RESUMO
RNA velocity has been rapidly adopted to guide interpretation of transcriptional dynamics in snapshot single-cell data; however, current approaches for estimating RNA velocity lack effective strategies for quantifying uncertainty and determining the overall applicability to the system of interest. Here, we present veloVI (velocity variational inference), a deep generative modeling framework for estimating RNA velocity. veloVI learns a gene-specific dynamical model of RNA metabolism and provides a transcriptome-wide quantification of velocity uncertainty. We show that veloVI compares favorably to previous approaches with respect to goodness of fit, consistency across transcriptionally similar cells and stability across preprocessing pipelines for quantifying RNA abundance. Further, we demonstrate that veloVI's posterior velocity uncertainty can be used to assess whether velocity analysis is appropriate for a given dataset. Finally, we highlight veloVI as a flexible framework for modeling transcriptional dynamics by adapting the underlying dynamical model to use time-dependent transcription rates.
Assuntos
RNA , Transcriptoma , RNA/genética , AprendizagemRESUMO
Direct comparison of bulk gene expression profiles is complicated by distinct cell type mixtures in each sample that obscure whether observed differences are actually caused by changes in the expression levels themselves or are simply a result of differing cell type compositions. Single-cell technology has made it possible to measure gene expression in individual cells, achieving higher resolution at the expense of increased noise. If carefully incorporated, such single-cell data can be used to deconvolve bulk samples to yield accurate estimates of the true cell type proportions, thus enabling one to disentangle the effects of differential expression and cell type mixtures. Here, we propose a generative model and a likelihood-based inference method that uses asymptotic statistical theory and a novel optimization procedure to perform deconvolution of bulk RNA-seq data to produce accurate cell type proportion estimates. We show the effectiveness of our method, called RNA-Sieve, across a diverse array of scenarios involving real data and discuss extensions made uniquely possible by our probabilistic framework, including a demonstration of well-calibrated confidence intervals.
Assuntos
RNA , Transcriptoma , Perfilação da Expressão Gênica/métodos , Funções Verossimilhança , RNA-Seq , Análise de Sequência de RNA , Análise de Célula Única/métodosRESUMO
PURPOSE: Aging is a risk factor for several debilitating conditions including those related to chronic back pain and intervertebral disc degeneration, both of which have no cure. Mouse models are useful tools for studying disc degeneration and chronic back pain in a tightly controlled and clinically relevant aging environment. Moreover, mice offer the advantage of carrying out longitudinal studies to understand the etiology and progression of disc pathology induced by genetic or surgical strategies. Previously, age-related behavioral trends of discomfort and enhanced nociception in mice were reported; however, whether these measures are mediated by structural and pathological changes in the disc is unknown. METHODS: The goal of the present observational study was to identify behavioral correlates of age-related degenerative changes in the disc. Towards this, we collected radiographs from 150 mice (77 females) between three and 23 months of age and measured the disc height index for each level of lumbar disc. Behavioral measures were collected on several of these mice which included rearing and distance travelled in an open field test; time spent in rearing, reaching, immobile, and self-suspended in the tail suspension test; bilateral hind paw licking in response to cold allodynia using acetone; and unilateral hind paw licking in response to heat hyperalgesia using capsaicin. RESULTS: Results show that the lower lumbar discs lose height with age and these changes are independent of body composition measures including body weight, bone mineral density, fat mass, lean weight mass, percent fat mass, and percent lean mass. Disc height positively correlates with rearing and mobility in the open field test, immobility in the tail suspension test, and thermal hyperalgesia. Disc height negatively correlates with cold allodynia and rearing in the tail suspension test. Furthermore, mediation analysis shows that the lumbosacral disc significantly mediates the effect of age on rearing in the open field test, but not cold allodynia, suggesting this behavior is a useful measure of age-related axial discomfort due to disc degeneration. CONCLUSION: In summary, the findings from the current study show that disc height are associated with measures of axial discomfort and nociception in mice.
