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Despite the understanding that renal clearance is pivotal for driving the pharmacokinetics of numerous therapeutic proteins and peptides, the specific processes that occur following glomerular filtration remain poorly defined. For instance, sites of catabolism within the proximal tubule can occur at the brush border, within lysosomes following endocytosis, or even within the tubule lumen itself. The objective of the current study was to address these limitations and develop methodology to study the kidney disposition of a model therapeutic protein. Exenatide is a peptide used to treat type 2 diabetes mellitus. Glomerular filtration and ensuing renal catabolism have been shown to be its principal clearance pathway. Here, we designed and validated a Förster resonance energy transfer-quenched exenatide derivative to provide critical information on the renal handling of exenatide. A combination of in vitro techniques was used to confirm substantial fluorescence quenching of intact peptide that was released upon proteolytic cleavage. This evaluation was then followed by an assessment of the in vivo disposition of quenched exenatide directly within kidneys of living rats via intravital two-photon microscopy. Live imaging demonstrated rapid glomerular filtration and identified exenatide metabolism occurred within the subapical regions of the proximal tubule epithelia, with subsequent intracellular trafficking of cleaved fragments. These results provide a novel examination into the real-time, intravital disposition of a protein therapeutic within the kidney and offer a platform to build upon for future work.
Assuntos
Diabetes Mellitus Tipo 2 , Exenatida , Rim , Animais , Ratos , Diabetes Mellitus Tipo 2/metabolismo , Exenatida/metabolismo , Exenatida/farmacocinética , Rim/metabolismo , Túbulos Renais Proximais/metabolismo , Peptídeos/metabolismoRESUMO
Megalin and cubilin, endocytic proteins present in the proximal tubule of the kidney, are responsible for reabsorbing filtered proteins from urine. Our hypothesis was that potential substrates of megalin/cubilin could be identified by examining urinary protein differences between control (WT) mice and kidney-specific megalin knockdown (KD) mice. Using the IonStar proteomics approach, 877 potential megalin/cubilin substrates were discovered, with 23 of these compounds representing known megalin/cubilin substrates. Some of the proteins with the largest fold changes in the urine between KD and WT included the known megalin substrates retinol-binding protein and vitamin D-binding protein. Of the total proteins identified as novel substrates, about three-quarters of compounds had molecular weights (MWs) below 69 kDa, the MW of albumin, and the remaining had higher MWs, with about 5% of the proteins having MWs greater than 150 kDa. Sex differences in the number of identified substrates occurred, but this may be due to differences in kidney megalin expression between both male and female megalin KD and WT animals, with the ratio of megalin between WT and KD being 2.76 and 2.14 for female and male mice, respectively. The top three ingenuity canonical pathways based on the urinary proteins in both female and male KD mice were acute phase response signaling, liver X receptor/retinoid X receptor activation, and intrinsic prothrombin activation pathways. In conclusion, analysis of urine samples from kidney-specific megalin KD and WT mice was found to be useful for the identification of potential endogenous substrates for megalin and cubilin.
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Proteína-2 Relacionada a Receptor de Lipoproteína de Baixa Densidade , Proteína de Ligação a Vitamina D , Albuminas , Animais , Endocitose/fisiologia , Feminino , Rim/metabolismo , Túbulos Renais Proximais/metabolismo , Receptores X do Fígado/metabolismo , Proteína-2 Relacionada a Receptor de Lipoproteína de Baixa Densidade/metabolismo , Masculino , Camundongos , Proteômica , Protrombina/metabolismo , Receptores de Superfície Celular , Receptores X de Retinoides/metabolismo , Proteínas de Ligação ao Retinol/metabolismo , Proteína de Ligação a Vitamina D/metabolismoRESUMO
This publication represents the first to report global information on characteristics and requirements of doctoral programs in the pharmaceutical sciences in schools/colleges of Pharmacy. Survey responses (140 responses) were received from doctoral programs in 23 countries, with the greatest number of responses obtained from Japan, followed by India and the United States. Program characteristics and requirements, and student and faculty information, including graduate placement, in programs in Asia, North America, Europe, Africa and Australia were compared. Survey responses indicated differences in entrance requirements for doctoral programs with minimum requirements being a bachelor's degree, pharmacy degree or master's degree, including a M.Phil. degree. Programs differed widely in size in all geographical areas, but there was a similar emphasis on core educational learning outcomes (core competencies) and Ph.D. graduation requirements including qualifying examinations, thesis defense with internal and external reviewers and requirements for peer-reviewed publications. Additionally, three-quarters of programs indicated that there was external review of their programs every 2-4 or 5-7 years. Female students and female faculty mentors represented about 50% of students/faculty in programs in most geographical areas. Placement of students after graduation indicated that the highest percentage went into the pharmaceutical industry in Asia (predominantly India) and North America, with a lower percentage in Europe, Africa and Australia.
