Preclinical and Clinical Pharmacokinetics and Bioavailability in Healthy Volunteers of a Novel Formulation of the Selective P2X3 Receptor Antagonist Eliapixant.

BACKGROUND AND OBJECTIVES: The potent, selective P2X3 receptor antagonist eliapixant (BAY 1817080) is under development for conditions characterized by neuronal hypersensitization. As prominent food effects and limited bioavailability in the fasted state were observed with immediate-release eliapixant tablets, a novel formulation was needed. Accordingly, several novel eliapixant formulations were assessed by in vitro and animal studies in a structured way. The most promising of the formulations was then investigated in a phase I study designed to assess its pharmacokinetics, food effect, and bioavailability in healthy volunteers. METHODS: In vitro non-sink dissolution tests were performed with two amorphous solid dispersion (ASD) granule prototypes compared with pure crystalline eliapixant as a surrogate for the immediate-release formulation. Subsequently, the drug exposure of novel eliapixant formulations under fed and fasted conditions in rats and dogs was assessed to confirm improvements in bioavailability versus the suspension-based formulation. A novel Kollidon VA64®-based eliapixant formulation was identified from the preclinical studies and compared with the original tablet formulation in an open-label, partially randomized, threefold, crossover phase I study, in which healthy males received single oral doses (25-400 mg, fasted/fed). Pharmacokinetic parameters, absolute bioavailability (using an intravenous [13C715N]-eliapixant microdose), relative bioavailability (novel versus original formulation), effect of food, and adverse events (AEs) were evaluated. RESULTS: The non-sink dissolution test demonstrated that the two ASD formulations had an improved dissolution rate compared with pure crystalline eliapixant, with a Kollidon VA64-based prototype having the highest dissolution rate. Further testing of this prototype in animal studies confirmed an approximately twofold higher bioavailability compared with the suspension-based formulation. In the phase I study, 30 subjects were randomized. With the novel Kollidon VA64® formulation (400 mg; fasted), area under the concentration-time curve (AUC) and maximum plasma concentration (Cmax) were up to 3.1-fold and 1.7-fold higher, respectively, than with the original formulation (fed). AUC increased dose proportionally between 25 and 100 mg, and less than dose proportionally from 100 to 400 mg. Food had no clinically relevant effect on the novel formulation, with AUC increasing 1.3-fold and Cmax 2.1-2.4-fold (time to maximum concentration was delayed by 1.5-2.25 h). Absolute bioavailability with the novel formulation (100 mg) was 50%. AEs occurred in 57% of patients; most were mild in severity. CONCLUSIONS: The novel eliapixant formulation substantially improved bioavailability compared with immediate-release eliapixant and may be administered with/without food. CLINICAL TRIAL REGISTRATION: Clinicaltrials.gov: NCT03773068 (initial registration: 12 December 2018).

Effect of hepatic impairment on the pharmacokinetics of vilaprisan: An open-label, single-dose, parallel-group study.

AIMS: The study objective was to evaluate the pharmacokinetics of the selective progesterone receptor modulator vilaprisan in participants with hepatic impairment. Additionally, the safety and tolerability of vilaprisan were investigated. METHODS: In this phase 1, open-label, nonrandomised, parallel-group, pharmacokinetic study, men and women with mild or moderate hepatic impairment (Child-Pugh grade A or B) and control participants with normal hepatic function matched by age, weight and sex received a single oral 2 mg dose of vilaprisan. Key pharmacokinetic parameters, relationships between parameters and safety outcomes were measured. RESULTS: Thirty-six participants completed the study: 9 with mild hepatic impairment, 9 with moderate hepatic impairment and 18 matched control participants with normal hepatic function. Vilaprisan reached maximum plasma concentrations after 1-2 hours. Unbound vilaprisan exposure was 1.44-fold higher for participants with mild hepatic impairment vs controls (90% confidence interval: 0.91-2.26), and 1.74-fold higher for participants with moderate impairment vs controls (90% confidence interval: 1.09-2.78). The maximum observed unbound peak concentrations were similar for participants with hepatic impairment and matched controls. Vilaprisan 2 mg was well tolerated and the incidence of treatment-emergent adverse events was similar across cohorts. CONCLUSION: Only mild increases of <1.75-fold in exposure were observed in participants with mild or moderate hepatic impairment compared with control participants. No safety concern was identified. These data, alongside the excellent safety profile observed in phase 1 and 2 studies, do not indicate that a dose adjustment would be required in patients with mild or moderate hepatic impairment.

