showed an increased plasma AUC0-30min and a markedly decreased accumulation and tissue-to-plasma ratio in the small intestinal content when Abcb1a/1b and Abcg2 were absent. A large fraction of oral EAI045 was converted to its hydrolyzed metabolite PIA, but Abcb1a/1b, Abcg2, and Oatp1a/1b had little impact on PIA pharmacokinetics. Mouse Cyp3a knockout or transgenic human CYP3A4 overexpression did not significantly affect oral EAI045 pharmacokinetics. Our results show that blood-brain barrier ABCB1 can markedly limit EAI045 brain accumulation. Moreover, elacridar coadministration can effectively reverse this process.

Combination of Elacridar with Imatinib Modulates Resistance Associated with Drug Efflux Transporters in Chronic Myeloid Leukemia.

Multidrug resistance (MDR) development has emerged as a complication that compromises the success of several chemotherapeutic agents. In chronic myeloid leukemia (CML), imatinib resistance has been associated with changes in BCR-ABL1 and intracellular drug concentration, controlled by SLC and ABC transporters. We evaluate the therapeutic potential of a P-glycoprotein and BCRP inhibitor, elacridar, in sensitive (K562 and LAMA-84) and imatinib-resistant (K562-RC and K562-RD) CML cell lines as monotherapy and combined with imatinib. Cell viability was analyzed by resazurin assay. Drug transporter activity, cell death, cell proliferation rate, and cell cycle distribution were analyzed by flow cytometry. Both resistant models presented an increased activity of BCRP and P-gP compared to K562 cells. Elacridar as monotherapy did not reach IC50 in any CML models but activated apoptosis without cytostatic effect. Nevertheless, the association of elacridar (250 nM) with imatinib overcomes resistance, re-sensitizing K562-RC and K562-RD cells with five and ten times lower imatinib concentrations, respectively. Drug combination induced apoptosis with increased cleaved-caspases-3, cleaved-PARP and DNA damage, reduced cell proliferation rate, and arrested CML cells in the S phase. These data suggest that elacridar combined with imatinib might represent a new therapeutic option for overcoming TKI resistance involving efflux transporters.

ABCB1 limits brain exposure of the KRASG12C inhibitor sotorasib, whereas ABCB1, CYP3A, and possibly OATP1a/1b restrict its oral availability.

Sotorasib (Lumakras™) is the first FDA-approved KRASG12C inhibitor for treatment of patients with non-small cell lung cancer (NSCLC) carrying this mutation. Using genetically modified mouse models, we studied the influence of the efflux transporters ABCB1 and ABCG2, the OATP1a/1b uptake transporters, and the CYP3A drug-metabolizing enzyme complex on the plasma pharmacokinetics and tissue distribution of oral sotorasib. In vitro, sotorasib was a potent substrate for human ABCB1 and a modest substrate for mouse Abcg2, but not for human ABCG2. In vivo, the brain-to-plasma ratio of sotorasib (40 mg/kg) was highly increased in Abcb1a/1b-/- (5.9-fold) and Abcb1a/1b;Abcg2-/- (7.6-fold) compared to wild-type mice, but not in single Abcg2-/- mice. Upon coadministering elacridar, an ABCB1/ABCG2 inhibitor, sotorasib brain accumulation increased 7.5-fold, approaching the levels observed in Abcb1a/1b-deficient mice. No acute CNS toxicity emerged upon boosting of the sotorasib exposure. In Oatp1a/1b-deficient mice, we observed a 2-fold reduction in liver disposition compared to wild-type mice, although these uptake transporters had no noticeable impact on sotorasib plasma exposure. However, plasma exposure was limited by mouse Cyp3a and human CYP3A4, as the AUC0-4 h in Cyp3a-/- mice was increased by 2.5-fold compared to wild-type mice, and subsequently strongly decreased (by 3.9-fold) in Cyp3aXAV mice transgenically overexpressing human CYP3A4 in liver and intestine. Collectively, the oral availability of sotorasib was markedly limited by CYP3A and possibly also by ABCB1 and OATP1a/b, whereas its brain accumulation was strongly restricted by ABCB1. The obtained results may help to further optimize the safety and efficacy of sotorasib in clinical use.

Comprehensive Evaluation of Multiple Approaches Targeting ABCB1 to Resensitize Docetaxel-Resistant Prostate Cancer Cell Lines.