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Educação em Farmácia , Feminino , Humanos , África , Europa (Continente) , Inquéritos e Questionários , Estados Unidos , América do Norte , ÁsiaRESUMO
Information on master's programs in the pharmaceutical sciences is lacking; this manuscript addresses this gap in the literature, by reporting on the results of an international survey performed in 2021 of master's programs in the pharmaceutical sciences offered at Schools/Colleges of Pharmacy. Ninety-six responses were received from universities from 23 countries, with the greatest number of responses obtained from India, followed by the United States and Japan. Master's programs in the pharmaceutical sciences are generally full time and 2 years in duration. Only 3% of programs were reported to be examination-based, while the remaining 97% had a research component with 70% of programs having a thesis defense with external and/or internal examiners. Master's programs tended to be larger in Asia and Europe than in North America; as well, programs in North America tended to have more international students. Didactic coursework was included in 96% of master's programs in North America, but only in 38% of Asian and 58% of European programs. The predominant placement of graduates from master's programs in Asia was in the pharmaceutical industry (70%); this contrasted with programs in Europe, Africa and North America where 28-36% enter careers in the pharmaceutical industry and higher percentages enter Ph.D. programs. The major challenge identified by programs was funding of faculty and of graduate students, although decreasing career opportunities was identified as a challenge in Asia and Africa.
Assuntos
Farmácia , Humanos , Estados Unidos , Inquéritos e Questionários , Preparações Farmacêuticas , Europa (Continente) , ÁfricaRESUMO
High doses of the partial agonist of the GABA B receptor, γ-hydroxybutyric acid (GHB), causes respiratory depression that can lead to death. Previously, it has been shown that GABAB- receptor antagonism is able to prevent respiratory depression and sedation when inhibitors are pre-administered. In order to treat GHB overdoses, safety and efficacy of a treatment strategy at various times after GHB administration is necessary, in order to more closely replicate a true overdose situation. Preliminary studies developed an assay for SGS742 and determined its pharmacokinetics in rats. The effects of SGS742 on GHB-induced respiratory depression were evaluated when SGS742 administration was delayed 1 and 2 hours after intravenous or oral administration of GHB or γ-butyrolactone, a GHB prodrug. SGS742 reversed GHB-induced respiratory depression in a dose-dependent manner at both time points tested, with no effects on its toxicokinetics. However, some of the dosing paradigms resulted in toxicity in the form of tremors, seizures or abnormal movements. The tremors/seizures occurred in a manner that was dependent on both the dose and timing of SGS742 administration, and were not altered with pretreatment with gabazine, a GABAA receptor inhibitor, and only partially reduced with pretreatment with NCS382, a selective GHB receptor antagonist. Additional studies with a second GABAB antagonist SCH50911 demonstrated similar effects, producing reversal of respiratory depression but producing tremors and abnormal movements. Further studies are necessary in order to identify the potential use of GABAB antagonism as a treatment strategy for GHB overdoses. Significance Statement There is no current treatment for overdoses of the drug of abuse γ-hydroxybutyric acid (GHB). Since the toxicodynamic effects of GHB, including respiratory depression and lethality, are mediated through GABAB receptor agonism, GABAB receptor antagonists may represent a therapeutic strategy to treat overdoses. This study demonstrates that while GABAB receptor antagonists are effective as a pretreatment, they are less effective when administered at times after GHB administration and their administration is also associated with time- and dose-associated toxicity.
