RESUMO
CPX-351, a liposomal encapsulation of cytarabine and daunorubicin at a synergistic 5:1 molar ratio, is indicated for adults with newly diagnosed, therapy-related acute myeloid leukemia or acute myeloid leukemia with myelodysplasia-related changes. In preclinical species, this article demonstrated (1) similar release of cytarabine and daunorubicin by CPX-351 in plasma; (2) similar patterns of metabolism of cytarabine and daunorubicin following administration of CPX-351 versus non-liposomal cytarabine/daunorubicin combination; (3) prolonged tissue exposure to CPX-351; (4) dramatically different tissue distribution of cytarabine and daunorubicin following administration of CPX-351 versus non-liposomal combination (tissue:plasma ratios generally <1 versus >1, respectively); and (5) dramatically lower unbound plasma and tissue concentrations of cytarabine and daunorubicin following administration of CPX-351 versus non-liposomal combination. Together, these results provide insight into the safety profile of CPX-351, as well as mechanisms that drive the improved efficacy observed for CPX-351 versus the conventional 7â¯+â¯3 cytarabine/daunorubicin regimen in clinical studies.
Assuntos
Antineoplásicos/farmacocinética , Citarabina/farmacocinética , Daunorrubicina/farmacocinética , Animais , Área Sob a Curva , Bile/química , Cromatografia Líquida de Alta Pressão/métodos , Citarabina/sangue , Citarabina/urina , Daunorrubicina/sangue , Daunorrubicina/urina , Cães , Combinação de Medicamentos , Fezes/química , Feminino , Meia-Vida , Limite de Detecção , Masculino , Camundongos , Ratos , Espectrometria de Fluorescência/métodos , Espectrofotometria Ultravioleta/métodos , Distribuição TecidualRESUMO
UNLABELLED: Caffeine is one of the world's most consumed drugs. Recently, several studies showed that its consumption is associated with lower risk for nonalcoholic fatty liver disease (NAFLD), an obesity-related condition that recently has become the major cause of liver disease worldwide. Although caffeine is known to stimulate hepatic fat oxidation, its mechanism of action on lipid metabolism is still not clear. Here, we show that caffeine surprisingly is a potent stimulator of hepatic autophagic flux. Using genetic, pharmacological, and metabolomic approaches, we demonstrate that caffeine reduces intrahepatic lipid content and stimulates ß-oxidation in hepatic cells and liver by an autophagy-lysosomal pathway. Furthermore, caffeine-induced autophagy involved down-regulation of mammalian target of rapamycin signaling and alteration in hepatic amino acids and sphingolipid levels. In mice fed a high-fat diet, caffeine markedly reduces hepatosteatosis and concomitantly increases autophagy and lipid uptake in lysosomes. CONCLUSION: These results provide novel insight into caffeine's lipolytic actions through autophagy in mammalian liver and its potential beneficial effects in NAFLD.
Assuntos
Autofagia/efeitos dos fármacos , Cafeína/farmacologia , Metabolismo dos Lipídeos/efeitos dos fármacos , Fígado/efeitos dos fármacos , Fígado/metabolismo , Lisossomos/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Animais , Autofagia/fisiologia , Cafeína/uso terapêutico , Linhagem Celular Tumoral , Dieta Hiperlipídica/efeitos adversos , Regulação para Baixo/efeitos dos fármacos , Fígado Gorduroso/induzido quimicamente , Fígado Gorduroso/metabolismo , Fígado Gorduroso/prevenção & controle , Células Hep G2 , Humanos , Técnicas In Vitro , Lipólise/efeitos dos fármacos , Lipólise/fisiologia , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Lisossomos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Modelos Animais , Oxirredução/efeitos dos fármacos , Transdução de Sinais/fisiologia , Serina-Treonina Quinases TOR/metabolismoRESUMO
Hepatic gluconeogenesis is a concerted process that integrates transcriptional regulation with hormonal signals. A major regulator is thyroid hormone (TH), which acts through its nuclear receptor (TR) to induce the expression of the hepatic gluconeogenic genes, phosphoenolpyruvate carboxykinase (PCK1) and glucose-6-phosphatase (G6PC). Forkhead transcription factor FoxO1 also is an important regulator of these genes; however, its functional interactions with TR are not known. Here, we report that TR-mediated transcriptional activation of PCK1 and G6PC in human hepatic cells and mouse liver was FoxO1-dependent and furthermore required FoxO1 deacetylation by the NAD(+)-dependent deacetylase, SirT1. siRNA knockdown of FoxO1 decreased, whereas overexpression of FoxO1 increased, TH-dependent transcriptional activation of PCK1 and G6PC in cultured hepatic cells. FoxO1 siRNA knockdown also decreased TH-mediated transcription in vivo. Additionally, TH was unable to induce FoxO1 deacetylation or hepatic PCK1 gene expression in TH receptor ß-null (TRß(-/-)) mice. Moreover, TH stimulated FoxO1 recruitment to the PCK1 and G6PC gene promoters in a SirT1-dependent manner. In summary, our results show that TH-dependent deacetylation of a second metabolically regulated transcription factor represents a novel mechanism for transcriptional integration of nuclear hormone action with cellular energy status.
