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1.
Birth Defects Res ; 115(14): 1284-1293, 2023 08 15.
Artigo em Inglês | MEDLINE | ID: mdl-37140214

RESUMO

OBJECTIVES: The objective of this study was to examine the fetal skeletons using both alizarin red stain and micro-computed tomography (CT) images; investigate differences, and to determine if the conclusions of the study were the same regardless of the examination method. METHODS: A candidate drug was given orally by gavage to pregnant New Zealand White rabbits on gestation day (GD) 7 to GD 19 (mating = GD 0) at doses of 0 (control), 0.02, 0.5, 5, and 15 mg/kg/day. Maternal toxicity was evident at ≥0.02 mg/kg/day. The 199 fetal skeletons (totaling 50,546 skeletal elements) obtained at cesarean delivery on GD29 were first stained with Alizarin Red S, then imaged by a Siemens Inveon micro-CT scanner. All fetal skeletons were examined by both methods, without knowledge of dose group, and the results were compared. RESULTS: In total, 33 types of skeletal abnormalities were identified. There was 99.8% concordance of results comparing stain to micro-CT. Ossification of the middle phalanx of the forepaw digit 5 showed the greatest difference between the two methods. CONCLUSION: Overall, micro-CT imaging is a realistic, and robust alternative to skeletal staining to examine fetal rabbit skeletons in developmental toxicity studies.


Assuntos
Osso e Ossos , Cesárea , Gravidez , Feminino , Coelhos , Animais , Microtomografia por Raio-X/métodos , Osso e Ossos/diagnóstico por imagem , Coloração e Rotulagem
2.
Mol Imaging Biol ; 25(3): 528-540, 2023 06.
Artigo em Inglês | MEDLINE | ID: mdl-36266600

RESUMO

PURPOSE: The presence and functional competence of intratumoral CD8+ T cells is often a barometer for successful immunotherapeutic responses in cancer. Despite this understanding and the extensive number of clinical-stage immunotherapies focused on potentiation (co-stimulation) or rescue (checkpoint blockade) of CD8+ T cell antitumor activity, dynamic biomarker strategies are often lacking. To help fill this gap, immuno-PET nuclear imaging has emerged as a powerful tool for in vivo molecular imaging of antibody targeting. Here, we took advantage of immuno-PET imaging using 89Zr-IAB42M1-14, anti-mouse CD8 minibody, to characterize CD8+ T-cell tumor infiltration dynamics following ICOS (inducible T-cell co-stimulator) agonist antibody treatment alone and in combination with PD-1 blocking antibody in a model of mammary carcinoma. PROCEDURES: Female BALB/c mice with established EMT6 tumors received 10 µg, IP of either IgG control antibodies, ICOS agonist monotherapy, or ICOS/PD-1 combination therapy on days 0, 3, 5, 7, 9, 10, or 14. Imaging was performed at 24 and 48 h post IV dose of 89Zr IAB42M1-14. In addition to 89Zr-IAB42M1-14 uptake in tumor and tumor-draining lymph node (TDLN), 3D radiomic features were extracted from PET/CT images to identify treatment effects. Imaging mass cytometry (IMC) and immunohistochemistry (IHC) was performed at end of study. RESULTS: 89Zr-IAB42M1-14 uptake in the tumor was observed by day 11 and was preceded by an increase in the TDLN as early as day 4. The spatial distribution of 89Zr-IAB42M1-14 was more uniform in the drug treated vs. control tumors, which had spatially distinct tracer uptake in the periphery relative to the core of the tumor. IMC analysis showed an increased percentage of cytotoxic T cells in the ICOS monotherapy and ICOS/PD-1 combination group compared to IgG controls. Additionally, temporal radiomics analysis demonstrated early predictiveness of imaging features. CONCLUSION: To our knowledge, this is the first detailed description of the use of a novel immune-PET imaging technique to assess the kinetics of CD8+ T-cell infiltration into tumor and lymphoid tissues following ICOS agonist and PD-1 blocking antibody therapy. By demonstrating the capacity for increased spatial and temporal resolution of CD8+ T-cell infiltration across tumors and lymphoid tissues, these observations underscore the widespread potential clinical utility of non-invasive PET imaging for T-cell-based immunotherapy in cancer.


Assuntos
Linfócitos T CD8-Positivos , Neoplasias , Animais , Camundongos , Feminino , Linfócitos T CD8-Positivos/patologia , Tomografia por Emissão de Pósitrons combinada à Tomografia Computadorizada , Receptor de Morte Celular Programada 1 , Neoplasias/patologia , Tomografia por Emissão de Pósitrons/métodos , Imunoglobulina G , Linhagem Celular Tumoral , Proteína Coestimuladora de Linfócitos T Induzíveis
3.
J Control Release ; 352: 199-210, 2022 12.
Artigo em Inglês | MEDLINE | ID: mdl-36084816

