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1.
AAPS PharmSciTech ; 22(5): 204, 2021 Jul 13.
Artigo em Inglês | MEDLINE | ID: mdl-34258696

RESUMO

Hirsutism is a dermatological condition that refers to the excessive growth of hair in androgen-sensitive areas in women. Recently, the enhancement of the visible signs of a hairy female has taken special concern that affected the quality of life. The present study was developed to compare the follicular targeting effect of topical spironolactone (SP) or progesterone (PG)-loaded nanostructured lipid carrier (NLC) on the management of hirsutism. Four NLC formulations were prepared using cold homogenization techniques and pharmaceutically evaluated. SP-NLC and PG-NLC topical hydrogels were prepared to explore their pharmacological effect on letrozole induced polycystic ovarian syndrome (PCOS) in rats. Inflammatory mediators, antioxidant, and hormonal parameters were assayed. Additionally, histopathological examination was carried out to confirm the successful induction of PCOS. Results confirmed that all NLC formulations have a spherical shape with particle size ranged from 225.92 ± 0.41 to 447.80 ± 0.66 nm, entrapment efficiency > 75%, and zeta potential (- 31.4 to - 36.5 mV). F1 and F3 NLCs were considered as selected formulations for SP and PG, respectively. Female Wistar rats treated with F1 formulation for 3 weeks displayed better outcomes as manifested by the measured parameters as compared to the other tested groups. A significant reduction in hair follicle diameter and density was observed after topical application of SP or PG nano-gels. Finally, the outcomes pose a strong argument that the development of topically administered SP-NLC can be explored as a promising carrier over PG-NLC for more effectual improvement in the visible sign of hirsutism.


Assuntos
Portadores de Fármacos/administração & dosagem , Hirsutismo/sangue , Hirsutismo/tratamento farmacológico , Nanoestruturas/administração & dosagem , Progesterona/administração & dosagem , Espironolactona/administração & dosagem , Animais , Portadores de Fármacos/síntese química , Avaliação Pré-Clínica de Medicamentos/métodos , Feminino , Hidrogéis/administração & dosagem , Hidrogéis/síntese química , Inflamação/tratamento farmacológico , Inflamação/patologia , Nanoestruturas/química , Tamanho da Partícula , Progesterona/síntese química , Ratos , Ratos Wistar , Espironolactona/síntese química
2.
Int J Mol Sci ; 22(13)2021 Jun 24.
Artigo em Inglês | MEDLINE | ID: mdl-34202634

RESUMO

Novel psychoactive substances (NPS) are synthetic substances belonging to diverse groups, designed to mimic the effects of scheduled drugs, resulting in altered toxicity and potency. Up to now, information available on the pharmacology and toxicology of these new substances is very limited, posing a considerable challenge for prevention and treatment. The present in vitro study investigated the possible mechanisms of toxicity of two emerging NPS (i) 4'-methyl-alpha-pyrrolidinoexanophenone (3,4-MDPHP), a synthetic cathinone, and (ii) 2-chloro-4,5-methylenedioxymethamphetamine (2-Cl-4,5-MDMA), a phenethylamine. In addition, to apply our model to the class of synthetic opioids, we evaluated the toxicity of fentanyl, as a reference compound for this group of frequently abused substances. To this aim, the in vitro toxic effects of these three compounds were evaluated in dopaminergic-differentiated SH-SY5Y cells. Following 24 h of exposure, all compounds induced a loss of viability, and oxidative stress in a concentration-dependent manner. 2-Cl-4,5-MDMA activates apoptotic processes, while 3,4-MDPHP elicits cell death by necrosis. Fentanyl triggers cell death through both mechanisms. Increased expression levels of pro-apoptotic Bax and caspase 3 activity were observed following 2-Cl-4,5-MDMA and fentanyl, but not 3,4-MDPHP exposure, confirming the different modes of cell death.


Assuntos
Avaliação Pré-Clínica de Medicamentos/métodos , Neurônios/efeitos dos fármacos , Psicotrópicos/efeitos adversos , Apoptose/efeitos dos fármacos , Apoptose/genética , Proteínas Reguladoras de Apoptose/genética , Proteínas Reguladoras de Apoptose/metabolismo , Biomarcadores , Linhagem Celular , Linhagem Celular Tumoral , Células Cultivadas , Relação Dose-Resposta a Droga , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Estrutura Molecular , Psicotrópicos/química , Psicotrópicos/toxicidade , Relação Estrutura-Atividade
3.
Molecules ; 26(12)2021 Jun 16.
Artigo em Inglês | MEDLINE | ID: mdl-34208597