CYP3A4-mediated effects of rifampicin on the pharmacokinetics of vilaprisan and its UGT1A1-mediated effects on bilirubin glucuronidation in humans.

AIMS: The primary aim of the present study was to quantify the effects of rifampicin, a strong cytochrome P450 (CYP) 3A4 inducer, on the pharmacokinetics of the new selective progesterone receptor modulator, vilaprisan. In addition, the effects of rifampicin on the glucuronidation of bilirubin, an endogenous UDP-glucuronosyltransferase family 1 member A1 (UGT1A1) substrate, were explored. METHODS: This was an open-label, two-period study in 12 healthy postmenopausal women. Subjects received a single oral dose of vilaprisan 4 mg in each period. In period 2, administration of vilaprisan was preceded and followed by rifampicin 600 mg day-1 . A subtherapeutic dose of midazolam (1 mg) was coadministered with vilaprisan to monitor CYP3A4 induction. Details of the administration and sampling schedule were optimized by means of a physiologically based pharmacokinetic model. Plasma concentrations of vilaprisan, midazolam, and 1'- hydroxy-midazolam were measured and rifampicin-associated changes in the glucuronidation of bilirubin were determined. RESULTS: As predicted by our model, the coadministration of rifampicin was associated with a substantial decrease in exposure to vilaprisan and midazolam - indicated by the following point estimates (90% confidence intervals) for the area under the plasma concentration-time curve from zero to the time of the last quantifiable concentration ratio with or without rifampicin: 0.040 (0.0325, 0.0505) for vilaprisan and 0.144 (0.117, 0.178) for midazolam. Further, it was associated with an increase in bilirubin glucuronidation, indicating that UGT1A1 was induced. CONCLUSIONS: The exposure to vilaprisan was reduced by 96%. Such a reduction is likely to render the drug therapeutically ineffective. Therefore, it is recommended that the use of strong CYP3A4 inducers is avoided when taking vilaprisan.

111In-labeled trastuzumab-modified gold nanoparticles are cytotoxic in vitro to HER2-positive breast cancer cells and arrest tumor growth in vivo in athymic mice after intratumoral injection.