Assuntos
Degeneração do Disco Intervertebral , Disco Intervertebral , Dor Lombar , Feminino , Camundongos , Animais , Degeneração do Disco Intervertebral/patologia , Hiperalgesia/etiologia , Dor Lombar/patologia , Disco Intervertebral/diagnóstico por imagem , Disco Intervertebral/patologia , Região Lombossacral/patologiaRESUMO
Oxidation is an important degradation pathway of protein drugs. The susceptibility to oxidation is a common concern for therapeutic proteins as it may impact product efficacy and patient safety. In this work, we used 2,2'-azobis (2-amidinopropane) dihydrochloride (AAPH) as an oxidative stress reagent to evaluate the oxidation of therapeutic antibodies. In addition to the oxidation of methionine (Met) and tryptophan (Trp) residues, we also observed an increase of protein aggregation. Size-exclusion chromatography and multi-angle light scattering showed that the soluble aggregates induced by AAPH consist of dimer, tetramer, and higher-order aggregate species. Sodium dodecyl sulfate polyacrylamide gel electrophoresis indicated that inter-molecular disulfide bonds contributed to the protein aggregation. Furthermore, intrinsic fluorescence spectra suggested that dimerization of tyrosine (Tyr) residues could account for the non-reducible cross-links. An excipient screening study demonstrated that Trp, pyridoxine, or Tyr could effectively reduce protein aggregation due to oxidative stress. This work provides valuable insight into the mechanisms of oxidative-stress induced protein aggregation, as well as strategies to minimize such aggregate formation during the development and storage of therapeutic proteins.
Assuntos
Anticorpos Monoclonais/química , Estresse Oxidativo/genética , Proteínas/química , Proteólise/efeitos dos fármacos , Amidinas , Anticorpos Monoclonais/genética , Dimerização , Radicais Livres/química , Radicais Livres/metabolismo , Humanos , Oxidantes/química , Oxidantes/farmacologia , Oxirredução/efeitos dos fármacos , Agregados Proteicos/genética , Proteínas/genética , Proteínas/uso terapêutico , Triptofano/química , Triptofano/genéticaRESUMO
The 1-month Lupron Depot (LD) is a 75/25 acid-capped poly(lactic-co-glycolic acid) (PLGA) microsphere product encapsulating water-soluble leuprolide acetate with no generic products available in the U.S. Composition-equivalent PLGA microsphere formulations to the LD as a function of raw material and manufacturing variables were developed by using the solvent evaporation encapsulation method. The following variables were adjusted: polymer supplier/polymerization type, gelatin supplier/bloom number, polymer concentration, first homogenization speed and time, volume of primary water phase, second homogenization time, volume of secondary water phase, and stirring rate. The loading and encapsulation efficiency (EE) of leuprolide and gelatin were determined to identify a large number of composition-equivalent formulations within a ±10% specification of the LD. Key physical-chemical properties of the formulations (e.g., morphology, particle size distribution, glass transition temperature (Tg), residual moisture and solvent, and porosity) were characterized to determine the effect of manufacturing variables on the product attributes. The EE of gelatin across all formulations prepared (101 ± 1%) was observed to be much higher than the EE of leuprolide (57 ± 1%). Judicious adjustment of polymer concentration, second homogenization time, and volume of second water phase was key to achieving high EE of leuprolide, although EE higher than 70% was not easily achievable owing to the difficulty of emulsifying highly viscous primary emulsion into homogeneous small droplets that could prevent peptide loss during the second homogenization under the conditions and equipment used. The in vitro release kinetics of the formulations was highly similar to the LD in a zero-order manner after â¼20% initial burst release, indicating a critical role of the composition on peptide release in this formulation. The characterization of composition-equivalent formulations described here could be useful for further development of generic leuprolide PLGA microspheres and for guiding decisions on the influence of process variables on product physicochemical attributes and release performance.