RESUMO

Liposomes are promising targeted drug delivery systems with the potential to improve the efficacy and safety profile of certain classes of drugs. Though attractive, there are unique analytical challenges associated with the development of liposomal drugs including human dose prediction given these are multi-component drug delivery systems. In this study, we developed a multimodal imaging approach to provide a comprehensive distribution assessment for an antibacterial drug, GSK2485680, delivered as a liposomal formulation (Lipo680) in a mouse thigh model of bacterial infection to support human dose prediction. Positron emission tomography (PET) imaging was used to track the in vivo biodistribution of Lipo680 over 48 h post-injection providing a clear assessment of the uptake in various tissues and, importantly, the selective accumulation at the site of infection. In addition, a pharmacokinetic model was created to evaluate the kinetics of Lipo680 in different tissues. Matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry (IMS) was then used to quantify the distribution of GSK2485680 and to qualitatively assess the distribution of a liposomal lipid throughout sections of infected and non-infected hindlimb tissues at high spatial resolution. Through the combination of both PET and MALDI IMS, we observed excellent correlation between the Lipo680-radionuclide signal detected by PET with the GSK2485680 and lipid component signals detected by MALDI IMS. This multimodal translational method can reduce drug attrition by generating comprehensive biodistribution profiles of drug delivery systems to provide mechanistic insight and elucidate safety concerns. Liposomal formulations have potential to deliver therapeutics across a broad array of different indications, and this work serves as a template to aid in delivering future liposomal drugs to the clinic.


Assuntos
Doenças Transmissíveis , Lipossomos , Animais , Camundongos , Humanos , Lipossomos/química , Distribuição Tecidual , Antibacterianos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Tomografia por Emissão de Pósitrons , Imagem Multimodal , Lipídeos
4.
Front Immunol ; 13: 1081156, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36713458

RESUMO

The goal of this study was to utilize a multimodal magnetic resonance imaging (MRI) and positron emission tomography (PET) imaging approach to assess the local innate immune response in skeletal muscle and draining lymph node following vaccination in rats using two different vaccine platforms (AS01 adjuvanted protein and lipid nanoparticle (LNP) encapsulated Self-Amplifying mRNA (SAM)). MRI and 18FDG PET imaging were performed temporally at baseline, 4, 24, 48, and 72 hr post Prime and Prime-Boost vaccination in hindlimb with Cytomegalovirus (CMV) gB and pentamer proteins formulated with AS01, LNP encapsulated CMV gB protein-encoding SAM (CMV SAM), AS01 or with LNP carrier controls. Both CMV AS01 and CMV SAM resulted in a rapid MRI and PET signal enhancement in hindlimb muscles and draining popliteal lymph node reflecting innate and possibly adaptive immune response. MRI signal enhancement and total 18FDG uptake observed in the hindlimb was greater in the CMV SAM vs CMV AS01 group (↑2.3 - 4.3-fold in AUC) and the MRI signal enhancement peak and duration were temporally shifted right in the CMV SAM group following both Prime and Prime-Boost administration. While cytokine profiles were similar among groups, there was good temporal correlation only between IL-6, IL-13, and MRI/PET endpoints. Imaging mass cytometry was performed on lymph node sections at 72 hr post Prime and Prime-Boost vaccination to characterize the innate and adaptive immune cell signatures. Cell proximity analysis indicated that each follicular dendritic cell interacted with more follicular B cells in the CMV AS01 than in the CMV SAM group, supporting the stronger humoral immune response observed in the CMV AS01 group. A strong correlation between lymph node MRI T2 value and nearest-neighbor analysis of follicular dendritic cell and follicular B cells was observed (r=0.808, P<0.01). These data suggest that spatiotemporal imaging data together with AI/ML approaches may help establish whether in vivo imaging biomarkers can predict local and systemic immune responses following vaccination.


Assuntos
Infecções por Citomegalovirus , Fluordesoxiglucose F18 , Ratos , Animais , Vacinação , Imageamento por Ressonância Magnética/métodos , Tomografia por Emissão de Pósitrons , Citomegalovirus , Imunidade Inata , Músculo Esquelético/diagnóstico por imagem , Imagem Multimodal , Linfonodos/diagnóstico por imagem
5.
Br J Clin Pharmacol ; 88(4): 1655-1666, 2022 02.
Artigo em Inglês | MEDLINE | ID: mdl-34240449