Docetaxel (DTX) is a mainstay in the treatment of metastatic prostate cancer. Failure of DTX therapy is often associated with multidrug resistance caused by overexpression of efflux membrane transporters of the ABC family such as the glycoprotein ABCB1. This study investigated multiple approaches targeting ABCB1 to resensitize DTX-resistant (DTXR) prostate cancer cell lines. In DU145 DTXR and PC-3 DTXR cells as well as age-matched parental controls, the expression of selected ABC transporters was analyzed by quantitative PCR, Western blot, flow cytometry and immunofluorescence. ABCB1 effluxing activity was studied using the fluorescent ABCB1 substrate rhodamine 123. The influence of ABCB1 inhibitors (elacridar, tariquidar), ABCB1-specific siRNA and inhibition of post-translational glycosylation on DTX tolerance was assessed by cell viability and colony formation assays. In DTXR cells, only ABCB1 was highly upregulated, which was accompanied by a strong effluxing activity and additional post-translational glycosylation of ABCB1. Pharmacological inhibition and siRNA-mediated knockdown of ABCB1 completely resensitized DTXR cells to DTX. Inhibition of glycosylation with tunicamycin affected DTX resistance partially in DU145 DTXR cells, which was accompanied by a slight intracellular accumulation and decreased effluxing activity of ABCB1. In conclusion, DTX resistance can be reversed by various strategies with small molecule inhibitors representing the most promising and feasible approach.

Rifampin and ritonavir increase oral availability and elacridar enhances overall exposure and brain accumulation of the NTRK inhibitor larotrectinib.

INTRODUCTION: Larotrectinib is an FDA-approved oral small-molecule inhibitor for neurotrophic tropomyosin receptor kinase (NTRK) fusion-positive cancer treatment. Here larotrectinib pharmacokinetic behavior upon co-administration with prototypical inhibitors of the efflux transporters ABCB1/ABCG2 (elacridar), the SLCO1A/1B (OATP1A/1B) uptake transporters (rifampin), and the drug-metabolizing enzyme CYP3A (ritonavir), respectively, was investigated. METHODS: Inhibitors were orally administered prior to oral larotrectinib (10 mg/kg) to relevant genetically modified mouse models. Larotrectinib plasma and tissue homogenate concentrations were measured by a liquid chromatography-tandem mass spectrometric assay. RESULTS: Elacridar increased oral availability (2.7-fold) and markedly improved brain-to-plasma ratios (5.0-fold) of larotrectinib in wild-type mice. Mouse (m)Oatp1a/1b but not hepatic transgenic human (h)OATP1B1 or -1B3 restricted larotrectinib oral availability and affected its tissue distribution. Rifampin enhanced larotrectinib oral availability not only in wild-type mice (1.9-fold), but surprisingly also in Slco1a/1b-/- mice (1.7-fold). Similarly, ritonavir increased the larotrectinib plasma exposure in both wild-type (1.5-fold) and Cyp3a-/- mice (1.7-fold). Intriguingly, both rifampin and ritonavir decreased liver and/or intestinal larotrectinib levels in all related experimental groups, suggesting additional inhibition of enterohepatic Abcb1a/1b activity. CONCLUSIONS: Elacridar enhances both larotrectinib plasma and tissue exposure and especially relative brain penetration, which might be therapeutically relevant. Hepatic mOatp1a/1b but not hOATP1B1 or -1B3 transported larotrectinib. Additionally, rifampin enhances larotrectinib systemic exposure, most likely by inhibiting mOatp1a/1b, but probably also hepatic and/or intestinal mAbcb1. Similar to rifampin, dual-inhibition functions of ritonavir affecting both CYP3A enzymes and enterohepatic Abcb1 transporters enhanced larotrectinib oral availability. The obtained insights may be used to further optimize the clinical-therapeutic application of larotrectinib.

ABCB1 and ABCG2, but not CYP3A4 limit oral availability and brain accumulation of the RET inhibitor pralsetinib.

