Case report: Intraretinal hyperflow microinfiltration lesions on swept-source optical coherence tomography angiography as a potential biomarker of primary vitreoretinal lymphoma.

Primary vitreoretinal lymphoma (PVRL) is often associated with central nervous system involvement, contributing to a heightened mortality rate, thus imaging features that are characteristic enough to be potential biomarkers of PVRL are important, either in diagnosis or in assessment of disease activity. This report details the case of a 68-year-old male who presented with blurred vision in both eyes persisting for 2 months. Fundus examination demonstrated vitreous opacity and multiple subretinal yellow nodular lesions of varying sizes in the peripheral fundus of both eyes. Multiple vertical hyperreflective lesions in the neural retina of posterior pole, indistinct outer retina borders in the fovea, and hyperreflective lesions in the sub-retinal pigment epithelium (RPE) space of the peripheral retina were demonstrated on swept-source optical coherence tomography (SS-OCT) of the left eye. Hyperflow signals corresponding to the vertical hyperreflective lesions were detected on swept-source optical coherence tomography angiography (SS-OCTA) images of retinal deep capillary plexus (DCP) layer. Notably, the hyperflow signals, precisely located around retinal vessels from the nerve fiber layer to the outer plexiform layer, were postulated to stem from the dilation of infiltrated retinal vessels. Vitreous pathological results of the left eye confirmed the diagnosis of PVRL. Treatments with intravitreal methotrexate injections led to a marked improvement of best-corrected visual acuity (BCVA) and regression of the hyperflow microinfiltration lesions demonstrated on SS-OCTA. In conclusion, SS-OCTA effectively delineated the vertical hyperreflective lesions and corresponding hyperflow signals in the posterior pole macular region of a patient with PVRL. These lesions significantly diminished following intravitreal methotrexate injections. We speculated that the specific hyperflow signals on SS-OCTA could act as a potential biomarker of PVRL, and SS-OCTA holds promise in facilitating early diagnosis and monitoring therapeutic responses in PVRL cases.

Predicting the Stability of Formulations Containing Lyophilized Human Serum Albumin and Sucrose/Trehalose Using Solid-State NMR Spectroscopy.

Stabilization of proteins by disaccharides in lyophilized formulations depends on the interactions between the protein and the disaccharide (system homogeneity) and the sufficiently low mobility of the system. Human serum albumin (HSA) was lyophilized with disaccharides (sucrose and/or trehalose) in different relative concentrations. Solid-state nuclear magnetic resonance (ssNMR) spectroscopy 1H T1 and 1H T1ρ relaxation times were measured to determine the homogeneity of the lyophilized systems on 20-50 and 1-3 nm domains, respectively, with 1H T1 relaxation times also being used to determine the ß-relaxation rate. HSA/sucrose systems had longer 1H T1 relaxation times and were slightly more stable than HSA/trehalose systems in almost all cases shown. HSA/sucrose/trehalose systems have 1H T1 relaxation times between the HSA/sucrose and HSA/trehalose systems and did not result in a more stable system compared with binary systems. Inhomogeneity was evident in a sample containing relative concentrations of 10% HSA and 90% trehalose, suggesting trehalose crystallization during lyophilization. Under these stability conditions and with these ssNMR acquisition parameters, a 1H T1 relaxation time below 1.5 s correlated with an unstable sample, regardless of the disaccharide(s) used.

Vinyl and methyl-ester groups in the insoluble polymer drug patiromer identified and quantified by solid-state NMR.

