Journal of pharmaceutical sciences最新文献

{"title":"Amorphous solubility advantage: Theoretical considerations, experimental methods, and contemporary relevance.","authors":"Keisuke Ueda, Dana E Moseson, Lynne S Taylor","doi":"10.1016/j.xphs.2024.08.029","DOIUrl":"10.1016/j.xphs.2024.08.029","url":null,"abstract":"<p><p>Twenty-five years ago, Hancock and Parks asked a provocative question: \"what is the true solubility advantage for amorphous pharmaceuticals?\" Difficulties in determining the amorphous solubility have since been overcome due to significant advances in theoretical understanding and experimental methods. The amorphous solubility is now understood to be the concentration after the drug undergoes liquid-liquid or liquid-glass phase separation, forming a water-saturated drug-rich phase in metastable equilibrium with an aqueous phase containing molecularly dissolved drug. While crystalline solubility is an essential parameter impacting the absorption of crystalline drug formulations, amorphous solubility is a vital factor for considering absorption from supersaturating formulations. However, the amorphous solubility of drugs is complex, especially in the presence of formulation additives and gastrointestinal components, and concentration-based measurements may not indicate the maximum drug thermodynamic activity. This review discusses the concept of the amorphous solubility advantage, including a historical perspective, theoretical considerations, experimental methods for amorphous solubility measurement, and the contribution of supersaturation and amorphous solubility to drug absorption. Leveraging amorphous solubility and understanding the associated physicochemical principles can lead to more effective development strategies for poorly water-soluble drugs, ultimately benefiting therapeutic outcomes.</p>","PeriodicalId":16741,"journal":{"name":"Journal of pharmaceutical sciences","volume":" ","pages":"18-39"},"PeriodicalIF":3.7,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142120107","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":"Manufacture, characterization, and elucidation of drug release mechanisms of etonogestrel implants based on ethylene vinyl acetate.","authors":"Angela Ren, Ziyue Zhong, Yan Wang, Bin Qin, William Smith, Xiaoming Xu, Tony Listro, Feng Zhang","doi":"10.1016/j.xphs.2024.08.015","DOIUrl":"10.1016/j.xphs.2024.08.015","url":null,"abstract":"<p><p>In this work, etonogestrel implants were manufactured using coextrusion. The purpose of the study was to correlate changes in microstructure and transport properties that occurred in etonogestrel implants to drug release mechanisms. The implants consisted of an EVA 28 (28 % vinyl acetate) core containing dispersed and dissolved etonogestrel, and an EVA 15 (15 % vinyl acetate) skin. The drug release was determined to be via diffusion at a controlled rate and governed by implant dimensions. In-vitro release revealed evidence of supersaturation in the implant core and skin, likely from the intense mechanical energy input during the twin-screw manufacturing process. Subsequently during storage under ambient conditions, supersaturation resulted in recrystallization of drug crystals, preferentially in the implant core. Etonogestrel solubility and diffusivity in EVA were determined by permeation experiments and used for release modeling. Drug release from the EVA skin layer deviated from the predicted values due to 1) formation of a drug depletion zone in the core and 2) presence of a stagnant media layer adjacent to the skin. Drug release from implant ends was significantly faster than predicted. Air-filled pores were observed in the implant core using microCT which likely contributed to the faster release from implant ends.</p>","PeriodicalId":16741,"journal":{"name":"Journal of pharmaceutical sciences","volume":" ","pages":"199-209"},"PeriodicalIF":3.7,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142140325","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":"Improving oral absorption of a rapidly crystallizing parent drug using prodrug strategy: Comparison of phosphate versus glycine based prodrugs.","authors":"Anura S Indulkar, Russell Slade, Navendu Jana, Robin R Frey, Thomas D Penning, Albert Lai, Alix F Leblanc","doi":"10.1016/j.xphs.2024.09.012","DOIUrl":"10.1016/j.xphs.2024.09.012","url":null,"abstract":"<p><p>With an increasing number of Biopharmaceutical Classification