Journal of pharmaceutical sciences最新文献_第5页

Development of lacosamide-loaded in-situ gels through experimental design for evaluation of ocular irritation in vitro and in vivo 通过实验设计开发lacosamide负载原位凝胶用于体外和体内眼部刺激评价。

IF 3.7 3区医学 Q2 CHEMISTRY, MEDICINAL

Journal of pharmaceutical sciences

Pub Date : 2025-02-01 DOI: 10.1016/j.xphs.2024.11.027

Özlem Çoban , Sıla Gülbağ Pınar , Heybet Kerem Polat , Gülşah Gedik , Nasıf Fatih Karakuyu , Esra Pezik , Sedat Ünal , Behzad Mokhtare , Aleyna Akşit

Lacosamide (LCM) selectively increases the slow inactivation of voltage-gated sodium channels (VGSCs) and is a N-methyl d-aspartate acid (NMDA) receptor glycine site antagonist. Therefore, it can be used in dryness-related hyperexcitability of corneal cold receptor nerve terminals. Ocular in-situ gels remain in liquid form until they reach the target site, where they undergo a sol-gel transformation in response to specific stimuli. They can show mucoadhesive properties related to the polymer used and increase the residence time of the drug in the mucosa. In the presented study, ocular in-situ gel formulation of LCM, which has potential for use in ocular diseases and consists of hyaluronic acid and poloxamer 407 as polymers, was developed using cold method. The effect of formulation components on target product properties (pH, gelation temperature and viscosity) was evaluated by design of experiments (DoE) design. The optimized LCM-loaded in-situ gel had a pH value of 6.90 ± 0.01, showed pseudo-plastic flow with a viscosity of 562 ± 58 cP at 25 °C, gelled at 33 ± 0.47 °C, and released drugs via the Peppas-Sahlin mechanism. Ocular safety was confirmed via in vitro tests using two different cell lines (L929 and Arpe-19), along with in vivo Draize tests, histological examinations, and Hen's Egg Chario-Allontioc-Membrane (HET-CAM) analysis. In vitro studies confirmed the optimized LCM-loaded in-situ gel's suitability for ocular use, demonstrating long-acting effects through controlled release. In addition, ocular irritation and histological studies have supported that it will not show any toxic effect on the eye tissue.

Lacosamide （LCM）选择性地增加电压门控钠通道（VGSCs）的缓慢失活，是一种n -甲基d -天冬氨酸（NMDA）受体甘氨酸位点拮抗剂。因此，它可用于干燥相关性角膜冷受体神经末梢的高兴奋性。眼内原位凝胶在到达目标部位之前保持液体形式，在那里它们经历了响应特定刺激的溶胶-凝胶转化。它们可以显示与所用聚合物相关的黏附特性，并增加药物在粘膜中的停留时间。本研究以透明质酸和poloxam407为聚合物，采用冷法制备了具有治疗眼病潜力的LCM眼原位凝胶配方。通过实验设计（DoE）评价了配方组分对目标产品性能（pH、胶凝温度和粘度）的影响。优化后的lcm负载原位凝胶的pH值为6.90±0.01，在25℃条件下表现为562±58 cP的伪塑性流动，在33±0.47℃条件下成胶，并通过Peppas-Sahlin机制释放药物。通过使用两种不同细胞系（L929和Arpe-19）进行的体外试验，以及体内Draize试验、组织学检查和鸡蛋膜（HET-CAM）分析，证实了眼部安全性。体外研究证实了优化后的lcm负载原位凝胶适合眼部使用，并通过控释显示出长效效果。此外，眼刺激和组织学研究支持，它不会显示出任何毒性作用，对眼组织。

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引用次数: 0

Biowaiver monographs for immediate-release solid oral dosage forms: Lemborexant 速释口服固体制剂的生物豁免专论：Lemborexant.