Assuntos
Fatores de Transcrição Forkhead/metabolismo , Gluconeogênese/fisiologia , Fígado/metabolismo , Hormônios Tireóideos/metabolismo , Transcrição Gênica/fisiologia , Ativação Transcricional/fisiologia , Acetilação , Animais , Proteína Forkhead Box O1 , Fatores de Transcrição Forkhead/genética , Glucose-6-Fosfatase/biossíntese , Glucose-6-Fosfatase/genética , Células Hep G2 , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/biossíntese , Peptídeos e Proteínas de Sinalização Intracelular/genética , Masculino , Camundongos , Camundongos Knockout , Fosfoenolpiruvato Carboxiquinase (GTP)/biossíntese , Fosfoenolpiruvato Carboxiquinase (GTP)/genética , Regiões Promotoras Genéticas/fisiologia , Receptores dos Hormônios Tireóideos/genética , Receptores dos Hormônios Tireóideos/metabolismo , Sirtuína 1/genética , Sirtuína 1/metabolismo , Hormônios Tireóideos/genéticaRESUMO
Insulin and glucagon signaling in the liver are major contributors to glucose homeostasis. Patients with Type 1 and Type 2 diabetes have impaired glycemic control due, in part, to dysregulation of the opposing actions of these hormones. While hyperglucagonemia is a common feature in diabetes, its precise role in insulin resistance is not well understood. Recently, metformin, an AMPK activator, was shown to regulate hepatic glucose output via inhibition of glucagon-induced cAMP/PKA signaling; however, the mechanism for how PKA inhibition leads to AMPK activation in human hepatic cells is not known. Here we show that glucagon impairs insulin-mediated AKT phosphorylation in human hepatic cell line Huh7. This impairment of AKT activation by glucagon is due to PKA-mediated inhibition of AMPK via increased inhibitory phosphorylation of AMPK(Ser173) and reduced activating phosphorylation of AMPK(Thr172). In contrast, metformin decreases PKA activity, leading to decreased pAMPK(Ser173) and increased pAMPK(Thr172). These data support a novel mechanism involving PKA-dependent AMPK phosphorylation that provides new insight into how glucagon and metformin modulate hepatic insulin resistance.
Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Glucagon/metabolismo , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Hipoglicemiantes/farmacologia , Insulina/metabolismo , Metformina/farmacologia , Proteínas Quinases Ativadas por AMP/antagonistas & inibidores , Proteínas Quinases Ativadas por AMP/genética , Linhagem Celular , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Técnicas de Silenciamento de Genes , Humanos , Resistência à Insulina/fisiologia , Fosforilação , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-akt/metabolismo , RNA Interferente Pequeno/genética , Transdução de Sinais/efeitos dos fármacosRESUMO
OBJECTIVE: Crinophagy is a secretory granule-specific autophagic process that regulates hormone content and secretion in endocrine cells. However, despite being one of the earliest described autophagic processes, its mechanism of action and regulation in mammalian cells remains unclear. METHODS AND RESULTS: Here, we examined mammalian crinophagy and its modulation that regulate hormone secretion in a glucagon-producing mouse pancreatic α-cell line, alpha TC1 clone 9 (αTC9), and in vivo. Western blot, electron microscopy, and immunofluorescence analyses were performed to study crinophagy and glucagon secretion in αTC9 cells and C57BL/6 mice, in response to the mammalian target of rapamycin complex 1 (MTORC1) inhibitor rapamycin. Amino acid depletion and pharmacological inhibition of MTORC1 increased the shuttling of glucagon-containing secretory granules into lysosomes for crinophagic degradation to reduce glucagon secretion through a macroautophagy-independent mechanism. Furthermore, MTORC1 inhibition reduced both intracellular and secreted glucagon in rapamycin-treated mice, in response to hypoglycaemia. CONCLUSION: In summary, we have identified a novel crinophagic mechanism of intracellular glucagon turnover in pancreatic α-cells regulated by MTORC1 signalling.
Assuntos
Autofagia , Glucagon/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Vesículas Secretórias/metabolismo , Animais , Células Cultivadas , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos TransgênicosRESUMO
We demonstrate here that cytarabine and daunorubicin, a standard drug combination used in the treatment of leukaemia, exhibits drug ratio-dependent synergistic antitumor activity in vitro and in vivo. A cytarabine:daunorubicin molar ratio of 5:1 displayed the greatest degree of synergy and minimum antagonism in a panel of 15 tumor cell lines in vitro. Co-encapsulating cytarabine and daunorubicin inside liposomes maintained the synergistic drug ratio in plasma for 24h post-injection. Liposome-encapsulated cytarabine:daunorubicin combinations exhibited drug ratio-dependent in vivo efficacy with the 5:1 molar drug ratio (designated CPX-351) having the greatest therapeutic index, despite using sub-MTD daunorubicin doses. CPX-351 exhibited superior therapeutic activity compared to free-drug cocktails, with high proportions of long-term survivors, consistent with in vivo synergy. The therapeutic advantage of CPX-351 was associated with prolonged maintenance of synergistic drug ratios in bone marrow. These results indicate that in vitro informatics on cytarabine:daunorubicin cytotoxicity can be translated in vivo to optimize the efficacy of anticancer drug combinations by controlling the exposure of drug ratios with drug delivery vehicles.
Assuntos
Antineoplásicos/farmacologia , Citarabina/farmacologia , Daunorrubicina/farmacologia , Animais , Antineoplásicos/administração & dosagem , Antineoplásicos/farmacocinética , Linhagem Celular Tumoral , Proliferação de Células , Citarabina/administração & dosagem , Citarabina/farmacocinética , Daunorrubicina/administração & dosagem , Daunorrubicina/farmacocinética , Citometria de Fluxo , Humanos , Lipossomos , CamundongosRESUMO
A series of paclitaxel prodrugs designed for formulation in lipophilic nanoparticles are described. The hydrophobicity of paclitaxel was increased by conjugating a succession of increasingly hydrophobic lipid anchors to the drug using succinate or diglycolate cross-linkers. The prodrugs were formulated in well defined block copolymer-stabilized nanoparticles. These nanoparticles were shown to have an elimination half-life of approximately 24 h in vivo. The rate at which the prodrug was released from the nanoparticles could be controlled by adjusting the hydrophobicity of the lipid anchor, resulting in release half-lives ranging from 1 to 24 h. The diglycolate and succinate cross-linked prodrugs were 1-2 orders of magnitude less potent than paclitaxel in vitro. Nanoparticle formulations of the succinate prodrugs showed no evidence of efficacy in HT29 human colorectal tumor xenograph models. Efficacy of diglycolate prodrug nanoparticles increased as the anchor hydrophobicity increased. Long circulating diglycolate prodrug nanoparticles provided significantly enhanced therapeutic activity over commercially formulated paclitaxel at the maximum tolerated dose.