System (BCS) II/IV pipeline compounds, solubilizing and supersaturating formulation strategies are becoming prevalent. Beyond formulation and solid form strategies, prodrugs are also employed to overcome solubility-limited absorption of poorly water-soluble compounds. Prodrugs can potentially yield supersaturated systems upon conversion to the parent drug intraluminally and thus enhance absorption. However, supersaturation also increases the driving force for crystallization, resulting in low solution concentrations, which can potentially negate the advantage of prodrugs. In this work, two unique solubility-enhancing prodrugs, phosphate and glycine esters, were investigated for a rapidly crystallizing parent drug. Ex vivo absorption studies using rat tissue and in vivo studies in dogs were performed. Conversion rate of the phosphate prodrug to the parent was dependent on the milieu and increased ∼24-fold in the presence of intestinal contents as medium and tissue relative to neat buffer. In contrast, conversion of the glycine prodrug was minimal under any conditions tested, suggesting that the conversion occurs after absorption into the enterocytes. Phosphate prodrug showed a non-linear increase in parent drug absorptive flux across rat intestinal tissue with concentration when intestinal contents were used as donor media. This was attributed to rapid conversion and high supersaturation of the parent drug which subsequently resulted in crystallization at high doses in the donor chamber. Glycine prodrug did not undergo complete conversion at high doses and was absorbed unchanged on the basolateral side, indicating saturation of the converting enzymes in the enterocytes. The combined flux (parent drug and glycine) showed a linear increase with dose and crystallization was not observed. Under physiological conditions, glycine prodrug that is absorbed unchanged from the intestine can potentially undergo complete conversion in hepatocytes after absorption and make the parent drug systemically available. Thus, glycine prodrug provided overall higher absorption compared to phosphate prodrug. The observed flux levels for both the prodrugs were higher compared to the parent drug alone, highlighting an advantage to use of a prodrug strategy to improve absorption of such compounds. Oral dosing in a dog PK study revealed that the bioavailability using the phosphate prodrug was ∼50% whereas, it was ∼100% with glycine prodrug, supporting the in vitro observations.</p>","PeriodicalId":16741,"journal":{"name":"Journal of pharmaceutical sciences","volume":" ","pages":"279-288"},"PeriodicalIF":3.7,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142378010","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":"Molecular-properties based formulation guidance tree for amorphous and supersaturable mesoporous silica preparations of poorly soluble compounds.","authors":"Dipy M Vasa, Shih-Wen Wang, Matthew F Dunn, Erica Long, Suman A Luthra","doi":"10.1016/j.xphs.2024.10.040","DOIUrl":"10.1016/j.xphs.2024.10.040","url":null,"abstract":"<p><p>A huge majority of new chemical entities (NCEs) advancing through the drug discovery pipeline often have poor aqueous solubility. This requires formulation scientists to search for solubility enhancement strategies, within the constraints of time and material. To address these challenges, a strategic platform formulation is often required for a rapid compound screening to enable early exploratory PK and toxicology studies. Through this work, we present an option of a material-sparing, high yielding and solubility-enabling amorphous API and HPMCAS-L co-loaded mesoporous silica-based formulation. The usability of this platform formation strategy was assessed for a physico-chemically diverse set of eleven compounds. The formulation approach was successful in stabilizing the model compounds mesoporous silica. Additionally, through the presence of HPMCAS-L, the precipitation risk in supersaturable aqueous environment was significantly reduced. Finally, this manuscript provides fundamental, computational and experimental molecular-properties based formulation guidance tree to a priori gauge the (1) possibility of generating solid-state stable amorphous formulations and (2) sustaining in vitro supersaturation in extreme non-sink dissolution conditions. This unique and conceptual formulation guidance tree is believed to be extremely beneficial to drug discovery formulators to triage NCEs and streamline solubility-enabling formulation efforts.