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Monocarboxylate transporter 6 (MCT6; SLC16A5) is an orphan transporter protein with expression in multiple tissues. The endogenous function of MCT6 related to human health and disease remains unknown. Our previous transcriptomic and proteomic analyses in Mct6 knockout (KO) mice suggested that MCT6 may play a role in lipid and glucose homeostasis, but additional evidence is required. Thus, the objective of this study was to further explore the impact of MCT6 on metabolic function using untargeted metabolomic analysis in Mct6 KO mice. The plasma from male and female mice and livers from male mice were submitted for global metabolomics analysis to assess the relative changes in endogenous small molecules across the liver and systemic circulation associated with absence of Mct6. More than 782 compounds were detected with 101 and 51 metabolites significantly changed in plasma of male and female mice, respectively, and 100 metabolites significantly changed in the livers of male mice (p < .05). Significant perturbations in lipid metabolism were annotated in the plasma and liver metabolome, with additional alterations in the amino acid metabolism pathway in plasma samples from male and female mice. Elevated lipid diacylglycerol and altered fatty acid metabolite concentrations were found in liver and plasma samples of male Mct6 KO mice. Significant reduction of N-terminal acetylated amino acids was found in plasma samples of male and female Mct6 KO mice. In summary, the present study confirmed the significant role of MCT6 in lipid and amino acid homeostasis, suggesting its contribution in metabolic diseases.
Assuntos
Metabolômica , Proteômica , Aminoácidos , Animais , Feminino , Metabolismo dos Lipídeos , Lipídeos , Masculino , Camundongos , Camundongos KnockoutRESUMO
γ-hydroxybutyric acid (GHB) is widely abused alone and in combination with other club drugs such as ketamine. GHB exhibits nonlinear toxicokinetics, characterized by saturable metabolism, saturable absorption and saturable renal reabsorption mediated by monocarboxylate transporters (MCTs). In this research, we characterized the effects of ketamine on GHB toxicokinetics/toxicodynamics (TK/TD) and evaluated the use of MCT inhibition and specific receptor antagonism as potential treatment strategies for GHB overdose in the presence of ketamine. Adult male Sprague-Dawley rats were administered GHB 600 mg/kg i.v. alone or with ketamine (6 mg/kg i.v. bolus plus 1 mg/kg/min i.v. infusion). Plasma and urine samples were collected and respiratory parameters (breathing frequency, tidal and minute volume) continuously monitored using whole-body plethysmography. Ketamine co-administration resulted in a significant decrease in GHB total and metabolic clearance, with renal clearance remaining unchanged. Ketamine prevented the compensatory increase in tidal volume produced by GHB, and this resulted in a significant decline in minute volume when compared to GHB alone. Sleep time and lethality were also increased after ketamine co-administration when compared to GHB. L-lactate and AR-C155858 (potent MCT inhibitor) treatment resulted in an increase in GHB renal and total clearance and improvement in respiratory depression. AR-C155858 administration also resulted in a significant decrease in GHB brain/plasma ratio. SCH50911 (GABAB receptor antagonist), but not naloxone, improved GHB-induced respiratory depression in the presence of ketamine. In conclusion, ketamine ingestion with GHB can result in significant TK/TD interactions. MCT inhibition and GABAB receptor antagonism can serve as potential treatment strategies for GHB overdose when it is co-ingested with ketamine.
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Gamma hydroxybutyric acid (GHB) has been approved clinically to treat excessive daytime sleepiness and cataplexy in patients with narcolepsy, alcohol and opioid withdrawal, and as an anesthetic. The use of GHB clinically is limited due to its high abuse potential. The absorption, clearance and tissue uptake of GHB is mediated by proton-dependent and sodium-coupled monocarboxylate transporters (MCTs and SMCTs) and inhibition of these transporters may result in a change in GHB pharmacokinetics and pharmacodynamics. Previous studies have reported that non-steroidal anti-inflammatory drugs (NSAIDs) may inhibit these monocarboxylate transporters. Therefore, the purpose of this work was to analyze the interaction between GHB (at a dose of 600 mg/kg i. v.) and the NSAID, diclofenac, by examining the effects of this drug on the in vivo pharmacokinetics and pharmacodynamics in rat studies. The pharmacodynamic effect evaluated was respiratory depression, a measure of toxicity observed by GHB at this dose. There was an improvement in the respiratory rate with diclofenac administration suggesting an effect of diclofenac on GHB toxicity. In vitro studies with rat blood brain endothelial cells (RBE4) that express MCT1 indicated that diclofenac can inhibit GHB transport with an IC50 of 10.6 µM at pH 7.4. In vivo studies found a decrease in brain GHB concentrations and a decrease in the brain-to-plasma concentration ratio following diclofenac treatment. With this study we can conclude that diclofenac and potentially other NSAIDs can inhibit the transport of GHB into the brain, therefore decreasing GHB's pharmacodynamic effects and toxicity.