Patiromer (Veltassa®) is a crosslinked, insoluble co-polymer drug used as a nonabsorbent potassium binder, approved for treatment of hyperkalemia. Quantitative solid-state 13C nuclear magnetic resonance (NMR) analysis with comprehensive peak assignment, component quantification, and calculation of mole and weight fractions of monomer units was performed on three doses of patiromer. The workflow is documented in detail. Spectrally edited solid-state 13C NMR spectra of patiromer show =CHn peaks of matching intensity at 116 and 141 ppm, characteristic of -CH=CH2 vinyl groups. Similar spectral features can be observed in earlier studies but were previously ignored. In this study, the vinyl signals are well-resolved in a 2-s direct polarization (DP) spectrum without and with dipolar dephasing, which confirms that these sp2-hybridized carbons are bonded to hydrogen and partially mobile, consistent with vinyl side groups from incompletely reacted divinyl crosslinkers. The vinyl groups account for 1.6% of all carbon, 3% of the monomer units, and nearly 1/3 of the crosslinkers. Furthermore, an unexpected OCH3 moiety accounting for ∼1.2% of all carbons was identified by spectral editing; its chemical shift of 54 ppm is more consistent with a methyl ester than with a methyl ether. It can originate from incomplete hydrolysis of ∼6% of methyl-2-fluoroacrylate, the main monomer of patiromer. Characteristic cross peaks in two-dimensional 1H-13C heteronuclear correlation NMR confirm the presence of the vinyl and OCH3 groups. Trace amounts of xanthan gum are also detected. The quantitative 13C NMR spectrum of patiromer has been matched in a simulation using a model with five monomer units.

Probing Chemical Equilibrium in Frozen Sodium Phosphate Buffer Solution by 31P Solid-State NMR.

Phosphate buffers are crucial for cryopreservative stability in pharmaceuticals, food processing, biomedical sciences, and biology. However, their freeze concentrates lack quantitative characterization, especially regarding the physicochemical properties of phosphate salt species in equilibrium at subzero temperatures. This study employs 31P solid-state NMR (ssNMR) to analyze frozen sodium phosphate (NaP) solutions, providing insights into phase composition, ionic strength, and pH. For the first time, we have directly quantified phosphate species in frozen NaP buffer, including crystallized disodium phosphate dodecahydrate (Na2HPO4·12H2O) content and the concentrations of H2PO4- and HPO42- in the freeze concentrate. This enabled the calculation of the pH as well as the ionic strength in the freeze concentrate. Trehalose effectively mitigated pH shifts in buffer solutions by preventing the selective crystallization of salt, a spectroscopic phenomenon not previously observed experimentally.

Quantification of Residual Water in Pharmaceutical Frozen Solutions Via 1H Solid-State NMR.

Freezing is essential for the stability of biological drug substances and products, particularly in frozen solution formulations and during the primary drying of lyophilized preparations. However, the unfrozen segment within the frozen matrix can alter solute concentration, ionic strength, and stabilizer crystallization, posing risks of increased biophysical instability and faster chemical degradation. While quantifying the unfrozen water content is important for designing stable biopharmaceuticals, there is a lack of analytical techniques for in situ quantitative measurements. In this study, we introduce a 1H magic angle spinning NMR technique to identify the freezing point (Tice) and quantify mobile water content in frozen biologics, applying this method to analyze the freezing of a commercial high-concentration drug product, Dupixent®. Our results demonstrate that water freezing is influenced by buffer salt properties and formulation composition, including the presence of sugar cryoprotectants and protein concentration. Additionally, the 1H chemical shift can probe pH in the unfrozen phase, potentially predicting the microenvironmental acidity in the frozen state. Our proposed methodology provides fresh insights into the analysis of freeze-concentrated solutions, enhancing our understanding of the stability of frozen and lyophilized biopharmaceuticals.

Insights into Factors Affecting Ethylene-Vinyl Acetate Copolymer Crystallinity in Islatravir Implant.

Islatravir, a highly potent nucleoside reverse transcriptase translocation inhibitor (NRTTI) for the treatment of HIV, has great potential to be formulated as ethylene-vinyl acetate (EVA) copolymer-based implants via hot melt extrusion. The crystallinity of EVA determines its physical and rheological properties and may impact the drug-eluting implant performance. Herein, we describe the systematic analysis of factors affecting the EVA crystallinity in islatravir implants. Differential scanning calorimetry (DSC) on EVA and solid-state NMR revealed drug loading promoted EVA crystallization, whereas BaSO4 loading had negligible impact on EVA crystallinity. The sterilization through γ-irradiation appeared to significantly impact the EVA crystallinity and surface characteristics of the implants. Furthermore, DSC analysis of thin implant slices prepared with an ultramicrotome indicated that the surface layer of the implant was more crystalline than the core. These findings provide critical insights into factors affecting the crystallinity, mechanical properties, and physicochemical properties of the EVA polymer matrix of extruded islatravir implants.

