RESUMO
Prescription opioids are a mainstay in the treatment of acute moderate to severe pain. However, chronic use leads to a host of adverse consequences including tolerance and opioid-induced hyperalgesia (OIH), leading to more complex treatment regimens and diminished patient compliance. Patients with OIH paradoxically experience exaggerated nociceptive responses instead of pain reduction after chronic opioid usage. The development of OIH and tolerance tend to occur simultaneously and, thus, present a challenge when studying the molecular mechanisms driving each phenomenon. We tested the hypothesis that a G protein-biased µ-opioid peptide receptor (MOPR) agonist would not induce symptoms of OIH, such as mechanical allodynia, following chronic administration. We observed that the development of opioid-induced mechanical allodynia (OIMA), a model of OIH, was absent in ß-arrestin1-/- and ß-arrestin2-/- mice in response to chronic administration of conventional opioids such as morphine, oxycodone and fentanyl, whereas tolerance developed independent of OIMA. In agreement with the ß-arrestin knockout mouse studies, chronic administration of TRV0109101, a G protein-biased MOPR ligand and structural analog of oliceridine, did not promote the development of OIMA but did result in drug tolerance. Interestingly, following induction of OIMA by morphine or fentanyl, TRV0109101 was able to rapidly reverse allodynia. These observations establish a role for ß-arrestins in the development of OIH, independent of tolerance, and suggest that the use of G protein-biased MOPR ligands, such as oliceridine and TRV0109101, may be an effective therapeutic avenue for managing chronic pain with reduced propensity for opioid-induced hyperalgesia.
Assuntos
Analgésicos Opioides/administração & dosagem , Proteínas de Ligação ao GTP/agonistas , Hiperalgesia/tratamento farmacológico , Medição da Dor/efeitos dos fármacos , Receptores Opioides mu/agonistas , Animais , Relação Dose-Resposta a Droga , Esquema de Medicação , Proteínas de Ligação ao GTP/fisiologia , Células HEK293 , Humanos , Hiperalgesia/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Medição da Dor/métodos , Receptores Opioides mu/fisiologiaRESUMO
The concept of ligand bias at G protein-coupled receptors broadens the possibilities for agonist activities and provides the opportunity to develop safer, more selective therapeutics. Morphine pharmacology in ß-arrestin-2 knockout mice suggested that a ligand that promotes coupling of the µ-opioid receptor (MOR) to G proteins, but not ß-arrestins, would result in higher analgesic efficacy, less gastrointestinal dysfunction, and less respiratory suppression than morphine. Here we report the discovery of TRV130 ([(3-methoxythiophen-2-yl)methyl]({2-[(9R)-9-(pyridin-2-yl)-6-oxaspiro[4.5]decan-9-yl]ethyl})amine), a novel MOR G protein-biased ligand. In cell-based assays, TRV130 elicits robust G protein signaling, with potency and efficacy similar to morphine, but with far less ß-arrestin recruitment and receptor internalization. In mice and rats, TRV130 is potently analgesic while causing less gastrointestinal dysfunction and respiratory suppression than morphine at equianalgesic doses. TRV130 successfully translates evidence that analgesic and adverse MOR signaling pathways are distinct into a biased ligand with differentiated pharmacology. These preclinical data suggest that TRV130 may be a safer and more tolerable therapeutic for treating severe pain.
Assuntos
Analgésicos/farmacologia , Proteínas de Ligação ao GTP/metabolismo , Trato Gastrointestinal/efeitos dos fármacos , Morfina/farmacologia , Receptores Opioides mu/metabolismo , Sistema Respiratório/efeitos dos fármacos , Animais , Arrestinas/metabolismo , Linhagem Celular , Gastroenteropatias/induzido quimicamente , Gastroenteropatias/tratamento farmacológico , Gastroenteropatias/metabolismo , Células HEK293 , Humanos , Ligantes , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Ratos , Ratos Sprague-Dawley , Receptores Acoplados a Proteínas G/metabolismo , Doenças Respiratórias/induzido quimicamente , Doenças Respiratórias/tratamento farmacológico , Doenças Respiratórias/metabolismo , Transdução de Sinais/efeitos dos fármacos , beta-Arrestina 2 , beta-ArrestinasRESUMO
Biased G protein-coupled receptor ligands engage subsets of the receptor signals normally stimulated by unbiased agonists. However, it is unclear whether ligand bias can elicit differentiated pharmacology in vivo. Here, we describe the discovery of a potent, selective ß-arrestin biased ligand of the angiotensin II type 1 receptor. TRV120027 (Sar-Arg-Val-Tyr-Ile-His-Pro-D-Ala-OH) competitively antagonizes angiotensin II-stimulated G protein signaling, but stimulates ß-arrestin recruitment and activates several kinase pathways, including p42/44 mitogen-activated protein kinase, Src, and endothelial nitric-oxide synthase phosphorylation via ß-arrestin coupling. Consistent with ß-arrestin efficacy, and unlike unbiased antagonists, TRV120027 increased cardiomyocyte contractility in vitro. In rats, TRV120027 reduced mean arterial pressure, as did the unbiased antagonists losartan and telmisartan. However, unlike the unbiased antagonists, which decreased cardiac performance, TRV120027 increased cardiac performance and preserved cardiac stroke volume. These striking differences in vivo between unbiased and ß-arrestin biased ligands validate the use of biased ligands to selectively target specific receptor functions in drug discovery.