IF 3.7 3区医学 Q2 CHEMISTRY, MEDICINAL

Journal of pharmaceutical sciences

Pub Date : 2025-02-01 DOI: 10.1016/j.xphs.2024.10.030

Kristian Beran , Bertil Abrahamsson , Naseem Charoo , Rodrigo Cristofoletti , René Holm , Atsushi Kambayashi , Peter Langguth , Mehul Mehta , Alan Parr , James E. Polli , Vinod P. Shah , Jennifer Dressman

Lemborexant is a dual orexin receptor antagonist assigned to class II of the Biopharmaceutics Classification System (BCS). Thus, the ICH M9 Guideline excludes immediate-release (IR) solid oral dosage forms containing lemborexant from BCS-based biowaivers, irrespective of their in vitro dissolution behavior. By contrast, classification of lemborexant according to the refined Developability Classification System (rDCS) falls into class I, indicating few biopharmaceutics risks. Customized rDCS investigations identify dissolution as the main risk factor, in line with clinical data in humans which suggest that the absorption of lemborexant is limited neither by solubility nor by permeability. Instead, any risks lie in dissolution. Analysis by the rDCS coupled with biorelevant dissolution testing thus provides a way forward for manufacturers to mitigate the risks associated with changes in formulation or introduction of a generic version prior to running clinical bioequivalence (BE) studies. As a way forward regarding biowaivers for lemborexant and similar cases, where justifying BE based on the current BCS-based approach is not possible, a four-step pathway towards establishing BE virtually could be adopted as follows: (i) rDCS analysis to identify critical bioavailability attributes, (ii) comparative (biorelevant) dissolution testing, (iii) Physiologically Based Biopharmaceutics Modeling (PBBM), and (iv) virtual BE assessment.

伦博雷沙坦是一种双重奥曲肽受体拮抗剂，属于生物制药分类系统（BCS）的第二类。因此，无论其体外溶出行为如何，《ICH M9 指南》都将含有伦博雷生的速释（IR）口服固体制剂排除在基于 BCS 的生物豁免之外。与此相反，根据改进的可显影性分类系统（rDCS）对伦博雷康进行的分类属于 I 类，表明生物制药风险很小。定制的 rDCS 调查发现，溶解是主要的风险因素，这与人体临床数据一致，表明 Lemborexant 的吸收既不受溶解度的限制，也不受渗透性的限制。相反，任何风险都在于溶解。因此，在进行临床生物等效性（BE）研究之前，rDCS 分析与生物相关溶出度测试相结合，为生产商提供了一条降低制剂变化或引入仿制药相关风险的途径。在不可能根据目前基于生物相容性标准的方法证明生物等效性的情况下，可采用以下四步方法来确定生物等效性：(i) rDCS 分析以确定关键的生物利用度属性，(ii) 比较（生物相关）溶出试验，(iii) 基于生理学的生物药剂学建模 (PBBM)，(iv) 虚拟生物利用度评估。

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引用次数: 0

Evaluation of the correlation between nuclear localization levels and genome editing efficiencies of Cas12a fused with nuclear localization signals 评估与核定位信号融合的 Cas12a 的核定位水平与基因组编辑效率之间的相关性。

IF 3.7 3区医学 Q2 CHEMISTRY, MEDICINAL

Journal of pharmaceutical sciences

Pub Date : 2025-02-01 DOI: 10.1016/j.xphs.2024.10.029

Tomohito Tsukamoto , Haruna Mizuta , Eiko Sakai , Fuminori Sakurai , Hiroyuki Mizuguchi

Genome editing technology using the CRISPR-Cas system is attracting much attention not only as a promising experimental tool for analysis of genome functions, but also as a novel therapeutic approach for genetic disorders. Among the various types of Cas proteins, Cas12a is expected to be a promising gene editing tool due to its unique properties, including low off-target effects. As Cas proteins are of prokaryotic origin, they need to be fused with appropriate localization signals to perform their function in eukaryotic cells. Cas12a proteins fused with a nuclear localization signal (NLS) have been developed so far, but the relation between the nuclear localization activity and the genome editing efficiency has not been fully elucidated. Here, utilizing two Cas12a orthologs, AsCas12a and LbCas12a, with various number of NLSs derived from various origins, we revealed that the improved nuclear localization resulted in increased genome editing efficiencies when expressed using adenovirus (Ad) vector in cultured cells. However, when they were expressed in mouse liver, the improvement of the nuclear localization activity was not necessarily required to achieve the maximum genome editing efficiency four weeks after Ad vector administration. These data indicated that the optimized NLS modification of Cas12a proteins in vitro situations differed from that in vivo.

