In Vitro Interactions of Epacadostat and its Major Metabolites with Human Efflux and Uptake Transporters: Implications for Pharmacokinetics and Drug Interactions.

Epacadostat (EPAC) is a first-in-class, orally active inhibitor of the enzyme indoleamine 2,3-dioxygenase 1 and has demonstrated promising clinical activity. In humans, three major plasma metabolites have been identified: M9 (a glucuronide-conjugate), M11 (a gut microbiota metabolite), and M12 (a secondary metabolite formed from M11). It is proposed, based on the human pharmacokinetics of EPAC, that the biliary excretion of M9, the most abundant metabolite, leads to the enterohepatic circulation of EPAC. Using various in vitro systems, we evaluated in the present study the vitro interactions of EPAC and its major metabolites with major drug transporters involved in drug absorption and disposition. EPAC is a substrate for efflux transporters P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP), but it is not a substrate for hepatic uptake transporters [organic anion transporting polypeptides OATP1B1 and OATP1B3]. The low permeability of M9 suggests an essential role for transporters in its disposition. M9 is likely excreted from hepatocytes into bile via multidrug resistance-associated protein 2 (MRP2) and BCRP, excreted into blood via MRP3, and transported from blood back into hepatocytes via OATP1B1 and OATP1B3. M11 and M12 are not substrates for P-gp, OATP1B1 or OATP1B3, and M11, but not M12, is a substrate for BCRP. With respect to inhibition of drug transporters, the potential of EPAC, M9, M11, and M12 to cause clinical drug-drug interactions via inhibition of P-gp, BCRP, OATP1B1, OATP1B3, OAT1, OAT3, or organic cation transporter 2 was estimated to be low. The current investigation underlines the importance of metabolite-transporter interactions in the disposition of clinically relevant metabolites, which may have implications for the pharmacokinetics and drug interactions of parent drugs.

Enhanced passive pulmonary targeting and retention of PEGylated rigid microparticles in rats.

The current study examines the passive pulmonary targeting efficacy and retention of 6µm polystyrene (PS) microparticles (MPs) covalently modified with different surface groups [amine (A-), carboxyl (C-) and sulfate (S-)] or single (PEG(1)-) and double (PEG(2)-) layers of α,ω-diamino poly(ethylene glycol) attached to C-MPs. The ζ-potential of A-MPs (-44.0mV), C-MPs (-54.3mV) and S-MPs (-49.6mV) in deionized water were similar; however PEGylation increased the ζ-potential for both PEG(1)-MPs (-18.3mV) and PEG(2)-MPs (11.5mV). The biodistribution and retention of intravenously administered MPs to male Sprague-Dawley rats was determined in homogenized tissue by fluorescence spectrophotometry. PEG(1)-MPs and PEG(2)-MPs demonstrated enhanced pulmonary retention in rats at 48h after injection when compared to unmodified A-MPs (59.6%, 35.9% and 17.0% of the administered dose, respectively). While unmodified MPs did not significantly differ in lung retention, PEGylation of MPs unexpectedly improved passive lung targeting and retention by modifying surface properties including charge and hydrophobicity but not size.

Threshold size for optimal passive pulmonary targeting and retention of rigid microparticles in rats.

The relationship between microparticle (MP) size and lung targeting efficiency, intra-lung distribution and retention time was systematically studied after intravenous administration of rigid fluorescent polystyrene MPs of various sizes (2, 3, 6 and 10 microm) to Sprague Dawley rats. Total fluorescence was assessed and it was found that 2 microm and 3 microm MPs readily passed through the lung to the liver and spleen while 10 microm MPs were completely entrapped in the lung for the one-week duration of the study. Approximately 84% of 6 microm MPs that were initially entrapped in the lung were cleared over the next 2 days and 15% were cleared over the remaining 5 days. A Caliper IVIS 100 small animal imaging system confirmed that 3 microm MPs were not retained in the lung but that 6 microm and 10 microm MPs were widely distributed throughout the lung. Moreover, histologic examination showed MP entrapment in capillaries but not arterioles. These studies suggest that for rigid MPs the optimal size range required to achieve transient but highly efficiently targeting to pulmonary capillaries after IV injection is >6 microm but <10 microm in rats and that systemic administration of optimally sized MPs may be an efficient alternative to currently used inhalation-based delivery to the lung.