Assuntos
Angiotensina II/análogos & derivados , Angiotensina II/farmacologia , Arrestinas/metabolismo , Pressão Sanguínea/efeitos dos fármacos , Fenômenos Fisiológicos Cardiovasculares/efeitos dos fármacos , Receptor Tipo 1 de Angiotensina/agonistas , Transdução de Sinais/efeitos dos fármacos , Angiotensina II/metabolismo , Bloqueadores do Receptor Tipo 1 de Angiotensina II/farmacologia , Animais , Arrestinas/genética , Ligação Competitiva , Linhagem Celular Tumoral , Interações Medicamentosas , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Quinase 1 de Adesão Focal/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Células HEK293 , Humanos , Masculino , Camundongos , Contração Miocárdica/efeitos dos fármacos , Contração Miocárdica/fisiologia , Miócitos Cardíacos/citologia , Miócitos Cardíacos/efeitos dos fármacos , Óxido Nítrico Sintase Tipo III/metabolismo , Oligopeptídeos/metabolismo , Oligopeptídeos/farmacologia , Proteínas Proto-Oncogênicas c-jun/metabolismo , RNA Interferente Pequeno/genética , Ratos , Receptor Tipo 1 de Angiotensina/genética , Transdução de Sinais/fisiologia , Transfecção , Função Ventricular Esquerda/efeitos dos fármacos , Função Ventricular Esquerda/fisiologia , beta-Arrestinas , Quinases da Família src/metabolismoRESUMO
BACKGROUND: Tissue factor (TF) expression on islets can result in an instant blood-mediated inflammatory reaction (IBMIR) that contributes to early islet loss. We tested whether peritransplant protection of islets from IBMIR with a monoclonal anti-TF antibody (CNTO859) would enhance engraftment in our nonhuman primate marginal mass model. METHODS: Each of six pairs of cynomolgus monkeys (CM) with streptozotocin-induced diabetes was closely matched for metabolic control and was transplanted with 5,000 IEQ/kg allogeneic, ABO-compatible islets from the same donor under the cover of steroid-free immunosuppression. For each pair, experimental animals received islets cultured with 20 microg/mL anti-TF and were dosed with 6 mg/kg anti-TF intravenously, 10-25 min before islet infusion; control monkeys received an equal number of islets from the same preparation cultured without anti-TF and no in vivo treatment. RESULTS: Early fasting C-peptide (CP) values were different between (P<0.01), but not within, pairs and correlated with in vitro functional capacity of islets as assessed by perifusion (r=0.60; P=0.022). Compared to their matched controls, experimental animals had decreased posttransplant markers of coagulation, higher fasting CP levels (1 month posttransplant and end of study) and prolonged graft function. CONCLUSIONS: These data suggest that pretreatment of islets and the recipient with anti-TF may limit the effects of IBMIR, thereby enhancing islet engraftment and survival.
Assuntos
Sobrevivência de Enxerto/imunologia , Transplante das Ilhotas Pancreáticas/imunologia , Tromboplastina/fisiologia , Tolerância ao Transplante/imunologia , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Diabetes Mellitus Experimental/cirurgia , Relação Dose-Resposta a Droga , Fibrinólise/imunologia , Ilhotas Pancreáticas/efeitos dos fármacos , Ilhotas Pancreáticas/fisiologia , Macaca fascicularis , Modelos Biológicos , Estreptozocina , Tromboplastina/efeitos dos fármacosRESUMO
The syntheses, in vitro characterizations, and rat and monkey in vivo pharmacokinetic profiles of a series of 5-, 6-, and 7-methyl-substituted azepanone-based cathepsin K inhibitors are described. Depending on the particular regiochemical substitution and stereochemical configuration, methyl-substituted azepanones were identified that had widely varied cathepsin K inhibitory potency as well as pharmacokinetic properties compared to the 4S-parent azepanone analogue, 1 (human cathepsin K, K(i,app) = 0.16 nM, rat oral bioavailability = 42%, rat in vivo clearance = 49.2 mL/min/kg). Of particular note, the 4S-7-cis-methylazepanone analogue, 10, had a K(i,app) = 0.041 nM vs human cathepsin K and 89% oral bioavailability and an in vivo clearance rate of 19.5 mL/min/kg in the rat. Hypotheses that rationalize some of the observed characteristics of these closely related analogues have been made using X-ray crystallography and conformational analysis. These examples demonstrate the potential for modulation of pharmacological properties of cathepsin inhibitors by substituting the azepanone core. The high potency for inhibition of cathepsin K coupled with the favorable rat and monkey pharmacokinetic characteristics of compound 10, also known as SB-462795 or relacatib, has made it the subject of considerable in vivo evaluation for safety and efficacy as an inhibitor of excessive bone resorption in rat, monkey, and human studies, which will be reported elsewhere.