INTRODUCTION: Gold nanoparticles (AuNP; 30nm) were modified with polyethylene glycol (PEG) chains linked to trastuzumab for binding to HER2-positive breast cancer (BC) cells and diethylenetriaminepentaacetic acid (DTPA) for complexing the Auger electron-emitter, 111In (trastuzumab-AuNP-111In). Our objective was to determine the cytotoxicity of trastuzumab-AuNP-111In on HER2-positive BC cells in vitro and evaluate its tumor growth inhibition properties and normal tissue toxicity in vivo following intratumoral (i.t.) injection in mice with s.c. HER2-overexpressing BC xenografts. METHODS: Binding and internalization of trastuzumab-AuNP-111In or non-targeted AuNP-111In in SK-BR-3 (1-2×106 HER2/cell) and MDA-MB-361 (5×105 HER2/cell) human BC cells were studied. The surviving fraction (SF) of SK-BR-3 or MDA-MB-361 cells exposed to trastuzumab-AuNP-111In or AuNP-111In was determined. DNA double-strand breaks (DSBs) were assayed by probing for γ-H2AX. Tumor growth was monitored over 70days in CD1 athymic mice with s.c. MDA-MB-361 xenografts after i.t. injection of 10MBq (0.7mg; 2.6×1012 AuNP) of trastuzumab-AuNP-111In and normal tissue toxicity was assessed by monitoring body weight, complete blood cell (CBC) counts and serum alanine aminotransferase (ALT) and creatinine (Cr). RESULTS: Trastuzumab-AuNP-111In was specifically bound by SK-BR-3 and MDA-MB-361 cells. Trastuzumab-AuNP-111In was more efficiently internalized than AuNP-111In and localized to a peri-nuclear region. The SF fraction of SK-BR-3 cells was reduced by 1.8-fold by treatment with 3nM (7MBq/mL) of trastuzumab-AuNP-111In. The SF of MDA-MB-361 cells was reduced by 3.7-fold at 14.4nM (33.6MBq/mL). In comparison, non-targeted AuNP-111In at these concentrations reduced the SF of SK-BR-3 or MDA-MB-361 cells by 1.2-fold (P=0.03) and 1.7-fold (P<0.0001), respectively. DNA DSBs were greater in SK-BR-3 and MDA-MB-361 cells exposed to trastuzumab-AuNP-111In compared to AuNP-111In, but unlabeled trastuzumab-AuNP did not increase DNA DSBs. Local i.t. injection of trastuzumab-AuNP-111In in CD1 athymic mice with s.c. MDA-MB-361 tumors arrested tumor growth for 70days. There was no apparent normal tissue toxicity. The radiation absorbed dose deposited in the tumor by trastuzumab-AuNP-111In was 60.5Gy, while normal organs received <0.9Gy. CONCLUSION: These results are promising for further development of trastuzumab-AuNP-111In as a novel Auger electron-emitting radiation nanomedicine for local treatment of HER2-positive BC. ADVANCES IN KNOWLEDGE AND IMPLICATIONS FOR PATIENT CARE: A local radiation treatment for HER2-positive BC based on AuNP modified with trastuzumab and labeled with the Auger electron-emitter, 111In was developed and shown to arrest tumor growth with no normal tissue toxicity.

Recent advances of semiconducting polymer nanoparticles in in vivo molecular imaging.

Semiconducting polymer nanoparticles (SPNs) emerge as attractive molecular imaging nanoagents in living animals because of their excellent optical properties including large absorption coefficients, tunable optical properties and controllable dimensions, high photostability, and the use of organic and biologically inert components without toxic metals. This review summarizes the recent advances of these new organic nanoparticles in in vivo molecular imaging. The in vivo biocompatibility of SPNs is discussed first in details, followed by examples of their applications ranging from sentinel lymph node mapping and tumor imaging to long-term cell tracking, to drug toxicity and bacterial infection imaging for fluorescence, bioluminescence, chemiluminescence and photoacoustic imaging in living animals. The utility of SPNs for designing smart activatable probes for real-time in vivo imaging is also discussed.

Diketopyrrolopyrrole-Based Semiconducting Polymer Nanoparticles for In Vivo Photoacoustic Imaging.

Diketopyrrolopyrrole-based semiconducting polymer nanoparticles with high photostability and strong photoacoustic brightness are designed and synthesized, which results in 5.3-fold photoacoustic signal enhancement in tumor xenografts after systemic administration.

Investigation of the effects of cell model and subcellular location of gold nanoparticles on nuclear dose enhancement factors using Monte Carlo simulation.