使用CRISPR-Cas系统的基因组编辑技术不仅是分析基因组功能的一种有前途的实验工具，而且也是治疗遗传疾病的一种新方法，因此备受关注。在各种类型的 Cas 蛋白中，Cas12a 因其独特的特性，包括低脱靶效应，有望成为一种前景广阔的基因编辑工具。由于 Cas 蛋白来源于原核细胞，因此需要融合适当的定位信号才能在真核细胞中发挥作用。目前已开发出融合了核定位信号（NLS）的 Cas12a 蛋白，但其核定位活性与基因组编辑效率之间的关系尚未完全阐明。在这里，我们利用两种Cas12a直向同源物AsCas12a和LbCas12a，以及来自不同来源的不同数量的NLS，发现当使用腺病毒（Ad）载体在培养细胞中表达时，改进的核定位导致基因组编辑效率的提高。然而，当它们在小鼠肝脏中表达时，核定位活性的改善并不一定要求在使用腺病毒载体四周后达到最高的基因组编辑效率。这些数据表明，Cas12a蛋白在体外的优化NLS修饰与体内不同。

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引用次数: 0

Comprehensive analysis of deflazacort oxidative degradation: Insights into novel degradation products and mechanisms 地氟沙星氧化降解综合分析：洞察新型降解产物和机制。

IF 3.7 3区医学 Q2 CHEMISTRY, MEDICINAL

Journal of pharmaceutical sciences

Pub Date : 2025-02-01 DOI: 10.1016/j.xphs.2024.10.048

Airton G. Salles Jr. , Manoel T. Rodrigues Jr. , Bruno B. Guidotti , Paulo C.P. Rosa

The oxidative degradation pathways of deflazacort (DFL) were investigated to address the gap in understanding its degradation products, focusing on reactions with oxidative stressors such as hydrogen peroxide and 4,4′-azobis (4-cyanovaleric acid) (ACVA). Using HPLC-PDA, high-resolution mass spectrometry (HRMS), NMR and IR spectroscopy, four novel degradation products were identified and structurally characterized. Two of these products were isolated using preparative HPLC before characterization. Hydrogen peroxide led to the formation of three novel products (DP-1, DP-2, and DP-3), while ACVA resulted in a single novel product (DP-4). Mechanistic and kinetic experiments supported the proposed degradation pathways under the various oxidative stress conditions studied, revealing distinct rates of formation for the degradation products during the time-course study. The identification and detailed structural elucidation of these degradation products provide critical insights into the chemical stability and potential reactivity of DFL under oxidative stress. These findings underscore the importance of comprehensive stability testing for ensuring drug safety and efficacy, and offer valuable data for future research on the toxicity and pharmacological impact of DFL degradation products.

为了填补对其降解产物了解的空白，研究人员研究了去氯羟酸（DFL）的氧化降解途径，重点是与过氧化氢和 4,4'-偶氮双（4-氰基戊酸）（ACVA）等氧化应激源的反应。利用 HPLC-PDA、高分辨率质谱（HRMS）、核磁共振（NMR）和红外光谱，确定了四种新型降解产物，并对其进行了结构鉴定。其中两种产物在表征前使用制备型高效液相色谱进行了分离。过氧化氢导致了三种新型产物（DP-1、DP-2 和 DP-3）的生成，而 ACVA 则导致了一种新型产物（DP-4）的生成。机理和动力学实验支持了在各种氧化应激条件下的降解途径，揭示了降解产物在时间历程研究中的不同形成速率。对这些降解产物的鉴定和详细的结构阐释为深入了解 DFL 在氧化应激下的化学稳定性和潜在反应性提供了重要依据。这些发现强调了全面的稳定性测试对确保药物安全性和有效性的重要性，并为今后研究 DFL 降解产物的毒性和药理作用提供了宝贵的数据。