Assuntos
Azepinas/síntese química , Conservadores da Densidade Óssea/síntese química , Catepsinas/antagonistas & inibidores , Sulfonas/síntese química , Animais , Azepinas/química , Azepinas/farmacologia , Disponibilidade Biológica , Proteínas Sanguíneas/metabolismo , Conservadores da Densidade Óssea/química , Conservadores da Densidade Óssea/farmacologia , Catepsina K , Catepsinas/química , Linhagem Celular , Permeabilidade da Membrana Celular , Cristalografia por Raios X , Haplorrinos , Humanos , Conformação Molecular , Ligação Proteica , Ratos , Estereoisomerismo , Relação Estrutura-Atividade , Sulfonas/química , Sulfonas/farmacologiaRESUMO
The extension of a previously reported cathepsin K azepanone-based inhibitor template to the design and synthesis of potent and selective inhibitors of the homologous cysteine protease cathepsin L is detailed. Structure-activity studies examining the effect of inhibitor selectivity as a function of the P3 and P2 binding elements of the potent cathepsin K inhibitor 1 revealed that incorporation of either a P3 quinoline-8-carboxamide or a naphthylene-1-carboxamide led to increased selectivity for cathepsin L over cathepsin K. Substitution of the P2 leucine of 1 with either a phenylalanine or a beta-naphthylalanine also resulted in an increased selectivity for cathepsin L over cathepsin K. Molecular modeling studies with the inhibitors docked within the active sites of both cathepsins L and K have rationalized the observed selectivities. Optimization of cathepsin L binding by the combination of the P3 naphthylene-1-carboxamide with the P2 beta-naphthylalanine provided 15, which is a potent, selective, and competitive inhibitor of human cathepsin L with a K(i) = 0.43 nM.
Assuntos
Azepinas/síntese química , Catepsinas/antagonistas & inibidores , Catepsinas/química , Cisteína Endopeptidases/química , Inibidores de Cisteína Proteinase/síntese química , Sulfonas/síntese química , Amidas/química , Azepinas/química , Sítios de Ligação , Catepsina L , Inibidores de Cisteína Proteinase/química , Humanos , Modelos Moleculares , Quinolinas/química , Relação Estrutura-Atividade , Sulfonas/químicaRESUMO
Inhibition of bone resorption is a mechanism that has been clinically validated as a means to control bone loss in diseases such as postmenopausal osteoporosis. The development of marketable drugs in this area has resulted in significant clinical benefits; however, improvements can still be made. Several novel antiresorptive mechanisms are currently under consideration in the pharmaceutical industry, which will hopefully result in the development of improved bone antiresorptive therapies.
Assuntos
Reabsorção Óssea/tratamento farmacológico , Animais , Reabsorção Óssea/patologia , Reabsorção Óssea/fisiopatologia , Difosfonatos/uso terapêutico , HumanosRESUMO
Inhibition of the cyteine proteinase, cathepsin K (E.C. 3.4.22.38) has been postulated as a means to control osteoclast-mediated bone resorption. The preferred animal models for evaluation of antiresorptive activity are in the rat. However, the development of compounds that inhibit rat cathepsin K has proven difficult because the human and rat enzymes differ in key residues in the active site. In this study, a potent, nonpeptide inhibitor of rat cathepsin K (K(i) = 4.7 nmol/L), 5-(2-morpholin-4-yl-ethoxy)-benzofuran-2-carboxylic acid ((S)-3-methyl-1-(3-oxo-1-[2-(3-pyridin-2-yl-phenyl)-ethenoyl]-azepan-4-ylcarbanoyl)-butyl)-amide (SB 331750), is described, which is efficacious in rat models of bone resorption. SB 331750 potently inhibited human cathepsin K activity in vitro (K(i) = 0.0048 nmol/L) and was selective for human cathepsin K vs. cathepsins B (K(i) = 100 nmol/L), L (0.48 nmol/L), or S (K(i) = 14.3 nmol/L). In an in situ enzyme assay, SB 331750 inhibited osteoclast-associated cathepsin activity in tissue sections containing human osteoclasts (IC(50) approximately 60 nmol/L) and this translated into potent inhibition of human osteoclast-mediated bone resorption in vitro (IC(50) approximately 30 nmol/L). In vitro, SB 331750 partially, but dose-dependently, prevented the parathyroid hormone-induced hypercalcemia in an acute rat model of bone resorption. To evaluate the ability of SB 331750 to inhibit bone matrix degradation in vivo, it was administered for 4 weeks at 3, 10, or 30 mg/kg, intraperitoneally (i.p.), u.i.d. in the ovariectomized (ovx) rat. Both 10 and 30 mg/kg doses of compound prevented the ovx-induced elevation in urinary deoxypyridinoline and prevented the ovx-induced increase in percent eroded perimeter. Histological evaluation of the bones from compound-treated animals indicated that SB 331750 retarded bone matrix degradation in vivo at all three doses. The inhibition of bone resorption at the 10 and 30 mg/kg doses resulted in prevention of the ovx-induced reduction in percent trabecular area, trabecular number, and increase in trabecular spacing. These effects on bone resorption were also reflected in inhibition of the ovx-induced loss in trabecular bone volume as assessed using microcomputerized tomography (microCT; approximately 60% at 30 mg/kg). Together, these data indicate that the cathepsin K inhibitor, SB 331750, prevented bone resorption in vivo and this inhibition resulted in prevention of ovariectomy-induced loss in trabecular structure.