RESUMO

Several natural products (NPs) have displayed varying in vitro activities against methicillin-resistant Staphylococcus aureus (MRSA). However, few of these compounds have not been developed into potential antimicrobial drug candidates. This may be due to the high cost and tedious and time-consuming process of conducting the necessary preclinical tests on these compounds. In this study, cheminformatic profiling was performed on 111 anti-MRSA NPs (AMNPs), using a few orally administered conventional drugs for MRSA (CDs) as reference, to identify compounds with prospects to become drug candidates. This was followed by prioritizing these hits and identifying the liabilities among the AMNPs for possible optimization. Cheminformatic profiling revealed that most of the AMNPs were within the required drug-like region of the investigated properties. For example, more than 76% of the AMNPs showed compliance with the Lipinski, Veber, and Egan predictive rules for oral absorption and permeability. About 34% of the AMNPs showed the prospect to penetrate the blood-brain barrier (BBB), an advantage over the CDs, which are generally non-permeant of BBB. The analysis of toxicity revealed that 59% of the AMNPs might have negligible or no toxicity risks. Structure-activity relationship (SAR) analysis revealed chemical groups that may be determinants of the reported bioactivity of the compounds. A hit prioritization strategy using a novel "desirability scoring function" was able to identify AMNPs with the desired drug-likeness. Hit optimization strategies implemented on AMNPs with poor desirability scores led to the design of two compounds with improved desirability scores.


Assuntos
Produtos Biológicos/química , Produtos Biológicos/farmacologia , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Antibacterianos/farmacologia , Anti-Infecciosos/farmacologia , Quimioinformática/métodos , Bases de Dados Factuais , Avaliação Pré-Clínica de Medicamentos/métodos , Staphylococcus aureus Resistente à Meticilina/metabolismo , Testes de Sensibilidade Microbiana , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/metabolismo , Relação Estrutura-Atividade
4.
Nat Commun ; 12(1): 3309, 2021 06 03.
Artigo em Inglês | MEDLINE | ID: mdl-34083527

RESUMO

The ongoing pandemic caused by the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), necessitates strategies to identify prophylactic and therapeutic drug candidates for rapid clinical deployment. Here, we describe a screening pipeline for the discovery of efficacious SARS-CoV-2 inhibitors. We screen a best-in-class drug repurposing library, ReFRAME, against two high-throughput, high-content imaging infection assays: one using HeLa cells expressing SARS-CoV-2 receptor ACE2 and the other using lung epithelial Calu-3 cells. From nearly 12,000 compounds, we identify 49 (in HeLa-ACE2) and 41 (in Calu-3) compounds capable of selectively inhibiting SARS-CoV-2 replication. Notably, most screen hits are cell-line specific, likely due to different virus entry mechanisms or host cell-specific sensitivities to modulators. Among these promising hits, the antivirals nelfinavir and the parent of prodrug MK-4482 possess desirable in vitro activity, pharmacokinetic and human safety profiles, and both reduce SARS-CoV-2 replication in an orthogonal human differentiated primary cell model. Furthermore, MK-4482 effectively blocks SARS-CoV-2 infection in a hamster model. Overall, we identify direct-acting antivirals as the most promising compounds for drug repurposing, additional compounds that may have value in combination therapies, and tool compounds for identification of viral host cell targets.


Assuntos
Antivirais/farmacologia , COVID-19/tratamento farmacológico , Reposicionamento de Medicamentos/métodos , Pandemias , SARS-CoV-2 , Animais , COVID-19/prevenção & controle , COVID-19/virologia , Linhagem Celular , Citidina/administração & dosagem , Citidina/análogos & derivados , Citidina/farmacologia , Bases de Dados de Produtos Farmacêuticos , Descoberta de Drogas/métodos , Avaliação Pré-Clínica de Medicamentos/métodos , Células HeLa , Ensaios de Triagem em Larga Escala/métodos , Humanos , Hidroxilaminas/administração & dosagem , Hidroxilaminas/farmacologia , Mesocricetus , Nelfinavir/farmacologia , SARS-CoV-2/efeitos dos fármacos , SARS-CoV-2/fisiologia , Replicação Viral/efeitos dos fármacos
6.
Molecules ; 26(11)2021 May 31.
Artigo em Inglês | MEDLINE | ID: mdl-34073016

RESUMO

The pathogenesis of several neurodegenerative diseases such as Alzheimer's or Huntington's disease has been associated with metabolic dysfunctions caused by imbalances in the brain and cerebral spinal fluid levels of neuroactive metabolites. Kynurenine monooxygenase (KMO) is considered an ideal therapeutic target for the regulation of neuroactive tryptophan metabolites. Despite significant efforts, the known KMO inhibitors lack blood-brain barrier (BBB) permeability and upon the mimicking of the substrate binding mode, are subject to produce reactive oxygen species as a side reaction. The computational drug design is further complicated by the absence of complete crystal structure information for human KMO (hKMO). In the current work, we performed virtual screening of readily available compounds using several protein-ligand complex pharmacophores. Each of the pharmacophores accounts for one of three distinct reported KMO protein-inhibitor binding conformations. As a result, six novel KMO inhibitors were discovered based on an in vitro fluorescence assay. Compounds VS1 and VS6 were predicted to be BBB permeable and avoid the hydrogen peroxide production dilemma, making them valuable, novel hit compounds for further drug property optimization and advancement in the drug design pipeline.