High species diversity of Phintella and Phintella-like spiders (Araneae: Salticidae) in Vietnam revealed by DNA-based species delimitation analyses.

Salticidae (jumping spiders) usually exhibit pronounced sexual dimorphism in adult morphology, particularly body coloration and size and shape of the first legs. Consequently, the male and female from the same species might be erroneously assigned to different species or even different genera, which could generate synonymies in classification if only morphological data were used. Phintella is a species-rich genus of Salticidae, which currently exhibits 76 named species. However, the male-female counterpart is unknown for nearly half of the species. In this study, we used a molecular approach to delineate the species boundaries for Phintella and Phintella-like specimens collected in Vietnam, using morphological information as supporting data. We used three gene fragments (mitochondrial COI, 16S-ND1, and nuclear 28S) and biogeographical considerations for species delimitation. A total of 22 putative species were recognized: 18 species of the genus Phintella, one species of the genus Lechia (L. squamata), and three species of the genus Phinteloides. Eleven undescribed species were discovered, of which seven have a male-female combination, two species have only males, and two species have only females. The crown age of Phintella was estimated at the Serravallian stage of the Miocene after the increase of species number around 16 MYA. The crown ages of most putative species recognized in this study were estimated in the Pleistocene, and the divergence among sister species likely occurred from the mid-Miocene to the Pliocene. Our ancestral range reconstruction results showed that the diversification of our ingroup was governed by progressive dispersal events, i.e., Phintella and their related species in Vietnam diversified while expanding their range on the continent. Our results provide fundamental biodiversity data for a high-diversity genus in Vietnamese Phintella spiders.

Unraveling Pre-filled Syringe Needle Clogging: Exploring a Fresh Outlook Through Innovative Techniques.

OBJECTIVE: This study aimed to investigate the movement of liquid in the needle region of staked-in-needle pre-filled syringes using neutron imaging and synchrotron X-ray tomography. The objective was to gain insights into the dynamics of liquid presence and understand the factors contributing to needle clogging. METHODS: Staked-in-needle pre-filled syringes were examined using neutron radiography and synchrotron X-ray phase-contrast computed tomography. Neutron radiography provided a 2D visualization of liquid presence in the needle, while synchrotron X-ray tomography offered high-resolution 3D imaging to study detailed morphological features of the liquid. RESULTS: Neutron radiography revealed liquid presence in the needle region for as-received samples and after temperature and pressure cycling. Pressure cycling had a more pronounced effect on liquid formation. Synchrotron X-ray tomography confirmed the presence of liquid and revealed various morphologies, including droplets of different sizes, liquid segments blocking sections of the needle, and a thin layer covering the needle wall. Liquid presence was also observed between the steel needle and the glass barrel. CONCLUSIONS: The combination of neutron imaging and synchrotron X-ray tomography provided valuable insights into the dynamics of liquid movement in staked-in-needle pre-filled syringes. Temperature and pressure cycling were found to contribute to additional liquid formation, with pressure changes playing a significant role. The detailed morphological analysis enhanced the understanding of microstructural arrangements within the needle. This research contributes to addressing the issue of needle clogging and can guide the development of strategies to improve pre-filled syringe performance.

Probing Molecular Packing of Lipid Nanoparticles from 31P Solution and Solid-State NMR.