Assuntos
Encéfalo , Diclofenaco/farmacocinética , Interações Medicamentosas , Hidroxibutiratos/farmacocinética , Transportadores de Ácidos Monocarboxílicos , Insuficiência Respiratória , Simportadores , Anestésicos/farmacocinética , Anestésicos/toxicidade , Animais , Anti-Inflamatórios não Esteroides/farmacocinética , Transporte Biológico Ativo/efeitos dos fármacos , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Células Cultivadas , Relação Dose-Resposta a Droga , Células Endoteliais/metabolismo , Hidroxibutiratos/toxicidade , Transportadores de Ácidos Monocarboxílicos/antagonistas & inibidores , Transportadores de Ácidos Monocarboxílicos/metabolismo , Ratos , Ratos Sprague-Dawley , Insuficiência Respiratória/induzido quimicamente , Insuficiência Respiratória/tratamento farmacológico , Oxibato de Sódio/farmacocinética , Simportadores/antagonistas & inibidores , Simportadores/metabolismoRESUMO
The drug of abuse, γ-hydroxybutyric acid (GHB), is commonly co-ingested with ethanol, resulting in a high incidence of toxicity and death. Our laboratory has previously reported that GHB is a substrate for the monocarboxylate transporters (MCTs), necessary for its absorption, renal clearance, and tissue distribution, including across the blood-brain barrier. Our goal was to investigate the drug-drug interaction (DDI) between GHB and ethanol and to evaluate MCT1 inhibition as a strategy to reverse toxicity. The toxicokinetics of this DDI were investigated, including brain-to-plasma concentration ratios, in the presence and absence of ethanol. The toxicodynamic parameters examined were respiratory depression (breathing frequency, tidal volume) and sedation (time of return-of-righting reflex). Ethanol was administered (2 g/kg i.v.) 5 minutes before the intravenous or oral administration of GHB, and MCT1 inhibitors AZD-3965 and AR-C155858 (5 mg/kg i.v.) were administered 60 minutes after GHB administration. Ethanol administration did not alter the toxicokinetics or respiratory depression caused by GHB after intravenous or oral administration; however, it significantly increased the sedation effect, measured by return-to-righting time. AZD-3965 or AR-C155858 significantly decreased the effects of the co-administration of GHB and ethanol on respiratory depression and sedation of this DDI and decreased brain concentrations and the brain-to-plasma concentration ratio of GHB. The results indicate that ethanol co-administered with GHB increases toxicity and that MCT1 inhibition is effective in reversing toxicity by inhibiting GHB brain uptake when given after GHB-ethanol administration. SIGNIFICANCE STATEMENT: These studies investigated the enhanced toxicity observed clinically when γ-hydroxybutyric acid (GHB) is co-ingested with alcohol and evaluated strategies to reverse this toxicity. The effects of the novel monocarboxylate transporter 1 (MCT1) inhibitors AR-C155858 and AZD-3965 on this drug-drug interaction have not been studied before, and these preclinical studies indicate that MCT1 inhibitors can decrease brain concentrations of GHB by inhibiting brain uptake, even when administered at times after GHB-ethanol. AZD-3965 represents a potential treatment strategy for GHB-ethanol overdoses.
