Optimizing Process Parameters for Controlled Drug Delivery: A Quality by Design (QbD) Approach in Naltrexone Microspheres.

The formulation of microspheres involves a complex manufacturing process with multiple steps. Identifying the appropriate process parameters to achieve the desired quality attributes poses a significant challenge. This study aims to optimize the critical process parameters (CPPs) involved in the preparation of naltrexone microspheres using a Quality by Design (QbD) methodology. Additionally, the research aims to assess the drug release profiles of these microspheres under both in vivo and in vitro conditions. Critical process parameters (CPPs) and critical quality attributes (CQAs) were identified, and a Box-Behnken design was utilized to delineate the design space, ensuring alignment with the desired Quality Target Product Profile (QTPP). The investigated CPPs comprised polymer concentration, aqueous phase ratio to organic phase ratio, and quench volume. The microspheres were fabricated using the oil-in-water emulsion solvent extraction technique. Analysis revealed that increased polymer concentration was correlated with decreased particle size, reduced quench volume resulted in decreased burst release, and a heightened aqueous phase ratio to organic phase ratio improved drug entrapment. Upon analyzing the results, an optimal formulation was determined. In conclusion, the study conducted in vivo drug release testing on both the commercially available innovator product and the optimized test product utilizing an animal model. The integration of in vitro dissolution data with in vivo assessments presents a holistic understanding of drug release dynamics. The QbD approach-based optimization of CPPs furnishes informed guidance for the development of generic pharmaceutical formulations.

Thermosensitive biomaterial gels with chemical permeation enhancers for enhanced microneedle delivery of naltrexone for managing opioid and alcohol dependency.

Naltrexone (NTX) can be transdermally delivered using microneedles (MN) to treat opioid and alcohol misuse disorders, but delivery is blunted by rapid in vivo micropore closure. Poloxamer (P407), a thermosensitive biocompatible hydrogel, sustains NTX delivery through MN-treated skin by generating a drug depot within the micropores. Optimizing P407 formulations could maintain sustained delivery after micropore closure while reducing required patch sizes, which would be more discreet and preferred by most patients. Here we developed NTX-loaded P407 gels with chemical permeation enhancers (CPEs) and used these novel formulations alongside MN treatment to enhance NTX permeation, utilizing parallel micropore and intact skin transport pathways. We analyzed physicochemical and rheological properties of CPE-loaded P407 formulations and selected formulations with DMSO and benzyl alcohol for further study. In vitro permeation tests demonstrated more consistent and sustained NTX delivery through MN-treated porcine skin from 16% P407 formulations vs. aqueous solutions. P407 with 1% benzyl alcohol and 10% DMSO significantly, P < 0.05, increased flux through MN-treated skin vs. formulations with benzyl alcohol alone. This formulation would require a smaller size patch than previously used to deliver NTX in humans, with half the NTX concentration. This is the first time poloxamer biomaterials have been used in combination with CPEs to improve MN-assisted transdermal delivery of an opioid antagonist. Here we have demonstrated that P407 in combination with CPEs effectively sustains NTX delivery in MN-treated skin while requiring less NTX than previously needed to meet clinical goals.

Engineered PLGA microspheres for extended-release of naltrexone: in vitro, in vivo, and IVIVR.

Poly(lactide-co-glycolide) (PLGA)-based formulation is one of the most often used parenteral extended-release forms to deliver various therapeutics. VIVITROL® as a commercialized PLGA microsphere formulation encapsulates naltrexone, a narcotic antagonist for opioid addiction and alcohol dependency. However, no U.S. Food and Drug Administration-approved generic product of naltrexone PLGA microsphere formulation has entered the market. The availability of generic naltrexone PLGA microspheres in low-income countries will broaden patients' accessibility to the safe, effective, and more affordable drug. A major challenge in developing such generic forms is the sensitivity of the drug-loaded microspheres' critical characteristics to the small manufacturing changes, even in formulations with the same compositions as the reference product. In this study, we evaluated the different key manufacturing parameters on the physicochemical, in vitro and in vivo release characteristics of naltrexone microspheres to develop a generic form of naltrexone PLGA microspheres. The selected formulations demonstrated a significant similarity in physicochemical characteristics and release profiles (f2 > 50) to the reference product, VIVITROL®. A strong relationship was observed between in vitro release profile of naltrexone as against its corresponding in vivo profile. It helped to roughly predict the in vivo release behavior of the different manufactured formulations by their corresponding in vitro release profiles.