Assuntos
Benzofuranos/farmacologia , Reabsorção Óssea/tratamento farmacológico , Reabsorção Óssea/prevenção & controle , Catepsinas/antagonistas & inibidores , Inibidores de Cisteína Proteinase/farmacologia , Osteoclastos/efeitos dos fármacos , Piridinas/farmacologia , Animais , Sítios de Ligação/efeitos dos fármacos , Catepsina K , Catepsinas/química , Catepsinas/metabolismo , Inibidores de Cisteína Proteinase/química , Modelos Animais de Doenças , Feminino , Humanos , Técnicas In Vitro , Masculino , Osteoclastos/citologia , Ovariectomia , Paratireoidectomia , Ratos , Ratos Sprague-Dawley , TireoidectomiaRESUMO
Drug discovery targeting G protein-coupled receptors (GPCRs) is no longer limited to seeking agonists or antagonists to stimulate or block cellular responses associated with a particular receptor. GPCRs are now known to support a diversity of pharmacological profiles, a concept broadly referred to as functional selectivity. In particular, the concept of ligand bias, whereby a ligand stabilizes subsets of receptor conformations to engender novel pharmacological profiles, has recently gained increasing prominence. This review discusses how biased ligands may deliver safer, better tolerated, and more efficacious drugs, and highlights several biased ligands that are in clinical development. Biased ligands targeting the angiotensin II type 1 receptor and the µ opioid receptor illustrate the translation of the biased ligand concept from basic biology to clinical drug development.
Assuntos
Receptores Acoplados a Proteínas G/agonistas , Receptores Acoplados a Proteínas G/antagonistas & inibidores , Bloqueadores do Receptor Tipo 1 de Angiotensina II/farmacologia , Animais , Desenho de Fármacos , Humanos , Ligantes , Oligopeptídeos/farmacologia , Receptor Tipo 1 de Angiotensina/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Receptores Opioides mu/metabolismo , Compostos de Espiro/farmacologia , Tiofenos/farmacologiaRESUMO
Opioids provide powerful analgesia but also efficacy-limiting adverse effects, including severe nausea, vomiting, and respiratory depression, by activating µ-opioid receptors. Preclinical models suggest that differential activation of signaling pathways downstream of these receptors dissociates analgesia from adverse effects; however, this has not yet translated to a treatment with an improved therapeutic index. Thirty healthy men received single intravenous injections of the biased ligand TRV130 (1.5, 3, or 4.5mg), placebo, or morphine (10mg) in a randomized, double-blind, crossover study. Primary objectives were to measure safety and tolerability (adverse events, vital signs, electrocardiography, clinical laboratory values), and analgesia (cold pain test) versus placebo. Other measures included respiratory drive (minute volume after induced hypercapnia), subjective drug effects, and pharmacokinetics. Compared to morphine, TRV130 (3, 4.5mg) elicited higher peak analgesia (105, 116 seconds latency vs 75 seconds for morphine, P<.02), with faster onset and similar duration of action. More subjects doubled latency or achieved maximum latency (180 seconds) with TRV130 (3, 4.5mg). Respiratory drive reduction was greater after morphine than any TRV130 dose (-15.9 for morphine versus -7.3, -7.6, and -9.4 h*L/min, P<.05). More subjects experienced severe nausea after morphine (n=7) than TRV130 1.5 or 3mg (n=0, 1), but not 4.5mg (n=9). TRV130 was generally well tolerated, and exposure was dose proportional. Thus, in this study, TRV130 produced greater analgesia than morphine at doses with less reduction in respiratory drive and less severe nausea. This demonstrates early clinical translation of ligand bias as an important new concept in receptor-targeted pharmacotherapy.
Assuntos
Analgésicos Opioides/efeitos adversos , Morfina/efeitos adversos , Dor/tratamento farmacológico , Compostos de Espiro/efeitos adversos , Tiofenos/efeitos adversos , Adolescente , Adulto , Analgesia , Analgésicos Opioides/administração & dosagem , Analgésicos Opioides/uso terapêutico , Estudos Cross-Over , Tontura/induzido quimicamente , Relação Dose-Resposta a Droga , Método Duplo-Cego , Cefaleia/induzido quimicamente , Voluntários Saudáveis , Humanos , Masculino , Pessoa de Meia-Idade , Morfina/administração & dosagem , Morfina/uso terapêutico , Compostos de Espiro/administração & dosagem , Compostos de Espiro/uso terapêutico , Tiofenos/administração & dosagem , Tiofenos/uso terapêutico , Vômito/induzido quimicamente , Adulto JovemRESUMO
TRV130 is a G protein-biased ligand at the µ-opioid receptor. In preclinical studies it was potently analgesic while causing less respiratory depression and gastrointestinal dysfunction than morphine, suggesting unique benefits in acute pain management. A first-in-human study was conducted with ascending doses of TRV130 to explore its tolerability, pharmacokinetics, and pharmacodynamics in healthy volunteers. TRV130 was well-tolerated over the dose range 0.15 to 7 mg administered intravenously over 1 hour. TRV130 geometric mean exposure and Cmax were dose-linear, with AUC0-inf of 2.52 to 205.97 ng h/mL and Cmax of 1.04 to 102.36 ng/mL across the dose range tested, with half-life of 1.6-2.7 hours. A 1.5 mg dose of TRV130 was also well-tolerated when administered as 30, 15, 5, and 1 minute infusions. TRV130 pharmacokinetics were modestly affected by CYP2D6 phenotype: clearance was reduced by 53% in CYP2D6 poor metabolizers.TRV130 caused dose- and exposure-related pupil constriction, confirming central compartment µ-opioid receptor engagement. Marked pupil constriction was noted at 2.2, 4, and 7 mg doses. Nausea and vomiting observed at the 7 mg dose limited further dose escalation. These findings suggest that TRV130 may have a broad margin between doses causing µ-opioid receptor-mediated pharmacology and doses causing µ-opioid receptor-mediated intolerance.
