Purpose Poly (lactic-co-glycolic acid) (PLGA) microspheres are widely used long-acting injectable depots. However, their performance is highly sensitive to polymer material attributes that can influence microsphere structure, degradation, and drug release. While factors such as molecular weight and monomer ratio are well recognized, impact of residual species present in commercial PLGA has not been systematically evaluated. The novelty of the present study is the investigation of the effect of PLGA residual content on the physicochemical characteristics and in vitro performance of risperidone-loaded PLGA microspheres. Methods PLGA with varying amounts of residual content was obtained either through purification of a high-residual polymer by reprecipitation or by deliberately spiking PLGA with controlled quantities of monomeric residues (lactide or glycolide) or organic solvent. Risperidone was selected as the model drug, and microsphere formulations were prepared using PLGA with different amounts of residual content and compared with microspheres prepared using low residual PLGA. The micro-spheres were characterized for particle size, surface morphology, porosity, drug loading, polymer degradation kinetics, and in vitro drug release. Results Residual content was shown to significantly influence microsphere formation and performance. High solvent residue had a great impact on microsphere particle size, porosity, and drug loading, resulting in accelerated polymer degradation and drug release. A clear correlation was established between residual content, microsphere structure, degradation behavior, and release kinetics. Conclusions These findings identify residual species as a critical material attribute of PLGA microspheres and highlight the importance of polymer purification and raw-material control to ensure consistent performance of microsphere formulations.

In vitro/in vivo correlation (IVIVC) is of great importance to expedite the development life-cycles for pharmaceutical industry and possibly to reduce the burden for regulatory assessment, especially for long-acting injectable (LAI) drug products. However, owing to the inherent complexity and relative novelty, no regulatory guidance is available for IVIVC development of LAIs. Moreover, LAI dosage forms are prone to exhibit much higher inter-patient variability compared to conventional formulation types. Currently, relevant publications and an industry survey indicate most of the researchers tend to use mean (both in vitro and in vivo) data to establish IVIVCs, which may discount the inherent variability of LAIs, and hence may result in stringent IVIVCs with poor predictive performance. Therefore, it is critical to use statistical tools such as bootstrapping to represent highly variable in vivo pharmacokinetic profiles and more accurately develop IVIVC for various LAIs. The objective of this work is to use an innovative bootstrapping strategy for IVIVC development and evaluation to capture the inherent high inter-subject variabilities observed with four LAI aqueous suspensions. By using bootstrapping, developed IVIVC models were able to predict the in vivo performance of LAI suspensions. More importantly, it demonstrated the feasibility of using such a technique to reflect the variability of pharmacokinetic characteristics of LAI suspensions.

Microglia-mediated neuroinflammation is implicated in the pathogenesis of Parkinson’s disease (PD). Cystathionine β-synthase (CBS) and transsulfur metabolism modulate neuroinflammation. However, the regulation of Cbs transcription is poorly understood, and it remains unknown whether microglial CBS expression is affected by genetic factors in PD. Here, our in vitro study revealed that microglial CBS expression was downregulated by lipopolysaccharide (LPS)/interferon-γ (IFN-γ) but upregulated by interleukin (IL)-4/IL-13 stimulation. CBS and transsulfur metabolism not only inhibited inflammation but also promoted the anti-inflammatory transition of microglia. The mice with conditional microglial Cbs overexpression conferred resistance to LPS-induced neuroinflammation and dopaminergic neuron damage. A novel regulatory effect of the signal transducer and activator of transcription (STAT) family on Cbs transcription was identified: STAT1 as a suppressor, whereas STAT6 as an enhancer of Cbs transcription. PD-related gene DJ-1 knockdown (KD) decreased Cbs transcription through STAT1 activation in microglia. Moreover, Cbs overexpression alleviated the susceptibility of DJ-1 KD microglia to α-synuclein preformed fibrils (α-Syn PFFs) stimulation and the neurotoxicity to dopaminergic cells. Taken together, our findings reveal novel and opposite regulatory effects of STAT1 and STAT6 on microglial Cbs transcription in response to pro- and anti-inflammatory stimulation and demonstrate that CBS acts downstream of DJ-1, highlighting its role in PD.