Structure-Based Design and Development of Chemical Probes Targeting Putative MOR-CCR5 Heterodimers to Inhibit Opioid Exacerbated HIV-1 Infectivity.

Crystal structures of ligand-bound G-protein-coupled receptors provide tangible templates for rationally designing molecular probes. Herein, we report the structure-based design, chemical synthesis, and biological investigations of bivalent ligands targeting putative mu opioid receptor C-C motif chemokine ligand 5 (MOR-CCR5) heterodimers. The bivalent ligand VZMC013 possessed nanomolar level binding affinities for both the MOR and CCR5, inhibited CCL5-stimulated calcium mobilization, and remarkably improved anti-HIV-1BaL activity over previously reported bivalent ligands. VZMC013 inhibited viral infection in TZM-bl cells coexpressing CCR5 and MOR to a greater degree than cells expressing CCR5 alone. Furthermore, VZMC013 blocked human immunodeficiency virus (HIV)-1 entry in peripheral blood mononuclear cells (PBMC) cells in a concentration-dependent manner and inhibited opioid-accelerated HIV-1 entry more effectively in phytohemagglutinin-stimulated PBMC cells than in the absence of opioids. A three-dimensional molecular model of VZMC013 binding to the MOR-CCR5 heterodimer complex is constructed to elucidate its mechanism of action. VZMC013 is a potent chemical probe targeting MOR-CCR5 heterodimers and may serve as a pharmacological agent to inhibit opioid-exacerbated HIV-1 entry.

Nalmefene non-enantioselectively targets myeloid differentiation protein 2 and inhibits toll-like receptor 4 signaling: wet-lab techniques and in silico simulations.

Nalmefene is an opiate derivative having a similar structure to naltrexone. Recent evidence suggests that nalmefene, acting as the innate immune protein toll-like receptor 4 (TLR4) antagonist, effectively reduces the injury of lung ischemia-reperfusion and prevents neuroinflammation. However, the molecular recognition mechanism, especially the enantioselectivity, of nalmefene by the innate immune receptor is not well understood. Herein in vitro assays and in silico simulations were performed to dissect the innate immune recognition of nalmefene at the atomic, molecular, and cellular levels. Biophysical binding experiments and molecular dynamic simulations provide direct evidence that (-)-nalmefene and (+)-nalmefene bind to the hydrophobic cavity of myeloid differentiation protein 2 (MD-2) and behave similarly, which is primarily driven by hydrophobic interactions. The inhibition activity and the calculated binding free energies show that no enantioselectivity was observed during the interaction of nalmefene with MD-2 as well as the inhibition of TLR4 signaling. Interestingly, nalmefene showed ∼6 times better TLR4 antagonisic activity than naltrexone, indicating that the bioisosteric replacement with the methylene group is critical for the molecular recognition of nalmefene by MD-2. In all, this study provides molecular insight into the innate immune recognition of nalmefene, which demonstrates that nalmefene is non-enantioselectively sensed by MD-2.

Antiviral drug design based on the opening mechanism of spike glycoprotein in SARS-CoV-2.

The novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) enters the host cell after the receptor binding domain (RBD) of the virus spike (S) glycoprotein binds to the human angiotensin-converting enzyme 2 (hACE2). This binding requires the RBD to undergo a conformational change from a closed to an open state. In the present study, a key pair of salt bridges formed by the side chains of K537 and E619, residues at the interfaces of SD1 and SD2, respectively, was identified to promote the opening of the RBD. Mutations of K537Q and E619D reduced their side chain lengths and eliminated this pair of salt bridges; as a result, the opening of the RBD was not observed in the MD simulations. Thus, blocking the formation of this pair of salt bridges is a promising approach for treating novel coronavirus disease 2019 (COVID-19). FDA approved drug molecules were screened by their capabilities of blocking the formation of the key pair of salt bridges, achieved by their positional stabilities in the cavity containing the side chains of K537 and E619 formed in the interface between SD1 and SD2. Simeprevir, imatinib, and naldemedine were identified to possess the desired capability with the most favorable interaction energies.

Key Factor Study for Generic Long-Acting PLGA Microspheres Based on a Reverse Engineering of Vivitrol®.