BACKGROUND AND PURPOSE: Pralsetinib is an FDA-approved oral small-molecule inhibitor for treatment of rearranged during transfection (RET) proto-oncogene fusion-positive non-small cell lung cancer. We investigated how the efflux transporters ABCB1 and ABCG2, the SLCO1A/1B uptake transporters and the drug-metabolizing enzyme CYP3A influence pralsetinib pharmacokinetics. EXPERIMENTAL APPROACH: In vitro, transepithelial pralsetinib transport was assessed. In vivo, pralsetinib (10 mg/kg) was administered orally to relevant genetically modified mouse models. Pralsetinib concentrations in cell medium, plasma samples and organ homogenates were measured using liquid chromatography-tandem mass spectrometry. KEY RESULTS: Pralsetinib was efficiently transported by human (h)ABCB1 and mouse (m)Abcg2, but not hACBG2. In vivo, mAbcb1a/1b markedly and mAbcg2 slightly limited pralsetinib brain penetration (6.3-and 1.8-fold, respectively). Testis distribution showed similar results. Abcb1a/1b;Abcg2-/- mice showed 1.5-fold higher plasma exposure, 23-fold increased brain penetration, and 4-fold reduced recovery of pralsetinib in the small intestinal content. mSlco1a/1b deficiency did not affect pralsetinib oral availability or tissue exposure. Oral coadministration of the ABCB1/ABCG2 inhibitor elacridar boosted pralsetinib plasma exposure (1.3-fold) and brain penetration (19.6-fold) in wild-type mice. Additionally, pralsetinib was a modest substrate of mCYP3A, but not of hCYP3A4, which did not noticeably restrict the oral availability or tissue distribution of pralsetinib. CONCLUSIONS AND IMPLICATIONS: SLCO1A/1B and CYP3A4 are unlikely to affect the pharmacokinetics of pralsetinib, but ABCG2 and especially ABCB1 markedly limit its brain and testis penetration, as well as oral availability. These effects are mostly reversed by oral coadministration of the ABCB1/ABCG2 inhibitor elacridar. These insights may be useful in the further clinical development of pralsetinib.

Optimization of dose and route of administration of the P-glycoprotein inhibitor, valspodar (PSC-833) and the P-glycoprotein and breast cancer resistance protein dual-inhibitor, elacridar (GF120918) as dual infusion in rats.

Transporters can play a key role in the absorption, distribution, metabolism, and excretion of drugs. Understanding these contributions early in drug discovery allows for more accurate projection of the clinical pharmacokinetics. One method to assess the impact of transporters in vivo involves co-dosing specific inhibitors. The objective of the present study was to optimize the dose and route of administration of a P-glycoprotein (P-gp) inhibitor, valspodar (PSC833), and a dual P-gp/breast cancer resistance protein (BCRP) inhibitor, elacridar (GF120918), by assessing the transporters' impact on brain penetration and absorption. A dual-infusion strategy was implemented to allow for flexibility with dose formulation. The chemical inhibitor was dosed intravenously via the femoral artery, and a cassette of known substrates was infused via the jugular vein. Valspodar or elacridar was administered as 4.5-hour constant infusions over a range of doses. To assess the degree of inhibition, the resulting ratios of brain and plasma concentrations, Kp's, of the known substrates were compared to the vehicle control. These data demonstrated that doses greater than 0.9 mg/hr/kg valspodar and 8.9 mg/hr/kg elacridar were sufficient to inhibit P-gp- and BCRP-mediated efflux at the blood-brain barrier in rats without any tolerability issues. Confirmation of BBB restriction by efflux transporters in preclinical species allows for subsequent prediction in humans based upon the proteomic expression at rodent and human BBB. Overall, the approach can also be applied to inhibition of efflux at other tissues (gut absorption, liver clearance) or can be extended to other transporters of interest using alternate inhibitors.

Role of ATP-Binding Cassette Transporter Proteins in CNS Tumors: Resistance- Based Perspectives and Clinical Updates.

Despite gigantic advances in medical research and development, chemotherapeutic resistance remains a major challenge in complete remission of CNS tumors. The failure of complete eradication of CNS tumors has been correlated with the existence of several factors including overexpression of transporter proteins. To date, 49 ABC-transporter proteins (ABC-TPs) have been reported in humans, and the evidence of their strong association with chemotherapeutics' influx, dissemination, and efflux in CNS tumors, is growing. Research studies on CNS tumors are implicating ABC-TPs as diagnostic, prognostic and therapeutic biomarkers that may be utilised in preclinical and clinical studies. With the current advancements in cell biology, molecular analysis of genomic and transcriptomic interplay, and protein homology-based drug-transporters interaction, our research approaches are streamlining the roles of ABC-TPs in cancer and multidrug resistance. Potential inhibitors of ABC-TP for better clinical outcomes in CNS tumors have emerged. Elacridar has shown to enhance the chemo-sensitivity of Dasatanib and Imatinib in various glioma models. Tariquidar has improved the effectiveness of Temozolomide's in CNS tumors. Although these inhibitors have been effective in preclinical settings, their clinical outcomes have not been as significant in clinical trials. Thus, to have a better understanding of the molecular evaluations of ABC-TPs, as well as drug-interactions, further research is being pursued in research labs. Our lab aims to better comprehend the biological mechanisms involved in drug resistance and to explore novel strategies to increase the clinical effectiveness of anticancer chemotherapeutics, which will ultimately improve clinical outcomes.

