RESUMO
The pharmacokinetics (PK) of methylprednisolone (MPL) exhibited tissue-specific saturable binding and reversible conversion with its metabolite, methylprednisone (MPN). Blood and 11 tissues were collected in male rats after intravenous (i.v.) bolus doses of 50 mg/kg MPL and 20 mg/kg MPN and upon i.v. infusion of MPL and MPN at 0.3, 3, and 10 mg/h per kg. The concentrations of MPL and MPN were simultaneously measured. A comprehensive physiologically based pharmacokinetic (PBPK) model was applied to describe the plasma and tissue profiles and estimate PK parameters of the MPL/MPN interconversion system. Both dosed and formed MPL and MPN were in rapid equilibrium or achieved steady-state rapidly in plasma and tissues. MPL tissue partitioning was nonlinear, with highest capacity in liver (322.9 ng/ml) followed by kidney, heart, intestine, skin, spleen, bone, brain, muscle, and lowest in adipose (2.74 ng/ml) and displaying high penetration in lung. The tissue partition coefficient of MPN was linear but widely variable (0.15â¼5.38) across most tissues, with nonlinear binding in liver and kidney. The conversion of MPL to MPN occurred in kidney, lung, and intestine with total clearance of 429 ml/h, and the back conversion occurred in liver and kidney at 1342 ml/h. The irreversible elimination clearance of MPL was 789 ml/h from liver and that of MPN was 2758 ml/h with liver accounting for 44%, lung 35%, and kidney 21%. The reversible metabolism elevated MPL exposure in rats by 13%. This highly complex PBPK model provided unique and comprehensive insights into the disposition of a major corticosteroid. SIGNIFICANCE STATEMENT: Our dual physiologically based pharmacokinetic (PBPK) study and model of methylprednisolone/methylprednisone (MPL/MPN) with multiple complexities reasonably characterized and parameterized their disposition, and provided greater insights into the interpretation of their pharmacodynamics in rats. Drug knowledge gained in this study may be translatable to higher-order species to appreciate the clinical utility of MPL. The complex model itself is instructive for advanced PBPK analysis of drugs with reversible metabolism and/or nonlinear tissue partitioning features.
Assuntos
Metilprednisolona , Modelos Biológicos , Ratos Sprague-Dawley , Animais , Metilprednisolona/farmacocinética , Metilprednisolona/administração & dosagem , Metilprednisolona/metabolismo , Masculino , Ratos , Distribuição Tecidual , Fígado/metabolismoRESUMO
BACKGROUND: Several reports demonstrated anti-inflammatory properties of minocycline in various inflammatory disorders including colitis. We have experimental evidence suggesting synergistic anti-inflammatory effect of minocycline with methyl prednisolone in reducing colitis severity in mice, but if this effect is in part related to modulating the composition of colonic microbiota is still unknown. METHODS: the effect of vehicle (V), minocycline (M), methyl prednisolone (MP), or combination (C) regimen on the composition of the microbiota of mice in a state of colon inflammation compared to untreated (UT) healthy mice was determined using 16s metagenomic sequencing, and the taxonomic and functional profiles were summarized. RESULTS: Overall, the bacterial flora from the phylum Firmicutes followed by Bacteroidota were found to be predominant in all the samples. However, the composition of Firmicutes was decreased relatively in all the treatment groups compared to UT group. A relatively higher percentage of Actinobacteriota was observed in the samples from the C group. At the genus level, Muribaculaceae, Bacteroides, Bifidobacterium, and Lactobacillus were found to be predominant in the samples treated with both drugs (C). Whereas "Lachnospiraceae NK4A136 group" and Helicobacter in the M group, and Helicobacter in the MP group were found to be predominant. But, in the UT group, Weissella and Staphylococcus were found to be predominant. Eubacterium siraeum group, Clostridia vadinBB60 group, Erysipelatoclostridium and Anaeroplasma genera were identified to have a significant (FDR p < 0.05) differential abundance in V compared to C and UT groups. While at the species level, the abundance of Helicobacter mastomyrinus, Massiliomicrobiota timonensis and uncultured Anaeroplasma were identified as significantly low in UT, C, and M compared to V group. Functional categories related to amino acid, carbohydrate, and energy metabolism, cell motility and cell cycle control were dominated overall across all the samples. Methane metabolism was identified as an enriched pathway. For the C group, "Colitis (decrease)" was among the significant (p = 1.81E-6) associations based on the host-intrinsic taxon set. CONCLUSION: Combination regimen of minocycline plus methyl prednisolone produces a synergistic anti-inflammatory effect which is part related to alternation in the colonic microbiota composition.