Mitochondrial dysfunction is a critical contributor to neuronal damage in acute ischemic stroke (AIS), and targeting mitochondrial function represents a promising therapeutic strategy. This study unveils the pivotal role of Cend1 protein in ischemic stroke and elucidates its underlying mechanisms. Using Cend1 knockout (KO) mice, Cend1 deficiency was shown to exacerbate cerebral ischemia/reperfusion injury, as evidenced by enlarged infarct volume, worsened neurological deficits in motor coordination and grip strength, together with alterations in mitochondrial membrane potential (ΔΨm), mPTP opening, ATP content, and the activities of respiratory Complex I and V. Mechanistically, Cend1 forms dimers via conserved GXXXA motifs in its transmembrane domain to enhance ATP synthesis. Disruption of dimerization of Cend1 (such as G130P mutation) destabilized Cend1, accelerating its degradation and abolishing ATP-enhancing effects. Atp5f1b, a mitochondrial ATP synthase subunit, was found to interact with Cend1. Furthermore, screening identified the small-molecule compound Tianeptine (TNT), which stabilizes Cend1 dimers, elevates ATP levels, and confers neuroprotection in a Cend1-dependent manner. Notably, TNT's efficacy was abolished in Cend1 KO mice, highlighting its reliance on Cend1. The findings support the Cend1/Atp5f1b interaction as a potential mitochondria-targeted mechanism, offering innovative strategies to combat ischemic stroke by enhancing bioenergetic resilience.

Non-coding CGG repeat expansions within the 5' untranslated region are implicated in a range of neurological disorders, including fragile X-associated tremor/ataxia syndrome, oculopharyngeal myopathy with leukodystrophy, and oculopharyngodistal myopathy. This review outlined the general characteristics of diseases associated with non-coding CGG repeat expansions, detailing their clinical manifestations and neuroimaging patterns, which often overlap and indicate shared pathophysiological traits. We summarized the underlying molecular mechanisms of these disorders, providing new insights into the roles that DNA, RNA, and toxic proteins play. Understanding these mechanisms is crucial for the development of targeted therapeutic strategies. These strategies include a range of approaches, such as antisense oligonucleotides, RNA interference, genomic DNA editing, small molecule interventions, and other treatments aimed at correcting the dysregulated processes inherent in these disorders. A deeper understanding of the shared mechanisms among non-coding CGG repeat expansion disorders may hold the potential to catalyze the development of innovative therapies, ultimately offering relief to individuals grappling with these debilitating neurological conditions.

Epigenetic mechanisms such as DNA methylation and hydroxymethylation play a significant role in depression. This research has shown that Ten-eleven translocation 2 (Tet2) deficiency prompts depression-like behaviors, but Tet2's transcriptional regulation remains unclear. In the study, bioinformatics is used to identify nuclear receptor subfamily 2 group E member 3 (Nr2e3) as a potential Tet2 regulator. Nr2e3 is found to enhance Tet2's transcriptional activity by binding to its promoter region. Nr2e3 knockdown in mouse hippocampus leads to reduced Tet2 expression, depression-like behaviors, decreased hydroxymethylation of synaptic genes, and downregulation of synaptic proteins like postsynaptic density 95 KDa (PSD95) and N-methy-d-aspartate receptor 1 (NMDAR1). Fewer dendritic spines are also observed. Nr2e3 thus appears to play an antidepressant role under stress. In search of potential treatments, small molecule compounds to increase Nr2e3 expression are screened. Azacyclonal (AZA) is found to enhance the Nr2e3/Tet2 pathway and exhibited antidepressant effects in stressed mice, increasing PSD95 and NMDAR1 expression and dendritic spine density. This study illuminates Tet2's upstream regulatory mechanism, providing a new target for identifying early depression biomarkers and developing treatments.