</p>","PeriodicalId":16741,"journal":{"name":"Journal of pharmaceutical sciences","volume":" ","pages":"554-565"},"PeriodicalIF":3.7,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142558086","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":"Dissolution profiles of high-dose salt-form drugs in bicarbonate buffer and phosphate buffer.","authors":"Yuki Tarumi, Kiyohiko Sugano","doi":"10.1016/j.xphs.2024.10.025","DOIUrl":"10.1016/j.xphs.2024.10.025","url":null,"abstract":"<p><p>The purpose of the present study was to compare the dissolution profiles of high-dose salt-form drugs in bicarbonate buffer (BCB) and phosphate buffer (PPB) focusing on the pH changes in the bulk phase. The pH titration curves of BCB and PPB (pH 6.5, buffer capacity (β) = 4.4 mmol/L/pH unit) were first theoretically calculated and experimentally validated. For dissolution tests, six drug salts with an acid counterion, one drug salt with a weak base counterion, and one free acid drug were employed (125-800 mg clinical dose). The dose/fluid volume ratio (Dose/FV) was aligned with the clinical condition. In the pH titration study, the pH value decreased below pH 6.0 by adding HCl > 2.8 mmol/L (BCB) or > 1.6 mmol/L (PPB) and increased above pH 7.0 by adding NaOH > 2.0 mmol/L (BCB) or > 2.4 mmol/L (PPB). In the dissolution test, even though the initial pH and β values were the same, the pH value at 4 h was lower in PPB than in BCB in all cases. For the drug salts with an acid counterion, the area under the dissolution curve was 1.2 to 2.6-fold lower in BCB than in PPB. A marked precipitation process was observed in BCB, but less pronounced or absent in PPB. The results of this study suggest the use of BCB and a clinically equivalent Dose/FV may be valuable in predicting the oral absorption of high-dose drug salts.</p>","PeriodicalId":16741,"journal":{"name":"Journal of pharmaceutical sciences","volume":" ","pages":"477-485"},"PeriodicalIF":3.7,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142564220","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":"Complemental hard modeling in Raman spectroscopy: A case study on titanium dioxide-free coating in-line monitoring.","authors":"René Brands, Jens Bartsch, Markus Thommes","doi":"10.1016/j.xphs.2024.10.044","DOIUrl":"10.1016/j.xphs.2024.10.044","url":null,"abstract":"<p><p>Tablets are coated for taste or odor modification, for modified release profiles or as a protective layer to increase the stability. Here, titanium dioxide is frequently added as a coating component due to its opaque properties. Furthermore, its Raman activity makes it an integral part of in-line monitoring models. However, due to the carcinogenic potential of titanium dioxide, calcium carbonate is utilized as a substitute, exhibiting similar opaque properties. Calcium carbonate tends to exhibit overlapped peaks with carbon hydrates in the Raman spectrum. Consequently, new models based on e.g. hard modeling are required instead of peak integration. In this study, tablets were coated with a coating including calcium carbonate. Partial Least Squares Regression (PLS) and Complemental Hard Modeling (CHM) were examined as feasible in-line monitoring approaches. Furthermore, two different measurement positions in the coater were compared, orthogonal and tangential with respect to the moving tablet bed. Cross-validation exhibited improved CHM performance with reduced RMSECV values of about 5 %. The prediction of the coating mass growth occurred comparable with RMSEP values in a similar range of 2-5 %. Despite this, the CHM´s achieved improved performance with reduced training data quantity and quality. The different measurement positions indicated no process-relevant differences.