Assuntos
Colite , Minociclina , Camundongos , Animais , Minociclina/farmacologia , Minociclina/uso terapêutico , Minociclina/metabolismo , Sulfato de Dextrana/metabolismo , Sulfato de Dextrana/farmacologia , Sulfato de Dextrana/uso terapêutico , Metilprednisolona/metabolismo , Metilprednisolona/farmacologia , Metilprednisolona/uso terapêutico , Colo , Colite/tratamento farmacológico , Inflamação/tratamento farmacológico , Bactérias , Anti-Inflamatórios/farmacologia , Anti-Inflamatórios/uso terapêutico , Anti-Inflamatórios/metabolismo , Modelos Animais de Doenças , Camundongos Endogâmicos C57BLRESUMO
BACKGROUND: The engagement of the complement regulatory proteins CD46 and CD3 in human CD4+ T cells induces the type 1 regulatory T cells (Tr1) and interleukin-10 (IL-10) secretion. This study aimed to elucidate the molecular changes of Tr1 cells through CD46 cytoplasmic Cyt1 tail in lupus nephritis (LN) respond to intravenous methylprednisolone (ivMP) therapy. METHODS: We enrolled 40 pediatric patients with LN and 30 healthy controls. Clinical characteristics and peripheral blood mononuclear cells were collected before and 3 days after the administration of ivMP. Kidney specimens were taken from five LN and five minimal-change nephrotic syndrome patients. RESULTS: We found that defective CD46-mediated T-helper type 1 contraction (IL-10 switching) is present in active LN patients. The ivMP therapy enhanced LN remission, restored the production of IL-10, increased the CD46-Cyt1/Cyt2 ratio, AKT, and cAMP-responsive element-binding protein phosphorylation, and induced migration with the expression of chemokine receptor molecules CCR4, CCR6, and CCR7 of CD3/CD46-activated Tr1 cells. CONCLUSIONS: Pharmacologic interventions that alter the patterns of CD46-Cyt1/Cyt2 expression and the secretion of IL-10 by CD3/CD46-activated Tr1 cells can be used in patients with active LN. IMPACT: In patients with LN, ivMP was associated with increased IL-10 production and increased CD46-Cyt1/Cyt2 ratio and AKT phosphorylation by Tr1 cells, with enhanced potential to migration in response to CCL17. These results suggest that expression levels of CD46 isoforms Cyt1 and Cyt2 in CD4 + CD46 + Tr1 cells differ in patients with active LN but can be corrected by corticosteroid treatment. Enhancing the expression of functional CD4 + CD46 + Tr1 cells may be a useful therapeutic approach for LN.
Assuntos
Interleucina-10 , Nefrite Lúpica , Humanos , Criança , Interleucina-10/metabolismo , Linfócitos T Reguladores/metabolismo , Nefrite Lúpica/tratamento farmacológico , Proteína Cofatora de Membrana/metabolismo , Receptores CCR7/metabolismo , Leucócitos Mononucleares/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Linfócitos T CD4-Positivos , Metilprednisolona/farmacologia , Metilprednisolona/uso terapêutico , Metilprednisolona/metabolismo , Isoformas de Proteínas/metabolismo , Corticosteroides/uso terapêuticoRESUMO
Synthetic glucocorticoids such as methylprednisolone are compounds of fundamental interest to the pharmaceutical industry as their modifications within the sterane scaffold lead to higher inflammatory potency and reduced side effects compared with their parent compound cortisol. In methylprednisolone production, the complex chemical hydroxylation of its precursor medrane in position C21 exhibits poor stereo- and regioselectivity making the process unprofitable and unsustainable. By contrast, the use of a recombinant E. coli system has recently shown high suitability and efficiency. In this study, we aim to overcome limitations in this biotechnological medrane conversion yielding the essential methylprednisolone-precursor premedrol by optimizing the CYP21A2-based whole-cell system on a laboratory scale. We successfully improved the whole-cell process in terms of premedrol production by (a) improving the electron supply to CYP21A2; here we use the N-terminally truncated version of the bovine NADPH-dependent cytochrome P450 reductase (bCPR-27 ) and coexpression of microsomal cytochrome b5 ; (b) enhancing substrate access to the heme by modification of the CYP21A2 substrate access channel; and (c) circumventing substrate inhibition which is presumed to be the main limiting factor of the presented system by developing an improved fed-batch protocol. By overcoming the presented limitations in whole-cell biotransformation, we were able to achieve a more than 100% improvement over the next best system under equal conditions resulting in 691 mg·L-1 ·d-1 premedrol.
Assuntos
Escherichia coli/genética , Engenharia Metabólica/métodos , Metilprednisolona , Proteínas Recombinantes/metabolismo , Esteroide 21-Hidroxilase/metabolismo , Animais , Biotransformação , Bovinos , Escherichia coli/metabolismo , Hidroxilação , Metilprednisolona/análogos & derivados , Metilprednisolona/análise , Metilprednisolona/química , Metilprednisolona/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Esteroide 21-Hidroxilase/química , Esteroide 21-Hidroxilase/genéticaRESUMO
The plasma and tissue binding properties of two corticosteroids, dexamethasone (DEX) and methylprednisolone (MPL), were assessed in the rat in anticipation of developing physiologically based pharmacokinetic and pharmacokinetic/pharmacodynamic models. The tissue-to-plasma partition coefficients (K P) of DEX and MPL were measured in liver, muscle, and lung in vivo after steady-state infusion and bolus injection in rats. Since K P is often governed by reversible binding to macromolecules in blood and tissue, an attempt was made to assess K P values of DEX and MPL by in vitro binding studies using rat tissue homogenates and to compare these estimates to those obtained from in vivo kinetics after dosing. The K P values of both steroids were also calculated in rat tissues using mechanistic tissue composition-based equations. The plasma binding of DEX and MPL was linear with moderate binding (60.5% and 82.5%) in male and female rats. In vivo estimates of steroid uptake appeared linear across the tested concentrations and K P was highest in liver and lowest in muscle for both steroids. Assessment of hepatic binding of MPL in vitro was severely affected by drug loss at 37°C in male liver homogenates, whereas DEX was stable in both male and female liver homogenates. With the exception of MPL in liver, in vitro-derived K P estimates reasonably agreed with in vivo values. The mechanistic equations modestly underpredicted K P for both drugs. Tissue metabolism, saturable tissue binding, and active uptake are possible factors that can complicate assessments of in vivo tissue binding of steroids when using tissue homogenates. SIGNIFICANCE STATEMENT: Assuming the free hormone hypothesis, the ratio of the unbound drug fraction in plasma and in tissues defines the tissue-to-plasma partition coefficient (K P), an important parameter in physiologically based pharmacokinetic modeling that determines total drug concentrations within tissues and the steady-state volume of distribution. This study assessed the plasma and tissue binding properties of the synthetic corticosteroids, dexamethasone and methylprednisolone, in rats using ultrafiltration and tissue homogenate techniques. In vitro-in vivo and in silico-in vivo extrapolation of K P was assessed for both drugs in liver, muscle, and lung. Although the extrapolation was fairly successful across the tissues, in vitro homogenate studies severely underpredicted the K P of methylprednisolone in liver, partly attributable to the extensive hepatic metabolism.