Lipid nanoparticles (LNPs) are intricate multicomponent systems widely recognized for their efficient delivery of oligonucleotide cargo to host cells. Gaining insights into the molecular properties of LNPs is crucial for their effective design and characterization. However, analysis of their internal structure at the molecular level presents a significant challenge. This study introduces 31P nuclear magnetic resonance (NMR) methods to acquire structural and dynamic information about the phospholipid envelope of LNPs. Specifically, we demonstrate that the 31P chemical shift anisotropy (CSA) parameters serve as a sensitive indicator of the molecular assembly of distearoylphosphatidylcholine (DSPC) lipids within the particles. An analytical protocol for measuring 31P CSA is developed, which can be implemented using either solution NMR or solid-state NMR, offering wide accessibility and adaptability. The capability of this method is demonstrated using both model DSPC liposomes and real-world pharmaceutical LNP formulations. Furthermore, our method can be employed to investigate the impact of formulation processes and composition on the assembly of specifically LNP particles or, more generally, phospholipid-based delivery systems. This makes it an indispensable tool for evaluating critical pharmaceutical properties such as structural homogeneity, batch-to-batch reproducibility, and the stability of the particles.

In-situ biophysical characterization of high-concentration protein formulations using wNMR.

High-concentration protein formulation is of paramount importance in patient-centric drug product development, but it also presents challenges due to the potential for enhanced aggregation and increased viscosity. The analysis of critical quality attributes often necessitates the transfer of samples from their primary containers together with sample dilution. Therefore, there is a demand for noninvasive, in situ biophysical methods to assess protein drug products directly in primary sterile containers, such as prefilled syringes, without dilution. In this study, we introduce a novel application of water proton nuclear magnetic resonance (wNMR) to evaluate the aggregation propensity of a high-concentration drug product, Dupixent® (dupilumab), under stress conditions. wNMR results demonstrate a concentration-dependent, reversible association of dupilumab in the commercial formulation, as well as irreversible aggregation when exposed to accelerated thermal stress, but gradually reversible aggregation when exposed to freeze and thaw cycles. Importantly, these results show a strong correlation with data obtained from established biophysical analytical tools widely used in the pharmaceutical industry. The application of wNMR represents a promising approach for in situ noninvasive analysis of high-concentration protein formulations directly in their primary containers, providing valuable insights for drug development and quality assessment.

Deciphering molecular basis of pesticide-induced recurrent pregnancy loss: insights from transcriptomics analysis.

Recent studies have revealed a notable connection between pesticide exposure and Recurrent Pregnancy Loss (RPL), yet the precise molecular underpinning of this toxicity remains elusive. Through the alignment of Differentially Expressed Genes (DEGs) of healthy and RPL patients with the target genes of 9 pesticide components, we identified a set of 12 genes responsible for RPL etiology. Interestingly, biological process showed that besides RPL, those 12 genes also associated with preeclampsia and cardiovascular disease. Enrichment analysis showed the engagement of these genes associated with essential roles in the molecular transport of small molecules, as well as the aldosterone-regulated sodium reabsorption, endocrine and other factor-regulated calcium reabsorption, mineral absorption, ion homeostasis, and ion transport by P-type ATPases. Notably, the crosstalk targets between pesticide components played crucial roles in influencing RPL results, suggesting a role in attenuating pesticide agents that contribute to RPL. It is important to note that non-significant concentration of the pesticide components observed in both control and RPL samples should not prematurely undermine the potential for pesticides to induce RPL in humans. This study emphasizes the complexity of pesticide induced RPL and highlights avenues for further research and precautionary measures.

Design of a Reciprocal Injection Device for Stability Studies of Parenteral Biological Drug Products.

Formulation screening, essential for assessing the impact of physical, chemical, and mechanical stresses on protein stability, plays a critical role in biologics drug product development. This research introduces a Reciprocal Injection Device (RID) designed to accelerate formulation screening by probing protein stability under intensified stress conditions within prefilled syringes. This versatile device is designed to accommodate a broad spectrum of injection parameters and diverse syringe dimensions. A commercial drug product was employed as a model monoclonal antibody formulation. Our findings effectively highlight the efficacy of the RID in assessing concentration-dependent protein stability. This device exhibits significant potential to amplify the influences of interfacial interactions, such as those with buffer salts, excipients, air, metals, and silicone oils, commonly found in combination drug products, and to evaluate the protein stability under varied stresses.