Assuntos
Leuprolida/química , Microesferas , Copolímero de Ácido Poliláctico e Ácido Poliglicólico/química , Preparações de Ação Retardada , Composição de Medicamentos , Liberação Controlada de Fármacos , Gelatina/química , Leuprolida/administração & dosagem , Tamanho da Partícula , Copolímero de Ácido Poliláctico e Ácido Poliglicólico/administração & dosagem , PorosidadeRESUMO
Sandostatin LAR (SLAR) is an injectable long-acting release (LAR) microsphere formulation for octreotide based on a biodegradeable glucose star copolymer of d,l-lactic and glycolic acids (PLGA-glu), which is primarily used for the treatment of patients with acromegaly. There currently is no generic SLAR approved in the United States despite expiration of patent coverage. To understand better this important formulation, SLAR was assessed for its composition and physical-chemical properties. Octreotide release kinetics was monitored under physiological conditions over 56 days together with several bioerosion parameters [mass loss, water uptake, pH of release media, polymer molecular weight (Mw), and confocal microscopy after BODIPY uptake]. A significant increase in the amount of released peptide occurred after day 14. After 1 day of incubation in PBST, octreotide was not extractable completely from SLAR during 2 h of the extraction process, but complete extraction was accomplished after 24 h, which suggested that strong and noncovalent PLGA-octreotide interactions occurred beginning in the initial release phase. Leuprolide is considered as a cationic peptide competitor for octreotide-PLGA interactions and its presence in the release medium resulted in more continuous octreotide release from SLAR, which was linearly correlated with the mass loss from the polymer (i.e., an indication of erosion-controlled release). These data strongly suggest that octreotide forms a salt with acid end groups of linear PLGA chains that are either present as impurities in, and/or produced by the degradation of, the PLGA-Glu. This salt is expected to catalyze octreotide acylation and extend peptide release beyond that driven by erosion control. The characterization studies of physicochemical properties of SLAR described here could be useful for the development and regulatory evaluation of generic octreotide microspheres as well as new polymer formulations, in which the polymer strongly interacts with encapsulated peptides.
Assuntos
Portadores de Fármacos/química , Glucose/química , Microesferas , Octreotida/química , Copolímero de Ácido Poliláctico e Ácido Poliglicólico/química , Acilação , Composição de Medicamentos/métodos , Liberação Controlada de Fármacos , Estabilidade de Medicamentos , Medicamentos Genéricos/química , Cinética , Leuprolida/química , Peso Molecular , Porosidade , Temperatura de TransiçãoRESUMO
Cationic peptides are well known to readily bind poly(lactic-co-glycolic acids) (PLGAs) with a carboxylic acid (-COOH) end group, which poses a significant challenge to develop PLGA-based delivery systems for peptide therapeutics. This binding has been considered as a critical step leading to the peptide acylation within PLGA-based formulations, which is also known to affect microencapsulation and release. Herein, we utilized nano isothermal titration calorimetry (NanoITC) to investigate the thermodynamics of peptide-PLGA binding in dimethyl sulfoxide (DMSO) using a model cationic octapeptide, octreotide, which contains two primary amino groups located at its N-terminus and lysine side chain at position five. ITC results of PLGAs with different lactic acid to glycolic acid ratios (50:50 to 100:0) revealed that the extent of the interaction with the octreotide was solely dependent on the availability of the acid end group of the PLGA. The binding constants (Ka) at 37 °C were determined in a narrow range from 1.33 to 1.72 × 104 M-1 with 0.59 to 0.66 binding stoichiometries irrespective of the lactic/glycolic acid ratio in the PLGA-COOH. Over 25-65 °C, the octreotide-PLGA-COOH interactions were found to be enthalpically favored (ΔH < 0) and entropically unfavorable (ΔS < 0). Hence, the interactions were characterized as enthalpically driven. At different sodium chloride (NaCl) levels, the sensitivity of thermodynamics of the interactions to the charge screening effect contributed by the NaCl unveiled the actual driving force of the octreotide-PLGA-COOH interactions is simple ion-pairing.