Assuntos
Dexametasona/farmacologia , Dexametasona/farmacocinética , Metilprednisolona/farmacologia , Metilprednisolona/farmacocinética , Modelos Biológicos , Animais , Proteínas Sanguíneas/metabolismo , Simulação por Computador , Dexametasona/metabolismo , Estabilidade de Medicamentos , Feminino , Masculino , Metilprednisolona/metabolismo , Ratos , Ratos Wistar , Distribuição TecidualRESUMO
Our previous report examined the pharmacokinetics (PK) of methylprednisolone (MPL) and adrenal suppression after a 50 mg/kg IM bolus in male and female rats, and we described in detail the development of a minimal physiologically based pharmacokinetic/pharmacodynamic (mPBPK/PD) model. In continuation of such assessments, we investigated sex differences in genomic MPL responses (PD). Message expression of the glucocorticoid-induced leucine zipper (GILZ) was chosen as a multitissue biomarker of glucocorticoid receptor (GR)-mediated drug response. Potential time-dependent interplay between sex hormone and glucocorticoid signaling in vivo was assessed by comparing the enhancement of GILZ by MPL in the uterus [high estrogen receptor (ER) density] and in liver (lower ER density) from male and female rats dosed within the proestrus (high estradiol/progesterone) and estrus (low estradiol/progesterone) phases of the rodent estrous cycle. An expanded-systems PD model of MPL considering circadian rhythms, multireceptor (ER and GR) control, and estrous variations delineated the determinants controlling receptor/gene-mediated steroid responses. Hepatic GILZ response was â¼3-fold greater in females, regardless of estrous stage, compared with males, driven predominantly by increased MPL exposure in females and a negligible influence of estrogen interaction. In contrast, GILZ response in the uterus during proestrus in females was 60% of that observed in estrus-phased females, despite no PK or receptor differences, providing in vivo support to the hypothesis of estrogen-mediated antagonism of glucocorticoid signaling. The developed model offers a mechanistic platform to assess the determinants of sex and tissue specificity in corticosteroid actions and, in turn, reveals a unique PD drug-hormone interaction occurring in vivo. SIGNIFICANCE STATEMENT: Mechanisms relating to sex-based pharmacodynamic variability in genomic responses to corticosteroids have been unclear. Using combined experimental and systems pharmacology modeling approaches, sex differences in both pharmacokinetic and pharmacodynamic mechanisms controlling the enhancement of a sensitive corticosteroid-regulated biomarker, the glucocorticoid-induced leucine zipper (GILZ), were clarified in vivo. The multiscale minimal physiologically based pharmacokinetics/pharmacodynamic model successfully captured the experimental observations and quantitatively discerned the roles of the rodent estrous cycle (hormonal variation) and tissue specificity in mediating the antagonistic coregulation of GILZ gene synthesis. These findings collectively support the hypothesis that estrogens antagonize pharmacodynamic signaling of genomic corticosteroid actions in vivo in a time- and estrogen receptor-dependent manner.
Assuntos
Ciclo Estral/efeitos dos fármacos , Metilprednisolona/farmacologia , Metilprednisolona/farmacocinética , Modelos Biológicos , Receptores de Estrogênio/metabolismo , Fatores de Transcrição/antagonistas & inibidores , Animais , Estradiol/sangue , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Masculino , Metilprednisolona/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Caracteres Sexuais , Fatores de Transcrição/genéticaRESUMO
A novel drug delivery system mediated by ultrasound (US) combined with microbubbles (MBs) (US+MB) could improve local drug concentration to enhance its efficacy. To investigate the influence of US+MB on methylprednisolone (MP), the effect of US+MB combined with MP (US+MB+MP) on lipopolysaccharide (LPS)-induced human mesangial cells (HMCs) and the underlying mechanism were explored in this study. The results revealed that HMCs treated with LPS underwent significant proliferation and exhibited an increase in nuclear transcription factor-κB (NF-κB) and transforming growth factor-ß1 (TGF-ß1) expression and a decrease in cellular apoptosis. This effect was significantly inhibited by MP (30-100 µg/ml), US combined with MBs (3.22 × 107 and 8.05 × 107 bubbles/ml), and US combined with both MBs (1.29 × 107 bubbles/ml) and MP (12 µg/ml) (US+MB1+MP12). The effect of US+MB1+MP12 was better than the effect of 12 µg/ml of MP alone and was similar to the effect of 100 µg/ml of MP. Additionally, the intracellular free MP content was significantly higher in the US+MB1+MP12 group than in the MP12 group. US combined with MBs not only inhibited LPS-induced HMC proliferation and NF-κB and TGF-ß1 expression and increased cellular apoptosis but also synergized with the pharmacologic effect of MP. The mechanism is partially due to the US-assisted MB local drug delivery and the anti-inflammatory effect induced by US combined with MBs.