Assuntos
Etanol/toxicidade , Hidroxibutiratos/toxicidade , Transportadores de Ácidos Monocarboxílicos/antagonistas & inibidores , Pirimidinonas/farmacologia , Simportadores/antagonistas & inibidores , Tiofenos/farmacologia , Uracila/análogos & derivados , Animais , Interações Medicamentosas/fisiologia , Etanol/metabolismo , Hidroxibutiratos/metabolismo , Masculino , Transportadores de Ácidos Monocarboxílicos/metabolismo , Pirimidinonas/uso terapêutico , Ratos , Ratos Sprague-Dawley , Insuficiência Respiratória/induzido quimicamente , Insuficiência Respiratória/tratamento farmacológico , Insuficiência Respiratória/metabolismo , Simportadores/metabolismo , Tiofenos/uso terapêutico , Uracila/farmacologia , Uracila/uso terapêuticoRESUMO
Patients with chronic kidney disease (CKD) and end-stage renal disease suffer from increased cardiovascular events and cardiac mortality. Prior studies have demonstrated that a portion of this enhanced risk can be attributed to the accumulation of microbiota-derived toxic metabolites, with most studies focusing on the sulfonated form of p-cresol (PCS). However, unconjugated p-cresol (uPC) itself was never assessed due to rapid and extensive first-pass metabolism that results in negligible serum concentrations of uPC. These reports thus failed to consider the host exposure to uPC prior to hepatic metabolism. In the current study, not only did we measure the effect of altering the intestinal microbiota on lipid accumulation in coronary arteries, but we also examined macrophage lipid uptake and handling pathways in response to uPC. We found that atherosclerosis-prone mice fed a high-fat diet exhibited significantly higher coronary artery lipid deposits upon receiving fecal material from CKD mice. Furthermore, treatment with uPC increased total cholesterol, triglycerides, and hepatic and aortic fatty deposits in non-CKD mice. Studies employing an in vitro macrophage model demonstrated that uPC exposure increased apoptosis whereas PCS did not. Additionally, uPC exhibited higher potency than PCS to stimulate LDL uptake and only uPC induced endocytosis- and pinocytosis-related genes. Pharmacological inhibition of varying cholesterol influx and efflux systems indicated that uPC increased macrophage LDL uptake by activating macropinocytosis. Overall, these findings indicate that uPC itself had a distinct effect on macrophage biology that might have contributed to increased cardiovascular risk in patients with CKD.
Assuntos
Aorta/metabolismo , LDL-Colesterol/metabolismo , Doença da Artéria Coronariana/metabolismo , Cresóis/metabolismo , Microbioma Gastrointestinal , Fígado/metabolismo , Macrófagos/metabolismo , Pinocitose/fisiologia , Insuficiência Renal Crônica/metabolismo , Animais , Aorta/efeitos dos fármacos , Aorta/patologia , Colesterol/metabolismo , LDL-Colesterol/efeitos dos fármacos , Doença da Artéria Coronariana/patologia , Vasos Coronários/efeitos dos fármacos , Vasos Coronários/metabolismo , Vasos Coronários/patologia , Cresóis/farmacologia , Dieta Hiperlipídica , Transplante de Microbiota Fecal , Falência Renal Crônica/metabolismo , Falência Renal Crônica/microbiologia , Fígado/efeitos dos fármacos , Fígado/patologia , Macrófagos/efeitos dos fármacos , Camundongos , Pinocitose/efeitos dos fármacos , Insuficiência Renal Crônica/microbiologia , Triglicerídeos/metabolismoRESUMO
OBJECTIVE: To record the content and parental perceptions of family meetings in a Neonatal Intensive Care Unit (NICU) to improve existing frameworks for facilitating these meetings. STUDY DESIGN: A prospective, mixed-methods study. NICU family meetings were audio-recorded, transcribed, and analyzed by an iteratively derived coding framework until thematic saturation. We used descriptive statistics of parental post-meeting assessments. RESULTS: Qualitative analysis of 21 meetings identified both Communication Facilitators and Barriers. Facilitators included use of visual-aids and participation of social workers to clarify information for parents. Barriers included staff rarely eliciting parental comprehension (3 meetings) or concerns (5) before providing new information, resulting in 39% of parents reporting they didn't ask questions they wanted to ask. In 33% of meetings an important participant was absent. CONCLUSIONS: This novel qualitative and quantitative dataset of NICU family meetings highlights areas for improving communication. Attention to these components may improve parental perceptions of family meetings.