Pulmonary targeting microparticulate camptothecin delivery system: anticancer evaluation in a rat orthotopic lung cancer model.

Large (>6 microm) rigid microparticles (MPs) become passively entrapped within the lungs after intravenous (i.v.) injection making them an attractive and highly efficient alternative to inhalation for pulmonary delivery. In this study, PEGylated 6 microm polystyrene MPs with multiple copies of the norvaline (Nva) alpha-amino acid prodrug of camptothecin (CPT) were prepared. Surface morphology was characterized using a scanning electron microscope. CPT was released from the CPT-Nva-MPs over 24 h in rat plasma at 37 degrees C. In-vivo CPT plasma concentrations were low (approximately 1 ng/ml or less) and constant over a period of 4 days after a single i.v. injection of CPT-Nva-MPs as compared with high but short-lived systemic exposures after an i.v. injection of free CPT. This suggests that sustained local CPT concentrations were achieved in the lung after administration of the MP delivery system. Anticancer efficacy was evaluated in an orthotopic lung cancer animal model and compared with a bolus injection of CPT. Animals receiving free CPT (2 mg/kg) and CPT-Nva-MPs (0.22 mg/kg CPT and 100 mg/kg MPs) were found to have statistically significant smaller areas of lung cancer (P<0.05 and 0.01, respectively) than untreated animals. In addition, 40% of the animals receiving CPT-Nva-MPs were found to be free of cancer. The CPT dose using targeted MPs was 10 times lower than after i.v. injection of free CPT, but was more effective in reducing the amount of cancerous areas. In conclusion, CPT-Nva-MPs were able to achieve effective local lung and low systemic CPT concentrations at a dose that was 10 times lower than systemically administered CPT resulting in a significant improvement in anticancer efficacy in an orthotopic rat model of lung cancer.

Differential roles of P-glycoprotein, multidrug resistance-associated protein 2, and CYP3A on saquinavir oral absorption in Sprague-Dawley rats.

The objective of this investigation was to differentiate the roles of P-glycoprotein (Pgp), multidrug resistance-associated protein 2 (Mrp2), and CYP3A on saquinavir (SQV) oral absorption. With use of single-pass jejunal perfusion (in situ) and portal vein-cannulated rats (in vivo), SQV absorption was studied under chemical inhibition of Pgp [N-(4-[2-(1,2,3,4-tetrahydro-6,7-dimethoxy-2 isoquinolinyl)-ethyl]-phenyl)-9,10-dihydro-5-methoxy-9-oxo-4-acridine carboxamide (GF120918)], Mrp2 [(3-(((3-(2-(7-chloro-2-quinolinyl)-(E)-ethenyl)phenyl) ((3-(dimethylamino-3-oxopropyl)thio)methyl)-thio) propanoic acid (MK571)], and/or CYP3A (midazolam). Plasma concentrations of SQV and related metabolites were analyzed by liquid chromatography-tandem mass spectrometry. When given alone, SQV absorption was extremely low both in situ (F(a) = 0.07%) and in vivo [C(max) = 0.068 microg/ml; area under the curve (AUC) = 6.8 microg x min/ml]. Coadministration of GF120918 boosted SQV absorption by more than 20-fold with decreased variation in AUCs (percent coefficient of variation = 30% versus 100%). In contrast, coadministration of MK571 or midazolam increased SQV absorption only 2- to 3-fold without improving the variation in AUCs. SQV oral absorption was not further improved when it was given with GF120918 and midazolam or with GF120918 and MK571. The current results provide, for the first time, direct and explicit evidence that the low oral absorption of SQV is controlled by a secretory transporter, Pgp, and not by limited passive diffusion owing to its poor physicochemical properties. Pgp-mediated transport is also responsible for the highly variable oral bioavailability of SQV. In contrast, intestinal Mrp2 and intestinal CYP3A appear to play minor roles in SQV oral bioavailability. Given the differential and complex roles of Pgp and CYP3A in SQV oral absorption, the optimization of AIDS boosting regimens requires careful consideration to avoid therapy-limiting drug-drug transporter and enzyme interactions.