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引用次数: 0

Combating pharmaceutical folklore: No alkyl-sulfonate impurities formed during the synthesis of sulfonate salts 打击制药业的民间传说：磺酸盐合成过程中不会产生烷基磺酸盐杂质。

IF 3.7 3区医学 Q2 CHEMISTRY, MEDICINAL

Journal of pharmaceutical sciences

Pub Date : 2025-02-01 DOI: 10.1016/j.xphs.2024.11.002

David J. Snodin

Whilst an alcohol can be forced to react with a sulfonic acid, this reaction produces minimal ester conversion even under extreme conditions (anhydrous, very low pH) that bear no resemblance to the mild synthetic procedures typically used for the formation of sulfonate salts of basic drugs. The latter involve the addition of a molar equivalent of pharma-grade sulfonic acid to the base form of a drug substance (pKa ≥3.5), dissolved or suspended in an alcohol solvent, normally ethanol (pKa -2). All added acid is neutralized, and so there is no potential for ester formation. Many drug-substance base forms are polyamines, thus preventing the generation of acidic reaction conditions even in the presence of excess of sulfonic acid. Despite the experimental evidence, the perception that short-chain mutagenic alkyl sulfonates are “potential impurities” in sulfonate salts is widely held within regulatory bodies. This stance implies that a mechanistically-impossible reaction can occur: nucleophilic displacement by sulfonate anion of the hydroxyl group from a short-chain alcohol under non-acidic conditions. The European Pharmacopoeia (Ph.Eur.) and the British Pharmacopoeia (BP) include “production statements” in monographs for sulfonate-salt drug substances requiring a “risk assessment” of the production process. Neither body has provided supporting evidence. Information obtained from the BP via Freedom of Information requests showed that expert-group discussions were characterised by a range of ad-hoc opinions rather than an evidence-based evaluation of mechanism, kinetics and experimental data. Alternative sources of alkyl-sulfonate impurities such as methyl methanesulfonate (MMS) arising from the use of impure, reagent-grade methanesulfonic acid (MSA) were not considered. Both BP and Ph.Eur. production statements appear to be based on policy rather than scientific evidence and so should be discontinued.

虽然可以强迫酒精与磺酸发生反应，但即使在极端条件下（无水、pH 值极低），这种反应也只能产生极少的酯转化，与通常用于形成碱性药物磺酸盐的温和合成程序毫无相似之处。后者是在溶解或悬浮于醇溶剂（通常为乙醇，pKa -2）中的药物物质（pKa ≥3.5）的基础形式中加入摩尔当量的医药级磺酸。所有添加的酸都会被中和，因此没有形成酯的可能性。许多药物物质的碱基形式是多胺，因此即使在磺酸过量的情况下也不会产生酸性反应条件。尽管有实验证据，但监管机构普遍认为短链致突变烷基磺酸盐是磺酸盐中的 "潜在杂质"。这种立场意味着可能会发生一种机械上不可能发生的反应：在非酸性条件下，磺酸盐阴离子对短链醇羟基的亲核置换。欧洲药典》（Ph.Eur.）和《英国药典》（BP）在磺酸盐盐类药物的各论中列入了 "生产说明"，要求对生产过程进行 "风险评估"。这两个机构都没有提供支持证据。通过 "信息自由 "申请从英国药典获得的信息显示，专家组讨论的特点是提出一系列临时意见，而不是对机理、动力学和实验数据进行循证评估。没有考虑烷基磺酸盐杂质的其他来源，如使用不纯的试剂级甲磺酸 (MSA) 而产生的甲磺酸甲酯 (MMS)。英国石油公司和欧洲博士公司的生产声明似乎都是基于政策而非科学证据，因此应予停止。