</p>","PeriodicalId":16741,"journal":{"name":"Journal of pharmaceutical sciences","volume":" ","pages":"577-585"},"PeriodicalIF":3.7,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142710327","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":"Bioavailability improvement by atomic layer coating: Fenofibrate a case study.","authors":"Balaji Ganapathy, Vijayendra Redasani, Sujit Debnath, Neha Gupta, Ankur Kadam, Fei Wang, Pravin Narwankar","doi":"10.1016/j.xphs.2024.10.052","DOIUrl":"10.1016/j.xphs.2024.10.052","url":null,"abstract":"<p><p>Biopharmaceutical Classification Systems (BCS) class II drugs show poor solubility and high permeability in the body. Fenofibrate (FF) is a classic example of a BCS class II drug, used to treat high cholesterol and triglyceride (fat-like substances) levels in the blood. Atomic layer coating (ALC) is a surface engineering technology adapted from the semiconductor industry, where metal oxides are coated one atomic layer at a time over the active pharmaceutical ingredients (API) particles. ALC coating was proven to improve the processability, alter the hydrophilicity, improve the stability, and fine-tune the release of drugs. Herein, we report the intervention of ALC coating in enhancing the bioavailability of a poorly water-soluble drug (fenofibrate) in the animal model. The physical properties of uncoated fenofibrate were compared with those of zinc oxide-coated and silicon oxide-coated fenofibrate. Following the application of the coatings, the structural integrity (both chemical stability and solid-state stability) of the active pharmaceutical ingredient (API) remained uncompromised, as corroborated by ¹H NMR and powder X-ray diffraction analyses. Notably, zinc oxide-coated fenofibrate exhibited favorable flow characteristics, whereas no discernible enhancement in flow behavior was observed for silicon oxide-coated fenofibrate. The results from contact angle measurements suggest that the silicon oxide-coated fenofibrate exhibits superior wetting behavior, as indicated by a contact angle nearing 0°. The application of ALC demonstrates an enhanced dissolution rate when compared to the uncoated active pharmaceutical ingredient (API) while leaving its equilibrium solubility unaffected. Coating the API with silicon oxide improves particle hydrophilicity and wetting properties, whereas zinc oxide coating aids in particle de-agglomeration, thereby enhancing their interaction with an aqueous medium. In vivo bioavailability studies conducted on rodents and larger animal (dog) models indicate a substantial increase in bioavailability (approximately 2 times) for the silicon oxide-coated API in comparison to the uncoated API, as determined by the area under the curve (AUC). Furthermore, the C_max values for the silicon oxide-coated API also demonstrate a significant increase (approximately 3 times) over the uncoated API. Notably, an oral subacute toxicity study of ALC silicon-coated fenofibrate revealed no toxic effects attributable to the coating. This study underscores the potential of ALC in augmenting the bioavailability of BCS(II) drugs.</p>","PeriodicalId":16741,"journal":{"name":"Journal of pharmaceutical sciences","volume":" ","pages":"617-625"},"PeriodicalIF":3.7,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142569070","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":"Impact of surfactants on solution behavior and membrane transport of amorphous solid dispersions.","authors":"Amjad Alhalaweh, Mira El Sayed, Lucia Kovac, Christel A S Bergström","doi":"10.1016/j.xphs.2024.10.023","DOIUrl":"10.1016/j.xphs.2024.10.023","url":null,"abstract":"<p><p>The purpose of the study was to develop an amorphous solid dispersion (ASD) of a poorly soluble compound (AK100) and investigate the impact of different surfactants on its dissolution, supersaturation and membrane transport. The solubility of the AK100 was determined in crystalline and amorphous form in the absence and presence of three surfactants at different concentrations: sodium dodecyl sulphate (SDS), polysorbate 80 (PS80) and D-α-tocopherol polyethylene glycol succinate (TPGS). The relation between solubility and surfactant solubilization was evaluated using a computational model. The ASD powder was prepared by solvent evaporation for non-sink dissolution experiments with and without the pre-dissolved surfactants. A transport study with Caco-2 cells was conducted to evaluate the impact of surfactants-based formulation on membrane transport. Both the corresponding crystalline and amorphous solubility of AK100 increased linearly as a function of the surfactant concentrations. The supersaturation was maintained for at least three hours in absence of surfactant and in presence of TPGS, whereas supersaturation declined with SDS and PS80. As expected, the membrane flux of the AK100 was higher for the ASD than for the crystalline powder, and further increased with increased concentration of TPGS. The supersaturation ratio based on the activity-based calculation from Caco-2 cells study was always higher than that of the concentration-based one for the amorphous and crystalline forms of AK100. This study shows how additional solubilizing excipients during formulation development can improve the resulting dissolution and phase behavior of supersaturated drug solution.