Peritoneal macrophage uptake, pharmacokinetics and biodistribution of macrophage-targeted PEG-fMLF (N-formyl-methionyl-leucyl-phenylalanine) nanocarriers for improving HIV drug delivery.

PURPOSE: To assess in vivo macrophage targeting potential of PEG-fMLF nanocarriers and to investigate their biodistribution, peritoneal macrophage uptake, and pharmacokinetics. METHODS: Multiple copies of fMLF were conjugated to purchased and novel (branched, peptide-based) PEG nanocarriers. Peritoneal macrophage uptake was evaluated in mice 4 hours after IP administration of fluorescence-labeled PEG-fMLF nanocarriers. Pharmacokinetics and biodistribution were determined in rats after IV administration of tritiated PEG-fMLF nanocarriers. RESULTS: Attachment of one, two, or four fMLF copies increased uptake in macrophages by 3.8-, 11.3-, and 23.6-fold compared to PEG without fMLF. Pharmacokinetic properties and tissue distribution also differed between nanocarriers with and without fMLF. Attachment of fMLF residues increased the t(1/2) of PEG(5K) by threefold but decreased the t(1/2) of PEG(20K) by 40%. Attachment of fMLF increased accumulation of nanocarriers into macrophages of liver, kidneys and spleen. However, on a molar basis, penetration was equivalent suggesting nanocarrier size and targeting moieties are important determinants. CONCLUSIONS: These results demonstrate the feasibility for targeting macrophages, a primary HIV reservoir site. However, these studies also suggest that balancing peripheral tissue penetration (a size-dependent phenomenon) versus target cell uptake specificity remains a challenge to overcome.

Using novobiocin as a specific inhibitor of breast cancer resistant protein to assess the role of transporter in the absorption and disposition of topotecan.

PURPOSE: To investigate the role of intestinal breast cancer resistant protein (BCRP) in the absorption and disposition of topotecan (TPT) using novobiocin (NOV) as a specific inhibitor. METHODS: Transporter inhibition specificity of NOV was assessed in cells overexpressing BCRP or Pgp. Sprague-Dawley rats were orally or intravenously dosed with TPT (2 and 1 mg/kg for p.o. and i.v., respectively) with or without oral co-administration of NOV (50 mg/kg). Pharmacokinetic parameters of TPT were obtained by noncompartmental analysis. To assess the role of BCRP in TPT intestinal permeation, rat ileal segment was perfused with 10 microM TPT in the presence or absence of NOV (500 microM), TPT permeability was calculated based on drug appearance in mesenteric blood. To assess the role of BCRP in TPT intestinal secretion, rat ileal segment was perfused with saline in the presence or absence of NOV (500 microM), while TPT was i.v. infused into rat. Intestinal secretion of TPT was calculated based on drug appearance in the perfusate. RESULTS: NOV significantly inhibited efflux activity of BCRP, but not Pgp. Coadministration of NOV markedly increased oral TPT AUC(0-720) and Cmax by 3- and 4.5-fold, respectively, and decreased systemic clearance of i.v. injected TPT (from 44.40+/-7.28 without NOV to 29.44+/-1.99 ml/min/kg with NOV). The inclusion of NOV in perfusate significantly increased TPT permeability from 0.81+/-0.30 x10(-6) to 1.26+/-0.12 x10(-6) cm/s, while, the intestinal secretion of TPT was reduced by ~50% when NOV was included in perfusate. CONCLUSIONS: The present study establishes in vitro and in vivo inhibition potency and specificity of NOV on BCRP and provides direct evidence that intestinal BCRP plays an important role in limiting the oral absorption and influencing the systemic elimination of TPT.