PURPOSE: The authors' aims were to model how various factors influence radiation dose enhancement by gold nanoparticles (AuNPs) and to propose a new modeling approach to the dose enhancement factor (DEF). METHODS: The authors used Monte Carlo N-particle (MCNP 5) computer code to simulate photon and electron transport in cells. The authors modeled human breast cancer cells as a single cell, a monolayer, or a cluster of cells. Different numbers of 5, 30, or 50 nm AuNPs were placed in the extracellular space, on the cell surface, in the cytoplasm, or in the nucleus. Photon sources examined in the simulation included nine monoenergetic x-rays (10-100 keV), an x-ray beam (100 kVp), and (125)I and (103)Pd brachytherapy seeds. Both nuclear and cellular dose enhancement factors (NDEFs, CDEFs) were calculated. The ability of these metrics to predict the experimental DEF based on the clonogenic survival of MDA-MB-361 human breast cancer cells exposed to AuNPs and x-rays were compared. RESULTS: NDEFs show a strong dependence on photon energies with peaks at 15, 30/40, and 90 keV. Cell model and subcellular location of AuNPs influence the peak position and value of NDEF. NDEFs decrease in the order of AuNPs in the nucleus, cytoplasm, cell membrane, and extracellular space. NDEFs also decrease in the order of AuNPs in a cell cluster, monolayer, and single cell if the photon energy is larger than 20 keV. NDEFs depend linearly on the number of AuNPs per cell. Similar trends were observed for CDEFs. NDEFs using the monolayer cell model were more predictive than either single cell or cluster cell models of the DEFs experimentally derived from the clonogenic survival of cells cultured as a monolayer. The amount of AuNPs required to double the prescribed dose in terms of mg Au/g tissue decreases as the size of AuNPs increases, especially when AuNPs are in the nucleus and the cytoplasm. For 40 keV x-rays and a cluster of cells, to double the prescribed x-ray dose (NDEF = 2) using 30 nm AuNPs, would require 5.1 ± 0.2, 9 ± 1, 10 ± 1, 10 ± 1 mg Au/g tissue in the nucleus, in the cytoplasm, on the cell surface, or in the extracellular space, respectively. Using 50 nm AuNPs, the required amount decreases to 3.1 ± 0.3, 8 ± 1, 9 ± 1, 9 ± 1 mg Au/g tissue, respectively. CONCLUSIONS: NDEF is a new metric that can predict the radiation enhancement of AuNPs for various experimental conditions. Cell model, the subcellular location and size of AuNPs, and the number of AuNPs per cell, as well as the x-ray photon energy all have effects on NDEFs. Larger AuNPs in the nucleus of cluster cells exposed to x-rays of 15 or 40 keV maximize NDEFs.

Molecularly targeted gold nanoparticles enhance the radiation response of breast cancer cells and tumor xenografts to X-radiation.

The purpose of this study was to evaluate the effect of molecularly targeted gold nanoparticles (AuNPs) on tumor radiosensitization both in vitro and in vivo. Human Epidermal Growth Factor Receptor-2 (HER-2)-targeted AuNPs (Au-T) were synthesized by conjugating trastuzumab (Herceptin) to 30 nm AuNPs. In vitro, the cytotoxicity of Au-T or non-targeted AuNPs (Au-P) was assessed by γ-H2AX immunofluorescence microscopy for DNA damage and clonogenic survival assays. In vivo, athymic mice bearing subcutaneous MDA-MB-361 xenografts were treated with a single dose of 11 Gy of 100 kVp X-rays 24 h after intratumoral injection of Au-T (~0.8 mg of Au) or no X-radiation. Normal tissue toxicity was determined by hematology or biochemistry parameters. The combination of Au-P or Au-T with X-ray exposure increased the formation of γ-H2AX foci by 1.7 (P = 0.054) and 3.3 (P = 0.024) fold in comparison to X-radiation alone, respectively. The clonogenic survival of cells exposed to Au-T and X-rays was significantly lower from that of cells exposed to X-radiation alone, which translated to a dose enhancement factor of 1.6. In contrast, survival of cells exposed to Au-P and X-rays versus X-radiation alone were not significantly different. In vivo, the combination of Au-T and X-radiation resulted in regression of MDA-MB-361 tumors by 46 % as compared to treatment with X-radiation (16.0 % increase in tumor volume). No significant normal tissue toxicity was observed. Radiosensitization of breast cancer to X-radiation with AuNPs was successfully achieved with an optimized therapeutic strategy of molecular targeting of HER-2 and intratumoral administration.

Role of antibody-mediated tumor targeting and route of administration in nanoparticle tumor accumulation in vivo.