The purpose of this study was to develop an in vitro release testing (IVRT) strategy to predict the pre-clinical performance of single agent and combination long acting injectable (LAI) suspension products. Two accelerated IVRT methods were developed using USP apparatus 2 to characterize initial, intermediate, and terminal phases of drug release. Initial and intermediate phases were captured using a suspension cup with moderate agitation to ensure a constant, low surface area exposure of the LAI suspension to the release media. The terminal phase was obtained by exposing the LAI suspension to a high initial paddle speed. This resulted in smaller suspension particulates with high cumulative surface area that were dispersed throughout the release media, enabling rapid drug release. The in vitro release profiles obtained with these two methods in 48 h or less were independently time scaled to reflect the in vivo time scale of approximately 1800 h. Level-A in vitro in vivo correlations (IVIVCs) were separately developed for each method and active pharmaceutical ingredient (API) using in vivo absorption profiles obtained by deconvolution of rat plasma concentration-time profiles. The IVIVCs were successfully validated for each API. This work provides a framework for evaluating individual phases of drug release of complex LAIs to ultimately predict their in vivo performance.

Syphilis is a global public health concern. This study aimed to assess the global and regional burden of syphilis from 1990 to 2019. Disease burden was evaluated using disability-adjusted life-years (DALYs) and prevalence. Data were extracted from the 2019 global burden of disease Study, an open database available for download. Age-standardized rates (ASR) and estimated annual percentage changes (EAPC) were calculated to evaluate the syphilis burden over time. In 2019, the total number of prevalent cases of syphilis was 49.71 million worldwide. The ASR of prevalence was stable from 1990 to 2019 with an EAPC of 0.00 (95% CI − 0.10–0.11). The number of DALYs caused by syphilis was 7.36 million in 2019, reflecting a reduction of 16.38% compared with that in 1990 (8.80 million). The ASR of DALYs exhibited a decreasing trend from 1990 to 2019 (EAPC = − 1.01; 95% CI − 1.19 to − 0.84), with the highest rates observed in the younger age group (< 14 years old). In 2019, the highest ASR of DALYs was found in low sociodemographic index (SDI) regions (239.21/100,000), and the lowest in high SDI regions (3.14/100,000). Generally, the ASR of DALYs decreased as the SDI increased. The top three countries with the highest ASR of DALYs for syphilis were the Solomon Islands, Equatorial Guinea, and Liberia. While the global prevalence of syphilis remained persistently high from 1990 to 2019, there has been a recent decrease in the ASR of DALYs. Increased attention should be dedicated to younger populations and regions characterized by low SDIs.

Hydrophobically modified ethoxylated urethane (HEUR) with different structures was synthesized and confirmed by gel permeation chromatography, Fourier transform infrared spectroscopy and nuclear magnetic resonance (1H NMR). We propose the relationship between the temperature insensitivity model and the thickening mechanism of HEUR/latex/Fe2O3/Zn3(PO4)2/BaSO4 suspensions. Meanwhile, the temperature insensitivity of HEUR/C suspensions is the result of two main associations: intermolecular interactions bridging the hydrophobic tails of HEURs and the hydrophobic groups tightly adsorbing onto the latex particle surfaces. A smaller ratio of viscosity (Rv) at 1 s−1 from the steady state condition indicates the better temperature insensitivity of viscosity. The higher degree of crystallinity and rheological activation energy corresponds to a great extent with better temperature insensitivity due to stronger association. The temperature insensitivity is consistent with the longer hydrophobic chain, which was proven by hysteresis tests and oscillatory shear measurements. The storage stability was enhanced in the lockstep with a hydrophobic length of HEUR, which is consistent with the rougher surfaces of HEUR/C films. As an appealing method, the results are meaningful and instructive for coating storage and application. An intermolecular network model of HEUR/C is presented, which suggests intermolecular associations in the bridging of hydrophobic tails and the hydrophobic groups adsorbing onto the latex particle surfaces. A temperature insensitivity mechanism model combined with rheology results and AFM is also proposed to clarify the relationship between temperature insensitivity and storage stability and the hydrophobic tail length.

The lateral habenula (LHb) has been considered a moderator of social behaviors. However, it remains unknown how LHb regulates social interaction. Here, we show that the hydroxymethylase Tet2 is highly expressed in the LHb. Tet2 conditional knockout (cKO) mice exhibit impaired social preference; however, replenishing Tet2 in the LHb rescues social preference impairment in Tet2 cKO mice. Tet2 cKO alters DNA hydroxymethylation (5hmC) modifications in genes that are related to neuronal functions, as is confirmed by miniature two-photon microscopy data. Further, Tet2 knockdown in the glutamatergic neurons of LHb causes impaired social behaviors, but the inhibition of glutamatergic excitability restores social preference. Mechanistically, we identify that Tet2 deficiency reduces 5hmC modifications on the Sh3rf2 promoter and Sh3rf2 mRNA expression. Interestingly, Sh3rf2 overexpression in the LHb rescues social preference in Tet2 cKO mice. Therefore, Tet2 in the LHb may be a potential therapeutic target for social behavior deficit-related disorders such as autism.