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引用次数: 0

Top reviewers for 2024

IF 3.7 3区医学 Q2 CHEMISTRY, MEDICINAL

Journal of pharmaceutical sciences

Pub Date : 2025-02-01 DOI: 10.1016/j.xphs.2024.11.011

Kenneth L. Audus

引用次数: 0

Limitation of anion exchange chromatography and potential application of hydrophobic interaction chromatography for monitoring AAV9 capsid degradation upon thermal stress 阴离子交换色谱法的局限性和疏水相互作用色谱法在监测热应力下 AAV9 荚膜降解过程中的潜在应用

IF 3.7 3区医学 Q2 CHEMISTRY, MEDICINAL

Journal of pharmaceutical sciences

Pub Date : 2025-02-01 DOI: 10.1016/j.xphs.2024.11.005

Antonela Rodriguez , Ali Banazadeh , Amr Ali , Rajeeva Singh , Chen Zhou

Adeno-Associated Virus (AAV) is often selected as the vector of choice for gene therapy due to its superior clinical performance compared to other gene delivery systems. Currently the characterization of AAV degradation, especially the chemical degradation of capsid, has been limited due to lack of suitable methods. Our study using AAV9 as a model molecule shows that anion exchange chromatography (AEX) as a charge-based separation method has limitations in monitoring the chemical degradation of AAV9 capsid due to a confounding effect from DNA cargo ejection. We developed a hydrophobic interaction chromatography (HIC) method, free from DNA interference, that could serve as a quick and reliable alternative to resource-demanding peptide mapping method for monitoring AAV capsid chemical degradation. Compared with brief thermal stress at 75 °C, AAV9 capsid exhibited much higher levels of chemical degradation but slower capsid titer loss upon extended exposure for 4 weeks at 40 °C.

腺相关病毒（AAV）因其优于其他基因递送系统的临床表现，通常被选为基因治疗的首选载体。目前，由于缺乏合适的方法，AAV 的降解特性，尤其是囊膜的化学降解，一直受到限制。我们以 AAV9 为模型分子进行的研究表明，阴离子交换色谱法（AEX）作为一种基于电荷的分离方法，在监测 AAV9 荚膜的化学降解方面存在局限性，因为 DNA 货物喷射会产生混杂效应。我们开发了一种疏水相互作用色谱（HIC）方法，该方法不受DNA干扰，可快速可靠地替代对资源要求较高的肽图法来监测AAV囊壳的化学降解。与75 °C的短暂热应激相比，AAV9噬菌体在40 °C下暴露4周后表现出更高水平的化学降解，但噬菌体滴度的损失更慢。

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引用次数: 0

Recent advances in drying and development of solid formulations for stable mRNA and siRNA lipid nanoparticles 稳定的mRNA和siRNA脂质纳米颗粒固体配方的干燥和开发的最新进展。

IF 3.7 3区医学 Q2 CHEMISTRY, MEDICINAL

Journal of pharmaceutical sciences

Pub Date : 2025-02-01 DOI: 10.1016/j.xphs.2024.12.013

Kinnari Santosh Arte, Manlin Chen, Chanakya D. Patil, Yijing Huang, Li Qu Lily, Qi Zhou Tony

Current RNA lipid nanoparticle (LNP) based products are typically liquid formulations that require ultra-cold storage temperatures for stability. To address this limitation, recent efforts have focused on enhancing stability and enabling room temperature storage by converting these formulations into solid forms through drying processes such as lyophilization, spray drying, and spray-freeze drying. Nevertheless, the drying process itself can influence the stability of RNA/LNP formulations. Therefore, understanding the factors that contribute to instability during drying is essential. The choice of drying technique for LNPs depends on factors such as the mode of delivery, lipid components, and desired final product characteristics. Additionally, the drying mechanism and associated stresses must also be carefully considered. Drying methods involve a range of process parameters related to formulation, process settings, and the manufacturing environment. It is essential to understand how these parameters influence the final solid-state products’ attributes, including appearance, moisture content, flow properties, and reconstitution time, as these can significantly affect the physical and chemical stability of the formulation. This review focuses on various drying techniques and their impact on the stability of RNA/LNP-based systems.