Assuntos
Ácido Láctico , Octreotida , Calorimetria , Copolímero de Ácido Poliláctico e Ácido Poliglicólico , TermodinâmicaRESUMO
ABSTRACT: Medical provider's ability to detect, diagnose, and treat sport-related concussion has greatly improved in recent years. Although more is known about the biomechanical forces involved in concussion, it is still uncertain whether there are preventative measures athletes can take to prevent a sport-related concussion from occurring. The objective of this review was to determine if either neck size or neck strength is related to a decreased risk of sustaining a sport-related concussion. A literature review was conducted on Google Scholar and Ovid MEDLINE for pertinent articles. Findings indicate that isometric neck strength, but not neck size, has been shown to be a predictor for sport-related concussion prevention. Formal neck strengthening programs are feasible and lead to decreased sport-related concussion risk. Additionally, there may be greater opportunity to increase neck strength in amateur athletes compared to professional. In conclusion, cervical strengthening programs have been shown to be feasible and beneficial for athletes to decrease their risk of sustaining a sport-related concussion, though the optimal duration, intensity, and frequency has yet to be determined.
Assuntos
Traumatismos em Atletas , Concussão Encefálica , Força Muscular , Músculos do Pescoço , Humanos , Concussão Encefálica/prevenção & controle , Força Muscular/fisiologia , Traumatismos em Atletas/prevenção & controle , Músculos do Pescoço/fisiopatologia , Músculos do Pescoço/lesões , Atletas , Comportamento de Redução do Risco , PescoçoRESUMO
Over-activation of the epidermal growth factor receptor (EGFR) is a hallmark of glioblastoma. However, EGFR-targeted therapies have led to minimal clinical response. While delivery of EGFR inhibitors (EGFRis) to the brain constitutes a major challenge, how additional drug-specific features alter efficacy remains poorly understood. We apply highly multiplex single-cell chemical genomics to define the molecular response of glioblastoma to EGFRis. Using a deep generative framework, we identify shared and drug-specific transcriptional programs that group EGFRis into distinct molecular classes. We identify programs that differ by the chemical properties of EGFRis, including induction of adaptive transcription and modulation of immunogenic gene expression. Finally, we demonstrate that pro-immunogenic expression changes associated with a subset of tyrphostin family EGFRis increase the ability of T-cells to target glioblastoma cells.
RESUMO
ABSTRACT: The purpose of this narrative review was to assess work-related mild traumatic brain injury treatment approaches and outcomes. Literature indicates that incidence of work-related mild traumatic brain injury is high. Ability to return to work after injury is variable, with differences identified across industry sector, mechanisms of injury, sex, and timely treatment and referral. Additional challenges exist in the context of secondary gains (e.g., financial) and the potential for symptom exaggeration. Emerging evidence from studies outside the United States demonstrate the benefits of proactive assessment and treatment at the time of injury. These benefits can be further augmented by early referral to multidisciplinary treatment teams led by physical medicine and rehabilitation physicians. Opportunities for ongoing research and development of strategies to improve treatment, management, and more timely return to work for patients with occupational mild traumatic brain injury are discussed. It is concluded that challenges persist in treatment and management of patients with work-related mild traumatic brain injury as they present unique challenges not seen in those with nonwork-related mild traumatic brain injuries. The unique position of physical medicine and rehabilitation and the skills of physiatrists render them poised to lead multidisciplinary treatment teams for these patients and contribute to the development of a new guideline for return to work, with an emphasis on functional recovery.