RESUMO

AIM: Cabotegravir long-acting (LA) intramuscular (IM) injection is being investigated for HIV preexposure prophylaxis due to its potent antiretroviral activity and infrequent dosing requirement. A subset of healthy adult volunteers participating in a Phase I study assessing cabotegravir tissue pharmacokinetics underwent serial magnetic resonance imaging (MRI) to assess drug depot localization and kinetics following a single cabotegravir LA IM targeted injection. METHODS: Eight participants (four men, four women) were administered cabotegravir LA 600 mg under ultrasonographic-guided injection targeting the gluteal muscles. MRI was performed to determine injection-site location in gluteal muscle (IM), subcutaneous (SC) adipose tissue and combined IM/SC compartments, and to quantify drug depot characteristics, including volume and surface area, on Days 1 (≤2 hours postinjection), 3 and 8. Linear regression analysis examined correlations between MRI-derived parameters and plasma cabotegravir exposure metrics, including maximum observed concentration (Cmax ) and partial area under the concentration-time curve (AUC) through Weeks 4 and 8. RESULTS: Cabotegravir LA depot locations varied by participant and were identified in the IM compartment (n = 2), combined IM/SC compartments (n = 4), SC compartment (n = 1) and retroperitoneal cavity (n = 1). Although several MRI parameter and exposure metric correlations were determined, total depot surface area on Day 1 strongly correlated with plasma cabotegravir concentration at Days 3 and 8, Cmax and partial AUC through Weeks 4 and 8. CONCLUSION: MRI clearly delineated cabotegravir LA injection-site location and depot kinetics in healthy adults. Although injection-site variability was observed, drug depot surface area correlated with both plasma Cmax and partial AUC independently of anatomical distribution.


Assuntos
Fármacos Anti-HIV , Infecções por HIV , Imageamento por Ressonância Magnética Multiparamétrica , Adulto , Dicetopiperazinas , Feminino , Infecções por HIV/tratamento farmacológico , Humanos , Injeções Intramusculares , Cinética , Masculino , Piridonas , Voluntários
6.
Life Sci ; 272: 119267, 2021 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-33631173

RESUMO

AIMS: Indoxyl sulfate (IS), a protein-bound uremic toxin, is implicated in endothelial dysfunction, which contributes to adverse cardiovascular events in chronic kidney disease. Apoptosis signal regulating kinase 1 (ASK1) is a reactive oxygen species-driven kinase involved in IS-mediated adverse effects. This study assessed the therapeutic potential of ASK1 inhibition in alleviating endothelial effects induced by IS. MAIN METHODS: IS, in the presence and absence of a selective ASK1 inhibitor (GSK2261818A), was assessed for its effect on vascular reactivity in rat aortic rings, and cultured human aortic endothelial cells where we evaluated phenotypic and mechanistic changes. KEY FINDINGS: IS directly impairs endothelium-dependent vasorelaxation and endothelial cell migration. Mechanistic studies revealed increased production of reactive oxygen species-related markers, reduction of endothelial nitric oxide synthase and increased protein expression of tissue inhibitor of matrix metalloproteinase 1 (TIMP1). IS also increases angiopoietin-2 and tumour necrosis factor α gene expression and promotes transforming growth factor ß receptor abundance. Inhibition of ASK1 ameliorated the increase in oxidative stress markers, promoted autocrine interleukin 8 pro-angiogenic signalling and decreased anti-angiogenic responses at least in part via reducing TIMP1 protein expression. SIGNIFICANCE: ASK1 inhibition attenuated vasorelaxation and endothelial cell migration impaired by IS. Therefore, ASK1 is a viable intracellular target to alleviate uremic toxin-induced impairment in the vasculature.


Assuntos
Endotélio/metabolismo , MAP Quinase Quinase Quinase 5/antagonistas & inibidores , MAP Quinase Quinase Quinase 5/metabolismo , Animais , Apoptose/efeitos dos fármacos , Células Cultivadas , Células Endoteliais/metabolismo , Endotélio/efeitos dos fármacos , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/metabolismo , Indicã/efeitos adversos , Indicã/farmacologia , MAP Quinase Quinase Quinase 5/fisiologia , Masculino , NADPH Oxidases/metabolismo , Óxido Nítrico Sintase Tipo III/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/efeitos dos fármacos , Vasodilatação/efeitos dos fármacos
10.
Int J Cardiol ; 310: 128-136, 2020 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-32305147

RESUMO

BACKGROUND: Cardiorenal syndrome (CRS) is a major health burden worldwide in need of novel therapies, as current treatments remain suboptimal. The present study assessed the therapeutic potential of apoptosis signal-regulating kinase 1 (ASK1) inhibition in a rat model of CRS. METHODS: Adult male Sprague-Dawley rats underwent surgery for myocardial infarction (MI) (week 0) followed by 5/6 subtotal nephrectomy (STNx) at week 4 to induce to induce a combined model of heart and kidney dysfunction. At week 6, MI + STNx animals were randomized to receive either 0.5% carboxymethyl cellulose (Vehicle, n = 15, Sham = 10) or G226 (15 mg/kg daily, n = 11). Cardiac and renal function was assessed by echocardiography and glomerular filtration rate (GFR) respectively, prior to treatment at week 6 and endpoint (week 14). Haemodynamic measurements were determined at endpoint prior to tissue analysis. RESULTS: G226 treatment attenuated the absolute change in left ventricular (LV) fractional shortening and posterior wall thickness compared to Vehicle. G226 also attenuated the reduction in preload recruitable stroke work. Increased myocyte cross sectional area, cardiac interstitial fibrosis, immunoreactivity of cardiac collagen-I and III and cardiac TIMP-2 activation, were significantly reduced following G226 treatment. Although we did not observe improvement in GFR, G226 significantly reduced renal interstitial fibrosis, diminished renal collagen-I and -IV, kidney injury molecule-1 immunoreactivity as well as macrophage infiltration and SMAD2 phosphorylation. CONCLUSION: Inhibition of ASK1 ameliorated LV dysfunction and diminished cardiac hypertrophy and cardiorenal fibrosis in a rat model of CRS. This suggests that ASK1 is a critical pathway with therapeutic potential in the CRS setting.