Long-acting injectable (LAI) aqueous suspensions achieve extended drug release over a duration of weeks to months via slow dissolution of drug crystals with low solubility. There have been around ten LAI aqueous suspensions approved by the FDA to date and there are no generic equivalents for most of them. This may be largely due to the complex formulation development as well as the challenges in establishment of in vitro-in vivo correlation (IVIVC) for these products. Level A IVIVCs, using animal models, have been proven feasible for complex long-acting microsphere formulations with multiphasic release characteristics. Accordingly, it may be possible to develop IVIVCs for LAI aqueous drug suspensions since their release characteristics are relatively simple with only a drug dissolution phase. To establish IVIVCs for LAI drug suspensions, four compositionally equivalent medroxyprogesterone acetate LAIs with differences in processing and formulation factors (drug particle size and excipient source) were prepared using Depo-SubQ Provera 104 as the reference listed drug (RLD). Two in vitro release testing methods, modified based on USP apparatus 2 (with enhancer cells) and USP apparatus 4 (with semisolid adapters), were used. The in vivo release was investigated using a rabbit model. Level A IVIVCs were successfully established using the in vitro release profiles obtained with the USP apparatus 4. This is the first report of an IVIVC for LAI aqueous suspensions.

Introduction: Environmental stress promotes epigenetic alterations that impact gene expression and subsequently participate in the pathological processes of the disorder. Among epigenetic regulations, ten-eleven Translocation (Tet) enzymes oxidize 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) in DNA and RNA and function as critical players in the pathogenesis of diseases. Our previous results showed that chronic stress increases the expression of cytoplasmic Tet2 in the hippocampus of mice exposed to chronic mild stress (CMS). Whether the cytoplasmic Tet2 alters RNA 5hmC modification in chronic stress-related processes remains largely unknown.Methods: To explore the role of cytoplasmic Tet2 under CMS conditions, we established CMS mice model and detected the expression of RNA 5hmC by dot blot. We verified the interaction of Tet2 and its interacting protein by co-immunoprecipitation combined with mass spectrometry and screened downstream target genes by cluster analysis of Tet2 and upstream frameshift 1 (Upf1) interacting RNA. The expression of protein was detected by Western blot and the expression of the screened target genes was detected by qRT-PCR.Results: In this study, we found that increased cytoplasmic Tet2 expression under CMS conditions leads to increase in total RNA 5hmC modification. Tet2 interacted with the key non-sense-mediated mRNA decay (NMD) factor Upf1, regulated the stability of stress-related genes such as Unc5b mRNA, and might thereby affect neurodevelopment.Discussion: In summary, this study revealed that Tet2-mediated RNA 5hmC modification is involved in stress-related mRNA stability regulation and may serve as a potential therapeutic target for chronic stress-related diseases such as depression.

OBJECTIVETo explore the molecular pathological mechanism of liver metabolic disorder in severe spinal muscular atrophy (SMA).METHODSThe transgenic mice with type Ⅰ SMA (Smn-/- SMN20tg/2tg) and littermate control mice (Smn+/- SMN20tg/2tg) were observed for milk suckling behavior and body weight changes after birth. The mice with type Ⅰ SMA mice were given an intraperitoneal injection of 20% glucose solution or saline (15 μL/12 h), and their survival time was recorded. GO enrichment analysis was performed using the RNA-Seq data of the liver of type Ⅰ SMA and littermate control mice, and the results were verified using quantitative real-time PCR. Bisulfite sequencing was performed to examine CpG island methylation level in Fasn gene promoter region in the liver of the neonatal mice.RESULTSThe neonatal mice with type Ⅰ SMA showed normal milk suckling behavior but had lower body weight than the littermate control mice on the second day after birth. Intraperitoneal injection of glucose solution every 12 h significantly improved the median survival time of type Ⅰ SMA mice from 9±1.3 to 11± 1.5 days (P < 0.05). Analysis of the RNA-Seq data of the liver showed that the expression of the target genes of PPARα related to lipid metabolism and mitochondrial β oxidation were down-regulated in the liver of type Ⅰ SMA mice. Type Ⅰ SMA mice had higher methylation level of the Fasn promoter region in the liver than the littermate control mice (76.44% vs 58.67%). In primary cultures of hepatocytes from type Ⅰ SMA mice, treatment with 5-AzaC significantly up-regulated the expressions of the genes related to lipid metabolism by over 1 fold (P < 0.01).CONCLUSIONType Ⅰ SMA mice have liver metabolic disorder, and the down-regulation of the target genes of PPARα related to lipid and glucose metabolism due to persistent DNA methylation contributes to the progression of SMA.