Assuntos
Sistemas de Liberação de Medicamentos/métodos , Lipopolissacarídeos/farmacologia , Células Mesangiais/efeitos dos fármacos , Metilprednisolona/farmacologia , Microbolhas , Ondas Ultrassônicas , Apoptose/efeitos dos fármacos , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Espaço Intracelular/metabolismo , Células Mesangiais/citologia , Células Mesangiais/metabolismo , Metilprednisolona/metabolismo , NF-kappa B/metabolismo , Fator de Crescimento Transformador beta1/metabolismoRESUMO
BACKGROUND: Synthetic glucocorticoids like methylprednisolone (medrol) are of high pharmaceutical interest and represent powerful drugs due to their anti-inflammatory and immunosuppressive effects. Since the chemical hydroxylation of carbon atom 21, a crucial step in the synthesis of the medrol precursor premedrol, exhibits a low overall yield because of a poor stereo- and regioselectivity, there is high interest in a more sustainable and efficient biocatalytic process. One promising candidate is the mammalian cytochrome P450 CYP21A2 which is involved in steroid hormone biosynthesis and performs a selective oxyfunctionalization of C21 to provide the precursors of aldosterone, the main mineralocorticoid, and cortisol, the most important glucocorticoid. In this work, we demonstrate the high potential of CYP21A2 for a biotechnological production of premedrol, an important precursor of medrol. RESULTS: We successfully developed a CYP21A2-based whole-cell system in Escherichia coli by coexpressing the cDNAs of bovine CYP21A2 and its redox partner, the NADPH-dependent cytochrome P450 reductase (CPR), via a bicistronic vector. The synthetic substrate medrane was selectively 21-hydroxylated to premedrol with a max. yield of 90 mg L(-1) d(-1). To further improve the biocatalytic activity of the system by a more effective electron supply, we exchanged the CPR with constructs containing five alternative redox systems. A comparison of the constructs revealed that the redox system with the highest endpoint yield converted 70 % of the substrate within the first 2 h showing a doubled initial reaction rate compared with the other constructs. Using the best system we could increase the overall yield of premedrol to a maximum of 320 mg L(-1) d(-1) in shaking flasks. Optimization of the biotransformation in a bioreactor could further improve the premedrol gain to a maximum of 0.65 g L(-1) d(-1). CONCLUSIONS: We successfully established a CYP21-based whole-cell system for the biotechnological production of premedrol, a pharmaceutically relevant glucocorticoid, in E. coli and could improve the system by optimizing the redox system concerning reaction velocity and endpoint yield. This is the first step for a sustainable replacement of a complicated chemical low-yield hydroxylation by a biocatalytic cytochrome P450-based whole-cell system.
Assuntos
Escherichia coli/metabolismo , Engenharia Metabólica , Metilprednisolona/metabolismo , NADPH-Ferri-Hemoproteína Redutase/genética , Esteroide 21-Hidroxilase/genética , Animais , Biocatálise , Reatores Biológicos , Biotransformação , Bovinos , Escherichia coli/genética , Vetores Genéticos , NADPH-Ferri-Hemoproteína Redutase/metabolismo , Oxirredução , Esteroide 21-Hidroxilase/metabolismoRESUMO
Exserohilum rostratum caused a multistate fungal meningitis outbreak following iatrogenic inoculation of contaminated methylprednisolone in the United States. To gain insight into the immunopathogenesis of this infection, we studied the innate host responses of human neutrophils against E. rostratum conidia and hyphae with or without methylprednisolone. The neutrophil-induced percentage fungal damage against conidia and hyphae was effector-to-target ratio dependent (≤55%). While methylprednisolone did not affect neutrophil-induced fungal damage by treatment of Exserohilum or neutrophils, it compromised phagocytosis of conidia (P < 0.05). These findings suggest that methylprednisolone-treated neutrophils may have altered phagocytic clearance of Exserohilum conidia, reducing host capacity to contain the invasive process.
Assuntos
Ascomicetos/imunologia , Metilprednisolona/metabolismo , Neutrófilos/efeitos dos fármacos , Neutrófilos/imunologia , Fagocitose/efeitos dos fármacos , Células Cultivadas , Humanos , Hifas/imunologia , Esporos Fúngicos/imunologiaRESUMO
Molecular docking studies were performed on 18 17ß-carboxamide steroids in order to select compounds with potential local anti-inflammatory activity. These derivatives are amides of cortienic acids (obtained from hydrocortisone, prednisolone, and methylprednisolone) with methyl or ethyl esters of six amino acids. Interactions with the glucocorticoid receptor (GR), binding energies and ligand efficiency values of these compounds were compared with dexamethasone and cortienic acid obtained from prednisolone (inactive metabolite). On the basis of molecular docking studies, seven compounds were selected and their binding affinities for the GR were predicted by use of the exponential model created in this study. Subsequently, selected compounds were synthesized in good yields by use of modified N,N'-dicyclohexylcarbodiimide (DCC)/1-hydroxybenzotriazole (HOBt) coupling procedure. Finally, the local anti-inflammatory activity of the synthesized compounds was examined by use of the croton oil-induced ear edema test. In vivo evaluation of systemic side effects as well as in silico prediction of metabolism were performed on the derivative with the best local anti-inflammatory activity. The combination of molecular docking studies and the exponential model for the GR binding affinity prediction could be used as an in silico tool for the rational design of novel 17ß-carboxamide steroids with potentially better biological profile than dexamethasone.