Assuntos
Paclitaxel/administração & dosagem , Pró-Fármacos/administração & dosagem , Animais , Linhagem Celular Tumoral , Estabilidade de Medicamentos , Humanos , Interações Hidrofóbicas e Hidrofílicas , Lipídeos/química , Camundongos , Camundongos Nus , Micelas , Nanopartículas , Transplante de Neoplasias , Paclitaxel/química , Paclitaxel/farmacologia , Polietilenoglicóis , Poliestirenos , Pró-Fármacos/química , Pró-Fármacos/farmacologia , Transplante HeterólogoRESUMO
Thyroid hormone receptor ß1 (THRB1) and estrogen-related receptor α (ESRRA; also known as ERRα) both play important roles in mitochondrial activity. To understand their potential interactions, we performed transcriptome and ChIP-seq analyses and found that many genes that were co-regulated by both THRB1 and ESRRA were involved in mitochondrial metabolic pathways. These included oxidative phosphorylation (OXPHOS), the tricarboxylic acid (TCA) cycle, and ß-oxidation of fatty acids. TH increased ESRRA expression and activity in a THRB1-dependent manner through the induction of the transcriptional coactivator PPARGC1A (also known as PGC1α). Moreover, TH induced mitochondrial biogenesis, fission, and mitophagy in an ESRRA-dependent manner. TH also induced the expression of the autophagy-regulating kinase ULK1 through ESRRA, which then promoted DRP1-mediated mitochondrial fission. In addition, ULK1 activated the docking receptor protein FUNDC1 and its interaction with the autophagosomal protein MAP1LC3B-II to induce mitophagy. siRNA knockdown of ESRRA, ULK1, DRP1, or FUNDC1 inhibited TH-induced autophagic clearance of mitochondria through mitophagy and decreased OXPHOS. These findings show that many of the mitochondrial actions of TH are mediated through stimulation of ESRRA expression and activity, and co-regulation of mitochondrial turnover through the PPARGC1A-ESRRA-ULK1 pathway is mediated by their regulation of mitochondrial fission and mitophagy. Hormonal or pharmacologic induction of ESRRA expression or activity could improve mitochondrial quality in metabolic disorders.
Assuntos
Autofagia , Mitocôndrias/fisiologia , Dinâmica Mitocondrial , Mitofagia , Receptores de Estrogênio/metabolismo , Receptores beta dos Hormônios Tireóideos/fisiologia , Animais , Proteína Homóloga à Proteína-1 Relacionada à Autofagia/genética , Proteína Homóloga à Proteína-1 Relacionada à Autofagia/metabolismo , Células Cultivadas , Dinaminas/genética , Dinaminas/metabolismo , Humanos , Masculino , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/genética , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/metabolismo , Fosforilação , Receptores de Estrogênio/genética , Receptor ERRalfa Relacionado ao EstrogênioRESUMO
Lipotoxicity caused by saturated fatty acids (SFAs) induces tissue damage and inflammation in metabolic disorders. SCD1 (stearoyl-coenzyme A desaturase 1) converts SFAs to mono-unsaturated fatty acids (MUFAs) that are incorporated into triglycerides and stored in lipid droplets. SCD1 thus helps protect hepatocytes from lipotoxicity and its reduced expression is associated with increased lipotoxic injury in cultured hepatic cells and mouse models. To further understand the role of SCD1 in lipotoxicity, we examined the regulation of Scd1 in hepatic cells treated with palmitate, and found that NR1H/LXR (nuclear receptor subfamily 1 group H) ligand, GW3965, induced Scd1 expression and lipid droplet formation to improve cell survival. Surprisingly, ULK1/ATG1 (unc-51 like kinase) played a critical role in protecting hepatic cells from SFA-induced lipotoxicity via a novel mechanism that did not involve macroautophagy/autophagy. Specific loss of Ulk1 blocked the induction of Scd1 gene transcription by GW3965, decreased lipid droplet formation, and increased apoptosis in hepatic cells exposed to palmitate. Knockdown of ULK1 increased RPS6KB1 (ribosomal protein S6 kinase, polypeptide 1) signaling that, in turn, induced NCOR1 (nuclear receptor co-repressor 1) nuclear uptake, interaction with NR1H/LXR, and recruitment to the Scd1 promoter. These events abrogated the stimulation of Scd1 gene expression by GW3965, and increased lipotoxicity in hepatic cells. In summary, we have identified a novel autophagy-independent role of ULK1 that regulates NR1H/LXR signaling, Scd1 expression, and intracellular lipid homeostasis in hepatic cells exposed to a lipotoxic environment.
