AAPS Journal最新文献

{"title":"Estimating Intragastric Disintegration Times of Immediate Release Dose Units Administered After a High-Calorie, High-Fat Meal Using Standardized, Commercially Available Equipment and Materials.","authors":"Maria Vertzoni, Erato Androulaki, Shirin Dietrich, Marianna Gereoudaki, Paraskevi Karpasiti, Zoi Margiori, Ioannis Papazoglou, Ioanna Stavrinoudi, Christos Reppas","doi":"10.1208/s12248-026-01242-x","DOIUrl":"https://doi.org/10.1208/s12248-026-01242-x","url":null,"abstract":"<p><p>Intragastric disintegration times of immediate release (IR) dose units after high-calorie, high-fat meal would be useful in the assessment of drug absorption process and when considering waivers of bioequivalence assessment of non-high risk IR products in the fed state. We investigated the usefulness of standardized, commercially available equipment and materials in estimating intragastric disintegration times of IR dose units after high-calorie, high-fat meal. Disintegration times were estimated from the cumulative % drug in the medium vs. time data sets. When using 330 mL Level III FeSSGF-V3 and the compendial Apparatus II (60 rpm) with apex vessel without sinkers, the complete disintegration times of Panadol Actifast® tablets were close to previously estimated mean intragastric complete disintegration time (14 min), and the initial disintegration times of hard gelatine capsules were close to previously estimated mean intragastric initial disintegration time (11 min). Also, initial intragastric disintegration times of hard hypromellose capsules without gelling agents were predicted to be in line with intragastric initial disintegration times in the fasted state whereas disintegration of hard hypromellose capsules prepared with gelling agents was dramatically extended, in line with intragastric data after high-calorie, high-fat meal. Level III FeSSGF-V3 and the compendial Apparatus II (60 rpm) with apex vessel could be useful in estimating average intragastric disintegration times, in supporting justification for waiving in vivo bioequivalence studies, or in detecting non-IR characteristics of dose units after a high-calorie, high-fat meal.</p>","PeriodicalId":50934,"journal":{"name":"AAPS Journal","volume":"28 3","pages":""},"PeriodicalIF":3.7,"publicationDate":"2026-05-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147845825","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"High Concordance of Total Antibody and Transduction Inhibition Assays Enables Replacement of Cell-Based Antibody Testing for Preexisting Anti-AAV Immunity in Cynomolgus Monkey.","authors":"Florian Neff, Michael Antony, Julia Merz, Francesca Ros, Uwe Wessels, Kay-Gunnar Stubenrauch","doi":"10.1208/s12248-026-01239-6","DOIUrl":"https://doi.org/10.1208/s12248-026-01239-6","url":null,"abstract":"<p><p>Testing for preexisting anti-AAV immunity in non-human primates constitutes an essential part of screening procedures for nonclinical studies with gene therapy, which is customary performed using transduction inhibition (TI) assays. These assays cause high costs and a significant operational burden owing to their complexity and low throughput. However, broader implementation of more practical total antibody assays (TAb) is hindered by the lack of conclusive data demonstrating assays comparability in non-human primates. To determine whether TAb assays can serve as a viable alternative to TI assays for anti-AAV screening in monkeys, we conducted a comparative study for two AAV serotypes (AAV2 and AAV9), with tailored specificity control measures incorporated in both assay formats. Both the TI and TAb assays showed a high concordance in detection of preexisting anti-AAV immunity in cynomolgus monkey. The presented TAb assay offers a simple, cost- and time-saving alternative to TI assays for pre-study testing of non-human primates. The use of such TAb assays can thus result in concordant animal enrollment at lower cost and greater speed as compared to TI-based screening tests.