Assuntos
Anti-Inflamatórios/síntese química , Anti-Inflamatórios/farmacologia , Desenho de Fármacos , Edema/prevenção & controle , Glucocorticoides/síntese química , Glucocorticoides/farmacologia , Inflamação/prevenção & controle , Animais , Anti-Inflamatórios/metabolismo , Biotransformação , Óleo de Cróton , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Edema/induzido quimicamente , Glucocorticoides/metabolismo , Hidrocortisona/análogos & derivados , Hidrocortisona/síntese química , Hidrocortisona/metabolismo , Hidrocortisona/farmacologia , Inflamação/induzido quimicamente , Ligantes , Metilprednisolona/análogos & derivados , Metilprednisolona/síntese química , Metilprednisolona/metabolismo , Metilprednisolona/farmacologia , Modelos Biológicos , Simulação de Acoplamento Molecular , Estrutura Molecular , Prednisolona/análogos & derivados , Prednisolona/síntese química , Prednisolona/metabolismo , Prednisolona/farmacologia , Ratos , Receptores de Glucocorticoides/efeitos dos fármacos , Receptores de Glucocorticoides/metabolismo , Relação Estrutura-AtividadeRESUMO
BACKGROUND: Co-administration of a reduction inhibitor and a colon-specific prodrug of a glucocorticoid susceptible to colonic reductive metabolism is suggested as a strategy to circumvent the therapeutic loss of the glucocorticoid delivered to and acting locally at the large intestine. AIMS: We examined whether the strategy was feasible as a pharmacotherapy for treatment of inflammatory bowel disease. METHODS: Glycyrrhizin (GCZ), a reduction inhibitor, was tested for its inhibition of the colonic metabolism of methylprednisolone (MP). Therapeutic activity against TNBS-induced rat colitis and adrenal suppression were compared after oral administration of methylprednisolone 21-sulfate sodium (MPS), a colon-specific prodrug of MP, or MPS/GCZ to colitic rats. RESULTS: Upon incubation of MP with the cecal contents, MP disappeared, and this was delayed by addition of GCZ. In addition, more MP produced from MPS in the cecal contents accumulated in the presence of GCZ. Consistent with these results, upon oral administration of MPS/GCZ, MPS or MP, MP was detected at a greater level in the large intestine for MPS/GCZ. MPS/GCZ ameliorated TNBS-induced colitis of rats, and this therapeutic effect was superior to that of MPS and MP. Moreover, MPS/GCZ decreased the plasma levels of corticosterone and ACTH to a greater extent than MPS, but less than MP. CONCLUSIONS: Co-administration of GCZ, a reduction inhibitor, may be a plausible strategy to reduce the therapeutic loss of MP produced from MPS in the large intestine, thus improving the therapeutic property of the prodrug against inflammatory bowel disease.
Assuntos
Anti-Inflamatórios/uso terapêutico , Colite/tratamento farmacológico , Ácido Glicirrízico/uso terapêutico , Metilprednisolona/uso terapêutico , Glândulas Suprarrenais/efeitos dos fármacos , Animais , Anti-Inflamatórios/metabolismo , Colite/metabolismo , Avaliação Pré-Clínica de Medicamentos , Sinergismo Farmacológico , Masculino , Metilprednisolona/metabolismo , Pró-Fármacos/uso terapêutico , Ratos , Ratos Sprague-DawleyRESUMO
Spinal cord injury (SCI) can cause permanent loss of sensory and motor function, and there is no effective clinical treatment, to date. Due to the complex pathological process involved after injury, synergistic treatments are very urgently needed in clinical practice. We designed a nanofiber scaffold hyaluronic acid hydrogel patch to release both exosomes and methylprednisolone to the injured spinal cord in a non-invasive manner. This composite patch showed good biocompatibility in the stabilization of exosome morphology and toxicity to nerve cells. Meanwhile, the composite patch increased the proportion of M2-type macrophages and reduced neuronal apoptosis in an in vitro study. In vivo, the functional and electrophysiological performance of rats with SCI was significantly improved when the composite patch covered the surface of the hematoma. The composite patch inhibited the inflammatory response through macrophage polarization from M1 type to M2 type and increased the survival of neurons by inhibition neuronal of apoptosis after SCI. The therapeutic effects of this composite patch can be attributed to TLR4/NF-κB, MAPK, and Akt/mTOR pathways. Thus, the composite patch provides a medicine-exosomes dual-release system and may provide a non-invasive method for clinical treatment for individuals with SCI.