Assuntos
Unidades de Terapia Intensiva Neonatal , Pais , Comunicação , Humanos , Recém-Nascido , Estudos Prospectivos , Assistentes SociaisRESUMO
Therapeutic immunoglobulin G (IgG) antibodies comprise the largest class of protein therapeutics. Several factors that influence their overall disposition have been well-characterized, including target-mediated mechanics and convective flow. What remains poorly defined is the potential for non-targeted entry into various tissues or cell types by means of uptake via cell surface receptors at those sites. Megalin and cubilin are large endocytic receptors whose cooperative function plays important physiological roles at the tissues in which they are expressed. One such example is the kidney, where loss of either results in significant declines in proximal tubule protein reabsorption. Due to their diverse ligand profile and broad tissue expression, megalin and cubilin represent potential candidates for receptor-mediated uptake of IgG into various epithelia. Therefore, the objective of the current work was to determine if IgG was a novel ligand of megalin and/or cubilin. Direct binding was measured for human IgG with both megalin and the cubilin/amnionless complex. Additional work focusing on the megalin-IgG interaction was then conducted to build upon these findings. Cell uptake studies using megalin ligands for competitive inhibition or proximal tubule cells stably transduced with megalin-targeted shRNA constructs supported a role for megalin in the endocytosis of human IgG. Furthermore, a pharmacokinetic study using transgenic mice with a kidney-specific mosaic knockout of megalin demonstrated increased urinary excretion of human IgG in megalin knockout mice when compared to wild-type controls. These findings indicate that megalin is capable of binding and internalizing IgG via a high affinity interaction.
Assuntos
Imunoglobulina G/farmacologia , Túbulos Renais Proximais/metabolismo , Proteína-2 Relacionada a Receptor de Lipoproteína de Baixa Densidade/metabolismo , Animais , Linhagem Celular , Endocitose , Feminino , Técnicas de Silenciamento de Genes , Humanos , Imunoglobulina G/isolamento & purificação , Imunoglobulina G/uso terapêutico , Proteína-2 Relacionada a Receptor de Lipoproteína de Baixa Densidade/genética , Masculino , Camundongos , Camundongos Knockout , Gambás , Ratos , Eliminação RenalRESUMO
Gamma-hydroxybutyrate (GHB) is a short-chain fatty acid present endogenously in the brain and used therapeutically for the treatment of narcolepsy, as sodium oxybate, and for alcohol abuse/withdrawal. GHB is better known however as a drug of abuse and is commonly referred to as the "date-rape drug"; current use in popular culture includes recreational "chemsex," due to its properties of euphoria, loss of inhibition, amnesia, and drowsiness. Due to the steep concentration-effect curve for GHB, overdoses occur commonly and symptoms include sedation, respiratory depression, coma, and death. GHB binds to both GHB and GABAB receptors in the brain, with pharmacological/toxicological effects mainly due to GABAB agonist effects. The pharmacokinetics of GHB are complex and include nonlinear absorption, metabolism, tissue uptake, and renal elimination processes. GHB is a substrate for monocarboxylate transporters, including both sodium-dependent transporters (SMCT1, 2; SLC5A8; SLC5A12) and proton-dependent transporters (MCT1-4; SLC16A1, 7, 8, and 3), which represent significant determinants of absorption, renal reabsorption, and brain and tissue uptake. This review will provide current information of the pharmacology, therapeutic effects, and pharmacokinetics/pharmacodynamics of GHB, as well as therapeutic strategies for the treatment of overdoses. Graphical abstract.
Assuntos
Overdose de Drogas/terapia , Hidroxibutiratos/farmacocinética , Oxibato de Sódio/farmacocinética , Abuso Oral de Substâncias/terapia , Alcoolismo/complicações , Alcoolismo/tratamento farmacológico , Animais , Modelos Animais de Doenças , Avaliação Pré-Clínica de Medicamentos , Overdose de Drogas/etiologia , Humanos , Hidroxibutiratos/administração & dosagem , Hidroxibutiratos/toxicidade , Taxa de Depuração Metabólica , Narcolepsia/tratamento farmacológico , Oxibato de Sódio/administração & dosagem , Oxibato de Sódio/toxicidade , Abuso Oral de Substâncias/etiologia , Síndrome de Abstinência a Substâncias/tratamento farmacológicoRESUMO
Delivery of therapeutic agents to the central nervous system is challenged by the barriers in place to regulate brain homeostasis. This is especially true for protein therapeutics. Targeting the barrier formed by the choroid plexuses at the interfaces of the systemic circulation and ventricular system may be a surrogate brain delivery strategy to circumvent the blood-brain barrier. Heterogenous cell populations located at the choroid plexuses provide diverse functions in regulating the exchange of material within the ventricular space. Receptor-mediated transcytosis may be a promising mechanism to deliver protein therapeutics across the tight junctions formed by choroid plexus epithelial cells. However, cerebrospinal fluid flow and other barriers formed by ependymal cells and perivascular spaces should also be considered for evaluation of protein therapeutic disposition. Various preclinical methods have been applied to delineate protein transport across the choroid plexuses, including imaging strategies, ventriculocisternal perfusions, and primary choroid plexus epithelial cell models. When used in combination with simultaneous measures of cerebrospinal fluid dynamics, they can yield important insight into pharmacokinetic properties within the brain. This review aims to provide an overview of the choroid plexuses and ventricular system to address their function as a barrier to pharmaceutical interventions and relevance for central nervous system drug delivery of protein therapeutics. Protein therapeutics targeting the ventricular system may provide new approaches in treating central nervous system diseases.