Assuntos
Proteína Homóloga à Proteína-1 Relacionada à Autofagia/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Fígado/metabolismo , Correpressor 1 de Receptor Nuclear/metabolismo , Proteínas Quinases S6 Ribossômicas 70-kDa/metabolismo , Estearoil-CoA Dessaturase/metabolismo , Animais , Apoptose , Autofagia , Ácidos Graxos/metabolismo , Hepatócitos/metabolismo , Homeostase , Humanos , Lipídeos/química , Camundongos , Ácido Palmítico/metabolismo , Regiões Promotoras Genéticas , RNA Interferente Pequeno/metabolismoRESUMO
BACKGROUND: Type 1 and type 2 diabetes are characterized by loss of ß-cells; therefore, ß-cell regeneration has become one of the primary approaches to diabetes therapy. Resveratrol, a naturally occurring polyphenolic compound, has been shown to improve glycaemic control in diabetic patients, but its action on pancreatic α-cells is not well understood. FINDINGS: Using mouse α-cells (αTC9), we showed that resveratrol induces expression of pancreatic ß-cell genes such as Pdx1 and Ins2 in a SirT1-dependent manner. The mRNA and protein levels of insulin were further increased by histone deacetylase (HDAC) inhibition. CONCLUSION: In summary, we provide new mechanistic insight into the anti-diabetic action of resveratrol through its ability to express ß-cell genes in α-cells.
RESUMO
Pharmacokinetic modeling and simulation is a powerful tool for the prediction of drug concentrations in the absence of analytical techniques that allow for direct quantification. The present study applied this modeling approach to determine active drug release from a nanoparticle prodrug formulation. A comparative pharmacokinetic study of a nanoscale micellar docetaxel (DTX) prodrug, Procet 8, and commercial DTX formulation, Taxotere, was conducted in bile duct cannulated rats. The nanoscale (~40nm) size of the Procet 8 formulation resulted in confinement within the plasma space and high prodrug plasma concentrations. Ex vivo prodrug hydrolysis during plasma sample preparation resulted in unacceptable error that precluded direct measurement of DTX concentrations. Pharmacokinetic modeling of Taxotere and Procet 8 plasma concentrations, and their associated biliary metabolites, allowed for prediction of the DTX concentration profile and DTX bioavailability, and thereby evaluation of Procet 8 metabolism. Procet 8 plasma decay and in vitro plasma hydrolytic rates were identical, suggesting that systemic clearance of the prodrug was primarily metabolic. The Procet 8 and Taxotere plasma profiles, and associated docetaxel hydroxy-tert-butyl carbamate (HDTX) metabolite biliary excretion, were best fit by a two compartment model, with both linear and non-linear DTX clearance, and first order Procet 8 hydrolysis. The model estimated HDTX clearance rate agreed with in vitro literature values, supporting the predictability of the proposed model. Model simulation at the 10mg DTX equivalent/kg dose level predicted DTX formation rate-limited kinetics and a peak plasma DTX concentration of 39ng/mL at 4h for Procet 8, in comparison to 2826ng/mL for Taxotere. As a result of nonlinear DTX clearance, the DTX AUCinf for the Procet 8 formulation was predicted to be 2.6 times lower than Taxotere (775 vs. 2017h×ng/mL, respectively), resulting in an absolute bioavailability estimate of 38%. As DTX clearance in man is considered linear, this low bioavailability is likely species-dependent. These data support the use of pharmacokinetic modeling and simulation in cases of complex formulations, where analytical methods for direct measurement of free (released) drug concentrations are unavailable. Uses of such models may include interpretation of preclinical toxicology studies, selection of first in man dosing regimens, and PK/PD model development.