Assuntos
Analgésicos , Compostos de Espiro , Tiofenos , Administração Intravenosa , Adulto , Analgésicos/administração & dosagem , Analgésicos/efeitos adversos , Analgésicos/farmacocinética , Analgésicos/farmacologia , Estudos Cross-Over , Método Duplo-Cego , Voluntários Saudáveis , Humanos , Ligantes , Masculino , Pessoa de Meia-Idade , Náusea/induzido quimicamente , Receptores Opioides mu/metabolismo , Compostos de Espiro/administração & dosagem , Compostos de Espiro/efeitos adversos , Compostos de Espiro/farmacocinética , Compostos de Espiro/farmacologia , Tiofenos/administração & dosagem , Tiofenos/efeitos adversos , Tiofenos/farmacocinética , Tiofenos/farmacologia , Vômito/induzido quimicamente , Adulto JovemRESUMO
TRV027 is a novel ß-arrestin biased peptide ligand of the angiotensin II type 1 receptor (AT1R). The compound antagonizes G protein coupling while simultaneously stimulating ß-arrestin-mediated signaling. In preclinical studies, TRV027 reversibly reduced blood pressure while preserving renal function in a dog tachypaced heart failure model and stimulating cardiomyocyte contractility in vitro. This profile suggests that TRV027 may have unique benefits in acute heart failure, a condition associated with renin-angiotensin system activation. A first-time-in-human study was conducted with ascending doses of TRV027 to explore its tolerability, pharmacokinetics and pharmacodynamics in healthy volunteers. Subjects' salt intake was restricted to stimulate RAS activation. In this study TRV027 was safe and well tolerated with a short-half-life (ranging between 2.4 and 13.2 minutes) and dose-proportional increases in systemic exposure. Consistent with the pre-clinical findings, TRV027 reduced blood pressure to a greater degree in subjects with RAS activation, measured as elevated plasma renin activity, than in those with normal PRA levels. This study in sodium-restricted healthy subjects suggests that TRV027 will successfully target a core mechanism of acute heart failure pathophysiology. Further clinical studies with TRV027 in patients with heart failure are underway.
Assuntos
Bloqueadores do Receptor Tipo 1 de Angiotensina II/farmacologia , Anti-Hipertensivos/farmacologia , Oligopeptídeos/farmacologia , Adulto , Bloqueadores do Receptor Tipo 1 de Angiotensina II/sangue , Anti-Hipertensivos/sangue , Pressão Sanguínea/efeitos dos fármacos , Estudos Cross-Over , Dieta Hipossódica , Método Duplo-Cego , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Oligopeptídeos/sangue , Renina/sangueRESUMO
G protein-coupled receptors have been successfully targeted by numerous therapeutics including drugs that have transformed the management of cardiovascular disease. However, many GPCRs, when activated or blocked by drugs, elicit both beneficial and adverse pharmacology. Recent work has demonstrated that in some cases, the salutary and deleterious signals linked to a specific GPCR can be selectively targeted by "biased ligands" that entrain subsets of a receptor's normal pharmacology. This review briefly summarizes the advances and current state of the biased ligand field, focusing on an example: biased ligands targeting the angiotensin II type 1 receptor. These compounds exhibit unique pharmacology, distinct from classic agonists or antagonists, and one such molecule is now in clinical development for the treatment of acute heart failure.
Assuntos
Bloqueadores do Receptor Tipo 1 de Angiotensina II , Insuficiência Cardíaca , Receptores Acoplados a Proteínas G , Bloqueadores do Receptor Tipo 1 de Angiotensina II/metabolismo , Bloqueadores do Receptor Tipo 1 de Angiotensina II/farmacologia , Descoberta de Drogas , Insuficiência Cardíaca/tratamento farmacológico , Insuficiência Cardíaca/metabolismo , Humanos , Ligantes , Terapia de Alvo Molecular/efeitos adversos , Terapia de Alvo Molecular/métodos , Receptores Acoplados a Proteínas G/classificação , Receptores Acoplados a Proteínas G/uso terapêuticoRESUMO
BACKGROUND: TRV120027 is a novel ß-arrestin biased ligand of the angiotensin II type 1 receptor; it antagonizes canonical G-protein-mediated coupling while, in contrast to classical angiotensin II type 1 receptor antagonists, it engages ß-arrestin-mediated signaling. Consequently, TRV120027 inhibits angiotensin II-mediated vasoconstriction while, via ß-arrestin coupling, it increases cardiomyocyte contractility. We hypothesized that TRV120027 would elicit beneficial cardiorenal actions when added to furosemide in experimental heart failure. METHODS AND RESULTS: Two groups of anesthetized dogs (n=6 each) with tachypacing-induced heart failure were studied. After a baseline clearance, 1 group (F+V) received furosemide (1 mg/kg per hour) plus saline for 90 minutes, whereas the other (F+T) received the same dose of furosemide plus TRV120027 (0.3 and 1.5 µg/kg per minute for 45 minutes each); 2 clearances were done during drug infusion. After a washout, a postinfusion clearance was done; *P<0.05 between groups. F+V and F+T increased diuresis and natriuresis to a similar extent during drug administration, but urine flow* and urinary sodium excretion* were higher in the postinfusion clearance with F+T. Glomerular filtration rate was preserved in both groups. Renal blood flow increased with F+T but this was not significant versus F+V. Compared with F+V, F+T decreased mean arterial pressure*, systemic* and pulmonary* vascular resistances, and atrial natriuretic peptide*. Pulmonary capillary wedge pressure* decreased to a larger extent with F+T than with F+V. CONCLUSIONS: When added to furosemide, TRV120027, a novel ß-arrestin biased angiotensin II type 1 receptor ligand, preserved furosemide-mediated natriuresis and diuresis, while reducing cardiac preload and afterload. These results provide support for TRV120027 as a promising novel therapeutic for the treatment of heart failure.