Solid-State NMR Spectroscopy to Probe State and Phase Transitions in Frozen Solutions.

Freezing is commonly encountered during the processing and storage of biomacromolecule products. Therefore, understanding the phase and state transitions in pharmaceutical frozen solutions is crucial for the rational development of biopharmaceuticals. Solid-state nuclear magnetic resonance spectroscopy (ssNMR) was used to analyze solutions containing sodium phosphate buffer, histidine, and trehalose. Upon freezing, crystallization of disodium phosphate hydrogen dodecahydrate (Na2HPO4·12H2O, DPDH) and histidine was identified using 31P and 13C ssNMR, respectively, and confirmed by synchrotron X-ray diffractometry (SXRD). Using histidine as a molecular probe and based on the chemical shifts of atoms of interest, the pH of the freeze concentrate was measured. The unfrozen water content in freeze concentrates was quantified by 1H single pulse experiments. 13C-insensitive nuclei enhancement by polarization transfer (INEPT) and cross-polarization (CP) experiments were used as orthogonal tools to characterize the solutes in a "mobile" and a more "solid-like" state in the freeze-concentrated solutions, respectively. The above analyses were applied to a commercial monoclonal antibody (mAb) formulation of dupilumab. This work further establishes ssNMR spectroscopy as a highly capable biophysical tool to investigate the attributes of biopharmaceuticals and thereby provide insights into process optimization and formulation development.

Probing Molecular Packing of Amorphous Pharmaceutical Solids Using X-ray Atomic Pair Distribution Function and Solid-State NMR.

The structural investigation of amorphous pharmaceuticals is of paramount importance in comprehending their physicochemical stability. However, it has remained a relatively underexplored realm primarily due to the limited availability of high-resolution analytical tools. In this study, we utilized the combined power of X-ray pair distribution functions (PDFs) and solid-state nuclear magnetic resonance (ssNMR) techniques to probe the molecular packing of amorphous posaconazole and its amorphous solid dispersion at the molecular level. Leveraging synchrotron X-ray PDF data and employing the empirical potential structure refinement (EPSR) methodology, we unraveled the existence of a rigid conformation and discerned short-range intermolecular C-F contacts within amorphous posaconazole. Encouragingly, our ssNMR 19F-13C distance measurements offered corroborative evidence supporting these findings. Furthermore, employing principal component analysis on the X-ray PDF and ssNMR data sets enabled us to gain invaluable insights into the chemical nature of the intermolecular interactions governing the drug-polymer interplay. These outcomes not only furnish crucial structural insights facilitating the comprehension of the underlying mechanisms governing the physicochemical stability but also underscore the efficacy of synergistically harnessing X-ray PDF and ssNMR techniques, complemented by robust modeling strategies, to achieve a high-resolution exploration of amorphous structures.

Corrected solid-state 13 C nuclear magnetic resonance peak assignment and side-group quantification of hydroxypropyl methylcellulose acetyl succinate pharmaceutical excipients.

Hydroxypropyl methylcellulose acetyl succinate (HPMCAS) is widely used as a pharmaceutical excipient, making a detailed understanding of its tunable structure important for formulation design. Several recently reported peak assignments in the solid-state 13 C NMR spectrum of HPMCAS have been corrected here using peak integrals in quantitative spectra, spectral editing, empirical chemical-shift predictions based on solution NMR, and full spectrum simulation analogous to deconvolution. Unlike in cellulose, the strong peak at 84 ppm must be assigned to C2 and C3 methyl ethers, instead of regular C4 of cellulose. The proposed assignment of signals at <65 ppm to OCH sites, including C5 of cellulose, could not be confirmed. CH2 spectral editing showed two resolved OCH2 bands, a more intense one from O-CH2 ethers of C6 at >69 ppm and a smaller one from its esters and possibly residual CH2 -OH groups, near 63 ppm. The strong intensities of resolved signals of acetyl, succinoyl, and oxypropyl substituents indicated the substitution of >85% of the OH groups in HPMCAS. The side-group concentrations in three different grades of HPMCAS were quantified.