Assuntos
Antineoplásicos/metabolismo , Bile/metabolismo , Nanopartículas/metabolismo , Pró-Fármacos/metabolismo , Taxoides/metabolismo , Animais , Antineoplásicos/farmacocinética , Simulação por Computador , Docetaxel , Feminino , Hidrólise , Cinética , Masculino , Micelas , Modelos Biológicos , Pró-Fármacos/farmacocinética , Ratos , Ratos Sprague-Dawley , Taxoides/farmacocinéticaRESUMO
Diblock copolymer nanoparticles encapsulating a paclitaxel prodrug, Propac 7, have been used to demonstrate the usefulness of a nonmetabolizable radioactive marker, cholesteryl hexadecyl ether (CHE), to evaluate nanoparticle formulation variables. Since CHE did not exchange out of the nanoparticles, the rate of clearance of the CHE could be used as an indicator of nanoparticle stability in vivo. We simultaneously monitored prodrug circulation and carrier circulation in the plasma and the retention of CHE relative to the retention of prodrug in the plasma was used to distinguish prodrug release from nanoparticle plasma clearance. Nanoparticles labelled with CHE were also used to evaluate accumulation of nanoparticles in the tumour. This marker has provided relevant data which we have applied to optimise our nanoparticle formulations.
RESUMO
Essiac, a widely consumed, sparsely tested herbal tea, was evaluated for preparation consistency and antiproliferative effects on prostate cancer cells and xenografts. High performance liquid chromatography (HPLC) was used to compare different lots of Essiac and evaluate extraction consistency by comparing peak areas in concentrated preparations. Repeated analysis of one lot showed < 2% RSD between corresponding peaks. Absolute peak areas varied widely between lots, but similarity in relative size of corresponding peaks was observed. Cytotoxic effects of Essiac were tested in vitro by crystal violet assay and analysis of cell cycle distribution by flow cytometry, but no differences between control and treatment groups was observed. Paclitaxel was used as a positive control in cell cycle analysis and was the only treatment which showed significant effects on cell cycle distribution. Toxicity in nude mice was tested, and efficacy in inhibiting PC-3 xenograft growth. No toxicity or tumour size difference was observed dosing up to 240 mg/kg QD, over 28 days, excepting the positive control group treated with paclitaxel. Ki-67 and PCNA expression was analyzed in treated tumors, but no difference in expression of either marker was observed. These evaluations suggest Essiac has no marked antiproliferative effect on the models tested.
Assuntos
Antineoplásicos Fitogênicos/farmacologia , Ciclo Celular/efeitos dos fármacos , Extratos Vegetais/análise , Extratos Vegetais/farmacologia , Neoplasias da Próstata/tratamento farmacológico , Animais , Área Sob a Curva , Cromatografia Líquida de Alta Pressão/métodos , Relação Dose-Resposta a Droga , Citometria de Fluxo , Imuno-Histoquímica , Antígeno Ki-67/metabolismo , Masculino , Camundongos , Camundongos Nus , Paclitaxel/farmacologia , Extratos Vegetais/toxicidade , Antígeno Nuclear de Célula em Proliferação/metabolismo , Distribuição Aleatória , Transplante HeterólogoRESUMO
The methanol extract of the underground part of Rhodiola rosea was found to show inhibitory activity against Staphylococcus aureus. Bioactivity-guided fractionation of a 95% ethanol extract from the stems of R. rosea led to the isolation of five compounds: gossypetin-7-O-L-rhamnopyranoside (1), rhodioflavonoside (2), gallic acid (3), trans-p-hydroxycinnamic acid (4) and p-tyrosol (5). Their structures were elucidated by UV, IR, MS and NMR data, as well as by comparison with those of the literature. Compounds 1 and 2 were evaluated for their antibacterial and antiprostate cancer cell activities. Compounds 1 and 2 exhibited activity against Staphylococcus aureus with minimum inhibitory concentrations of 50 microg/mL and 100 microg/mL, respectively. Cytotoxicity studies of 1 and 2 also displayed activity against the prostate cancer cell line with IC(50) values of 50 microg/mL and 80 microg/mL, respectively.