Assuntos
Bloqueadores do Receptor Tipo 1 de Angiotensina II/farmacologia , Arrestinas/metabolismo , Diuréticos/farmacologia , Furosemida/farmacologia , Insuficiência Cardíaca/tratamento farmacológico , Coração/efeitos dos fármacos , Rim/efeitos dos fármacos , Oligopeptídeos/farmacologia , Receptor Tipo 1 de Angiotensina/efeitos dos fármacos , Bloqueadores do Receptor Tipo 1 de Angiotensina II/administração & dosagem , Animais , Pressão Arterial/efeitos dos fármacos , Fator Natriurético Atrial/sangue , Estimulação Cardíaca Artificial , Modelos Animais de Doenças , Diurese/efeitos dos fármacos , Diuréticos/administração & dosagem , Cães , Quimioterapia Combinada , Furosemida/administração & dosagem , Taxa de Filtração Glomerular/efeitos dos fármacos , Coração/fisiopatologia , Insuficiência Cardíaca/metabolismo , Insuficiência Cardíaca/fisiopatologia , Infusões Intravenosas , Rim/metabolismo , Rim/fisiopatologia , Ligantes , Masculino , Natriurese/efeitos dos fármacos , Oligopeptídeos/administração & dosagem , Pressão Propulsora Pulmonar/efeitos dos fármacos , Receptor Tipo 1 de Angiotensina/metabolismo , Circulação Renal/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Fatores de Tempo , Urodinâmica/efeitos dos fármacos , Resistência Vascular/efeitos dos fármacos , beta-ArrestinasRESUMO
BACKGROUND: The angiotensin II type 1 receptor (AT1R) plays a key role in regulating cardiorenal function. Classic "unbiased" AT1R antagonists block receptor coupling to both G(αq) and ß-arrestin-mediated signals, which desensitize G-protein signaling as well as transduce G-protein-independent signals. TRV120027 is a novel ß-arrestin-biased AT1R ligand, which engages ß-arrestins while blocking G-protein signaling. At the AT1R, TRV120027 can inhibit angiotensin II-mediated vasoconstriction, whereas, through ß-arrestin coupling, increase cardiomyocyte contractility. We defined for the first time the acute cardiorenal actions of TRV120027 in healthy and heart failure (HF) canines. METHODS AND RESULTS: Healthy and HF canines (induced by tachypacing) were anesthetized. After instrumentation and equilibration, a 30-minute baseline clearance was performed, followed by further clearance with escalating doses of intravenous TRV120027 (0.01, 0.1, 1, 10, and 100 µg/kg per minute) and a 30-minute washout. In healthy canines, TRV120027 decreased pulmonary capillary wedge pressure and systemic and renal vascular resistances, while increasing cardiac output, renal blood flow, glomerular filtration rate, and urinary sodium excretion. In HF canines, TRV120027 decreased mean arterial pressure, right atrial pressure, and pulmonary capillary wedge pressure, systemic and renal vascular resistances and increased cardiac output and renal blood flow. Glomerular filtration rate and urinary sodium excretion were maintained. CONCLUSIONS: We report for the first time the cardiorenal actions of the novel ß-arrestin-biased AT1R ligand TRV120027. In both normal and HF canines, TRV120027 demonstrated cardiac unloading actions while preserving renal function. With this beneficial pharmacological profile, TRV120027 represents a novel strategy for the treatment of HF.
Assuntos
Arrestinas/metabolismo , Insuficiência Cardíaca/tratamento farmacológico , Coração/efeitos dos fármacos , Rim/efeitos dos fármacos , Oligopeptídeos/farmacologia , Oligopeptídeos/uso terapêutico , Receptor Tipo 1 de Angiotensina/metabolismo , Animais , Pressão Sanguínea/efeitos dos fármacos , Pressão Sanguínea/fisiologia , Débito Cardíaco/efeitos dos fármacos , Débito Cardíaco/fisiologia , Modelos Animais de Doenças , Cães , Relação Dose-Resposta a Droga , Taxa de Filtração Glomerular/efeitos dos fármacos , Taxa de Filtração Glomerular/fisiologia , Coração/fisiopatologia , Insuficiência Cardíaca/metabolismo , Insuficiência Cardíaca/fisiopatologia , Rim/fisiopatologia , Ligantes , Masculino , Sódio/urina , Resultado do Tratamento , Resistência Vascular/efeitos dos fármacos , Resistência Vascular/fisiologia , beta-ArrestinasRESUMO
OBJECTIVE: Traumatic joint injury can damage cartilage and release inflammatory cytokines from adjacent joint tissue. The present study was undertaken to study the combined effects of compression injury, tumor necrosis factor alpha (TNFalpha), and interleukin-6 (IL-6) and its soluble receptor (sIL-6R) on immature bovine and adult human knee and ankle cartilage, using an in vitro model, and to test the hypothesis that endogenous IL-6 plays a role in proteoglycan loss caused by a combination of injury and TNFalpha. METHODS: Injured or uninjured cartilage disks were incubated with or without TNFalpha and/or IL-6/sIL-6R. Additional samples were preincubated with an IL-6-blocking antibody Fab fragment and subjected to injury and TNFalpha treatment. Treatment effects were assessed by histologic analysis, measurement of glycosaminoglycan (GAG) loss, Western blot to determine proteoglycan degradation, zymography, radiolabeling to determine chondrocyte biosynthesis, and Western blot and enzyme-linked immunosorbent assay to determine chondrocyte production of IL-6. RESULTS: In bovine cartilage samples, injury combined with TNFalpha and IL-6/sIL-6R exposure caused the most severe GAG loss. Findings in human knee and ankle cartilage were strikingly similar to those in bovine samples, although in human ankle tissue, the GAG loss was less severe than that observed in human knee tissue. Without exogenous IL-6/sIL-6R, injury plus TNFalpha exposure up-regulated chondrocyte production of IL-6, but incubation with the IL-6-blocking Fab significantly reduced proteoglycan degradation. CONCLUSION: Our findings indicate that mechanical injury potentiates the catabolic effects of TNFalpha and IL-6/sIL-6R in causing proteoglycan degradation in human and bovine cartilage. The temporal and spatial evolution of degradation suggests the importance of transport of biomolecules, which may be altered by overload injury. The catabolic effects of injury plus TNFalpha appeared partly due to endogenous IL-6, since GAG loss was partially abrogated by an IL-6-blocking Fab.