RESUMO
Graphical Abstract.
Assuntos
Farmacologia/história , Mulheres/história , Feminino , História do Século XX , História do Século XXI , Humanos , Pesquisadores/históriaRESUMO
Endocytosis by podocytes is gaining increased attention as a biologic means of removing large proteins such as serum albumin from the glomerular barrier. Some of this function has been attributed to the megalin/cubilin (Lrp2/Cubn) receptor complex and the albumin recycling protein FcRn (Fcgrt). However, whether other glomerular cells possess the potential to perform this same phenomenon or express these proteins remains uncharacterized. Mesangial cells are uniquely positioned in glomeruli and represent a cell type capable of performing several diverse functions. Here, the expression of megalin and FcRn in murine mesangial cells along with the megalin adaptor protein Dab-2 (Dab2) was shown for the first time. Cubilin mRNA expression was detected, but the absence of the cubilin partner amnionless (Amn) suggested that cubilin is minimally functional, if at all, in these cells. Mesangial cell endocytosis of albumin was characterized and shown to involve a receptor-mediated process. Albumin endocytosis was significantly impaired (p < 0.01) under inducible megalin knockdown conditions in stably transduced mesangial cells. The current work provides both the novel identification of megalin and FcRn in mesangial cells and the functional demonstration of megalin-mediated albumin endocytosis.
Assuntos
Endocitose , Proteína-2 Relacionada a Receptor de Lipoproteína de Baixa Densidade/metabolismo , Células Mesangiais/citologia , Soroalbumina Bovina/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Proteínas Reguladoras de Apoptose/metabolismo , Bovinos , Linhagem Celular , Antígenos de Histocompatibilidade Classe I/metabolismo , Células Mesangiais/metabolismo , Camundongos , Receptores Fc/metabolismoRESUMO
Monocarboxylate transporter 1 (MCT1) represents a potential therapeutic target in cancer. The objective of this study was to determine the efficacy of AZD3965 (a specific inhibitor of MCT1) and α-cyano-4-hydroxycinnamic acid (CHC, a nonspecific inhibitor of MCTs) in the murine 4T1 tumor model of triple-negative breast cancer (TNBC). Expression of MCT1 and MCT4 in 4T1 and mouse mammary epithelial cells were determined by Western blot. Inhibition of MCT1-mediated L-lactate uptake and cellular proliferation by AZD3965 and CHC was determined. Mice bearing 4T1 breast tumors were treated with AZD3965 100 mg/kg i.p. twice-daily or CHC 200 mg/kg i.p. once-daily. Tumor growth, metastasis, intra-tumor lactate concentration, immune function, tumor MCT expression, and concentration-effect relationships were determined. AZD3965 and CHC inhibited cell growth and L-lactate uptake in 4T1 cells. AZD3965 treatment resulted in trough plasma and tumor concentrations of 29.1 ± 13.9 and 1670 ± 946 nM, respectively. AZD3965 decreased the tumor proliferation biomarker Ki67 expression, increased intra-tumor lactate concentration, and decreased tumor volume, although tumor weight was not different from untreated controls. CHC had no effect on tumor volume and weight, or intra-tumor lactate concentration. AZD3965 treatment reduced the blood leukocyte count and spleen weight and increased lung metastasis, while CHC did not. These findings indicate AZD3965 is a potent MCT1 inhibitor that accumulates to high concentrations in 4T1 xenograft tumors, where it increases tumor lactate concentrations and produces beneficial effects on markers of TNBC; however, overall effects on tumor growth were minimal and lung metastases increased.