Assuntos
Exossomos , Traumatismos da Medula Espinal , Ratos , Animais , Metilprednisolona/farmacologia , Metilprednisolona/uso terapêutico , Metilprednisolona/metabolismo , Exossomos/metabolismo , Traumatismos da Medula Espinal/tratamento farmacológico , Traumatismos da Medula Espinal/patologia , Macrófagos/metabolismo , Neurônios/metabolismo , Medula Espinal/patologiaRESUMO
Pulmonary inflammation is one of the most reported tissue inflammations in clinic. Successful suppression of inflammation is vital to prevent further inevitably fatal lung degeneration. Glucocorticoid hormone, such as methylprednisolone (MP), is the most applied strategy to control the inflammatory progression yet faces the challenge of systemic side effects caused by the requirement of large-dosage and frequent administration. Highly efficient delivery of MP specifically targeted to inflammatory lung sites may overcome this challenge. Therefore, the present study develops an inflammation-targeted biomimetic nanovehicle, which hybridizes the cell membrane of mesenchymal stem cell with liposome, named as MSCsome. This hybrid nanovehicle shows the ability of high targeting specificity toward inflamed lung cells, due to both the good lung endothelium penetration and the high uptake by inflamed lung cells. Consequently, a single-dose administration of this MP-loaded hybrid nanovehicle achieves a prominent treatment of lipopolysaccharide-induced lung inflammation, and negligible treatment-induced side effects are observed. The present study provides a powerful inflammation-targeted nanovehicle using biomimetic strategy to solve the current challenges of targeted inflammation intervention.
Assuntos
Inflamação , Pneumonia , Humanos , Inflamação/tratamento farmacológico , Inflamação/metabolismo , Pneumonia/tratamento farmacológico , Pneumonia/metabolismo , Metilprednisolona/metabolismo , Metilprednisolona/farmacologia , Metilprednisolona/uso terapêutico , Pulmão/metabolismo , Lipossomos/farmacologiaRESUMO
OBJECTIVES: Methylprednisolone (mPSL) pulse therapy is an essential option for patients with active systemic lupus erythematosus, but there is a risk of adverse events related to microcirculation disorders, including idiopathic osteonecrosis of the femoral head (ONFH). Recent studies have revealed that excessive neutrophil extracellular traps (NETs) are involved in microcirculation disorders. This study aimed to demonstrate that mPSL pulse could induce NETs in lupus mice and identify the factors contributing to this induction. METHODS: Six mice with imiquimod (IMQ)-induced lupus-like disease and six normal mice were intraperitoneally injected with mPSL on days 39 to 41, and five mice with IMQ-induced lupus-like disease and six normal mice were injected with phosphate-buffered saline. Pathological examinations were conducted to evaluate the ischaemic state of the femoral head and tissue infiltration of NET-forming neutrophils. Proteome analysis was performed to extract plasma proteins specifically elevated in mPSL-administered mice with IMQ-induced lupus-like disease, and their effects on NET formation were assessed in vitro. RESULTS: Mice with IMQ-induced lupus-like disease that received mPSL pulse demonstrated ischaemia of the femoral head cartilage with tissue infiltration of NET-forming neutrophils. Proteome analysis suggested that prenylcysteine oxidase 1 (PCYOX1) played a role in this phenomenon. The reaction of PCYOX1-containing very low-density lipoproteins (VLDL) with its substrate farnesylcysteine (FC) induced NETs in vitro. The combined addition of IMQ and mPSL synergistically enhanced VLDL-plus-FC-induced NET formation. CONCLUSION: PCYOX1 and related factors are worthy of attention to understand the underlying mechanisms and create novel therapeutic strategies for mPSL-mediated microcirculation disorders, including ONFH.
Assuntos
Armadilhas Extracelulares , Lúpus Eritematoso Sistêmico , Camundongos , Humanos , Animais , Metilprednisolona/uso terapêutico , Metilprednisolona/metabolismo , Metilprednisolona/farmacologia , Cabeça do Fêmur/patologia , Imiquimode/metabolismo , Imiquimode/farmacologia , Imiquimode/uso terapêutico , Lúpus Eritematoso Sistêmico/induzido quimicamente , Lúpus Eritematoso Sistêmico/tratamento farmacológico , Proteoma/metabolismo , Proteoma/farmacologia , Cartilagem , Isquemia/metabolismo , Isquemia/patologiaRESUMO
RATIONALE: The metabolism of methylprednisolone is revisited in order to find new metabolites that could be important for distinguishing between different routes of administration. Recently developed liquid chromatography/tandem mass spectrometry (LC/MS/MS) strategies for the detection of corticosteroid metabolites have been applied to the study of methylprednisolone metabolism. METHODS: The structures of these metabolites were studied using two complementary mass spectrometric techniques: LC/MS/MS in product ion scan mode with electrospray ionization and gas chromatography/mass spectrometry (GC/MS) in full scan mode with electron ionization. Metabolites were also isolated by semipreparative liquid chromatography fractionation. Each fraction was divided into two aliquots; one was studied by LC/MS/MS and the other by GC/MS after methoxyamine-trimethylsilyl derivatization. RESULTS: The combination of all the structural information allowed us to propose a comprehensive picture of methylprednisolone metabolism in humans. Overall, 15 metabolites including five previously unreported compounds have been detected. Specifically, 16ß,17α,21-trihydroxy-6α-methylpregna-1,4-diene-3,11,20-trione, 17α,20ß,21-trihydroxy-6α-methylpregna-1,4-diene-3, 11-dione, 11ß,17α,21-trihydroxy-6α-hydroxymethylpregna-1,4-diene-3,20-dione, 11ß,17α,20ξ,21-tetrahydroxy-6α-hydroxymethylpregna-1,4-diene-3-one, and 17α,21-dihydroxy-6α-hydroxymethylpregna-1,4-diene-3,11,20-trione are proposed as feasible structures for the novel metabolites. In addition to the expected biotransformations: reduction of the C20 carbonyl, oxidation of the C11 hydroxy group, and further 6ß-hydroxylation, we propose that hydroxylation of the 6α-methyl group can also take place. CONCLUSIONS: New metabolites have been identified in urine samples collected after oral administration of 40 mg of methylprednisolone. All identified metabolites were found in all samples collected up to 36 h after oral administration. However, after topical administration of 5 g of methylprednisolone aceponate, neither the parent compound nor any of the metabolites were detected.