Assuntos
Concussão Encefálica , Lesões Encefálicas Traumáticas , Medicina Física e Reabilitação , Humanos , Estados Unidos , Concussão Encefálica/terapia , Concussão Encefálica/diagnóstico , Recuperação de Função Fisiológica , Incidência , Lesões Encefálicas Traumáticas/reabilitaçãoRESUMO
Spray-dried poly(lactic-co-glycolic acid) (PLGA) peptide-loaded microspheres have demonstrated similar long-term in vitro release kinetics compared to those produced by the solvent evaporation method and commercial products. However, the difficult-to-control initial burst release over the first 24 h after administration presents an obstacle to product development and establishing bioequivalence. Currently, detailed information about underlying mechanisms of the initial burst release from microspheres is limited. We investigated the mechanism and extent of initial burst release using 16 previously developed spray-dried microsphere formulations of the hormone drug, leuprolide acetate, with similar composition to the commercial 1-month Lupron Depot® (LD). The burst release kinetics was measured with a previously validated continuous monitoring system as well as traditional sample-and-separate methods. The changes in pore structure and polymer permeability were investigated by SEM imaging and the uptake of a bodipy-dextran probe. In vitro results were compared to pharmacokinetics in rats over the same interval. High-burst, spray-dried microspheres were differentiated in the well-mixed continuous monitoring system but reached an upper limit when measured by the sample-and-separate method. Pore-like occlusions observed by confocal microscopy in some formulations indicated that particle swelling may have contributed to probe diffusion through the polymer phase and showed the extensive internal pore structure of spray-dried particles. Continuous monitoring revealed a rapid primary (1°) phase followed by a constant-rate secondary (2°) release phase, which comprised â¼80% and 20% of the 24-hr release, respectively. The ratio of 1° phase duration (t1°) and the characteristic probe diffusion time (τ) was highly correlated to 1° phase release for spray dried particles. Of the four spray-dried formulations administered in vivo, three spray-dried microspheres with similar polymer density showed nearly ideal linear correlation between in vivo absorption and well-mixed in vitro release kinetics over the first 24 h. By contrast, the more structurally dense LD and a more-dense in-house formulation showed a slight lag phase in vivo relative to in vitro. Furthermore, in vitro dimensionless times (tburst/τ) were highly correlated with pharmacokinetic parameters for spray-dried microspheres but not for LD. While the correlation of increases in effective probe diffusion and 1° phase release strongly suggests diffusion through the polymer matrix as a major release mechanism both in vitro and in vivo, a fixed lower limit for this release fraction implies an alternative release mechanism. Overall, continuous monitoring release and probe diffusion appears to have potential in differentiating between leuprolide formulations and establishing relationships between in vitro release and in vivo absorption during the initial burst period.
Assuntos
Leuprolida , Polímeros , Ratos , Animais , Leuprolida/química , Copolímero de Ácido Poliláctico e Ácido Poliglicólico/química , Microesferas , Polímeros/química , Solventes , Tamanho da PartículaRESUMO
The transition from notochord to vertebral column is a crucial milestone in chordate evolution and in prenatal development of all vertebrates. As ossification of the vertebral bodies proceeds, involutions of residual notochord cells into the intervertebral discs form the nuclei pulposi, shock-absorbing structures that confer flexibility to the spine. Numerous studies have outlined the developmental and evolutionary relationship between notochord and nuclei pulposi. However, the knowledge of the similarities and differences in the genetic repertoires of these two structures remains limited, also because comparative studies of notochord and nuclei pulposi across chordates are complicated by the gene/genome duplication events that led to extant vertebrates. Here we show the results of a pilot study aimed at bridging the information on these two structures. We have followed in different vertebrates the evolutionary trajectory of notochord genes identified in the invertebrate chordate Ciona, and we have evaluated the extent of conservation of their expression in notochord cells. Our results have uncovered evolutionarily conserved markers of both notochord development and aging/degeneration of the nuclei pulposi.
Assuntos
Cordados , Núcleo Pulposo , Animais , Notocorda/metabolismo , Projetos Piloto , Expressão GênicaRESUMO
Pooled genetic screens are powerful tools to study gene function in a high-throughput manner. Typically, sequencing-based screens require cell lysis, which limits the examination of critical phenotypes such as cell morphology, protein subcellular localization, and cell-cell/tissue interactions. In contrast, emerging optical pooled screening methods enable the investigation of these spatial phenotypes in response to targeted CRISPR perturbations. In this study, we report a multi-omic optical pooled CRISPR screening method, which we have named CRISPRmap. Our method combines a novel in situ CRISPR guide identifying barcode readout approach with concurrent multiplexed immunofluorescence and in situ RNA detection. CRISPRmap barcodes are detected and read out through combinatorial hybridization of DNA oligos, enhancing barcode detection efficiency, while reducing both dependency on third party proprietary sequencing reagents and assay cost. Notably, we conducted a multi-omic base-editing screen in a breast cancer cell line on core DNA damage repair genes involved in the homologous recombination and Fanconi anemia pathways investigating how nucleotide variants in those genes influence DNA damage signaling and cell cycle regulation following treatment with ionizing radiation or DNA damaging agents commonly used for cancer therapy. Approximately a million cells were profiled with our multi-omic approach, providing a comprehensive phenotypic assessment of the functional consequences of the studied variants. CRISPRmap enabled us to pinpoint likely-pathogenic patient-derived mutations that were previously classified as variants of unknown clinical significance. Furthermore, our approach effectively distinguished barcodes of a pooled library in tumor tissue, and we coupled it with cell-type and molecular phenotyping by cyclic immunofluorescence. Multi-omic spatial analysis of how CRISPR-perturbed cells respond to various environmental cues in the tissue context offers the potential to significantly expand our understanding of tissue biology in both health and disease.