In this study, we have looked at enhancing tumor uptake and intracellular delivery of gold nanoparticles (AuNPs) while reducing the systemic exposure by systematic evaluation of the impact of targeting and route of administration on organ distribution. High-resolution microSPECT/CT imaging was used to track the in vivo fate of (111)In-labeled nontargeted and human epidermal growth factor receptor-2 (HER-2) targeted AuNPs following intravenous (i.v.) or intratumoral (i.t.) injection. For i.v. injection, the effects of GdCl3 (for deactivation of macrophages) and nonspecific (anti-CD20) antibody rituximab (for blocking of Fc mediated liver and spleen uptake) were studied. It was found that HER-2 targeting via attachment of trastuzumab paradoxically decreased tumor uptake as a result of faster elimination of the targeted AuNPs from the blood while improving internalization in HER-2-positive tumor cells as compared to nontargeted AuNPs. I.T. injections with HER-2 targeted AuNPs resulted in high tumor retention with low systemic exposure and represents an attractive delivery strategy. Our results provide a strategy for optimizing tumor delivery and quantifying organ distribution of this widely studied class of nanomaterial.

Optimized digital counting colonies of clonogenic assays using ImageJ software and customized macros: comparison with manual counting.

PURPOSE: To develop a digital method for counting colonies that highly replicates manual counting. MATERIALS AND METHODS: Breast cancer cells were treated with trastuzumab-conjugated gold nanoparticles in combination with X-ray irradiation, (111)In labeled trastuzumab, or γ-radiation, followed by clonogenic assays. Colonies were counted manually or digitally using ImageJ software with customized macros. Key parameters, intensity threshold and minimum colony size, were optimized based on three preliminary manual counts or blindly chosen. The correlation of digital and manual counting and inter- and intra-experimenter variability were examined by linear regression. Survival curves derived from digital and manual counts were compared by F-test (P < 0.05). RESULTS: Using optimized parameters, digital counts corresponded linearly to manual counts with slope (S) and R(2) value close to 1 and a small y-intercept (y(0)): SK-BR-3 (S = 0.96 ± 0.02, R(2) = 0.969, y(0) = 5.9 ± 2.2), MCF-7/HER2-18 (S = 0.98 ± 0.03, R(2) = 0.952, y(0) = 0.74 ± 0.47), and MDA-MB-231 cells (S = 1.00 ± 0.02, R(2) = 0.995, y(0) = 3.3 ± 4.5). Both reproducibility and repeatability of digital counts were better than the manual method. Survival curves generated from digital and manual counts were not significantly different; P-values were 0.3646 for SK-BR-3 cells and 0.1818 for MCF-7/HER2-18 cells. Using blind parameters, survival curves generated by both methods showed some differences: P-values were 0.0897 for SK-BR-3 cells and 0.0024 for MCF-7/HER2-18 cells. CONCLUSIONS: The colony counting using ImageJ and customized macros with optimized parameters was a reliable method for quantifying the number of colonies.

Design and characterization of HER-2-targeted gold nanoparticles for enhanced X-radiation treatment of locally advanced breast cancer.