Pharmacokinetic and pharmacodynamic evaluation of a novel in situ forming poly(ethylene glycol)-based hydrogel for the controlled delivery of the camptothecins.

Inadequate drug delivery, due to problems associated with achieving constant therapeutic blood levels, has hampered the use of anticancer agents of the camptothecin (CPT) class. The objective of the current studies was to develop a depot delivery system for the water-soluble analog of CPT, topotecan (TPT). In this study, a 2-phase drug depot consisting of TPT-loaded liposomes entrapped in a poly(ethylene glycol) hydrogel was designed. Physically entrapped unaltered TPT displayed a rapid release rate from the hydrogel. Controlled release was demonstrated in vitro and in vivo from the 2-phase system with constant blood levels being achieved for several days in rats. Cytotoxicity and antitumor activity were also evaluated in rats inoculated with syngeneic MAT B III breast cancer cells. Rats treated with the liposome-loaded hydrogel displayed significantly longer tumor growth suppression and did not exhibit body weight loss compared to those treated with other delivery modes. These experiments constitute a proof-of-principle of the 2-phase depot concept and its potential value for enhancing safety and efficacy in chemotherapy.

Delineating the contribution of secretory transporters in the efflux of etoposide using Madin-Darby canine kidney (MDCK) cells overexpressing P-glycoprotein (Pgp), multidrug resistance-associated protein (MRP1), and canalicular multispecific organic anion transporter (cMOAT).

Multidrug resistance conferred to cancer cells is often mediated by the expression of efflux transporter "pumps". It is also believed that many of the same transporters are involved in drug efflux from numerous normal endothelial and epithelial cell types in the intestine, brain, kidney, and liver. Etoposide transport kinetics were characterized in Caco-2 cells and in well established Madin-Darby canine kidney (MDCKII) cell lines that were stably-transfected with a human cDNA encoding P-glycoprotein (Pgp), human multidrug resistance protein (MRP1), or the canalicular multispecific organic anion (cMOAT) transporters to determine the roles of these transporters in etoposide efflux. Etoposide transport kinetics were concentration-dependent in the MDCKII-MDR1 and MDCKII-cMOAT cells. The apparent secretory Michaelis constant (Km) and carrier-mediated permeability (Pc) values for Pgp and cMOAT were 254.96 +/- 94.39 microM and 5.96 +/- 0.41 x 10(-6) cm/s and 616.54 +/- 163.15 microM and 1.87 +/- 0.10 x 10(-5) cm/s, respectively. The secretory permeability of etoposide decreased significantly in the basal to apical (B to A) (i.e., efflux) direction, whereas the permeability increased 2.3-fold in the apical to basal (A to B) direction in MDCKII-MDR1 cells in the presence of elacridar (GF120918). Moderate inhibition of etoposide efflux by leukotriene C4 (LTC4) was observed in MDCKII-cMOAT cells. Furthermore, etoposide inhibited LTC4 efflux, confirming the involvement of cMOAT. The flux of etoposide in MDCKII-MRP1 cells was similar to that in MDCKII/wt control cells. The current results demonstrate that the secretory transport mechanism of etoposide involves multiple transporters, including Pgp and cMOAT but not MRP1. These results demonstrate that Pgp and cMOAT are involved in the intestinal secretory transport of etoposide. Since the intestinal secretion of etoposide was previously reported in the literature, it also suggests that they may be involved in the in vivo intestinal secretion of etoposide; however, mechanistic in vivo studies are required to confirm this.