目前基于RNA脂质纳米颗粒（LNP）的产品通常是液体配方，需要超低温储存才能保持稳定性。为了解决这一限制，最近的努力集中在通过干燥过程（如冻干、喷雾干燥和喷雾冷冻干燥）将这些配方转化为固体形式来提高稳定性和实现室温储存。然而，干燥过程本身会影响RNA/LNP配方的稳定性。因此，了解干燥过程中导致不稳定的因素是至关重要的。LNPs干燥技术的选择取决于输送方式、脂质成分和期望的最终产品特性等因素。此外，干燥机制和相关应力也必须仔细考虑。干燥方法涉及一系列与配方、工艺设置和制造环境相关的工艺参数。了解这些参数如何影响最终固态产品的属性是至关重要的，包括外观、水分含量、流动特性和重构时间，因为这些参数会显著影响配方的物理和化学稳定性。本文综述了各种干燥技术及其对RNA/ lnp基系统稳定性的影响。

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引用次数: 0

Pharmacokinetics and safety of pirfenidone in individuals with chronic kidney disease stage G2 and G3a: A single-dose, Phase I, bridging study 吡非尼酮在慢性肾脏疾病（CKD） G2期和G3a期患者中的药代动力学和安全性：单剂量I期桥接研究

IF 3.7 3区医学 Q2 CHEMISTRY, MEDICINAL

Journal of pharmaceutical sciences

Pub Date : 2025-02-01 DOI: 10.1016/j.xphs.2024.11.020

Dianwen Yu , Rui Zhang , Jinping Zhou, Pengpeng Guo, Peixia Li, Menghan Ye, Yani Liu, Shaojun Shi

Background and Objective

Pirfenidone is an inhibitor of transforming growth factor-beta 1 (TGF-β1) and is being developed for the treatment of diabetic kidney disease (DKD). We assessed the pharmacokinetics (PK) and safety of a single dose of pirfenidone in individuals with CKD stages G2/G3a.

Methods

In this phase I bridging study, patients with CKD stages G2 or G3a, aged 18–70 years, with a body mass index of 18–26 kg/m², and glomerular filtration rate (eGFR) ranging from 45 to 89 ml/min/1.73 m², received a single oral dose of 400 mg pirfenidone capsules 30 min after a standard breakfast. The pharmacokinetic parameters of the two groups were measured and compared after blood and urine collection. The co-primary endpoints were the area under the plasma concentration-time curve from time zero to 36 h (AUC₀–₃₆) and the maximum observed plasma concentration (C_max) of pirfenidone. Safety was a secondary endpoint. The trial has been registered on ClinicalTrials.gov (ChiCTR2300077297).

Results

A total of 20 subjects participated in this study. There were no significant differences between the control group and the patient group (CKD stages G2/G3a) in terms of plasma C_max, the time to reach the maximum observed concentration (T_max), and elimination half-life(t_1/2). However, the Vz/F of the patient group (CKD G2 stage) was significantly higher than that of the control group. Renal accumulation rate, renal clearance rate (CLr), and urine drug concentration also showed no significant differences. No severe adverse events occurred during the trial.

Conclusions

These results indicate that the PK and safety of pirfenidone are not influenced by renal function. Individuals with renal impairment may not require dose adjustments.