Assuntos
Quinurenina 3-Mono-Oxigenase/antagonistas & inibidores , Quinurenina 3-Mono-Oxigenase/metabolismo , Barreira Hematoencefálica/metabolismo , Encéfalo/metabolismo , Biologia Computacional/métodos , Desenho de Fármacos , Avaliação Pré-Clínica de Medicamentos/métodos , Inibidores Enzimáticos/farmacologia , Humanos , Cinurenina/metabolismo , Quinurenina 3-Mono-Oxigenase/química , Simulação de Acoplamento Molecular/métodos , Doenças Neurodegenerativas/tratamento farmacológico , Conformação Proteica
7.
AAPS PharmSciTech ; 22(5): 170, 2021 Jun 03.
Artigo em Inglês | MEDLINE | ID: mdl-34085150

RESUMO

A novel nanofiber insert was prepared with a modified electrospinning method to enhance the ocular residence time of ofloxacin (OFX) and to provide a sustained release pattern by covering hydrophilic polymers, chitosan/polyvinyl alcohol (CS/PVA) nanofibers, with a hydrophobic polymer, Eudragit RL100 in layers, and by glutaraldehyde (GA) cross-linking of CS-PVA nanofibers for the treatment of infectious conjunctivitis. The morphology of the prepared nanofibers was studied using scanning electron microscopy (SEM). The average fiber diameter was found to be 123 ± 23 nm for the single electrospun nanofiber with no cross-linking (OFX-O). The single nanofibers, cross-linked for 10 h with GA (OFX-OG), had an average fiber diameter of 159 ± 30 nm. The amount of OFX released from the nanofibers was measured in vitro and in vivo using UV spectroscopy and microbial assay methods against Staphylococcus aureus, respectively. The antimicrobial efficiency of OFX formulated in cross-linked and non-cross-linked nanofibers was affirmed by observing the inhibition zones of Staphylococcus aureus and Escherichia coli. In vivo studies using the OFX nanofibrous inserts on a rabbit eye confirmed a sustained release pattern for up to 96 h. It was found that the cross-linking of the nanofibers by GA vapor could reduce the burst release of OFX from OFX-loaded CS/PVA in one layer and multi-layered nanofibers. In vivo results showed that the AUC0-96 for the nanofibers was 9-20-folds higher compared to the OFX solution. This study thus demonstrates the potential of the nanofiber technology is being utilized to sustained drug release in ocular drug delivery systems.


Assuntos
Resinas Acrílicas/química , Administração Oftálmica , Quitosana/química , Nanofibras/química , Ofloxacino/química , Álcool de Polivinil/química , Resinas Acrílicas/administração & dosagem , Resinas Acrílicas/farmacocinética , Animais , Antibacterianos/química , Química Farmacêutica/métodos , Quitosana/administração & dosagem , Quitosana/farmacocinética , Preparações de Ação Retardada/administração & dosagem , Preparações de Ação Retardada/química , Preparações de Ação Retardada/farmacocinética , Sistemas de Liberação de Medicamentos/métodos , Avaliação Pré-Clínica de Medicamentos/métodos , Escherichia coli/efeitos dos fármacos , Escherichia coli/fisiologia , Nanofibras/administração & dosagem , Ofloxacino/administração & dosagem , Ofloxacino/farmacocinética , Álcool de Polivinil/administração & dosagem , Álcool de Polivinil/farmacocinética , Coelhos , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/fisiologia
8.
AAPS PharmSciTech ; 22(5): 180, 2021 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-34129135

RESUMO

Oleogel consists of hydrophobic solvent and an oleogelator. In this study, attempts were made to study the influence of Celecoxib solubility, concentration and dispersability on its release, absorption, and biological performance. Oleogels were prepared to study the formulation variables on its stability and release. Castor oil was selected as the oil and the oleogelator concentration was 4.5% w/w. F3 revealed the highest release and stability compared to other formulae. The percent permeated across the rat intestine showed a 7.5-fold increase over free Celecoxib, and its lifetime was found to be greater than 18 months. The efficacy of free Celecoxib and oleogel formulae to treat rats with ulcerative colitis was done via the induction of ulcerative colitis (UC) through administration of 5% dextran sodium sulphate (DSS). Celecoxib besides its formulae significantly reduced the release of Leucine rich 2 glycoprotein (LRG), Myeloperoxidase (MPO), Tumor necrosis factor-α (TNF-α), proinflammatory cytokine expression, High mobility group box 1 (HMGB1), Nuclear factor kappa B (NF-ΚB), Trefoil Factor 3 (TFF3), Metalloproteinase-3 (MMP3), and miRNA31. Moreover, F3 significantly increased the colonic cAMP in DSS treated rats and reduced the intestinal inflammation beside healing of mucosa and restitution of the epithelium of the gastrointestinal tract.