Assuntos
Síndrome Cardiorrenal , Disfunção Ventricular Esquerda , Animais , Síndrome Cardiorrenal/tratamento farmacológico , Fibrose , Hipertrofia Ventricular Esquerda , MAP Quinase Quinase Quinase 5 , Masculino , Ratos , Ratos Sprague-Dawley
11.
J Pharmacol Exp Ther ; 370(3): 786-795, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-30936291

RESUMO

Autosomal dominant polycystic kidney disease (ADPKD) is a leading monogenetic cause of end-stage renal disease with limited therapeutic repertoire. A targeted drug delivery strategy that directs a small molecule to renal niches around cysts could increase the safety margins of agents that slow the progression of ADPKD but are poorly tolerated due to extrarenal toxicity. Herein, we determined whether previously characterized lysine-based and glutamic acid-based megalin-binding peptides can achieve renal-specific localization in the juvenile cystic kidney (JCK) mouse model of polycystic kidney disease and whether the distribution is altered compared with control mice. We performed in vivo optical and magnetic resonance imaging studies using peptides conjugated to the VivoTag 680 dye and demonstrated that megalin-interacting peptides distributed almost exclusively to the kidney cortex in both normal and JCK mice. Confocal analysis demonstrated that the peptide-dye conjugate distribution overlapped with megalin-positive renal proximal tubules. However, in the JCK mouse, the epithelium of renal cysts did not retain expression of the proximal tubule markers aquaporin 1 and megalin, and therefore these cysts did not retain peptide-dye conjugates. Furthermore, human kidney tumor tissues were evaluated by immunohistochemistry and revealed significant megalin expression in tissues from patients with renal cell carcinoma, raising the possibility that these tumors could be treated using this drug delivery strategy. Taken together, our data suggest that linking a small-molecule drug to these carrier peptides could represent a promising opportunity to develop a new platform for renal enrichment and targeting in the treatment of ADPKD and certain renal carcinomas.


Assuntos
Sistemas de Liberação de Medicamentos/métodos , Rim/efeitos dos fármacos , Peptídeos/administração & dosagem , Doenças Renais Policísticas/tratamento farmacológico , Animais , Aquaporina 1/metabolismo , Corantes , Desenho de Fármacos , Epitélio/metabolismo , Ácido Glutâmico/química , Humanos , Córtex Renal/diagnóstico por imagem , Córtex Renal/metabolismo , Neoplasias Renais/metabolismo , Túbulos Renais Proximais/metabolismo , Proteína-2 Relacionada a Receptor de Lipoproteína de Baixa Densidade/metabolismo , Lisina/química , Imageamento por Ressonância Magnética , Camundongos , Peptídeos/química , Peptídeos/farmacocinética , Doenças Renais Policísticas/diagnóstico por imagem , Distribuição Tecidual
12.
Heart Lung Circ ; 28(3): 495-504, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-29277546

RESUMO

BACKGROUND: Cardiac remodelling is a dynamic process whereby structural and functional changes occur within the heart in response to injury or inflammation. Recent studies have demonstrated reactive oxygen species sensitive MAPK, apoptosis signal-regulating kinase 1 (ASK1) plays a critical role in cardiac remodelling. This study aims to determine the effectiveness of small molecule ASK1 inhibitors on these processes and their therapeutic potential. METHODS: Neonatal rat cardiac fibroblasts (NCF) were pre-treated with ASK1 inhibitors, G2261818A (G226) and G2358939A (G235), for 2hours before stimulated with 100nM angiotensin II (AngII), 10µM indoxyl sulphate (IS) or 10ng/ml transforming growth factor ß1 (TGFß1) for 48hours. Neonatal rat cardiac myocytes (NCM) were pre-treated with G226 and G235 for 2hours before being stimulated with 100nM AngII for 60hours, 10µM IS, 10ng/ml interleukin 1ß (IL-1ß) or tumour necrosis factor α (TNFα) for 48hours. 3H-proline and 3H-leucine incorporation was used to assess collagen turnover and hypertrophy, respectively. Pro-fibrotic, pro-hypertrophic and THP-1 inflammatory cytokine gene expressions were determined by RT-PCR. RESULTS: Both G226 and G235 dose-dependently attenuated AngII-, IS-, IL-1ß- and TNFα-stimulated NCM hypertrophy and hypertrophic gene expression, IS-, AngII- and TGFß1-stimulated NCF collagen synthesis and AngII- and TGFß1-stimulated pro-fibrotic gene expression. Inhibition of ASK1 by G226 and G235 inhibited lipopolysaccharides-stimulated inflammatory cytokine gene expression in THP-1 cells. CONCLUSIONS: Selective ASK1 inhibition confers anti-hypertrophic and anti-fibrotic effects in cardiac cells, and anti-inflammation in monocytic cells. ASK1 inhibitors may represent novel therapeutic agents to alleviate cardiac remodelling post cardiac injury where hypertrophy, fibrosis and inflammation play critical roles.