The influence of the coadministration of the p-glycoprotein modulator elacridar on the pharmacokinetics of lapatinib and its distribution in the brain and cerebrospinal fluid.

Background Lapatinib is a small-molecule tyrosine kinase inhibitor of human epidermal receptor 2 (HER2) and EGFR that has currently been approved for the treatment of HER2-positive advanced and metastatic breast cancer (BC). The ATP-binding cassette (ABC) family of transporters includes P-glycoprotein (P-gp; ABCB1) and breast cancer resistance protein (BCRP; ABCG2), which substantially restrict the penetration of drugs, including chemotherapeutics, through the blood-brain barrier and blood-cerebrospinal fluid barrier. The aim of this study was to investigate the effects of elacridar, an ABCB1 and ABCG2 inhibitor, on the brain and cerebrospinal fluid uptake of lapatinib. Methods Rats were divided into two groups: one group received 5 mg/kg elacridar and 100 mg/kg lapatinib (an experimental group), and the other group received 100 mg/kg lapatinib (a control group). Lapatinib concentrations in the blood plasma (BP), cerebrospinal fluid (CSF) and brain tissue (BT) were measured by liquid chromatography coupled with tandem mass spectrometry. Results Elacridar significantly increased lapatinib penetration into the CSF and BT (Cmax increase of 136.4% and 54.7% and AUC0-∞ increase of 53.7% and 86.5%, respectively). The Cmax of lapatinib in BP was similar in both experimental groups (3057.5 vs. 3257.5 ng/mL, respectively). Conclusion This study showed that elacridar influenced the pharmacokinetics of lapatinib. The inhibition of ABCB1 and ABCG2 transporters by elacridar substantially enhanced the penetration of lapatinib into the CSF and BT. The blocking of protein transporters could become indispensable in the treatment of patients with breast cancer and brain metastases.

Codelivery of doxorubicin and elacridar to target both liver cancer cells and stem cells by polylactide-co-glycolide/d-alpha-tocopherol polyethylene glycol 1000 succinate nanoparticles.

PURPOSE: Liver cancer is the third leading cause of cancer-related deaths worldwide. Liver cancer stem cells (LCSCs) are a subpopulation of cancer cells that are responsible for the initiation, progression, drug resistance, recurrence, and metastasis of liver cancer. Recent studies have suggested that the eradication of both LCSCs and liver cancer cells is necessary because the conversion of cancer stem cells (CSCs) to cancer cells occasionally occurs. As ATP-binding cassette (ABC) transporters are overexpressed in both CSCs and cancer cells, combined therapies using ABC transporter inhibitors and chemotherapy drugs could show superior therapeutic efficacy in liver cancer. In this study, we developed poly(lactide-co-glycolide)/d-alpha-tocopherol polyethylene glycol 1000 succinate nanoparticles to accomplish the simultaneous delivery of an optimized ratio of doxorubicin (DOX) and elacridar (ELC) to target both LCSCs and liver cancer cells. METHODS: Median-effect analysis was used for screening of DOX and ELC for synergy in liver cancer cells (HepG2 cells) and LCSCs (HepG2 tumor sphere [HepG2-TS]). Then, nanoparticles loaded with DOX and ELC at the optimized ratio (NDEs) were prepared by nanoprecipitation method. The cytotoxicity and colony and tumor sphere formation ability of nanoparticles were investigated in vitro, and the tissue distribution and antitumor activity of nanoparticles were evaluated in vivo. RESULTS: We demonstrated that a DOX/ELC molar ratio of 1:1 was synergistic in HepG2 cells and HepG2-TS. NDEs were shown to exhibit significantly increased cytotoxic effects against both HepG2 and HepG2-TS compared with DOX-loaded nanoparticles (NDs) or ELC-loaded nanoparticles (NEs) in vitro. In vivo studies demonstrated that the nanoparticles exhibited better tumor targeting, with NDE showing the strongest antitumor activity with lower systemic toxicity. CONCLUSION: These results suggested that NDE represented a promising combination therapy against liver cancer by targeting both liver cancer cells and CSCs.