Our purpose was to develop a human epidermal growth factor receptor-2 (HER-2) targeted nanotechnology-based radiosensitizer. HER-2 is overexpressed in 20-30% of all breast cancers and up to 2-fold higher in locally advanced disease (LABC). Trastuzumab was derivatized with a polyethylene glycol (OPSS-PEG-SVA) cross-linker to produce trastuzumab-PEG-OPSS. These immunoconjugates were analyzed by SDS-PAGE, and their immunoreactivity was assessed by flow cytometry using HER-2 overexpressing SK-BR-3 breast cancer cells. Reacting trastuzumab with increasing ratios of PEG resulted in an increase in molecular weight from approximately 148 kDa to 243 kDa, associated with increasing PEG substitution (0.6 to 18.9 PEG chains per trastuzumab). Attachment of approximately 7 PEG chains per trastuzumab resulted in 56% retention in immunoreactivity assessed by flow cytometry. The conjugates were then linked to 30 nm AuNPs. Using a novel (123)iodine-radiotracer based assay that overcomes the current limitations of spectrophotometric quantification of biological molecules on AuNPs we estimate 14.3 ± 2.7 antibodies were attached to each AuNP when 2 × 10(11) AuNPs were reacted with 20 µg of trastuzumab-PEG-OPSS. Specificity of trastuzumab-PEG-AuNPs for HER-2 and internalization in SK-BR-3 cells was demonstrated by comparing the uptake of trastuzumab-PEG-AuNPs or PEG-AuNPs by darkfield microscopy. The ability of trastuzumab-PEG-AuNPs in combination with 300 kVp X-rays to enhance DNA double strand breaks (DSBs) in SK-BR-3 cells was assessed by immunofluorescence using the γ-H2AX assay. γ-H2AX assay results revealed 5.1-fold higher DNA-DSBs with trastuzumab-PEG-AuNPs and X-radiation as compared to treatment with X-radiation alone. The trastuzumab-PEG-AuNPs are a promising targeted nanotechnology-based radiosensitizer for improving LABC therapy. The design and systematic approaches taken to surface modify and characterize trastuzumab-PEG-AuNPs described in this study would have application to other molecularly targeted AuNPs for cancer treatment.

Nanotechnology applications for improved delivery of antiretroviral drugs to the brain.

Human immunodeficiency virus (HIV) can gain access to the central nervous system during the early course of primary infection. Once in the brain compartment the virus actively replicates to form an independent viral reservoir, resulting in debilitating neurological complications, latent infection and drug resistance. Current antiretroviral drugs (ARVs) often fail to effectively reduce the HIV viral load in the brain. This, in part, is due to the poor transport of many ARVs, in particular protease inhibitors, across the blood-brain barrier (BBB) and blood-cerebrospinal fluid barrier (BCSBF). Studies have shown that nanocarriers including polymeric nanoparticles, liposomes, solid lipid nanoparticles (SLN) and micelles can increase the local drug concentration gradients, facilitate drug transport into the brain via endocytotic pathways and inhibit the ATP-binding cassette (ABC) transporters expressed at the barrier sites. By delivering ARVs with nanocarriers, significant increase in the drug bioavailability to the brain is expected to be achieved. Recent studies show that the specificity and efficiency of ARVs delivery can be further enhanced by using nanocarriers with specific brain targeting, cell penetrating ligands or ABC-transporters inhibitors. Future research should focus on achieving brain delivery of ARVs in a safe, efficient, and yet cost-effective manner.

Solid lipid nanoparticles enhance the delivery of the HIV protease inhibitor, atazanavir, by a human brain endothelial cell line.

PURPOSE: Protease inhibitors (PIs) exhibit low brain permeability. As a result, unchallenged HIV viral replication can lead to HIV-encephalitis and antiretroviral drug resistance. The objective of this study was to develop and evaluate a lipid nanoparticle system for enhanced brain delivery of the potent and frequently used HIV PI, atazanavir, using a well characterized human brain microvessel endothelial cell line (hCMEC/D3) representative of the blood-brain barrier. METHODS: Solid lipid nanoparticles (SLNs) were prepared by a thin film hydration technique and analyzed for atazanavir encapsulation efficiency, particle size, morphology, zeta potential and drug release. Cell viability experiments demonstrate that SLNs exhibit no toxicity in hCMEC/D3 cells up to a concentration corresponding to 200 nM of atazanavir. RESULTS: Spherical SLNs with an average particle size of approximately 167 nm were formulated. Delivery of [3H]-atazanavir by SLNs led to a significantly higher accumulation by the endothelial cell monolayer as compared to the drug aqueous solution. Furthermore, release of Rhodamine-123 (a fluorescent probe) by SLNs also resulted in a higher cellular accumulation. CONCLUSIONS: These data suggest that SLNs could be a promising drug delivery system to enhance brain uptake of atazanavir and potentially other PIs.