RESUMO
Glucagon-like peptide-1 receptor (GLP-1R) agonists are efficacious antidiabetic medications that work by enhancing glucose-dependent insulin secretion and improving energy balance. Currently approved GLP-1R agonists are peptide based, and it has proven difficult to obtain small-molecule activators possessing optimal pharmaceutical properties. We report the discovery and mechanism of action of LY3502970 (OWL833), a nonpeptide GLP-1R agonist. LY3502970 is a partial agonist, biased toward G protein activation over ß-arrestin recruitment at the GLP-1R. The molecule is highly potent and selective against other class B G protein-coupled receptors (GPCRs) with a pharmacokinetic profile favorable for oral administration. A high-resolution structure of LY3502970 in complex with active-state GLP-1R revealed a unique binding pocket in the upper helical bundle where the compound is bound by the extracellular domain (ECD), extracellular loop 2, and transmembrane helices 1, 2, 3, and 7. This mechanism creates a distinct receptor conformation that may explain the partial agonism and biased signaling of the compound. Further, interaction between LY3502970 and the primate-specific Trp33 of the ECD informs species selective activity for the molecule. In efficacy studies, oral administration of LY3502970 resulted in glucose lowering in humanized GLP-1R transgenic mice and insulinotropic and hypophagic effects in nonhuman primates, demonstrating an effect size in both models comparable to injectable exenatide. Together, this work determined the molecular basis for the activity of an oral agent being developed for the treatment of type 2 diabetes mellitus, offering insights into the activation of class B GPCRs by nonpeptide ligands.
Assuntos
Receptor do Peptídeo Semelhante ao Glucagon 1/agonistas , Hipoglicemiantes/farmacologia , Domínios Proteicos/genética , Administração Oral , Aminopiridinas/farmacologia , Animais , Fármacos Antiobesidade/farmacologia , Benzamidas/farmacologia , Microscopia Crioeletrônica , Receptor do Peptídeo Semelhante ao Glucagon 1/genética , Receptor do Peptídeo Semelhante ao Glucagon 1/metabolismo , Receptor do Peptídeo Semelhante ao Glucagon 1/ultraestrutura , Células HEK293 , Humanos , Incretinas/farmacologia , Macaca fascicularis , Masculino , Camundongos , Camundongos Transgênicos , Modelos Moleculares , Mutagênese Sítio-Dirigida , Ratos , Especificidade da Espécie , Suínos , Triptofano/genéticaRESUMO
BACKGROUND: Phosphate is absorbed in the small intestine via passive flow and active transport.NaPi-IIb, a type II sodium-dependent phosphate transporter, is considered to mediate active phosphate transport in rodents. To study the regulation of intestinal phosphate transport in chronic kidney disease (CKD), we analyzed the expression levels of NaPi-IIb, pituitary-specific transcription factor 1 (PiT-1) and PiT-2 and the kinetics of intestinal phosphate transport using two CKD models. METHODS: CKD was induced in rats via adenine orThy1 antibody injection. Phosphate uptake by intestinal brush border membrane vesicles (BBMV) and the messenger RNA (mRNA) expression of NaPi-IIb, PiT-1 and PiT-2 were analyzed. The protein expression level of NaPi-IIb was measured by mass spectrometry (e.g. liquid chromatography tandem mass spectrometry). RESULTS: In normal rats, phosphate uptake into BBMV consisted of a single saturable component and its Michaelis constant (Km) was comparable to that of NaPi-IIb. The maximum velocity (Vmax) correlated with mRNA and protein levels of NaPi-IIb. In the CKD models, intestinal phosphate uptake consisted of two saturable components. The Vmax of the higher-affinity transport, which is thought to be responsible for NaPi-IIb, significantly decreased and the decrease correlated with reduced NaPi-IIb expression. The Km of the lower-affinity transport was comparable to that of PiT-1 and -2. PiT-1 mRNA expression was much higher than that of PiT-2, suggesting that PiT-1 was mostly responsible for phosphate transport. CONCLUSIONS: This study suggests that the contribution of NaPi-IIb to intestinal phosphate absorption dramatically decreases in rats with CKD and that a low-affinity alternative to NaPi-IIb, in particular PiT-1, is upregulated in a compensatory manner in CKD.