Assuntos
Anti-Inflamatórios não Esteroides/uso terapêutico , Celecoxib/uso terapêutico , Colite Ulcerativa/tratamento farmacológico , Animais , Anti-Inflamatórios não Esteroides/síntese química , Anti-Inflamatórios não Esteroides/farmacocinética , Celecoxib/síntese química , Celecoxib/farmacocinética , Colite Ulcerativa/induzido quimicamente , Colite Ulcerativa/metabolismo , Colo/efeitos dos fármacos , Colo/metabolismo , Sulfato de Dextrana/toxicidade , Avaliação Pré-Clínica de Medicamentos/métodos , Inflamação/induzido quimicamente , Inflamação/tratamento farmacológico , Inflamação/metabolismo , Masculino , NF-kappa B/metabolismo , Compostos Orgânicos/síntese química , Compostos Orgânicos/farmacocinética , Compostos Orgânicos/uso terapêutico , Ratos , Ratos Wistar , Fator de Necrose Tumoral alfa/metabolismo
9.
AAPS PharmSciTech ; 22(5): 181, 2021 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-34129154

RESUMO

In this study, two hydrophilic polymers hydroxypropyl methyl cellulose and beta-cyclodextrin (ß-CD) are used to synthesize highly responsive and spongy polymeric matrices. Porous and stimulus-responsive polymeric network was developed to improve the solubility of acyclovir (ACV) at significant level. Grafting was successfully carried out by free radical polymerization technique. Spongy matrices were characterized by percentage entrapment efficiency, drug loading, solubility studies, FTIR, powder X-ray diffraction, TGA, DSC, XRD, SEM, swelling studies, and in vitro studies. Acute oral toxicity studies were conducted to determine the safety of oral administration of prepared HPMC-ßCD-g-poly(AMPS) formulation. Porous and spongy structures were depicted in SEM images. Complex formation and thermal stability of constituents and drug (ACV) were analyzed by FTIR, TGA, and DSC spectra. XRD analysis revealed reduction in acyclovir crystallinity in spongy matrices. Particle size of optimized formulation was found in the range of 197 ± 2.55 nm. The momentous difference with reference product committed that drug solubility and release characteristics were markedly enhanced by the developed spongy matrices. Toxicity studies endorsed that developed spongy matrices were non-toxic and compatible to biological system. The efficient method of preparation, enhanced solubility, excellent physico-chemical characteristics, high dissolution, and non-toxic HPMC-ßCD-g-poly(AMPS) spongy matrices may be a promising approach for oral delivery of poorly soluble drugs.


Assuntos
Acrilamidas/síntese química , Aciclovir/síntese química , Alcanossulfonatos/síntese química , Derivados da Hipromelose/síntese química , Polimerização , beta-Ciclodextrinas/síntese química , Acrilamidas/administração & dosagem , Aciclovir/administração & dosagem , Administração Oral , Alcanossulfonatos/administração & dosagem , Animais , Antivirais/administração & dosagem , Antivirais/síntese química , Avaliação Pré-Clínica de Medicamentos/métodos , Derivados da Hipromelose/administração & dosagem , Masculino , Tamanho da Partícula , Polímeros/administração & dosagem , Polímeros/síntese química , Coelhos , Solubilidade , Espectroscopia de Infravermelho com Transformada de Fourier/métodos , Difração de Raios X/métodos , beta-Ciclodextrinas/administração & dosagem
10.
AAPS PharmSciTech ; 22(5): 167, 2021 Jun 02.
Artigo em Inglês | MEDLINE | ID: mdl-34080078