Assuntos
Cardiomegalia/genética , Colágeno/biossíntese , Regulação da Expressão Gênica no Desenvolvimento , MAP Quinase Quinase Quinase 5/genética , Miócitos Cardíacos/patologia , RNA/genética , Animais , Animais Recém-Nascidos , Cardiomegalia/metabolismo , Cardiomegalia/patologia , Células Cultivadas , Modelos Animais de Doenças , Fibroblastos/metabolismo , MAP Quinase Quinase Quinase 5/antagonistas & inibidores , MAP Quinase Quinase Quinase 5/biossíntese , Miócitos Cardíacos/metabolismo , Reação em Cadeia da Polimerase , Ratos , Ratos Sprague-Dawley , Transdução de Sinais
13.
PLoS One ; 13(5): e0197213, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29771932

RESUMO

Drug-induced liver injury (DILI) is a leading cause of acute liver failure and transplantation. DILI can be the result of impaired hepatobiliary transporters, with altered bile formation, flow, and subsequent cholestasis. We used gadoxetate dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI), combined with pharmacokinetic modelling, to measure hepatobiliary transporter function in vivo in rats. The sensitivity and robustness of the method was tested by evaluating the effect of a clinical dose of the antibiotic rifampicin in four different preclinical imaging centers. The mean gadoxetate uptake rate constant for the vehicle groups at all centers was 39.3 +/- 3.4 s-1 (n = 23) and 11.7 +/- 1.3 s-1 (n = 20) for the rifampicin groups. The mean gadoxetate efflux rate constant for the vehicle groups was 1.53 +/- 0.08 s-1 (n = 23) and for the rifampicin treated groups was 0.94 +/- 0.08 s-1 (n = 20). Both the uptake and excretion transporters of gadoxetate were statistically significantly inhibited by the clinical dose of rifampicin at all centers and the size of this treatment group effect was consistent across the centers. Gadoxetate is a clinically approved MRI contrast agent, so this method is readily transferable to the clinic. CONCLUSION: Rate constants of gadoxetate uptake and excretion are sensitive and robust biomarkers to detect early changes in hepatobiliary transporter function in vivo in rats prior to established biomarkers of liver toxicity.


Assuntos
Meios de Contraste , Gadolínio DTPA , Fígado , Imageamento por Ressonância Magnética , Animais , Transporte Biológico Ativo/efeitos dos fármacos , Biomarcadores/metabolismo , Meios de Contraste/farmacocinética , Meios de Contraste/farmacologia , Avaliação Pré-Clínica de Medicamentos , Gadolínio DTPA/farmacocinética , Gadolínio DTPA/farmacologia , Fígado/diagnóstico por imagem , Fígado/metabolismo , Masculino , Ratos , Ratos Wistar
14.
Birth Defects Res ; 110(3): 276-298, 2018 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-29159925

RESUMO

OBJECTIVES: In our laboratory we evaluated the use of micro-computed tomography (micro-CT) using a high resolution acquisition protocol and fetuses obtained on Gestation Day (GD) 29 (mating = GD 0). METHODS: To show concordance between traditional Alizarin Red S stain and micro-CT skeletal examination methods, 103 fetuses from 19 untreated Dutch belted rabbits were obtained by cesarean section and stored frozen. The fetuses were thawed, imaged and examined digitally by micro-CT, then stained and re-examined using traditional methods. RESULTS: A total of 12 individual malformations and 35 unique variations were detected by both methods. Differences in the extent of ossification were found in only 51 of 26,196 bones while 99.8% of the observations were identical. Of the 51 differences, 31 were an unossified fifth medial phalanx of the forepaw indicating that very low-density skeletal bones may be visible by Alizarin Red stain but not by micro-CT scan. To establish this methodology under pharmaceutical testing conditions, we obtained and imaged by micro-CT Alizarin Red S stained abnormal fetal rabbit skeletons previously exposed to a drug candidate associated with craniofacial malformations in humans. All of the types of skeletal abnormalities first identified by staining were also detected by micro-CT examination. Representative images of these 66 different fetal skeletal abnormalities were characterized, and compiled to illustrate visual concordance between micro-CT scanned and traditional Alizarin Red S stained skeletons. CONCLUSION: Micro-CT imaging is an accurate, reliable and robust method that can be used as an alternative to stain when examining fetal rabbit skeletons in developmental toxicity studies.