The FDA (U.S. Food and Drug Administration) has approved only a negligible number of poly(lactide-co-glycolide) (PLGA)-based microsphere formulations, indicating the difficulty in developing a PLGA microsphere. A thorough understanding of microsphere formulations is essential to meet the challenge of developing innovative or generic microspheres. In this study, the key factors, especially the key process factors of the marketed PLGA microspheres, were revealed for the first time via a reverse engineering study on Vivitrol® and verified by the development of a generic naltrexone-loaded microsphere (GNM). Qualitative and quantitative similarity with Vivitrol®, in terms of inactive ingredients, was accomplished by the determination of PLGA. Physicochemical characterization of Vivitrol® helped to identify the critical process parameters in each manufacturing step. After being prepared according to the process parameters revealed by reverse engineering, the GNM demonstrated similarity to Vivitrol® in terms of quality attributes and in vitro release (f2 = 65.3). The research on the development of bioequivalent microspheres based on the similar technology of Vivitrol® will benefit the development of other generic or innovative microspheres.

Exploring the putative mechanism of allosteric modulations by mixed-action kappa/mu opioid receptor bitopic modulators.

The modulation and selectivity mechanisms of seven mixed-action kappa opioid receptor (KOR)/mu opioid receptor (MOR) bitopic modulators were explored. Molecular modeling results indicated that the 'message' moiety of seven bitopic modulators shared the same binding mode with the orthosteric site of the KOR and MOR, whereas the 'address' moiety bound with different subdomains of the allosteric site of the KOR and MOR. The 'address' moiety of seven bitopic modulators bound to different subdomains of the allosteric site of the KOR and MOR may exhibit distinguishable allosteric modulations to the binding affinity and/or efficacy of the 'message' moiety. Moreover, the 3-hydroxy group on the phenolic moiety of the seven bitopic modulators induced selectivity to the KOR over the MOR.

Effects of N-Substituents on the Functional Activities of Naltrindole Derivatives for the δ Opioid Receptor: Synthesis and Evaluation of Sulfonamide Derivatives.

We have recently reported that N-alkyl and N-acyl naltrindole (NTI) derivatives showed activities for the δ opioid receptor (DOR) ranging widely from full inverse agonists to full agonists. We newly designed sulfonamide-type NTI derivatives in order to investigate the effects of the N-substituent on the functional activities because the side chain and S=O part in the sulfonamide moiety located in spatially different positions compared with those in the alkylamine and amide moieties. Among the tested compounds, cyclopropylsulfonamide 9f (SYK-839) was the most potent full inverse agonist for the DOR, whereas phenethylsulfonamide 9e (SYK-901) showed full DOR agonist activity with moderate potency. These NTI derivatives are expected to be useful compounds for investigation of the molecular mechanism inducing these functional activities.

Naldemedine: Peripherally Acting Opioid Receptor Antagonist for Treating Opioid-induced Adverse Effects.

Opioids are widely used for pain management in moderate-to-severe pain. However, opioids are associated with adverse events, such as constipation and emesis/vomiting. To reduce these undesired effects, a structure-activity relationship study of morphinan derivatives was conducted, and a promising lead compound with inhibitory effects on opioid receptors was obtained. Further improvement in the potency and pharmacokinetic profiles of the lead compound led to the discovery of naldemedine, which showed anti-constipation and anti-emetic effects against these adverse events that were induced by morphine without influencing morphine's analgesic effect. Naldemedine was launched in Japan and the USA in 2017 and in the EU in 2019, for treating opioid-induced constipation.

Single-molecule analysis reveals agonist-specific dimer formation of µ-opioid receptors.

G-protein-coupled receptors (GPCRs) are key signaling proteins that mostly function as monomers, but for several receptors constitutive dimer formation has been described and in some cases is essential for function. Using single-molecule microscopy combined with super-resolution techniques on intact cells, we describe here a dynamic monomer-dimer equilibrium of µ-opioid receptors (µORs), where dimer formation is driven by specific agonists. The agonist DAMGO, but not morphine, induces dimer formation in a process that correlates both temporally and in its agonist- and phosphorylation-dependence with ß-arrestin2 binding to the receptors. This dimerization is independent from, but may precede, µOR internalization. These data suggest a new level of GPCR regulation that links dimer formation to specific agonists and their downstream signals.