RESUMO
INTRODUCTION: Dysphagia is a common side effect after botulinum toxin injections for cervical dystonia, with an incidence of 10-40%, depending upon the study and dose used. METHODS: Our study consisted of 5 preselected women who met criteria for cervical dystonia and subsequent dysphagia after electromyography (EMG)-guided injections. Injections were performed with ultrasound (US) imaging, and the effects on swallowing were examined. Separately, sternocleidomastoid (SCM) thickness in healthy controls and treated patients was measured. RESULTS: There were 34 episodes of dysphagia over 98 injection sessions using EMG guidance for a cumulative rate of 34.7%. Using US plus EMG guidance, there was 0% dysphagia across 27 injection sessions. SCM thickness was <1.1 cm. CONCLUSION: US combined with EMG guidance eliminated recurrent dysphagia after botulinum toxin treatment, possibly by keeping the injectate within the SCM.
Assuntos
Toxinas Botulínicas/administração & dosagem , Transtornos de Deglutição/diagnóstico por imagem , Transtornos de Deglutição/tratamento farmacológico , Torcicolo/tratamento farmacológico , Ultrassonografia de Intervenção/métodos , Adulto , Idoso , Antidiscinéticos/administração & dosagem , Feminino , Humanos , Pessoa de Meia-Idade , Estudos RetrospectivosRESUMO
The pH inside the aqueous pores of poly(lactic-co-glycolic acid) (PLGA) microspheres, often termed microclimate pH (µpH), has been widely evaluated in vitro and shown to commonly be deleterious to pH-labile encapsulated drug molecules. However, whether the in vitro µpH is representative of the actual in vivo values has long been remained a largely unresolved issue. Herein we quantitatively mapped, for the first time, the in vivo µpH distribution kinetics inside degrading PLGA microspheres by combining two previously validated techniques, a cage implant system and confocal laser scanning microscopy. PLGA (50/50, Mw = 24-38 kDa, acid-end capped and ester-capped) microsphere formulations with and without encapsulating exenatide, a pH-labile peptide that is known to be unstable when pH > 4.5, were administered to rats subcutaneously via cage implants for up to 6 weeks. The results were compared with two different in vitro conditions. Strikingly, the in vivo µpH developed similarly to the low microsphere concentration in vitro condition with 1-µm nylon bags but very different from conventional high microsphere concentration sample-and-separate conditions. Improved maintenance of stable external pH in the release media for the former condition may have been one important factor. Stability of exenatide remaining inside microspheres was evaluated by mass spectrometry and found that it was steadily degraded primarily via pH-dependent acylation with a trend that slightly paralleled the changes in µpH. This methodology may be useful to elucidate pH-triggered instability of PLGA encapsulated drugs in vivo and for improving in vivo-predictive in vitro conditions for assessing general PLGA microsphere performance.
Assuntos
Ácido Láctico , Ácido Poliglicólico , Animais , Ratos , Exenatida , Concentração de Íons de Hidrogênio , Ácido Láctico/química , Microesferas , Ácido Poliglicólico/química , Copolímero de Ácido Poliláctico e Ácido PoliglicólicoRESUMO
Poly(lactic-co-glycolic acid) (PLGA) long-acting release depots are effective for extending the duration of action of peptide drugs. We describe efficient organic-solvent-free remote encapsulation based on the capacity of common uncapped PLGA to bind and absorb into the polymer phase net positively charged peptides from aqueous solution after short exposure at modest temperature. Leuprolide encapsulated by this approach in low-molecular-weight PLGA 75/25 microspheres slowly and continuously released peptide for over 56 days in vitro and suppressed testosterone production in rats in an equivalent manner as the 1-month Lupron Depot®. The technique is generalizable to encapsulate a number of net cationic peptides of various size, including octreotide, with competitive loading and encapsulation efficiencies to traditional methods. In certain cases, in vitro and in vivo performance of remote-loaded PLGA microspheres exceeded that relative to marketed products. Remote absorption encapsulation further removes the need for a critical organic solvent removal step after encapsulation, allowing for simple and cost-effective sterilization of the drug-free microspheres before encapsulation of the peptide.