RESUMO

In developing countries, populations have employed herbal medicines for primary health care because they are believed to be more appropriate to the human body and have less side effects than chemically synthesized drugs. The present study aimed to develop and evaluate herbal tablets incorporated with a Thai traditional medicinal extract, U-pa-ri-waat (URW), using microwave-assisted extraction (MAE). The extraction efficiency for URW using MAE and traditional solvent extraction was compared based on the percent yield after spray drying. URW tablets were prepared using the dry granulation method. The optimized products were assessed using standard characterization methods based on the United States and British Pharmacopeias. DPPH and ABTS radical scavenging assays were performed to analyze the antioxidant capacity of the microwave-assisted extracts. The results revealed that the flowability of the dry granule with added maltodextrin was improved compared to a granule without additives, as indicated by an angle of repose of 33.69 ± 2.0°, a compressibility index of 15.38 ± 0.66, and a Hausner's ratio of 1.18 ± 0.06. The resulting formulation produced flat tablets with uniform weight variation, hardness, thickness, friability, and optimum disintegration time. The URW extracts showed antioxidant activity and MAE with maltodextrin carrier displayed the strongest DPPH and ABTS radical activities with IC50 values of 1.60 ± 0.02 µg/mL and 4.02 ± 0.24 µg/mL, respectively. The URW tablet formulation passed the quality control tests. Storage of the formulation tablets for 90 days under accelerated conditions had minimal effects on tablet characteristics.


Assuntos
Química Farmacêutica/métodos , Micro-Ondas , Compostos Fitoquímicos/síntese química , Preparações de Plantas/síntese química , Administração Oral , Antioxidantes/administração & dosagem , Antioxidantes/síntese química , Antioxidantes/farmacocinética , Avaliação Pré-Clínica de Medicamentos/métodos , Sequestradores de Radicais Livres/administração & dosagem , Sequestradores de Radicais Livres/síntese química , Sequestradores de Radicais Livres/farmacocinética , Medicina Herbária/métodos , Humanos , Compostos Fitoquímicos/administração & dosagem , Compostos Fitoquímicos/farmacocinética , Preparações de Plantas/administração & dosagem , Preparações de Plantas/farmacocinética , Comprimidos , Tailândia
11.
Nat Commun ; 12(1): 3932, 2021 06 24.
Artigo em Inglês | MEDLINE | ID: mdl-34168145

RESUMO

Chemical descriptors encode the physicochemical and structural properties of small molecules, and they are at the core of chemoinformatics. The broad release of bioactivity data has prompted enriched representations of compounds, reaching beyond chemical structures and capturing their known biological properties. Unfortunately, bioactivity descriptors are not available for most small molecules, which limits their applicability to a few thousand well characterized compounds. Here we present a collection of deep neural networks able to infer bioactivity signatures for any compound of interest, even when little or no experimental information is available for them. Our signaturizers relate to bioactivities of 25 different types (including target profiles, cellular response and clinical outcomes) and can be used as drop-in replacements for chemical descriptors in day-to-day chemoinformatics tasks. Indeed, we illustrate how inferred bioactivity signatures are useful to navigate the chemical space in a biologically relevant manner, unveiling higher-order organization in natural product collections, and to enrich mostly uncharacterized chemical libraries for activity against the drug-orphan target Snail1. Moreover, we implement a battery of signature-activity relationship (SigAR) models and show a substantial improvement in performance, with respect to chemistry-based classifiers, across a series of biophysics and physiology activity prediction benchmarks.


Assuntos
Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologia , Relação Estrutura-Atividade , Linhagem Celular Tumoral , Bases de Dados de Produtos Farmacêuticos , Avaliação Pré-Clínica de Medicamentos/métodos , Humanos , Fatores de Transcrição da Família Snail/antagonistas & inibidores , Fatores de Transcrição da Família Snail/genética , Fatores de Transcrição da Família Snail/metabolismo
12.
Nucleic Acids Res ; 49(11): 6456-6473, 2021 06 21.
Artigo em Inglês | MEDLINE | ID: mdl-34107032

RESUMO

RNA-protein interactions are central to all gene expression processes and contribute to a variety of human diseases. Therapeutic approaches targeting RNA-protein interactions have shown promising effects on some diseases that are previously regarded as 'incurable'. Here, we developed a fluorescent on-bead screening platform, RNA Pull-Down COnfocal NAnoscanning (RP-CONA), to identify RNA-protein interaction modulators in eukaryotic cell extracts. Using RP-CONA, we identified small molecules that disrupt the interaction between HuR, an inhibitor of brain-enriched miR-7 biogenesis, and the conserved terminal loop of pri-miR-7-1. Importantly, miR-7's primary target is an mRNA of α-synuclein, which contributes to the aetiology of Parkinson's disease. Our method identified a natural product quercetin as a molecule able to upregulate cellular miR-7 levels and downregulate the expression of α-synuclein. This opens up new therapeutic avenues towards treatment of Parkinson's disease as well as provides a novel methodology to search for modulators of RNA-protein interaction.