Assuntos
Cartilagem Articular/metabolismo , Interleucina-6/metabolismo , Articulações/lesões , Proteoglicanas/metabolismo , Receptores de Interleucina-6/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Adulto , Animais , Traumatismos do Tornozelo/metabolismo , Traumatismos do Tornozelo/patologia , Fenômenos Biomecânicos , Cartilagem Articular/efeitos dos fármacos , Cartilagem Articular/patologia , Bovinos , Células Cultivadas , Condrócitos/metabolismo , Condrócitos/patologia , Modelos Animais de Doenças , Feminino , Glicosaminoglicanos/metabolismo , Humanos , Interleucina-6/farmacologia , Traumatismos do Joelho/metabolismo , Traumatismos do Joelho/patologia , Masculino , Pessoa de Meia-Idade , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
INTRODUCTION: Fibronectin fragments have been found in the articular cartilage and synovial fluid of patients with osteoarthritis and rheumatoid arthritis. These matrix fragments can stimulate production of multiple mediators of matrix destruction, including various cytokines and metalloproteinases. The purpose of this study was to discover novel mediators of cartilage destruction using fibronectin fragments as a stimulus. METHODS: Human articular cartilage was obtained from tissue donors and from osteoarthritic cartilage removed at the time of knee replacement surgery. Enzymatically isolated chondrocytes in serum-free cultures were stimulated overnight with the 110 kDa alpha5beta1 integrin-binding fibronectin fragment or with IL-1, IL-6, or IL-7. Cytokines and matrix metalloproteinases released into the media were detected using antibody arrays and quantified by ELISA. IL-7 receptor expression was evaluated by flow cytometry, immunocytochemical staining, and PCR. RESULTS: IL-7 was found to be produced by chondrocytes treated with fibronectin fragments. Compared with cells isolated from normal young adult human articular cartilage, increased IL-7 production was noted in cells isolated from older adult tissue donors and from osteoarthritic cartilage. Chondrocyte IL-7 production was also stimulated by combined treatment with the catabolic cytokines IL-1 and IL-6. Chondrocytes were found to express IL-7 receptors and to respond to IL-7 stimulation with increased production of matrix metalloproteinase-13 and with proteoglycan release from cartilage explants. CONCLUSION: These novel findings indicate that IL-7 may contribute to cartilage destruction in joint diseases, including osteoarthritis.
Assuntos
Cartilagem Articular/metabolismo , Condrócitos/efeitos dos fármacos , Condrócitos/metabolismo , Interleucina-7/metabolismo , Interleucina-7/farmacologia , Metaloproteinase 13 da Matriz/biossíntese , Cartilagem Articular/citologia , Células Cultivadas , Sinergismo Farmacológico , Fibronectinas/metabolismo , Fibronectinas/farmacologia , Humanos , Técnicas In Vitro , Integrina alfa5beta1/metabolismo , Interleucina-1/farmacologia , Interleucina-6/farmacologia , Interleucinas/farmacologia , Articulação do Joelho , Fragmentos de Peptídeos/farmacologia , Regulação para CimaRESUMO
An excess of thyroid hormone results in increased bone turnover and loss of bone mass in humans. Exogenous administration of thyroid hormone to rats has served as a model of human hyperthyroidism in which antiresorptive therapies have been tested. We have further refined this model of thyroxine (T4)-induced turnover in the rat. Daily administration of T4 to aged rats for as short as 1 week resulted in elevated bone resorption determined by significantly higher urinary deoxypyridinoline (Dpd) compared with vehicle controls or animals receiving T4 plus estradiol. Three weeks of daily administration of T4 led to significantly lower bone mineral density compared with untreated controls or animals receiving T4 plus estradiol. In a follow-up study, a depot formulation of T4 caused an increase in Dpd identical to that achieved with a bolus dose. SB-273005 [(4S)-2,3,4,5-tetrahydro-8-[2-[6-(methylamino)-2-pyridinyl] ethoxy]-3-oxo-2-(2,2,2-trifluoroethyl)-1H-2-benzazepine-4- acetic acid] a potent antagonist of the integrins alpha(v)beta(3) and alpha(v)beta(5), has been shown previously to inhibit bone resorption in cultures of human osteoclasts and to protect bone in ovariectomized rats. The effect of SB-273005 by oral administration was evaluated in this thyroxine-induced turnover model. Dose-dependent inhibition of resorption was seen with SB-273005 after 7 days of dosing using Dpd as a measure of bone resorption. In summary, it has been demonstrated that the antiresorptive activity of a vitronectin receptor antagonist can be measured after only 7 days of treatment in this refined rat model of thyroxine-induced bone turnover. These data suggest that SB-273005 may be useful for the treatment of metabolic bone diseases, including those resulting from hyperthyroidism.