R-loop, an RNA-DNA hybrid structure, arises as a transcriptional by-product and has been implicated in DNA damage and genomic instability when excessive R-loop is accumulated. Although previous study demonstrated that R-loop is associated with ten-eleven translocation (Tet) proteins, which oxidize 5-methylcytosine to 5-hydroxymethylcytosine (5hmC), the sixth base of DNA. However, the relationship between R-loop and DNA 5hmC modification remains unclear. In this study, we found that chronic restraint stress increased R-loop accumulation and decreased 5hmC modification in the prefrontal cortex (PFC) of the stressed mice. The increase of DNA 5hmC modification by vitamin C was accompanied with the decrease of R-loop levels; on the contrary, the decrease of DNA 5hmC modification by a small compound SC-1 increased the R-loop levels, indicating that 5hmC modification inversely regulates R-loop accumulation. Further, we showed that Tet deficiency-induced reduction of DNA 5hmC promoted R-loop accumulation. In addition, Tet proteins immunoprecipitated with Non-POU domain-containing octamer-binding (NONO) proteins. The deficiency of Tet proteins or NONO increased R-loop levels, but silencing Tet proteins and NONO did not further increase the increase accumulation, suggesting that NONO and Tet proteins formed a complex to inhibit R-loop formation. It was worth noting that NONO protein levels decreased in the PFC of stressed mice with R-loop accumulation. The administration of antidepressant fluoxetine to stressed mice increased NONO protein levels, and effectively decreased R-loop accumulation and DNA damage. In conclusion, we showed that DNA 5hmC modification negatively regulates R-loop accumulation by the NONO-Tet complex under stress. Our findings provide potential therapeutic targets for depression.

Poly(lactic-co-glycolic acid) (PLGA) microspheres are a sustained-release drug delivery system with several successful commercial products used for the treatment of a variety of diseases. By utilizing PLGA polymers with different compositions, therapeutic agents can be released over durations varying from several weeks to several months. However, precise quality control of PLGA polymers and a fundamental understanding of all the factors associated with the performance of PLGA microsphere formulations remains challenging. This knowledge gap can hinder product development of both innovator and generic products. In this review, variability of the key release controlling excipient (PLGA), as well as advanced physicochemical characterization techniques for the PLGA polymer and PLGA microspheres are discussed. The relative merits and challenges of different in vitro release testing methods, in vivo pharmacokinetic studies, and in vitro-in vivo correlation development are also summarized. This review is intended to provide an in-depth understanding of long-acting microsphere products and consequently facilitate the development of these complex products.

Brain development requires a delicate balance between self-renewal and differentiation in neural stem cells (NSC), which rely on the precise regulation of gene expression. Ten-eleven translocation 2 (TET2) modulates gene expression by the hydroxymethylation of 5-methylcytosine in DNA as an important epigenetic factor and participates in the neuronal differentiation. Yet, the regulation of TET2 in the process of neuronal differentiation remains unknown. Here, the protein level of TET2 was reduced by the ubiquitin-proteasome pathway during NSC differentiation, in contrast to mRNA level. We identified that TET2 physically interacts with the core subunits of the glucose-induced degradation-deficient (GID) ubiquitin ligase complex, an evolutionarily conserved ubiquitin ligase complex and is ubiquitinated by itself. The protein levels of GID complex subunits increased reciprocally with TET2 level upon NSC differentiation. The silencing of the core subunits of the GID complex, including WDR26 and ARMC8, attenuated the ubiquitination and degradation of TET2, increased the global 5-hydroxymethylcytosine levels, and promoted the differentiation of the NSC. TET2 level increased in the brain of the Wdr26+/- mice. Our results illustrated that the GID complex negatively regulates TET2 protein stability, further modulates NSC differentiation, and represents a novel regulatory mechanism involved in brain development.