Assuntos
Antraquinonas/química , Osso e Ossos , Feto , Coloração e Rotulagem , Microtomografia por Raio-X/métodos , Animais , Osso e Ossos/anormalidades , Osso e Ossos/diagnóstico por imagem , Osso e Ossos/embriologia , Feto/anormalidades , Feto/diagnóstico por imagem , Feto/embriologia , Coelhos
15.
PLoS One ; 12(11): e0187459, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-29107962

RESUMO

Intracellular accumulation of protein-bound uremic toxins in the setting of cardiorenal syndrome leads to adverse effects on cardiorenal cellular functions, where cardiac hypertrophy and cardiorenal fibrosis are the hallmarks. In this study, we sought to determine if Apoptosis Signal-Regulated Kinase 1 (ASK1), an upstream regulator of cellular stress response, mediates cardiac hypertrophy and cardiorenal fibrosis induced by indoxyl sulfate (IS) and p-cresol sulfate (PCS) in vitro, and whether ASK1 inhibition is beneficial to ameliorate these cellular effects. PCS augmented cardiac myocyte hypertrophy and fibroblast collagen synthesis (as determined by 3H-leucine and 3H-proline incorporation, respectively), similar to our previous finding with IS. IS and PCS also increased collagen synthesis of proximal tubular cells and renal mesangial cells. Pro-hypertrophic (α-skeletal muscle actin and ß-MHC) and pro-fibrotic genes (TGF-ß1 and ctgf) were induced by both IS and PCS. Western blot analyses revealed the activation of ASK1 and downstream mitogen activated protein kinases (MAPKs) (p38MAPK and ERK1/2) as well as nuclear factor-kappa B (NF-κB) by IS and PCS. ASK1, OAT1/3, ERK1/2 and p38MAPK inhibitors suppressed all these effects. In summary, IS and PCS exhibit pro-hypertrophic and pro-fibrotic properties, at least in part, via the activation of ASK1 and its downstream pathways. ASK1 inhibitor is an effective therapeutic agent to alleviate protein-bound uremic toxin-induced cardiac hypertrophy and cardiorenal fibrosis in vitro, and may be translated further for cardiorenal syndrome therapy.


Assuntos
Síndrome Cardiorrenal/patologia , Cardiomegalia/prevenção & controle , MAP Quinase Quinase Quinase 5/antagonistas & inibidores , Toxinas Biológicas/toxicidade , Animais , Células Cultivadas , Cresóis/farmacologia , Fibrose , Indicã/farmacologia , Proteínas Quinases Ativadas por Mitógeno/metabolismo , NF-kappa B/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Ratos , Ésteres do Ácido Sulfúrico/farmacologia
16.
J Control Release ; 268: 102-112, 2017 Dec 28.
Artigo em Inglês | MEDLINE | ID: mdl-29042321

RESUMO

Long-Acting Parenterals (LAPs) have been used in the clinic to provide sustained therapeutic drug levels at a target site, and thereby reducing the frequency of dosing required. In an effort to understand the factors associated with long-acting cabotegravir (GSK1265744 LAP) pharmacokinetic variability, the current study was designed to investigate the temporal relationship between intramuscular (IM) or subcutaneous (SC) drug depot morphology and distribution kinetics with plasma pharmacokinetics. Therefore, a multi-modal molecular imaging (MRI & MALDI IMS) approach was employed to examine the temporal GSK1265744 LAP biodistribution in rat following either IM or SC administration. Serial MRI was performed immediately post drug administration, and then at day 1 (24h post), 2, 3, 4, 7, and 14. In a separate cohort of rats, an MRI contrast agent, Feraheme® (USPIO), was administered 2days post IM drug injection in order to investigate the potential involvement of macrophages trafficking to the GSK1265744 LAP and Vehicle depot sites. The GSK1265744 LAP depot volume increased rapidly by day 2 in the IM injected rats (~3-7 fold) compared with a ~1 fold increase in the SC injected rats. In addition, the USPIO contrast agent labeled macrophages were shown to be present in the depot region of the GSK1265744 LAP injected gastrocnemius while the Vehicle injected gastrocnemius appeared to show reduced uptake. Matrix-assisted laser desorption ionization (MALDI) imaging mass spectrometry (IMS) of muscle and abdominal tissue sections identified the drug content primarily within the depot. Co-registration of the GSK1265744 ion images with immunohistochemical images established that the drug was taken up by macrophages associated with the depot. Linear regression analysis demonstrated that the drug depot characteristics including volume, surface area, and perimeter assessed by MRI at day 2 correlated with early time point plasma drug concentrations. In summary, a multimodal molecular imaging approach was used to identify the drug depot location and volumetric/physiologic changes in both IM and SC locations following GSK1265744 LAP administration. The IM depot volume increased rapidly to a maximum volume at 2days post-GSK1265744 LAP administration, while the Vehicle depot did not suggesting that the active drug substance and/or related particle was a key driver for drug depot evolution. The depot expansion was associated with an increase in macrophage infiltration and edema in and around the depot region and was correlated to plasma drug concentration at early time points (0-4days). Consequently, molecular imaging approaches may be used in patients to help understand the biodistribution of GSK1265744 LAP and its associated pharmacokinetics.