Assuntos
Proteína Semelhante a ELAV 1/antagonistas & inibidores , MicroRNAs/antagonistas & inibidores , Quercetina/farmacologia , alfa-Sinucleína/metabolismo , Avaliação Pré-Clínica de Medicamentos/métodos , Proteína Semelhante a ELAV 1/metabolismo , Células HEK293 , Células HeLa , Humanos , MicroRNAs/metabolismo , Microscopia Confocal , RNA Mensageiro/metabolismo , alfa-Sinucleína/genética
13.
Methods Mol Biol ; 2277: 69-89, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34080145

RESUMO

The mitochondrial calcium uniporter (MCU ) is an essential protein of the inner mitochondrial membrane that mediates the uptake of calcium into mitochondria of virtually all mammalian tissues, regulating cell metabolism, signaling, and death. MCU-mediated calcium uptake has been shown to play a pathophysiological role in diverse human disease contexts, which qualifies this channel as a druggable target for therapeutic intervention.Here, we present a protocol to perform drug screens to identify effective and specific MCU-targeting inhibitors. The methodology is based on the use of cryopreserved mitochondria that are isolated from a yeast strain engineered to express the human MCU and its essential regulator EMRE together with the luminescence calcium sensor aequorin. Yeast mitochondria with a functionally reconstituted MCU-mediated calcium uptake are then employed as a ready-to-use screening reagent. False discovery rate is further minimized by energizing mitochondria with D-lactate in a mannitol/sucrose-based medium, which provides a mean to discriminate between direct and secondary effects of drugs on mitochondrial calcium uptake. This screening assay is sensitive and robust and can be easily implemented in any laboratory.


Assuntos
Bloqueadores dos Canais de Cálcio/farmacologia , Canais de Cálcio/metabolismo , Avaliação Pré-Clínica de Medicamentos/métodos , Mitocôndrias/efeitos dos fármacos , Equorina/farmacologia , Cálcio/metabolismo , Canais de Cálcio/genética , Descoberta de Drogas/métodos , Humanos , Ácido Láctico/farmacologia , Mitocôndrias/metabolismo , Mitoxantrona/farmacologia , Saccharomyces cerevisiae/citologia
14.
Methods Mol Biol ; 2277: 133-142, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34080149

RESUMO

Mitochondria are targets of newly synthesized drugs and being tested for the treatment of various diseases caused or accompanied by disruption of cellular bioenergetics. In drug development, it is necessary to test for drug-induced changes in mitochondrial enzyme activity that may be related to therapeutic or adverse drug effects. Measurement of drug effect on mitochondrial oxygen consumption kinetics and/or protective effects of drugs against calcium-induced inhibition of the mitochondrial respiration can be used for the study mitochondrial toxicity and neuroprotective effects of drugs. Supposing that the drug-induced inhibition of the mitochondrial respiratory rate and/or individual mitochondrial complexes is associated with adverse drug effects, the effects of drugs on mitochondrial respiration in isolated mitochondria allow selection of novel molecules that are relatively safe for mitochondrial toxicity.


Assuntos
Avaliação Pré-Clínica de Medicamentos/métodos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Animais , Encéfalo/citologia , Avaliação Pré-Clínica de Medicamentos/instrumentação , Complexo I de Transporte de Elétrons/metabolismo , Complexo III da Cadeia de Transporte de Elétrons/metabolismo , Suínos
15.
Antiviral Res ; 192: 105122, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34186107

RESUMO

There are, besides remdesivir, no approved antivirals for the treatment of SARS-CoV-2 infections. To aid in the search for antivirals against this virus, we explored the use of human tracheal airway epithelial cells (HtAEC) and human small airway epithelial cells (HsAEC) grown at the air-liquid interface (ALI). These cultures were infected at the apical side with one of two different SARS-CoV-2 isolates. Each virus was shown to replicate to high titers for extended periods of time (at least 8 days) and, in particular an isolate with the D614G in the spike (S) protein did so more efficiently at 35 °C than 37 °C. The effect of a selected panel of reference drugs that were added to the culture medium at the basolateral side of the system was explored. Remdesivir, GS-441524 (the parent nucleoside of remdesivir), EIDD-1931 (the parent nucleoside of molnupiravir) and IFN (ß1 and λ1) all resulted in dose-dependent inhibition of viral RNA and infectious virus titers collected at the apical side. However, AT-511 (the free base form of AT-527 currently in clinical testing) failed to inhibit viral replication in these in vitro primary cell models. Together, these results provide a reference for further studies aimed at selecting SARS-CoV-2 inhibitors for further preclinical and clinical development.