Nociceptin/Orphanin FQ Peptide Receptor-Related Ligands as Novel Analgesics.

Despite similar distribution patterns and intracellular events observed in the nociceptin/ orphanin FQ peptide (NOP) receptor and other opioid receptors, NOP receptor activation displays unique pharmacological profiles. Several researchers have identified a variety of peptide and nonpeptide ligands to determine the functional roles of NOP receptor activation and observed that NOP receptor- related ligands exhibit pain modality-dependent pain processing. Importantly, NOP receptor activation results in anti-nociception and anti-hypersensitivity at the spinal and supraspinal levels regardless of the experimental settings in non-human primates (NHPs). Given that the NOP receptor agonists synergistically enhance mu-opioid peptide (MOP) receptor agonist-induced anti-nociception, it has been hypothesized that dual NOP and MOP receptor agonists may display promising functional properties as analgesics. Accumulating evidence indicates that the mixed NOP/opioid receptor agonists demonstrate favorable functional profiles. In NHP studies, bifunctional NOP/MOP partial agonists (e.g., AT-121, BU08028, and BU10038) exerted potent anti-nociception via NOP and MOP receptor activation; however, dose-limiting adverse effects associated with the MOP receptor activation, including respiratory depression, itch sensation, physical dependence, and abuse liability, were not observed. Moreover, a mixed NOP/opioid receptor agonist, cebranopadol, presented promising outcomes in clinical trials as a novel analgesic. Collectively, the dual agonistic actions on NOP and MOP receptors, with appropriate binding affinities and efficacies, may be a viable strategy to develop innovative and safe analgesics.

Release rate determination from in situ gel forming PLGA implant: a novel 'shape-controlled basket in tube' method.

OBJECTIVES: This study aimed to examine the impact of syringe-needle assembly differences in making implants of different shapes as well as its influence on the release kinetics and investigate the release kinetics of the in situ forming implant under various release arrangements. METHODS: PLGA in situ forming implant was prepared in different shape and then subjected to in vitro release testing. Mathematical modelling was used to investigate drug release mechanisms. KEY FINDINGS: The in situ forming implant was investigated for the first time how implant shapes can affect release results. It was demonstrated that implant shape differences could lead to significant variation in the release data. Here, we addressed this issue by developing a shape-controlled method to provide a consistent surface to volume ratio and, therefore, a reliable release result. Injectability in the in vitro release was discussed for the first time. Comparisons between various release methods were also evaluated. The release arrangement was found to be of great importance in release kinetics. CONCLUSIONS: The developed 'shape-controlled basket in tube' method can provide the most reproducible release profiles by minimizing implant adhesion to the release vessels or movement without sacrificing full contact between the release medium and the implant surface.

Structure-Activity Relationship between Thiol Group-Trapping Ability of Morphinan Compounds with a Michael Acceptor and Anti-Plasmodium falciparum Activities.

7-Benzylidenenaltrexone (BNTX) and most of its derivatives showed in vitro antimalarial activities against chloroquine-resistant and -sensitive Plasmodium falciparum strains (K1 and FCR3, respectively). In addition, the time-dependent changes of the addition reactions of the BNTX derivatives with 1-propanethiol were examined by 1H-NMR experiments to estimate their thiol group-trapping ability. The relative chemical reactivity of the BNTX derivatives to trap the thiol group of 1-propanethiol was correlated highly with the antimalarial activity. Therefore, the measurements of the thiol group-trapping ability of the BNTX derivatives with a Michael acceptor is expected to become an alternative method for in vitro malarial activity and related assays.

In vitro evaluation of Naltrexone HCl 1% Topical Cream in XemaTop™ for psoriasis.