Assuntos
Piridonas/administração & dosagem , Piridonas/farmacocinética , Animais , Meios de Contraste/administração & dosagem , Preparações de Ação Retardada/administração & dosagem , Preparações de Ação Retardada/farmacocinética , Óxido Ferroso-Férrico/administração & dosagem , Injeções Intramusculares , Injeções Subcutâneas , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Imageamento por Ressonância Magnética , Masculino , Imagem Multimodal , Piridonas/sangue , Ratos Sprague-Dawley , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Distribuição Tecidual
17.
PLoS One ; 12(4): e0176075, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28448604

RESUMO

The purpose of this work was to use various molecular imaging techniques to non-invasively assess GSK2849330 (anti HER3 ADCC and CDC enhanced 'AccretaMab' monoclonal antibody) pharmacokinetics and pharmacodynamics in human xenograft tumor-bearing mice. Immuno-PET biodistribution imaging of radiolabeled 89Zr-GSK2849330 was assessed in mice with HER3 negative (MIA-PaCa-2) and positive (CHL-1) human xenograft tumors. Dose dependency of GSK2849330 disposition was assessed using varying doses of unlabeled GSK2849330 co-injected with 89Zr-GSK2849330. In-vivo NIRF optical imaging and ex-vivo confocal microscopy were used to assess the biodistribution of GSK2849330 and the HER3 receptor occupancy in HER3 positive xenograft tumors (BxPC3, and CHL-1). Ferumoxytol (USPIO) contrast-enhanced MRI was used to investigate the effects of GSK2849330 on tumor macrophage content in CHL-1 xenograft bearing mice. Immuno-PET imaging was used to monitor the whole body drug biodistribution and CHL-1 xenograft tumor uptake up to 144 hours post injection of 89Zr-GSK2849330. Both hepatic and tumor uptake were dose dependent and saturable. The optical imaging data in the BxPC3 xenograft tumor confirmed the tumor dose response finding in the Immuno-PET study. Confocal microscopy showed a distinguished cytoplasmic punctate staining pattern within individual CHL-1 cells. GSK2849330 inhibited tumor growth and this was associated with a significant decrease in MRI signal to noise ratio after USPIO injection and with a significant increase in tumor macrophages as confirmed by a quantitative immunohistochemistry analysis. By providing both dose response and time course data from both 89Zr and fluorescently labeled GSK2849330, complementary imaging studies were used to characterize GSK2849330 biodistribution and tumor uptake in vivo. Ferumoxytol-enhanced MRI was used to monitor aspects of the immune system response to GSK2849330. Together these approaches potentially provide clinically translatable, non-invasive techniques to support dose optimization, and assess immune activation and anti-tumor responses.


Assuntos
Anticorpos Monoclonais Humanizados/farmacocinética , Anticorpos Monoclonais/farmacocinética , Macrófagos/imunologia , Compostos Radiofarmacêuticos/farmacocinética , Receptor ErbB-3/imunologia , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/uso terapêutico , Anticorpos Monoclonais Humanizados/química , Anticorpos Monoclonais Humanizados/uso terapêutico , Linhagem Celular Tumoral , Feminino , Óxido Ferroso-Férrico/química , Humanos , Imuno-Histoquímica , Marcação por Isótopo , Macrófagos/citologia , Macrófagos/patologia , Camundongos , Camundongos Nus , Neoplasias/diagnóstico por imagem , Neoplasias/tratamento farmacológico , Radioisótopos , Compostos Radiofarmacêuticos/química , Compostos Radiofarmacêuticos/uso terapêutico , Receptor ErbB-3/metabolismo , Distribuição Tecidual , Transplante Heterólogo , Zircônio/química
18.
Oncotarget ; 7(26): 39861-39871, 2016 Jun 28.
Artigo em Inglês | MEDLINE | ID: mdl-27223434

RESUMO

Fibroblast growth factor (FGF) ligand-dependent signaling has a fundamental role in cancer development and tumor maintenance. GSK3052230 (also known as FP-1039) is a soluble decoy receptor that sequesters FGFs and inhibits FGFR signaling. Herein, the efficacy of this molecule was tested in models of mesothelioma, a tumor type shown to express high levels of FGF2 and FGFR1. GSK3052230 demonstrated antiproliferative activity across a panel of mesothelioma cell lines and inhibited growth of tumor xenografts in mice. High expression of FGF2 and FGFR1 correlated well with response to FGF pathway inhibition. GSK3052230 inhibited MAPK signaling as evidenced by decreased phospho-ERK and phospho-S6 levels in vitro and in vivo. Additionally, dose-dependent and statistically-significant reductions in tumor vessel density were observed in GSK3052230-treated tumors compared to vehicle-treated tumors. These data support the role of GSK3052230 in effectively targeting FGF-FGFR autocrine signaling in mesothelioma, demonstrate its impact on tumor growth and angiogenesis, and provide a rationale for the current clinical evaluation of this molecule in mesothelioma patients.