Assuntos
Cromatografia Gasosa-Espectrometria de Massas , Glucocorticoides/metabolismo , Glucocorticoides/urina , Metilprednisolona/metabolismo , Metilprednisolona/urina , Espectrometria de Massas em Tandem , Administração Oral , Administração Tópica , Cromatografia Líquida/métodos , Cromatografia Gasosa-Espectrometria de Massas/métodos , Glucocorticoides/administração & dosagem , Humanos , Masculino , Metilprednisolona/administração & dosagem , Espectrometria de Massas em Tandem/métodosRESUMO
OBJECTIVES: In this study, we aimed to evaluate the antioxidant, anti-apoptotic, osteoblastic and hypolipidemic effects of thymoquinone (TQ) treatment on the steroid-induced osteonecrosis of femoral head (ONFH) model in rats. MATERIALS AND METHODS: A total of 24 rats were randomly divided into four groups: the control group administered saline; the TQ group administered 10 mg/kg/day TQ orally; lipopolysaccharide/methylprednisolone (LPS/MPS) group administered 20 µg/kg intraperitoneally LPS and 40 mg/kg intramuscularly MPS to establish ONFH model; and the LPS/ MPS+TQ group administered both LPS/MPS and, then, TQ once daily for four weeks. All rats were sacrificed after intracardiac blood collection and their right femurs were removed. RESULTS: Micro-computed tomography showed a higher bone mineral density and lower porosity, Tr. Sp and Tr. Sep data were detected in the LPS/MPS+TQ group. In histopathology, osteonecrosis increased significantly in the LPS/MPS group and osteonecrosis decreased in the LPS/MPS+TQ group compared to the LPS/MPS group (p=0.0077). Histomorphometric examination revealed that the percentage of BV/TV in the LPS/MPS group was significantly lower compared to control and other groups (p<0.01 and p<0.05, respectively), while it reached normal rates in the LPS/MPS+TQ group. Immunohistochemically, antioxidant, anti-apoptotic, and angiogenesis indicators (8-hydroxy-20- deoxyguanosine [8-OHdG], malondialdehyde [MDA], B-cell lymphoma [Bcl-2], caspase-3, vascular endothelial growth factor [VEGF]) were significantly improved in tissue and serum with TQ. Furthermore, TQ significantly reduced low- and high-density lipoprotein cholesterol ratio and carboxy-terminal type 1 collagen crosslink (CTX) in serum. CONCLUSION: Vascular and hematopoietic cell damages that occur due to steroid-induced deoxyribonucleic acid (DNA) oxidative and lipid peroxidative damages in an ONFH model can be successfully ameliorated by TQ administration. This antioxidant and anti-apoptotic effects of TQ may be a promising treatment option for early stage of osteonecrosis.
Assuntos
Necrose da Cabeça do Fêmur , Cabeça do Fêmur , Animais , Ratos , Antioxidantes , Modelos Animais de Doenças , Cabeça do Fêmur/patologia , Necrose da Cabeça do Fêmur/induzido quimicamente , Necrose da Cabeça do Fêmur/diagnóstico por imagem , Necrose da Cabeça do Fêmur/tratamento farmacológico , Lipopolissacarídeos/metabolismo , Metilprednisolona/metabolismo , Esteroides/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Microtomografia por Raio-XRESUMO
OBJECTIVE: Improvement in ex vivo lung perfusion protocols could increase the number of donors available for transplantation and protect the lungs from primary graft dysfunction. We hypothesize that perfusate adsorption during ex vivo lung perfusion reconditions the allograft to ischemia-reperfusion injury after lung transplantation. METHODS: Donor pig lungs were preserved for 24 hours at 4°C, followed by 6 hours of ex vivo lung perfusion according to the Toronto protocol. The perfusate was additionally adsorbed through a CytoSorb adsorber (CytoSorbents, Berlin, Germany) in the treatment group, whereas control lungs were perfused according to the standard protocol (n = 5, each). Ex vivo lung perfusion physiology and biochemistry were monitored. Upon completion of ex vivo lung perfusion, a left single lung transplantation was performed. Oxygenation function and lung mechanics were assessed during a 4-hour reperfusion period. The inflammatory response was determined during ex vivo lung perfusion and reperfusion. RESULTS: The cytokine concentrations in the perfusate were markedly lower with the adsorber, resulting in improved ex vivo lung perfusion physiology and biochemistry during the 6-hour perfusion period. Post-transplant dynamic lung compliance was markedly better during the 4-hour reperfusion period in the treatment group. Isolated allograft oxygenation function and dynamic compliance continued to be superior in the adsorber group at the end of reperfusion, accompanied by a markedly decreased local inflammatory response. CONCLUSIONS: Implementation of an additional cytokine adsorber has refined the standard ex vivo lung perfusion protocol. Furthermore, cytokine removal during ex vivo lung perfusion improved immediate post-transplant graft function together with a less intense inflammatory response to reperfusion in pigs. Further studies are warranted to understand the beneficial effects of perfusate adsorption during ex vivo lung perfusion in the clinical setting.