Psoriasis is a multifactorial skin disease involving abnormal cell proliferation and inflammation; an efficacious topical treatment is yet to be identified. A formulation containing 1% Naltrexone HCl in XemaTop™ base was compounded, characterized and evaluated in vitro as a possible treatment for psoriasis. A three-dimensional psoriasis tissue model was exposed to the formulation for 2 or 5 days and analyzed for the level of markers of cellular proliferation, and inflammatory cytokine IL-6. Using immunohistochemical staining, the level of Ki67 protein significantly decreased in the drug-treated tissues. Western blot analysis showed 86% and 53% down-regulation of other proliferation markers PCNA and CYCLIN D1, respectively, after 5-day exposure. The pro-survival Wnt/ß-catenin pathway was compromised as indicated by 57% decrease in the level of ß-CATENIN and down-regulation of its down-stream targets including CYCLIN D1 (decreased by 53%), c-MYC (63%), c-JUN (92%) and MET (96%) proteins. Likewise, the PI3K/AKT/mTOR pathway was significantly inhibited by 1% Naltrexone HCl in XemaTop™, suggesting protein synthesis was affected. The production of IL-6 was inhibited by 70% in drug-treated tissues. These results suggest that the compounded drug is efficacious in down-regulating molecular markers associated with the pathogenesis of psoriasis. Low-dose Naltrexone in XemaTop™ was stable within 180 days when stored under refrigerated or ambient conditions. These results provide a basis for a clinical evaluation of 1% Naltrexone HCl in XemaTop™ in psoriasis patients.

Hydrogels for diabetic eyes: Naltrexone loading, release profiles and cornea penetration.

Naltrexone (NTX) is a potent opioid growth factor receptor (OGFR) antagonist proved to be useful for treatment of ocular surface complications. The aim of this work was to explore the feasibility of designing NTX-imprinted 2-hydroxyethyl methacrylate-based hydrogels for sustained drug release on the ocular surface. Acrylic acid (AAc) and benzyl methacrylate (BzMA) were chosen as functional monomers able to form binding cavities mimicking OGFR binding sites for NTX. Imprinted hydrogels containing functional monomers loaded higher amounts of NTX compared to non-imprinted ones by simple soaking in drug aqueous solution. In addition, possibility of carrying out the loading and sterilization processes in one step was investigated. NTX release was evaluated both under agitated sink conditions and in a microfluidic flow chamber mimicking the hydrodynamic conditions of the eye, namely the small volume of lachrymal fluid and its renovation rate. Sustained release profiles together with adequate swelling degree (46 to 57% w/w), light transparency (over 85%) and oxygen permeability may make these hydrogels suitable candidates to NTX-eluting contact lenses. NTX-loaded and non-loaded discs successfully passed the chorioallantoic membrane test for potential ocular irritation and were cytocompatible with human mesenchymal stem cells. Finally, NTX-imprinted hydrogels tested in the bovine corneal permeability assay provided therapeutically relevant amounts of NTX inside the cornea, reaching drug levels similar to those attained with a concentrated aqueous solution in spite the discs showed sustained release.

Stereochemistry and innate immune recognition: (+)-norbinaltorphimine targets myeloid differentiation protein 2 and inhibits toll-like receptor 4 signaling.

Deregulation of innate immune TLR4 signaling contributes to various diseases including neuropathic pain and drug addiction. Naltrexone is one of the rare TLR4 antagonists with good blood-brain barrier permeability and showing no stereoselectivity for TLR4. By linking 2 naltrexone units through a rigid pyrrole spacer, the bivalent ligand norbinaltorphimine was formed. Interestingly, (+)-norbinaltorphimine [(+)-1] showed ∼25 times better TLR4 antagonist activity than naltrexone in microglial BV-2 cell line, whereas (-)-norbinaltorphimine [(-)-1] lost TLR4 activity. The enantioselectivity of norbinaltorphimine was further confirmed in primary microglia, astrocytes, and macrophages. The activities of meso isomer of norbinaltorphimine and the molecular dynamic simulation results demonstrate that the stereochemistry of (+)-1 is derived from the (+)-naltrexone pharmacophore. Moreover, (+)-1 significantly increased and prolonged morphine analgesia in vivo. The efficacy of (+)-1 is long lasting. This is the first report showing enantioselective modulation of the innate immune TLR signaling.-Zhang, X., Peng, Y., Grace, P. M., Metcalf, M. D., Kwilasz, A. J., Wang, Y., Zhang, T., Wu, S., Selfridge, B. R., Portoghese, P. S., Rice, K. C., Watkins, L. R., Hutchinson, M. R., Wang, X. Stereochemistry and innate immune recognition: (+)-norbinaltorphimine targets myeloid differentiation protein 2 and inhibits toll-like receptor 4 signaling.

Simultaneous Transdermal Delivery of Buprenorphine Hydrochloride and Naltrexone Hydrochloride by Iontophoresis.