</p>","PeriodicalId":50934,"journal":{"name":"AAPS Journal","volume":"28 3","pages":""},"PeriodicalIF":3.7,"publicationDate":"2026-05-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147845761","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Control Strategy Considerations for the Continuous Manufacturing of Low-dose Oral Solid Dosage Formulations.","authors":"Melanie Dumarey, Cristina Ruiz Samblás, Manel Bautista, Sangah S Kim, Gregory D Doddridge, Andrew P Shier","doi":"10.1208/s12248-026-01250-x","DOIUrl":"https://doi.org/10.1208/s12248-026-01250-x","url":null,"abstract":"<p><p>The adoption of Continuous Manufacturing (CM) for Oral Solid Dosages (OSD) is often challenged by the limited sensitivity of traditional Process Analytical Technology (PAT), such as Near-infrared (NIR) and Raman spectroscopy, to provide sufficient accuracy for process monitoring and control of low-dose or fixed-dose formulations. This manuscript explores solutions by highlighting advanced control strategies and alternative manufacturing technologies. These strategies include enhanced spectroscopic methods (e.g., Spatially resolved-NIRS, Light-induced fluorescence) to provide improved accuracy/precision, the use of process data and process models (Residence Time Distribution, Multivariate Statistical Process Control) as soft sensors, hybrid PAT and process models and more traditional at-line/off-line monitoring using NIR, Raman or high-sensitivity liquid chromatography with stratified sampling and bracketing. Alternatively, several technologies inherently ensure high content uniformity, such as semi-Continuous Manufacturing (sCM) with accurate mini-batch dispensing and Dry Coating Technology. For Twin-Screw Hot Melt Extrusion (HME) molecular-level mixing delivers more uniform blends, but current low-dose applications still require pre-blending of the drug substance with suitable excipients. When fed with a uniform powder blend, twin screw wet granulation also ensures compliant content uniformity without the need for PAT monitoring. In conclusion, a successful CM of low dose products may be possible when strategically combining advanced spectral and data approaches, modelling, and innovative platforms to build robust and validated process controls. This has been demonstrated across multiple peer reviewed studies and is now gradually being incorporated into control strategies for the commercial manufacture of pharmaceutical products.</p>","PeriodicalId":50934,"journal":{"name":"AAPS Journal","volume":"28 3","pages":""},"PeriodicalIF":3.7,"publicationDate":"2026-05-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147845759","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Raltegravir Plasma Exposure: A Machine Learning-Based Model for its Prediction Using Limited Sampling Strategy.","authors":"Matheus De Lucca Thomaz, Kathley Lanna Rezende de Azevedo, Tiago Antunes Paz, Glauco Henrique Balthazar Nardotto, Fernanda de Lima Moreira, Geraldo Duarte, Jean-Baptiste Woillard, Vera Lucia Lanchote, Joao Paulo Bianchi Ximenez","doi":"10.1208/s12248-026-01248-5","DOIUrl":"https://doi.org/10.1208/s12248-026-01248-5","url":null,"abstract":"<p><p>Recent studies have applied machine learning (ML)-based limited sampling strategies (LSS) to predict drug exposure (AUC), achieving low prediction error and performance comparable to or better than multiple linear regression and population pharmacokinetics LSS. This study aimed to develop and validate a machine learning-based limited sampling strategy capable of predicting raltegravir (RAL) exposure. Four machine learning algorithms (XGBoost, Random Forest, GLMNet, and SVM) were trained using pharmacokinetic profiles generated via Monte Carlo simulation from a population pharmacokinetic (POPPK) model. Data were divided into training (75%) and test (25%) sets. All possible combinations of sampling times, pairs and triplets, in steady-state, up to 12 h post-dose were evaluated. Model performance was assessed by the lowest root mean square error (RMSE) in the cross-validation, and the best performing model was evaluated in the test set and externally validated using simulated PK profiles from an independent POPPK model and patient data from a clinical study. XGBoost trained with concentrations at 0.5, 2, and 4 h showed the best predictive performance. The model achieved excellent accuracy in the test set (bias/RMSE: 0.8%/8.7%) and in the independent simulation (1.9%/14.3%). Performance decreased in real patient data (5.0%/24.1%), highlighting the need for caution when extrapolating predictions to populations whose characteristics differ from those represented in the training datasets. A machine learning model using only three sampling timepoints has been developed and validated in different datasets, enabling accurate estimation of RAL AUC₀-₁₂. This approach provides a tool for pharmacokinetic and PK/PD studies and reduces intensive sampling need in clinical settings.