Assuntos
Neoplasias da Mama/tratamento farmacológico , Ácidos Cumáricos/administração & dosagem , Transportadores de Ácidos Monocarboxílicos/antagonistas & inibidores , Pirimidinonas/administração & dosagem , Simportadores/antagonistas & inibidores , Tiofenos/administração & dosagem , Animais , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Ácidos Cumáricos/metabolismo , Relação Dose-Resposta a Droga , Feminino , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/secundário , Camundongos , Camundongos Endogâmicos BALB C , Transportadores de Ácidos Monocarboxílicos/metabolismo , Pirimidinonas/metabolismo , Simportadores/metabolismo , Tiofenos/metabolismo , Resultado do Tratamento , Carga Tumoral/efeitos dos fármacos , Carga Tumoral/fisiologia , Ensaios Antitumorais Modelo de Xenoenxerto/métodosRESUMO
Bumetanide, a sulfamyl loop diuretic, is used for the treatment of edema in association with congestive heart failure. Being a polar, anionic compound at physiologic pH, bumetanide uptake and efflux into different tissues is largely transporter-mediated. Of note, organic anion transporters (SLC22A) have been extensively studied in terms of their importance in transporting bumetanide to its primary site of action in the kidney. The contribution of one of the less-studied bumetanide transporters, monocarboxylate transporter 6 (MCT6; SLC16A5), to bumetanide pharmacokinetics (PK) and pharmacodynamics (PD) has yet to be characterized. The affinity of bumetanide for murine Mct6 was evaluated using Mct6-transfected Xenopus laevis oocytes. Furthermore, bumetanide was intravenously and orally administered to wild-type mice (Mct6+/+) and homozygous Mct6 knockout mice (Mct6-/-) to elucidate the contribution of Mct6 to bumetanide PK/PD in vivo. We demonstrated that murine Mct6 transports bumetanide at a similar affinity compared with human MCT6 (78 and 84 µM, respectively, at pH 7.4). After bumetanide administration, there were no significant differences in plasma PK. Additionally, diuresis was significantly decreased by â¼55% after intravenous bumetanide administration in Mct6-/- mice. Kidney cortex concentrations of bumetanide were decreased, suggesting decreased Mct6-mediated bumetanide transport to its site of action in the kidney. Overall, these results suggest that Mct6 does not play a major role in the plasma PK of bumetanide in mice; however, it significantly contributes to bumetanide's pharmacodynamics due to changes in kidney concentrations. SIGNIFICANCE STATEMENT: Previous evidence suggested that MCT6 transports bumetanide in vitro; however, no studies to date have evaluated the in vivo contribution of this transporter. In vitro studies indicated that mouse and human MCT6 transport bumetanide with similar affinities. Using Mct6 knockout mice, we demonstrated that murine Mct6 does not play a major role in the plasma pharmacokinetics of bumetanide; however, the pharmacodynamic effect of diuresis was attenuated in the knockout mice, likely because of the decreased bumetanide concentrations in the kidney.
Assuntos
Bumetanida/farmacocinética , Diurese/efeitos dos fármacos , Transportadores de Ácidos Monocarboxílicos/metabolismo , Inibidores de Simportadores de Cloreto de Sódio e Potássio/farmacocinética , Administração Intravenosa , Administração Oral , Animais , Bumetanida/administração & dosagem , Avaliação Pré-Clínica de Medicamentos , Córtex Renal/efeitos dos fármacos , Córtex Renal/metabolismo , Masculino , Camundongos , Camundongos Knockout , Transportadores de Ácidos Monocarboxílicos/genética , Oócitos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Inibidores de Simportadores de Cloreto de Sódio e Potássio/administração & dosagem , Xenopus laevisRESUMO
Given the burden of HIV and other sexually transmitted infections among adolescents who are legal minors, it is critical that they be included in biomedical sexual health trials to ensure that new prevention and treatment interventions are safe, effective, and acceptable for their use. However, adolescents are often not well represented in clinical trials. We provide an overview of the available evidence regarding adolescent and parent willingness for adolescents to participate in biomedical sexual health trials, parental involvement in the permission-consent process, management of differences and discord among adolescents and parents, and parental involvement throughout the study period. We also outline recommendations for current practice and areas for future research.