Assuntos
Ácido Láctico , Ácido Poliglicólico , Animais , Preparações de Ação Retardada , Glicóis , Microesferas , Tamanho da Partícula , Peptídeos , Copolímero de Ácido Poliláctico e Ácido Poliglicólico , Ratos , SolventesRESUMO
Sandostatin long-acting release® (SLAR) is a long-acting injectable somatostatin analogue formulation composed of octreotide encapsulated in glucose-initiated poly(lactic-co-glycolic acid) (PLGA) microspheres. Despite the end of patent protection, SLAR remains resistant to generic competition likely due to complexity of production process, the uniqueness of the glucose star polymer, and the instability of octreotide in the formulation. Here, we describe development of glucose-PLGA-based composition-equivalent to SLAR formulations prepared by double emulsion-solvent evaporation method and the effect of variations in encapsulation variables on release kinetics and other formulation characteristics. The following encapsulation variables were adjusted at constant theoretical loading of 7.0% peptide: PLGA concentration, pH of inner water phase, and stirring rate. After final drying, the microspheres were examined with and without annealing at 50 °C under vacuum for 3 days. The loading and encapsulation efficiency (EE) of octreotide acetate, manufacturing yield, and in vitro drug release kinetics in PBStc (10 mM phosphate-buffered saline (PBS) with 1% triethyl citrate and 0.02% sodium azide at pH 7.4) were determined by UPLC. The in vitro release and acylation kinetics of octreotide for the solvent evaporation formulations prepared were similar to SLAR although the initial burst was slightly higher. Key formulation steps identified to maximize microsphere yield and minimize residual solvent and initial burst release included (a) addition of acetic acid to the peptide before preparation and (b) annealing the microspheres under vacuum after drying. Controlled release octreotide formulations prepared and investigated in this study could provide a better understanding of the effect of production variables on release performance and supply information useful for making progress in manufacturing of SLAR generic equivalents.
Assuntos
Octreotida , Ácido Poliglicólico , Preparações de Ação Retardada , Glucose/química , Ácido Láctico/química , Microesferas , Octreotida/química , Tamanho da Partícula , Ácido Poliglicólico/química , Copolímero de Ácido Poliláctico e Ácido Poliglicólico/química , SolventesRESUMO
Purpose/Objective: Older adults with a history of traumatic brain injury (TBI) remain an understudied population, resulting in a paucity of geriatric-specific guidelines. Given an increased vascular risk among older adults with TBI, we aimed to examine distal predictors of vascular health in this population. Specifically, we sought to compare levels of perceived discrimination in Black and White older adults with a history of complicated mild, moderate, or severe TBI, and to examine the relationship between levels of discrimination and pulse pressure, a measure of vascular health. Research Method/Design: Self-report measures of everyday discrimination (ED) and major experiences of discrimination (MED) were completed by 106 individuals aging with TBI (27 identified as Black, 79 identified as White). Resting blood pressure was collected during the assessment. Results: MED, but not ED, was significantly higher among Black individuals versus White individuals aging with TBI. Greater MED was significantly associated with higher pulse pressure independent of race and antihypertensive medication status. There was a marginally significant race by MED interaction, where the association between MED and pulse pressure was observed in Black individuals but not White individuals. Injury severity was not associated with pulse pressure, nor were there significant severity by discrimination interactions on pulse pressure. Conclusions/Implications: Discrimination, which may arise from multiple sources of bias (e.g., related to race, disability), is associated with vascular burden. These findings suggest that patients' experiences of discrimination should be addressed as a factor that contributes to health and well-being in brain injury rehabilitation. (PsycInfo Database Record (c) 2021 APA, all rights reserved).