</p>","PeriodicalId":50934,"journal":{"name":"AAPS Journal","volume":"28 3","pages":""},"PeriodicalIF":3.7,"publicationDate":"2026-05-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147845838","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Correction: Multicomponent Interactive Release (MIR) Model for Herbal Extract/Multidrug-Loaded Electrospun Nanofibers: A Mechanistic Framework Integrating Diffusion, Swelling, and Erosion.","authors":"Pitt Supaphol","doi":"10.1208/s12248-026-01252-9","DOIUrl":"https://doi.org/10.1208/s12248-026-01252-9","url":null,"abstract":"","PeriodicalId":50934,"journal":{"name":"AAPS Journal","volume":"28 3","pages":""},"PeriodicalIF":3.7,"publicationDate":"2026-05-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147845823","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Evaluating the Utility and Constraints of the Competitive Counter Flow (CCF) Assay in OATP1B1 Substrate Profiling.","authors":"Sumathy Mathialagan, Mark A West, Emi Yamaguchi, Hannah M Moulton, Brendon Kapinos, Sasan Paryad Zanjani, David A Tess, Emi Kimoto, Manthena V S Varma, Sook Wah Yee","doi":"10.1208/s12248-026-01251-w","DOIUrl":"https://doi.org/10.1208/s12248-026-01251-w","url":null,"abstract":"<p><p>The organic anion transporting polypeptide 1B1 (OATP1B1) plays a critical role in hepatic uptake of a wide range of high- and low-permeability, anionic molecules, particularly those in Extended Clearance Classification System (ECCS) Classes 1 and 3. However, conventional uptake assays using HEK293 cells overexpressing OATP1B1 can yield false-negative results, especially for highly permeable, lipophilic anionic or zwitterionic compounds, where passive diffusion and nonspecific binding may mask transporter-mediated uptake. In this study, we applied the competitive counterflow (CCF) assay to evaluate 58 compounds across different ECCS classes. The assay measures steady-state changes in intracellular [3H]-estradiol-17β-glucuronide retention following exposure to test compounds across multiple concentrations. Several known OATP1B1 substrates, exhibited more than 50% loss of intracellular [3H]-estradiol-17β-glucuronide at one or more concentrations compared to control. Using a three-tier classification based on tracer retention across multiple concentrations, the assay demonsrated high sensitivity for detecting compounds that are OATP1B1 substrates. Notably, the CCF assay enabled identification of four xenobiotics-bumetanide, candesartan, naringin and sulfasalazine-not previously recognized as OATP1B1 substrates using OATP1B1 uptake assay. Overall, this work provides a comprehensive evaluation of the CCF assay as a complementary, early‑stage screening and triage tool for OATP1B1 substrate assessment. The findings underscore the importance of an integrative strategy that combines CCF data with orthogonal in vitro and in vivo evidence to mitigate the risk of both false‑negative and false‑positive conclusions, particularly for ECCS Class 1B compounds where traditional overexpression systems show limited sensitivity.</p>","PeriodicalId":50934,"journal":{"name":"AAPS Journal","volume":"28 3","pages":""},"PeriodicalIF":3.7,"publicationDate":"2026-05-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147845795","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Foreign Comparator Drug Products, Manufacturing & Supply Chain Considerations for Otic, Ophthalmic, And Parenteral Solutions - Generic Drugs Perspective.","authors":"Andre Nana, Jonathan Hughes, Suman Dandamudi, Li Gong, April C Braddy","doi":"10.1208/s12248-026-01243-w","DOIUrl":"https://doi.org/10.1208/s12248-026-01243-w","url":null,"abstract":"<p><p>In the United States (US), only the Food and Drug Administration (FDA)-approved drug products serve as comparator products in bioequivalence (BE) studies. Using foreign comparator drug products raises concerns about comparability to FDA-approved Reference Listed Drugs (RLD), particularly regarding quality standards and therapeutic equivalence. There is potential for differences in BE that may result from even slight differences between the foreign comparator and the US-approved RLD. Such discrepancies can compromise safety, efficacy, and product performance, undermining market access