Assuntos
Fatores de Crescimento de Fibroblastos/metabolismo , Mesotelioma/metabolismo , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/metabolismo , Transdução de Sinais , Animais , Comunicação Autócrina , Linhagem Celular , Proliferação de Células , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Imunoglobulina G/química , Ligantes , Imageamento por Ressonância Magnética , Camundongos , Camundongos SCID , Transplante de Neoplasias , Neovascularização Patológica , Proteínas de Fusão Oncogênica/química , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/química , Proteínas Recombinantes de Fusão
19.
Toxicol Pathol ; 44(1): 51-60, 2016 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-26516164

RESUMO

To determine if amiodarone induces hepatic phospholipidosis (PLD) sufficient to detect changes in hepatobiliary transporter function as assessed by gadoxetate dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI), rats were orally dosed with vehicle (1% methyl cellulose) or amiodarone (300 mg/kg/day) for 7 consecutive days. Gadoxetate DCE-MRI occurred at baseline, day 7, and following a 2-week washout of amiodarone. At day 7, the gadoxetate washout rate was significantly decreased compared to the vehicle group. Blood chemistry analysis revealed no significant changes in liver enzymes (alanine aminotransferase [ALT]/aspartate aminotransferase [AST]/alkaline phosphatase [ALP]), bilirubin, or bile acids between vehicle or amiodarone groups. Hepatic PLD was confirmed in all rats treated with amiodarone at day 7 by transmission electron microscopy. Following the 2-week washout, there was no ultrastructural evidence of hepatic PLD in rats and the gadoxetate washout rate returned to baseline levels. This is the first study to show the application of gadoxetate DCE-MRI to detect hepatobiliary functional changes associated with PLD and offer a potential new technique with clinical utility in patients suspected of having PLD. These results also suggest PLD itself has functional consequences on hepatobiliary function in the absence of biomarkers of toxicity, given the cause/effect relationship between PLD and function has not been fully established.


Assuntos
Sistema Biliar/fisiopatologia , Gadolínio DTPA/farmacocinética , Lipidoses/fisiopatologia , Fígado/fisiopatologia , Imageamento por Ressonância Magnética/métodos , Fosfolipídeos/metabolismo , Amiodarona/toxicidade , Animais , Sistema Biliar/metabolismo , Sistema Biliar/patologia , Lipidoses/induzido quimicamente , Lipidoses/metabolismo , Fígado/metabolismo , Fígado/patologia , Masculino , Ratos , Ratos Sprague-Dawley
20.
PLoS One ; 10(6): e0130894, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26098939

RESUMO

Previous studies have shown that glucagon-like peptide-1 (GLP-1) provides cardiovascular benefits independent of its role on peripheral glycemic control. However, the precise mechanism(s) by which GLP-1 treatment renders cardioprotection during myocardial ischemia remain unresolved. Here we examined the role for GLP-1 treatment on glucose and fatty acid metabolism in normal and ischemic rat hearts following a 30 min ischemia and 24 h reperfusion injury, and in isolated cardiomyocytes (CM). Relative carbohydrate and fat oxidation levels were measured in both normal and ischemic hearts using a 1-13C glucose clamp coupled with NMR-based isotopomer analysis, as well as in adult rat CMs by monitoring pH and O2 consumption in the presence of glucose or palmitate. In normal heart, GLP-1 increased glucose uptake (↑64%, p<0.05) without affecting glycogen levels. In ischemic hearts, GLP-1 induced metabolic substrate switching by increasing the ratio of carbohydrate versus fat oxidation (↑14%, p<0.01) in the LV area not at risk, without affecting cAMP levels. Interestingly, no substrate switching occurred in the LV area at risk, despite an increase in cAMP (↑106%, p<0.05) and lactate (↑121%, p<0.01) levels. Furthermore, in isolated CMs GLP-1 treatment increased glucose utilization (↑14%, p<0.05) and decreased fatty acid oxidation (↓15%, p<0.05) consistent with in vivo finding. Our results show that this benefit may derive from distinct and complementary roles of GLP-1 treatment on metabolism in myocardial sub-regions in response to this injury. In particular, a switch to anaerobic glycolysis in the ischemic area provides a compensatory substrate switch to overcome the energetic deficit in this region in the face of reduced tissue oxygenation, whereas a switch to more energetically favorable carbohydrate oxidation in more highly oxygenated remote regions supports maintaining cardiac contractility in a complementary manner.


Assuntos
Cardiotônicos/farmacologia , Metabolismo Energético/efeitos dos fármacos , Peptídeo 1 Semelhante ao Glucagon/farmacologia , Miocárdio/metabolismo , Traumatismo por Reperfusão/metabolismo , Animais , Isótopos de Carbono/metabolismo , Cardiotônicos/administração & dosagem , AMP Cíclico/metabolismo , Metabolismo Energético/fisiologia , Ácidos Graxos/metabolismo , Peptídeo 1 Semelhante ao Glucagon/administração & dosagem , Glucose/metabolismo , Concentração de Íons de Hidrogênio , Espectroscopia de Ressonância Magnética , Miócitos Cardíacos/metabolismo , Consumo de Oxigênio/fisiologia , Ratos
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