背景与目的：吡非尼酮是一种转化生长因子-β1 （TGF-β1）抑制剂，目前正被开发用于治疗糖尿病肾病（DKD）。我们评估了单剂量吡非尼酮在G2/G3a期CKD患者中的药代动力学（PK）和安全性。方法：在这项I期桥接研究中，年龄为18-70岁、体重指数为18-26 kg/m2、肾小球滤过率（eGFR）为45 - 89 ml/min/1.73m2的G2或G3a期CKD患者在标准早餐后30分钟口服400 mg吡非尼酮胶囊。采集血、尿后测定两组药代动力学参数并进行比较。共同主要终点为0-36小时血浆浓度-时间曲线下面积（AUC0-36）和观察到的吡非尼酮最大血浆浓度（Cmax）。安全性是次要终点。该试验已在ClinicalTrials.gov注册（ChiCTR2300077297）。结果：共有20名受试者参与本研究。对照组与患者组（CKD分期G2/G3a）在血浆Cmax、达到最大观察浓度时间（Tmax）和消除半衰期（t1/2）方面无显著差异。但患者组（CKD G2期）Vz/F明显高于对照组。肾积存率、肾清除率（CLr）、尿药浓度差异无统计学意义。试验期间未发生严重不良事件。结论：吡非尼酮的PK和安全性不受肾功能的影响。有肾脏损害的人可能不需要调整剂量。

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引用次数: 0

Formulation development and feasibility of AAV5 as a lyophilized drug product AAV5冻干制剂的配方开发及可行性研究。

IF 3.7 3区医学 Q2 CHEMISTRY, MEDICINAL

Journal of pharmaceutical sciences

Pub Date : 2025-02-01 DOI: 10.1016/j.xphs.2025.01.004

Stephanie K. Vargas , Farrokh Sharifi , Reinard Nambayan , Saeed Moshashaee , Teruna J. Siahaan

The majority of adeno-associated virus (AAV) gene therapies are currently developed as frozen formulations (e.g., ≤ - 60 °C) that are challenging to maintain and distribute world-wide. Lyophilization can allow for long-term refrigerated storage and improved shelf-life that lowers long-term cost. Here, we performed a lyophilization feasibility study to assess the ability of several different excipients to stabilize AAV5 during lyophilization and on storage stability. A range of biophysical techniques were used to assess capsid integrity on a molecular level including quantification of externalized DNA, capsid particle size, and capsid monomer percent area. Additionally, transmission electron microscopy was used for the first time to monitor the size and integrity of the capsids subjected to the lyophilization process, and the results supported other characterization methods used in this study. A formulation containing hydroxyectoine and trehalose stabilized capsid structure directly after lyophilization, as observed directly by 5.0 % of internally stained capsids (empty) and indirectly with 7.5 % external DNA. A recombinant human albumin and trehalose formulation stabilized capsid structure on stability as observed by improved external DNA and monomer profiles overtime. Adversely, mannitol crystallization negatively affected capsid structure. Our findings indicate that lyophilization is a viable option to frozen formulation for stabilizing AAV5 drug products.

目前，大多数腺相关病毒（AAV）基因疗法都是冷冻制剂（例如≤- 60°C），难以维持和在全球范围内分发。冻干可以允许长期冷藏储存，提高货架寿命，降低长期成本。在这里，我们进行了一项冻干可行性研究，以评估几种不同赋形剂在冻干过程中稳定AAV5的能力和储存稳定性。一系列生物物理技术被用于在分子水平上评估衣壳的完整性，包括外化DNA的量化、衣壳粒度和衣壳单体面积百分比。此外，透射电镜首次用于监测冻干过程中衣壳的大小和完整性，结果支持本研究中使用的其他表征方法。含有羟基外托因和海藻糖的配方在冻干后直接稳定衣壳结构，通过5.0%的内部染色衣壳（空）直接观察，通过7.5%的外部DNA间接观察。重组人白蛋白和海藻糖配方稳定了衣壳结构的稳定性，这是通过改善外部DNA和单体谱观察到的。相反，甘露醇结晶对衣壳结构有负面影响。我们的研究结果表明，冻干是稳定AAV5药物产品的冷冻制剂的可行选择。

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引用次数: 0