Assuntos
Antivirais/farmacologia , COVID-19/tratamento farmacológico , SARS-CoV-2/efeitos dos fármacos , Replicação Viral/efeitos dos fármacos , Animais , Linhagem Celular , Chlorocebus aethiops , Avaliação Pré-Clínica de Medicamentos/métodos , Células Epiteliais/virologia , Humanos , RNA Viral , SARS-CoV-2/isolamento & purificação , Células Vero
16.
J Chromatogr A ; 1651: 462305, 2021 Aug 16.
Artigo em Inglês | MEDLINE | ID: mdl-34147833

RESUMO

Indoleamine 2,3-dioxygenase 1 (IDO1) has been shown to play an important role in the immune escape process of tumors, and therefore is considered as a promising target for tumor immunotherapy. In this study, off-line and on-line capillary electrophoresis methods were developed for IDO1 inhibitors screening from natural product extracts. The optimized separation conditions of CE were achieved with 32 mM sodium tetraborate (pH 9.22) as background electrolyte, using a separation voltage of 21 kV. The off-line CE method was verified by the determination of enzymatic kinetic parameters and inhibitory mechanisms of two known inhibitors. A partial filling on-line CE method combined with rapid polarity switching was used for rapid screening of IDO1 inhibitors. The whole reaction and separation process was completed within 5 min. The on-line CE screening results showed that six of 18 natural products had inhibitory effect on IDO1, namely Carthamus tinctorius, Schisandra chinensis, Raisin, Coffee, Hawthorn and Radix angelicae sinensis. The results of on-line CE experiments were consistent with the off-line results, which proved the practicability and effectiveness of the method for inhibitors screening.


Assuntos
Técnicas de Química Analítica/métodos , Avaliação Pré-Clínica de Medicamentos/métodos , Eletroforese Capilar , Inibidores Enzimáticos/isolamento & purificação , Indolamina-Pirrol 2,3,-Dioxigenase/antagonistas & inibidores , Produtos Biológicos/química , Ativação Enzimática/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Humanos
17.
Methods Mol Biol ; 2268: 137-147, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34085266

RESUMO

Here we describe the stepwise application of bioluminescence resonance energy transfer (BRET)-based conformational receptor biosensors to study GPCR activation in intact cells. This technology can be easily adopted to various plate reader devices and microtiter plate formats. Due to the high sensitivity of these BRET-based receptor biosensors and their ability to quantify simultaneously receptor activation/de-activation kinetics as well as compound efficacy and potency, these optical tools provide the most direct and unbiased approach to monitor GPCR activity in a high-throughput-compatible assay format, representing a novel promising tool for the discovery of potential GPCR therapeutics.


Assuntos
Técnicas de Transferência de Energia por Ressonância de Bioluminescência/métodos , Avaliação Pré-Clínica de Medicamentos/métodos , Corantes Fluorescentes/química , Ensaios de Triagem em Larga Escala/métodos , Luciferases/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Células HEK293 , Humanos , Conformação Proteica , Receptores Acoplados a Proteínas G/química
18.
AAPS PharmSciTech ; 22(5): 183, 2021 Jun 16.
Artigo em Inglês | MEDLINE | ID: mdl-34132921

RESUMO

The current study evaluated the effect of location and amount of various superdisintegrants on the properties of tablets made by twin-screw melt granulation (TSMG). Sodium-croscarmellose (CCS), crospovidone (CPV), and sodium starch glycolate (SSG) were used in various proportions intra- and extra-granular. Tabletability, compactibility, compressibility as well as friability, disintegration, and dissolution performance were assessed. The extra-granular addition resulted in the fasted disintegration and dissolution. CPV performed superior to CCS and SSG. Even if the solid fraction (SF) of the granules was lower for CPV, only a minor decrease in tabletability was observed, due to the high plastic deformation of the melt granules. The intra-granular addition of CPV resulted in a more prolonged dissolution profile, which could be correlated to a loss in porosity during tableting. The 100% intra-granular addition of the CPV resulted in a distinct decrease of the disintegration efficiency, whereas the performance of SSG was unaffected by the granulation process. CCS was not suitable to be used for the production of an immediate-release formulation, when added in total proportion into the granulation phase, but its efficiency was less impaired compared to CPV. Shortest disintegration (78 s) and dissolution (Q80: 4.2 min) was achieved with CPV extra-granular. Using CPV and CCS intra-granular resulted in increased disintegration time and Q80. However, at a higher level of appx. 500 s and appx. 15 min, only SSG showed a process and location independent disintegration and dissolution performance.