Six injectable, long-acting in situ forming implant drug products based on poly(lactide-co-glycolide) (PLGA) and N-Methyl-2-Pyrrolidone (NMP) are available on the market. However, generic products, which would likely be more affordable for patients, are not yet available. This is partially due to the unique complexity of these formulations as well as the inherent heterogeneity of PLGA and the challenges in the manufacture and characterization of this polymer. This article focuses on a comprehensive characterization of Perseris (risperidone) in situ forming implant drug product, and the development of compositionally equivalent formulations. The molecular weight (MW), lactide/glycolide (L/G) ratio, end group, blockiness and glass transition temperature (Tg) of PLGA, as well as the crystal form and particle size of risperidone powder used in Perseris were identified through reverse engineering. The dissolved/suspended drug ratio in the final implant suspension for administration, as well as the real-time drug solid state in the solidified Perseris drug depot were investigated. Two compositionally equivalent formulations prepared using customized PLGA polymers with similar properties to the Perseris PLGA showed similar in vitro release and swelling behavior to Perseris as demonstrated using a novel adapter-based dissolution method. The novelty of this dissolution method lies in its ability to control implant shape, generate reproducible data, distinguish different release phases, as well as identify formulation changes. The knowledge gained in this work and the methodology established for characterization of the implant formulations are important for implant formulation development.

Poly(lactic-co-glycolic acid) PLGA (release controlling excipient) plays a dominant role on the performance of PLGA based long-acting parenterals. These types of drug products typically exhibit complex multi-phasic in vitro/in vivo release/absorption characteristics. In particular, owing to their large size, charged state, and hydrophilicity, peptide loaded microspheres can exhibit more complex release mechanisms. Accordingly, it is challenging to develop Level A in vitro-in vivo correlations (IVIVCs) for such complex long-acting parenterals. With the objective of gaining a better understanding of how to achieve IVIVCs for peptide loaded PLGA microspheres, formulations with similar as well as different release characteristics were prepared with PLGAs from different sources. Leuprolide acetate was selected as the model drug. Owning to the different physicochemical properties of the PLGAs (such as inherent viscosity, molecular weight and blockiness), the formulations exhibited significant differences in their critical quality attributes (such as particle size, porosity and pore size) and consequently had different in vitro and in vivo performance. Affirmative conventional IVIVCs were developed that were able to predict the in vivo performance using the corresponding in vitro release profiles. In addition, the developed conventional IVIVCs were able to discriminate between formulations with comparable in vitro/in vivo performance and those that had dissimilar in vitro/in vivo performance. The present work provides a comprehensive understanding of the influence of PLGA source variations on IVIVC development and predictability for peptide loaded PLGA microspheres.

Due to the high clinical heterogeneity of neuronal intranuclear inclusion disease (NIID), it is easy to misdiagnose this condition and is considered to be a rare progressive neurodegenerative disease. More evidence demonstrates that NIID involves not only the central nervous system but also multiple systems of the body and shows a variety of symptoms, which makes a clinical diagnosis of NIID more difficult. This review summarizes the clinical symptoms in different systems and demonstrates that NIID is a multiple-system intranuclear inclusion disease. In addition, the core triad symptoms in the central nervous system, such as dementia, parkinsonism, and psychiatric symptoms, are proposed as an important clue for the clinical diagnosis of NIID. Recent studies have demonstrated that expanded GGC repeats in the 5′-untranslated region of the NOTCH2NLC gene are the cause of NIID. The genetic advances and possible underlying mechanisms of NIID (expanded GGC repeat-induced DNA damage, RNA toxicity, and polyglycine-NOTCH2NLC protein toxicity) are briefly summarized in this review. Interestingly, inflammatory cell infiltration and inflammation were observed in the affected tissues of patients with NIID. As a downstream pathological process of NIID, inflammation could be a therapeutic target for NIID.