by disrupting manufacturing and supply chain operations for generic drug products. This paper examined biowaiver considerations, product-specific guidances, labeling requirements, and manufacturing guidelines from nine regulatory agencies: FDA, ANVISA (Brazil), COFEPRIS (Mexico), EMA (European Union), Health Canada (Canada), MHLW/PMDA (Japan), SAHPRA (South Africa), Swissmedic (Switzerland), and TGA (Australia). The analysis focused on foreign comparator drug product considerations for parenteral, otic, and ophthalmic solutions, identifying regulatory similarities and differences across jurisdictions. Generally, biowaivers for in vivo BE studies are considered acceptable for these drug products across jurisdictions. However, criteria for qualitative (Q1) and quantitative (Q2) sameness between generic and RLD formulations vary among regulatory agencies. Labeling requirements also differ, though Q1 information is typically mandated for inclusion. Regarding quality-related regulations-including specifications, container and closure systems, stability, storage conditions, and CMC post-approval changes-most regulatory authorities align with international guidelines, particularly ICH quality guidance documents. This alignment suggests potential harmonization opportunities while acknowledging jurisdictional variations in specific requirements for the utilization of foreign comparator products to establish BE for generic drug products.</p>","PeriodicalId":50934,"journal":{"name":"AAPS Journal","volume":"28 3","pages":""},"PeriodicalIF":3.7,"publicationDate":"2026-05-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147845812","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Blood-glucose Profile Evaluation with a Model-based Approach using Continuous Glucose Monitoring Data.","authors":"Hanna Kunina, Jenny Y Chien, Parag Garhyan, Jeanne S Geiser, Maria C Kjellsson","doi":"10.1208/s12248-026-01245-8","DOIUrl":"https://doi.org/10.1208/s12248-026-01245-8","url":null,"abstract":"<p><p>Continuous glucose monitoring (CGM) is widely used in type 1 diabetes management. Although less common in type 2 diabetes (T2D), its application is increasing, especially among patients with T2D on insulin therapy. CGM provides detailed, continuous glucose data that reveal daily glycemic fluctuations and help mitigate hyper- and hypoglycemic episodes. However, missing information on meal size and timing complicates the interpretation of data. To address these challenges, we propose a pharmacometric modeling approach that describes blood glucose profiles in patients with T2D receiving basal insulin in the absence of exact meal inputs. In this study, 73 individuals with T2D receiving insulin glargine plus oral antidiabetic medications (OAMs) underwent CGM assessments at four visits (Visit 3 on OAMs alone; Visits 13, 16, 20 on OAMs + insulin). Building upon the existing Integrated Glucose-Insulin (IGI) model, we incorporated a population meal model and an insulin glargine pharmacokinetic model, creating a comprehensive \"meal-IGI-insulin\" framework. The model identified three daily meal intakes, modeled as the sum of a surge function and a maximum bioavailable glucose amount of 7.83 g/hour. The model evaluation indicated adequate performance in predicting fasting blood glucose and HbA1c, though some discrepancies arose in forecasting hypoglycemic events. The developed modeling framework can facilitate prospective simulations of diverse meal patterns and insulin regimens, potentially accelerating antidiabetic drug development, simplify closed-loop automated insulin delivery algorithms, and optimize clinical strategies for patients with T2D.</p>","PeriodicalId":50934,"journal":{"name":"AAPS Journal","volume":"28 3","pages":""},"PeriodicalIF":3.7,"publicationDate":"2026-05-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147845821","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"De-risking Biopharma Asset Acquisition: Towards a Quantitative Framework for Strategic Decision-making.","authors":"Xiaotang Ma, Zheng Lu, Yating Zhao, Jennifer Sheng","doi":"10.1208/s12248-026-01246-7","DOIUrl":"https://doi.org/10.1208/s12248-026-01246-7","url":null,"abstract":"<p><p>The impending patent cliff projected between 2028-2030 poses significant commercial and strategic challenges for innovative pharmaceutical and biotechnology companies. To sustain growth and maintain