Assuntos
Carboximetilcelulose Sódica/síntese química , Química Farmacêutica/métodos , Composição de Medicamentos/métodos , Povidona/síntese química , Carboximetilcelulose Sódica/farmacocinética , Avaliação Pré-Clínica de Medicamentos/métodos , Excipientes/síntese química , Excipientes/farmacocinética , Excipientes Farmacêuticos/síntese química , Excipientes Farmacêuticos/farmacocinética , Porosidade , Povidona/farmacocinética , Solubilidade , Comprimidos , Resistência à Tração
19.
AAPS PharmSciTech ; 22(5): 172, 2021 Jun 07.
Artigo em Inglês | MEDLINE | ID: mdl-34100150

RESUMO

Vaccination development and production was an essential question for the prevention and global control of COVID-19. The strong support from governing authorities such as Operation Warp Speed and robust funding has led to the development and authorization of the tozinameran (BNT162b2) vaccine. The BNT162b2 vaccine is a lipid nanoparticle-encapsulated mRNA that encodes for SARS-CoV-2 spike protein, the main site for neutralizing antibodies. Once it binds with the host cells, the lipid nanoparticles enable the transfer of the RNA, causing S antigens' expression of the SARS-CoV-2, conferring immunity. The vaccine is administered as a 2-dose regime 21 days apart for individuals 16 years and older. Pfizer-BioNTech's BNT162b2 vaccine was the first candidate to receive FDA-Emergency Use Authorization (EUA) on December 11, 2020. During phase 2/3 clinical trials, 95% efficacy was reported among 37,706 participants over the age of 16 who received the BNT162b2 vaccination; additionally, 52% efficacy was noted 12 days following the administration of the first dose of BNT162b2, reflecting early protection of COVID-19. The BNT162b2 vaccine has exhibited 100% efficacy in clinical trials of adolescents between the ages of 12 and 15. Clinical trials in pregnant women and children under the age of 12 are expected to also exhibit promising results. This review article encompasses tozinameran (BNT162b2) vaccine journey, summarizing the BNT162b1 and BNT162b2 vaccines from preclinical studies, clinical trial phases, dosages, immune response, adverse effects, and FDA-EUA.


Assuntos
Vacinas contra COVID-19/administração & dosagem , COVID-19/prevenção & controle , Ensaios Clínicos como Assunto/métodos , Aprovação de Drogas/métodos , SARS-CoV-2/efeitos dos fármacos , Animais , Anticorpos Neutralizantes/efeitos dos fármacos , Anticorpos Neutralizantes/metabolismo , COVID-19/epidemiologia , COVID-19/metabolismo , Vacinas contra COVID-19/efeitos adversos , Vacinas contra COVID-19/metabolismo , Ensaios Clínicos como Assunto/legislação & jurisprudência , Aprovação de Drogas/legislação & jurisprudência , Avaliação Pré-Clínica de Medicamentos/métodos , Exantema/induzido quimicamente , Feminino , Humanos , Masculino , SARS-CoV-2/metabolismo , Glicoproteína da Espícula de Coronavírus/efeitos dos fármacos , Glicoproteína da Espícula de Coronavírus/metabolismo , Vacinação/legislação & jurisprudência , Vacinação/métodos
20.
Int J Mol Sci ; 22(10)2021 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-34063491

RESUMO

Phosphodiesterases (PDEs) hydrolyze cyclic nucleotides to modulate multiple signaling events in cells. PDEs are recognized to actively associate with cyclic nucleotide receptors (protein kinases, PKs) in larger macromolecular assemblies referred to as signalosomes. Complexation of PDEs with PKs generates an expanded active site that enhances PDE activity. This facilitates signalosome-associated PDEs to preferentially catalyze active hydrolysis of cyclic nucleotides bound to PKs and aid in signal termination. PDEs are important drug targets, and current strategies for inhibitor discovery are based entirely on targeting conserved PDE catalytic domains. This often results in inhibitors with cross-reactivity amongst closely related PDEs and attendant unwanted side effects. Here, our approach targeted PDE-PK complexes as they would occur in signalosomes, thereby offering greater specificity. Our developed fluorescence polarization assay was adapted to identify inhibitors that block cyclic nucleotide pockets in PDE-PK complexes in one mode and disrupt protein-protein interactions between PDEs and PKs in a second mode. We tested this approach with three different systems-cAMP-specific PDE8-PKAR, cGMP-specific PDE5-PKG, and dual-specificity RegA-RD complexes-and ranked inhibitors according to their inhibition potency. Targeting PDE-PK complexes offers biochemical tools for describing the exquisite specificity of cyclic nucleotide signaling networks in cells.


Assuntos
Avaliação Pré-Clínica de Medicamentos/métodos , Inibidores de Fosfodiesterase/farmacologia , Extratos Vegetais/farmacologia , Proteínas Quinases/metabolismo , 3',5'-AMP Cíclico Fosfodiesterases/metabolismo , Domínio Catalítico , AMP Cíclico/metabolismo , GMP Cíclico/metabolismo , Polarização de Fluorescência , Terapia de Alvo Molecular , Complexos Multiproteicos/metabolismo , Nucleotídeos Cíclicos/metabolismo , Diester Fosfórico Hidrolases/metabolismo , Bibliotecas de Moléculas Pequenas/farmacologia , Especificidade por Substrato
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