Assuntos
Reabsorção Óssea/prevenção & controle , Piridinas/farmacologia , Receptores de Vitronectina/antagonistas & inibidores , Tiroxina/antagonistas & inibidores , Aminoácidos/urina , Animais , Biomarcadores , Densidade Óssea/efeitos dos fármacos , Reabsorção Óssea/induzido quimicamente , Masculino , Osteocalcina/sangue , Ratos , Tiroxina/metabolismo , Tiroxina/toxicidade , Tri-Iodotironina/metabolismoRESUMO
OBJECTIVE: Acute joint injury leads to increased risk for osteoarthritis (OA). Although the mechanisms underlying this progression are unclear, early structural, metabolic, and compositional indicators of OA have been reproduced using in vitro models of cartilage injury. This study was undertaken to determine whether glycosaminoglycan (GAG) loss following in vitro cartilage injury is mediated by cellular biosynthesis, activation of enzymatic activity, or mechanical disruption of the cartilage extracellular matrix. METHODS: Immature bovine cartilage was cultured for up to 10 days. After 3 days, groups of samples were subjected to injurious mechanical compression (single uniaxial unconfined compression to 50% thickness, strain rate 100% per second). GAG release to the medium was measured, and levels were compared with those in location-matched, uninjured controls. The effects of medium supplementation with inhibitors of biosynthesis (cycloheximide), of matrix metalloproteinase (MMP) activity (CGS 27023A or GM 6001), and of aggrecanase activity (SB 703704) on GAG release after injury were assessed. RESULTS: GAG release from injured cartilage was highest during the first 4 hours after injury, but remained higher than that in controls during the first 24 hours postinjury, and was not affected by inhibitors of biosynthesis or degradative enzymes. GAG release during the period 24-72 hours postinjury was similar to that in uninjured controls, but the MMP inhibitor CGS 27023A reduced cumulative GAG loss from injured samples between 1 day and 7 days postinjury. Other inhibitors of enzymatic degradation or biosynthesis had no significant effect on GAG release. CONCLUSION: Injurious compression of articular cartilage induces an initially high rate of GAG release from the tissue, which could not be inhibited, consistent with mechanical damage. However, the finding that MMP inhibition reduced GAG loss in the days following injury suggests a potential therapeutic intervention.
Assuntos
Cartilagem Articular/lesões , Glicosaminoglicanos/metabolismo , Animais , Cartilagem Articular/patologia , Cartilagem Articular/fisiopatologia , Bovinos , Sobrevivência Celular , Dipeptídeos/farmacologia , Endopeptidases/efeitos dos fármacos , Ácidos Hidroxâmicos/farmacologia , Técnicas In Vitro , Indenos/farmacologia , Cinética , Pressão , Inibidores de Proteases/farmacologia , Pirazinas/farmacologia , Sulfonamidas/farmacologia , Fatores de Tempo , Ferimentos e Lesões/etiologia , Ferimentos e Lesões/metabolismo , Ferimentos e Lesões/patologia , Ferimentos e Lesões/fisiopatologiaRESUMO
OBJECTIVE: Traumatic joint injury leads to an increased risk of osteoarthritis (OA), but the progression to OA is not well understood. We undertook this study to measure aspects of proteoglycan (PG) degradation after in vitro injurious mechanical compression, including up-regulation of enzymatic degradative expression and cytokine-stimulated degradation. METHODS: Articular cartilage tissue explants were obtained from newborn bovine femoropatellar groove and from adult normal human donor knee and ankle tissue. Following injurious compression of the cartilage, matrix metalloproteinase 3 (MMP-3) and MMP-13 messenger RNA (mRNA) expression levels were measured by Northern analysis, and PG loss to the medium after cartilage injury was measured in the presence and absence of added exogenous cytokine (interleukin-1alpha [IL-1alpha] or tumor necrosis factor alpha [TNFalpha]). RESULTS: During the first 24 hours after injury in bovine cartilage, MMP-3 mRNA levels increased 10-fold over the levels in control cartilage (n = 3 experiments), whereas MMP-13 mRNA levels were unchanged. PG loss was significantly increased after injury, but only by 2% of the total PG content and only for the first 3 days following injury. However, compared with injury alone or cytokine treatment alone, treatment of injured tissue with either 1 ng/ml IL-1alpha or 100 ng/ml TNFalpha caused marked increases in PG loss (35% and 54%, respectively, of the total cartilage PG content). These interactions between cytokine treatment and injury were statistically significant. In human knee cartilage, the interaction was also significant for both IL-1alpha and TNFalpha, although the magnitude of increase in PG loss was lower than that in bovine cartilage. In contrast, in human ankle cartilage, there was no significant interaction between injury and IL-1alpha. CONCLUSION: The cytokines IL-1alpha and TNFalpha can cause a synergistic loss of PG from mechanically injured bovine and human cartilage. By attempting to incorporate interactions with other joint tissues that may be sources of cytokines, in vitro models of mechanical cartilage injury may explain aspects of the interactions between mechanical forces and degradative pathways which lead to OA progression.