competitive positioning, organizations are increasingly relying on strategic mergers, acquisitions, and partnerships to replenish pipelines. However, systematic quantitative strategies and framework for asset evaluation remain limited. This review outlines how clinical pharmacology and pharmacometrics (CPP) can support asset evaluation and decision-making during the asset due diligence. First, CPP spans the entire drug development continuum, providing a quantitative framework for evaluating external assets, including pharmacological plausibility, dosing feasibility, and overall development risks. Second, Model-informed drug development (MIDD) approaches can be applied to predict human pharmacokinetics, inform dose selection, and estimate the probability of technical and regulatory success. Third, we examine the emerging role of artificial intelligence and machine learning in asset evaluation and portfolio decision-making, by discovering prognostic and predictive factors, and identifying the patient sub-group. We also introduce NewCo as an emerging drug-development and business model, where quantitative strategies may be deployed. Further, we address cognitive biases, such as confirmation bias and sunk cost fallacies that can influence acquisition outcomes. Importantly, we propose the development of a bias-aware, fit-for-purpose corporate template to integrate CPP and MIDD insights, standardize evaluation criteria, and support cross-functional decision-making during asset due diligence. Embedding quantitative and bias-mitigated CPP frameworks into due diligence workflows, can help identify high-value opportunities, de-risk development uncertainties, and accelerate delivery of innovative therapies to patients with unmet medical needs.</p>","PeriodicalId":50934,"journal":{"name":"AAPS Journal","volume":"28 3","pages":""},"PeriodicalIF":3.7,"publicationDate":"2026-05-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147845827","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Quantitative Investigation of ATP Switch Concept of STA551 Through Combined In Vivo Tissue Distribution Studies and Physiologically Based Pharmacokinetic Modeling.","authors":"Takayuki Nemoto, Haruka Kuroi, Meiri Shida-Kawazoe, Tatsuhiko Tachibana","doi":"10.1208/s12248-026-01238-7","DOIUrl":"10.1208/s12248-026-01238-7","url":null,"abstract":"<p><p>STA551 is an anti-human CD137 (hCD137) switch antibody with adenosine triphosphate (ATP)-dependent antigen binding, developed to achieve potent anti-tumor effects and superior safety. Based on several reports, unlike in normal tissues, high concentrations of ATP are considered to exist in the interstitial space of tumors, thus STA551 is expected to exhibit tumor-selective antigen (hCD137) binding (ATP switch concept). This study aims to quantitatively investigate this ATP switch concept in vivo by combining tissue distribution studies and physiologically based pharmacokinetic (PBPK) modeling. An iodinated conventional anti-hCD137 antibody, ¹²⁵I-Ure-mIgG1, was administered as a tracer to tumor-bearing hCD137 knock-in (KI) mice. Co-administration of excess unlabeled Ure-mIgG1 at 20 mg/kg increased plasma tracer radioactivity levels and decreased the tissue-to-plasma (T/P) ratios in several normal tissues and tumors. However, co-administration of unlabeled Sta-MB (mouse surrogate antibody of STA551) at 1 or 20 mg/kg did not change the plasma tracer radioactivity levels but clearly reduced T/P ratios, mainly in tumors, strongly suggesting tumor-selective binding of Sta-MB. A PBPK model was developed to explain this distribution data, and parameters including non-specific clearance, interstitial uptake clearance, target-related parameters, and ATP switch molecule concentrations were estimated. Importantly, the ATP switch molecule concentration in the tumor interstitial space was estimated to reach approximately hundreds μM, with much lower concentrations in non-tumor tissues. In conclusion, this combination approach successfully demonstrated the ATP switch concept of STA551 in vivo. These findings will help guide clinical trials for STA551 and the development of future switch antibodies.</p>","PeriodicalId":50934,"journal":{"name":"AAPS Journal","volume":"28 3","pages":""},"PeriodicalIF":3.7,"publicationDate":"2026-04-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147788252","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}