The opioids buprenorphine hydrochloride (BUP) and naltrexone hydrochloride (NTX) show promise as a combination treatment for addiction, but no means of delivering the two compounds in one medicine currently exist. In this paper, we report sufficient input rates of both these drugs from one iontophoretic transdermal drug delivery system. Experiments were performed using dermatomed pig skin mounted in glass side-bi-side cells. BUP and NTX were iontophoretically delivered together from the anode using direct constant current from Ag/AgCl electrodes. The transdermal drug fluxes and the masses of drugs in both the stratum corneum and the underlying epidermis/dermis were measured. The apparent electroosmotic flow was quantified using a neutral marker (acetaminophen). The effects of donor composition (drug concentration/molar fraction and pH), current density and profile, and the choice of receptor solution were assessed. Iontophoresis dramatically increased the flux of both drugs compared to passive control values. Target fluxes (calculated from literature clearance values and required therapeutic plasma concentrations) were greatly exceeded for NTX and were met for BUP. The latter accumulated in the skin and suppressed electroosmotic flow, inhibiting both its own flux and that of NTX. NTX, in turn, negatively influenced the flux of BUP via co-ion competition. Lowering current density by increasing the delivery area resulted in increased electroosmotic flow but did not significantly affect current-normalized drug fluxes. Delivering the drugs from both electrodes and reversing the polarity for every 2 h did not increase the flux of either compound. In summary, during iontophoresis, BUP and NTX inhibited each other's flux by two distinct mechanisms. While the more complex behavior of BUP complicates the optimization of this drug combination, iontophoresis nevertheless appears to be a feasible approach for the controlled codelivery of NTX and BUP through the skin.

Study on structural insight of the analysis of negative effects of opioids analgesics in naltrexone with TLR4 Mutations.

Chronic pain has been defined as the persistence that remained for more than three months. The extent of previous time duration with the normal time of natural healing phase becomes poor and results in reduced life quality and morbidity. Opioids are well recognized therapy for pain management and the clinical prescriptions based on opioids have been defined with increasing implicating behavior among patients suffering with chronic pain. The association between the pain and immunity has long been established since the involvement of interleukin-1ß (IL-1ß) in sickness that is considered with the induced hyperalgesia. In the context of pharmacodynamics Toll like receptors (TLRs) are involved in the negative effects of opioids as analgesics. The soluble factors released by immune cells as well as from the disruptive cells bind to TLRs. This binding leads the pre and post-synaptic ends on endothelial and microglial cells that exhibit the activation of complex inhibitory and excitatory process at the synapses site. In TLRs, TLR4 is mostly reported that is strongly associated in specifically in areas of T cells and macrophages. The current study is designed to investigate the structural insights of the opioids and TLR4 interactions by using computational approach in the aspect of recognizing the chemical combinatorial factors that are involved in the pain management. This study targets that how opioids interact with TLR4 and the process of chemical interaction that leads to negative effects of opioids at neuroimmune interface as well as to investigate the extent of particular naltrexone that mediates with the negative effects of opioids.

Minimal contribution of P-gp on the low brain distribution of naldemedine, a peripherally acting µ-opioid receptor antagonist.

Naldemedine tosylate, a peripherally acting µ-opioid receptor antagonist, is indicated for treatment of opioid induced constipation in both Japan and US. Naldemedine has limited ability to affect the central analgesic effect of opioid analgesics. In this study, we investigated the contribution of P-glycoprotein (P-gp) on the brain distribution of naldemedine. Naldemedine tosylate showed acceptable oral absorption in rats. Following a single oral administration of [14C]-naldemedine tosylate to rats and ferrets, little radioactivity was detected in the region protected by the blood-brain barrier (BBB). In the assessment using Caco-2 cells, it was determined that naldemedine is a substrate for P-gp. The contribution of P-gp to the brain distribution of naldemedine was assessed using multidrug resistance 1a/b (mdr1a/b) knockout mice. While the brain-to-plasma concentration ratio (brain Kp) of naldemedine in the mdr1a/b knockout mice was 4-fold of that in the wild-type mice, the brain Kp in the mdr1a/b knockout mice was quite low (brain Kp < 0.1). These results suggest that the low brain distribution of naldemedine was due to the limited ability to cross the BBB rather than efflux by P-gp and therefore brain distribution of naldemedine would not be affected by concomitant administration of P-gp inhibitors or functional disorder of P-gp.