Assuntos
Lesões Encefálicas Traumáticas , Lesões Encefálicas , Pessoas com Deficiência , Idoso , Envelhecimento , Pressão Sanguínea , HumanosRESUMO
Bydureon® (Bdn) is a once-weekly injectable long-acting release (LAR) product for adults with type 2 diabetes based on PLGA microspheres encapsulating the glucagon like peptide (GLP-1) analog, exenatide. Despite its widespread use in type 2 diabetes treatment, little information has been published concerning the physical-chemical aspects and exenatide stability in this product. Here, we developed and validated methods to evaluate attributes and performance of Bdn such as particle size/size distribution and residual levels of moisture and organic solvent(s). The reverse engineering of the exenatide LAR was also performed to identify and quantify principal components in the product. Stability-indicating UPLC and LC-MS methods were applied to characterize exenatide degradation (such as oxidation, deamidation and acylation products) during in vitro release evaluation. The 55-µm volume-median Bdn microspheres slowly released the exenatidein vitroover two months with a very low initial burst release to avoid unwanted side effects. Residual organic solvent levels (methylene chloride, ethanol, heptane, and silicon oil) also met the USP criteria. Peptide acylation was the most prominent peptide reaction during both encapsulation and in vitro release, and the acylated peptide steadily increased during release relative to parent exenatide, becoming the most abundant peptide species extracted from the microspheres at later release stages. The presence of peptide impurities during the release period, which are not extractable in the polymer and likely insoluble in water, might be one potential cause for immunogenicity. Further evaluation will be needed to confirm this hypothesis. Release of peptide was minimal over the first 2â¯weeks before the microspheres steadily released peptide for more than 28â¯days. The rigorous technical approach discussed in this paper may provide critical information for both companies and the FDA for developing generic exenatide-PLGA formulations and other important PLGA microsphere products.
Assuntos
Portadores de Fármacos/química , Exenatida/administração & dosagem , Hipoglicemiantes/administração & dosagem , Microesferas , Preparações de Ação Retardada/administração & dosagem , Preparações de Ação Retardada/farmacocinética , Diabetes Mellitus Tipo 2/tratamento farmacológico , Composição de Medicamentos/métodos , Liberação Controlada de Fármacos , Exenatida/farmacocinética , Humanos , Hipoglicemiantes/farmacocinética , Copolímero de Ácido Poliláctico e Ácido Poliglicólico/química , Equivalência TerapêuticaRESUMO
A spray drying technique was developed to prepare injectable and biodegradable poly(lactic-co-glycolic acid) (PLGA) microspheres encapsulating a model luteinizing hormone-releasing hormone agonist (LHRHa)-based peptide, leuprolide. Various spray drying parameters were evaluated to prepare 1-month controlled release formulations with a similar composition to the commercial Lupron Depot® (LD). A single water-in-oil emulsion of aqueous leuprolide/gelatin solution in PLGA 75/25 acid capped (13â¯kDaâ¯Mw) dissolved in methylene chloride (DCM) was spray-dried before washing the microspheres in cold ddH2O and freeze-drying. The spray-drying microencapsulation was characterized by: particle size/distribution (span), morphology, drug/gelatin loading, encapsulation efficiency, and residual DCM and water content. Long-term release was tested over 9â¯weeks in PBSâ¯+â¯0.02% Tween 80â¯+â¯0.02% sodium azide pHâ¯7.4 (PBST) at 37⯰C. Several physical-chemical parameters were monitored simultaneously for selected formulations, including: water uptake, mass loss, dry and hydrated glass transition temperature, to help understand the related long-term release profiles and explore the underlying controlled-release mechanisms. Compared with the commercial LD microspheres, some of the in-house spray-dried microspheres presented highly similar or even improved long-term release profiles, providing viable long-acting release (LAR) alternatives to the LD. The in vitro release mechanism of the peptide was shown to be controlled either by kinetics of polymer mass loss or by a second process, hypothesized to involve peptide desorption from the polymer. These data indicate spray drying can be optimized to prepare commercially relevant PLGA microsphere formulations for delivery of peptides, including the LHRHa, leuprolide.