Assuntos
Transplante de Pulmão/métodos , Pulmão/metabolismo , Perfusão/métodos , Adsorção , Animais , Citocinas/metabolismo , Feminino , Humanos , Pulmão/patologia , Pulmão/fisiologia , Meropeném/metabolismo , Metilprednisolona/metabolismo , Suínos , Resultado do TratamentoRESUMO
Sepsis has become a major public health problem worldwide. Methylprednisolone sodium succinate (MP) is a commonly used drug to prevent inflammation. However, the role and underlying mechanism of MP in sepsis remain vague. MP inhibited the lipopolysaccharide (LPS)-induced production of tumor necrosis factor-alpha (TNF-α) and interleukin (IL)-17 and suppressed cell growth in alveolar type II epithelial cells (ATII cells). Small nucleolar RNA host gene 5 (SNHG5) expression was inhibited by LPS and restored by MP. Upregulation of SNHG5 inhibited the cellular role of LPS in ATII cells, and further, downregulation of SNHG5 inhibited the cellular role of MP in ATII cells under LPS conditions. SNHG5 elevated the expression of Copine 1 (CPNE1) by enhancing the mRNA stability of CPNE1. Increasing CPNE1 expression restored the silenced SNHG5-induced inhibitor role of MP in ATII cells under LPS conditions. Finally, MP attenuated lung injury and TNF-α and IL-17 secretion in an LPS-induced sepsis mouse model. Overall, this study investigated the mechanism underlying the effect of MP treatment in sepsis and, for the first time, revealed the important role of the SNHG5/CPNE1 pathway in the development and treatment of sepsis and the potential to serve as a diagnostic and therapeutic target for sepsis.
Assuntos
Proteínas de Ligação ao Cálcio/metabolismo , Metilprednisolona/farmacologia , Sepse/tratamento farmacológico , Células Epiteliais Alveolares/efeitos dos fármacos , Células Epiteliais Alveolares/metabolismo , Animais , Apoptose/efeitos dos fármacos , Proteínas de Ligação ao Cálcio/efeitos dos fármacos , Proteínas de Ligação ao Cálcio/genética , Proteínas de Transporte , Ciclo Celular/efeitos dos fármacos , Proliferação de Células/genética , Feminino , Inflamação , Lipopolissacarídeos/farmacologia , Metilprednisolona/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , MicroRNAs/genética , RNA Longo não Codificante/genética , RNA Nucleolar Pequeno/genética , Sepse/metabolismo , Transdução de Sinais/efeitos dos fármacosRESUMO
A new, simple, sensitive and specific liquid chromatography/electrospray ionization tandem mass spectrometric (LC/ESI-MS/MS) method in precursor ion scanning (PIS) mode has been developed for the rapid detection of methylprednisolone acetate (MPA) and its metabolites in rat urine. A suitable product ion specific for methylprednisolone (MP) and MPA was selected after a fragmentation study on 20 (cortico)steroids at different collision energies (5-40 eV). Urine samples were simply treated with acetonitrile then dried in a SpeedVac system. The method was validated and compared with other PIS methods for detecting corticosteroids in human urine. It was more sensitive, with limit of detection (LOD) and lower limit of quantitation (LLOQ), respectively, of 5 and 10 ng mL(-1). The method was applied for the analysis of rat urine collected before and after (24, 48, 72 h) intra-articular (IA) injection of a marketed formulation of MPA (Depo-Medrol(R)). MS/MS acquisitions were taken at different collision energies for the precursor ions of interest, detected in PIS mode, to verify the MP-related structure. Six different metabolites were detected in rat urine, and their chemical structures were assigned with a computational study.
Assuntos
Metilprednisolona/análogos & derivados , Espectrometria de Massas por Ionização por Electrospray/métodos , Espectrometria de Massas em Tandem/métodos , Animais , Humanos , Masculino , Metilprednisolona/química , Metilprednisolona/metabolismo , Metilprednisolona/urina , Acetato de Metilprednisolona , Estrutura Molecular , Ratos , Ratos Wistar , Sensibilidade e EspecificidadeRESUMO
Synthetic glucocorticoids are generally preferred over their natural counterparts as these compounds exhibit improved anti-inflammatory potency and glucocorticoid receptor selectivity. However, the biotechnological production of these molecules is often subject to limitations inferred by restricted enzyme stability, selectivity or inhibition thereof. The latter is particularly important during 6α-methylprednisolone production, as the essential C21-hydroxylation of its precursor medrane appears to be hampered by product inhibition of the steroid-21-hydroxylase (CYP21A2). To circumvent this bottleneck, we established a two-step reaction for controlled mixed-culture fermentation, using recombinant E. coli. This process comprises the previously reported C21-hydroxylation of medrane by CYP21A2, followed by an instant derivatization of the hydroxylated product premedrol by chloramphenicol acetyl transferase 1 (CAT1). The CAT1-mediated C21-acetylation prevents the product from regaining access to the enzyme's active site which effectively shifts the chemical equilibrium toward premedrol formation. The successful circumvention of product inhibition at optimized conditions resulted in the formation of more than 1.5â¯g of product per liter which corresponds to an increase by more than 100 %. Taken together, we demonstrate an efficient system to enhance cytochrome P450-mediated biotransformations, holding great ecologic and economic potential to be applied in industrial processes.