Effect of ABC Theory Model on Negative Emotion of Young Patients with Breast Cancer During Treatment.

Objective: To evaluate the effect of emotional ABC theory on anxiety and depression in young patients with breast cancer. Methods: A total of 200 eligible young patients with breast cancer were randomly divided into control group (N = 100) and experimental group (N = 100). The control group received routine treatment, while the experimental group received emotional ABC theory intervention at the same time. Results: The Self Rating Anxiety Scale (SAS) and Self Rating Depression Scale (SDS) scores of the two groups were observed before and after nursing. There was no significant difference between the two groups before nursing (P > 0.05), but there was significant difference between the two groups after nursing, the control group was significantly higher than the experimental group (P < 0.05). The satisfaction degree of the control group was significantly lower than that of the experimental group (P < 0.05). Conclusion: Young patients with breast cancer using emotional ABC theory can effectively improve negative emotions, clinical can promote the nursing program.

Epidemiology-based wastewater monitoring for ecological risks of anti-tuberculosis drugs mixture effects.

First-line anti-tuberculosis (TB) drugs are commonly used to treat TB worldwide, leading to more contaminated wastewater being widely discharged into aquatic environments. However, studies of mixture interactions of anti-TB drugs and their residues in aquatic environments are scarce. This study aimed to determine the toxic interactions of anti-TB drugs-isoniazid (INH), rifampicin (RMP), and ethambutol (EMB)-in binary and ternary mixtures on Daphnia magna and used the epidemiology of TB history to construct epidemiology-based wastewater monitoring for assessing the environmental release of residues and related ecological risks. The acute immobilization of median effect concentrations (EC50) was 25.6 mg L-1 for INH, 80.9 mg L-1 for RMP, and 188.8 mg L-1 for EMB, as toxic units (TUs) for assessing mixture toxicity. The ternary mixture exhibited the lowest TUs at 50 % effects with 1.12, followed by 1.28 for RMP + EMB, 1.54 for INH + RMP, and 1.93 for INH + EMB, indicating antagonistic interactions. Nevertheless, the combination index (CBI) was used to examine the mixture toxicity in response to immobilization, revealing that the ternary mixture of CBI ranged from 1.01 to 1.08, tending to have a nearly additive effect when suffering >50 % effect (at high concentration levels). The forecasted environmentally relevant concentrations of anti-TB drugs have been on downward trends with ng L-1 level from 2020 to 2030 in Kaohsiung, Taiwan. Although ecotoxicological risks from the wastewater treatment plant and receiving water in the field were slightly greater than the prediction from epidemiology-based wastewater monitoring, there were no risk concerns. Here, we achieved the establishment of evidence that anti-TB drug mixtures' interaction and epidemiological-based monitoring support a systematic approach, resolving the absence of the mixture toxicity information for anti-TB mixture risk assessment in aquatic environments.

Characterizing Silicone Oil-Induced Protein Aggregation with Stimulated Raman Scattering Imaging.

Particles in biopharmaceutical products present high risks due to their detrimental impacts on product quality and safety. Identification and quantification of particles in drug products are important to understand particle formation mechanisms, which can help develop control strategies for particle formation during the formulation development and manufacturing process. However, existing analytical techniques such as microflow imaging and light obscuration measurement lack the sensitivity and resolution to detect particles with sizes smaller than 2 µm. More importantly, these techniques are not able to provide chemical information to determine particle composition. In this work, we overcome these challenges by applying the stimulated Raman scattering (SRS) microscopy technique to monitor the C-H Raman stretching modes of the proteinaceous particles and silicone oil droplets formed in the prefilled syringe barrel. By comparing the relative signal intensity and spectral features of each component, most particles can be classified as protein-silicone oil aggregates. We further show that morphological features are poor indicators of particle composition. Our method has the capability to quantify aggregation in protein therapeutics with chemical and spatial information in a label-free manner, potentially allowing high throughput screening or investigation of aggregation mechanisms.