</p>","PeriodicalId":16741,"journal":{"name":"Journal of pharmaceutical sciences","volume":" ","pages":"458-467"},"PeriodicalIF":3.7,"publicationDate":"2025-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142569089","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":"Review on macromolecule-based magnetic theranostic agents for biomedical applications: targeted therapy and diagnostic imaging.","authors":"Laila S Zeid, Heba A El-Masry, Hend H Mohamed, Amira Hathout, Ahmed S Younes, Aya A El-Kholy, Aya Ashraf Muhammad Abdel Hamid, Naira Ali Abd Elaziz, Fatma Shaban Hafez, May Emad Eldin Mostafa, Islam M M Omar, Tasneem Elsayed Ahmed, Mohamed S A Darwish","doi":"10.1016/j.xphs.2024.11.031","DOIUrl":"https://doi.org/10.1016/j.xphs.2024.11.031","url":null,"abstract":"<p><p>Clinical diagnostics and biological research are advanced by magnetic theranostic, which uses macromolecule-based magnetic theranostic agents for targeted therapy and diagnostic imaging. Within this review, the interaction of magnetic nanoparticles (MNPs) with biological macromolecules will be covered. The exciting potential of macromolecule-based magnetic theranostic agents to be used as a tool in drug delivery, photothermally therapy (PTT), gene therapy, hyperthermia therapy and photodynamic therapy (PDT) will be discussed. Innovative imaging technique: magnetic resonance imaging (MRI), magnetic particle imaging (MPI), fluorescence scanning, and photoacoustic scanning are revolutionizing biological diagnosis by potentially overcoming historical limitations. This review will cover the challenge of fabricating of macromolecule-based magnetic theranostic agents as a promising platform for theranostic that can combine therapies with diagnostics at subcellular level. Additionally, it looks at several chemical pathways leading to the process for generating MNPs, including the co-precipitation, the sol-gel, the hydrothermal synthesis, the polyol route, and the microemulsion technique. Eventually, the demands and prospects for magnetic theranostic are discussed, focusing on the requirement of further investigation to improve MNP structure towards biocompatible material and translation of these promising theranostic agents into clinical applications.</p>","PeriodicalId":16741,"journal":{"name":"Journal of pharmaceutical sciences","volume":" ","pages":""},"PeriodicalIF":3.7,"publicationDate":"2024-12-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142877501","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":"Enhancing physicochemical properties of Hydrochlorothiazide with zwitterionic L-proline and 5-Fluorocytosine cocrystals through mechanochemical synthesis.","authors":"Pollyana Pereira Firmino, Cecilia Carolina Pinheiro da Silva, Paulo Nunes, José Eduardo Gonçalves, Fabrizia Grepioni, Javier Ellena","doi":"10.1016/j.xphs.2024.12.004","DOIUrl":"https://doi.org/10.1016/j.xphs.2024.12.004","url":null,"abstract":"<p><p>Hydrochlorothiazide (HTZ) is a thiazide-type diuretic drug approved by the FDA in 1959 for treatment of hypertension and peripheral edema and has been used since. HTZ exhibits low solubility and low permeability, leading to variable oral bioavailability and limited intestinal drug permeability. For this reason, several attempts to improve HTZ physicochemical properties have been made during the past decades. In the broad frame of molecular crystal engineering, significant efforts and promising results in the quest for more effective solid/dosage forms of HTZ, including studies on polymorphism and cocrystals, are being developed. As part of these efforts, we report here two new cocrystals of HTZ with the zwitterionic L-proline and the prodrug 5-Fluorocytosine. Both cocrystals show improvement in solubility and permeability, suggesting that these new solid forms could be used as new drug candidates to deliver HTZ in the antihypertensive therapy.</p>","PeriodicalId":16741,"journal":{"name":"Journal of pharmaceutical sciences","volume":" ","pages":""},"PeriodicalIF":3.7,"publicationDate":"2024-12-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142877493","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}