Assuntos
Intestinos/fisiologia , Proteínas de Transporte de Fosfato/metabolismo , Fosfatos/metabolismo , Insuficiência Renal Crônica/metabolismo , Proteínas Cotransportadoras de Sódio-Fosfato Tipo IIb/metabolismo , Sódio/metabolismo , Adenina/toxicidade , Animais , Masculino , Ratos , Ratos Endogâmicos F344 , Ratos Wistar , Insuficiência Renal Crônica/induzido quimicamente , Insuficiência Renal Crônica/patologia , Proteínas Cotransportadoras de Sódio-Fosfato Tipo III/genética , Proteínas Cotransportadoras de Sódio-Fosfato Tipo III/metabolismo , Proteínas Cotransportadoras de Sódio-Fosfato Tipo IIb/classificação , Proteínas Cotransportadoras de Sódio-Fosfato Tipo IIb/genética , Fator de Transcrição Pit-1/genética , Fator de Transcrição Pit-1/metabolismoRESUMO
Scratching is an important factor exacerbating skin lesions through the so-called itch-scratch cycle in atopic dermatitis (AD). In mice, interleukin (IL)-31 and its receptor IL-31 receptor A (IL-31RA) are known to play a critical role in pruritus and the pathogenesis of AD; however, study of their precise roles in primates is hindered by the low sequence homologies between primates and mice and the lack of direct evidence of itch sensation by IL-31 in primates. We showed that administration of cynomolgus IL-31 induces transient scratching behaviour in cynomolgus monkeys and by that were able to establish a monkey model of scratching. We then showed that a single subcutaneous injection of 1 mg/kg nemolizumab, a humanized anti-human IL-31RA monoclonal antibody that also neutralizes cynomolgus IL-31 signalling and shows a good pharmacokinetic profile in cynomolgus monkeys, suppressed the IL-31-induced scratching for about 2 months. These results suggest that the IL-31 axis and IL-31RA axis play as critical a role in the induction of scratching in primates as in mice and that the blockade of IL-31 signalling by an anti-human IL-31RA antibody is a promising therapeutic approach for treatment of AD. Nemolizumab is currently under investigation in clinical trials.
Assuntos
Anticorpos Monoclonais Humanizados/farmacologia , Interleucinas/farmacologia , Prurido/induzido quimicamente , Receptores de Interleucina/metabolismo , Células A549 , Animais , Células CHO , Linhagem Celular , Cricetulus , DNA Complementar/metabolismo , Humanos , Cinética , Macaca fascicularis , Masculino , Camundongos , Prurido/metabolismo , Transdução de Sinais , Pele/imunologia , Pele/patologia , Dermatopatias/imunologia , Dermatopatias/patologiaRESUMO
Urate-lowering therapy is indispensable for the treatment of gout, but available drugs do not control serum urate levels tightly enough. Although the uricosurics benzbromarone and probenecid inhibit a urate reabsorption transporter known as renal urate transporter 1 (URAT1) and thus lower serum urate levels, they also inhibit other transporters responsible for secretion of urate into urine, which suggests that inhibiting URAT1 selectively would lower serum urate more effectively. We identified a novel potent and selective URAT1 inhibitor, UR-1102, and compared its efficacy with benzbromarone in vitro and in vivo. In human embryonic kidney (HEK)293 cells overexpressing URAT1, organic anion transporter 1 (OAT1), and OAT3, benzbromarone inhibited all transporters similarly, whereas UR-1102 inhibited URAT1 comparably to benzbromarone but inhibited OAT1 and OAT3 quite modestly. UR-1102 at 3-30 mg/kg or benzbromarone at 3-100 mg/kg was administered orally once a day for 3 consecutive days to tufted capuchin monkeys, whose low uricase activity causes a high plasma urate level. When compared with the same dosage of benzbromarone, UR-1102 showed a better pharmacokinetic profile, increased the fractional excretion of urinary uric acid, and reduced plasma uric acid more effectively. Moreover, the maximum efficacy of UR-1102 was twice that of benzbromarone, suggesting that selective inhibition of URAT1 is effective. Additionally UR-1102 showed lower in vitro potential for mechanisms causing the hepatotoxicity induced by benzbromarone. These results indicate that UR-1102 achieves strong uricosuric effects by selectively inhibiting URAT1 over OAT1 and OAT3 in monkeys, and could be a novel therapeutic option for patients with gout or hyperuricemia.
Assuntos
Benzobromarona/farmacologia , Transportadores de Ânions Orgânicos/antagonistas & inibidores , Proteínas de Transporte de Cátions Orgânicos/antagonistas & inibidores , Oxazinas/farmacologia , Piridinas/farmacologia , Uricosúricos/farmacologia , Animais , Cebus , Doença Hepática Induzida por Substâncias e Drogas/patologia , Relação Dose-Resposta a Droga , Feminino , Células HEK293 , Humanos , Masculino , Microssomos Hepáticos/efeitos dos fármacos , Microssomos Hepáticos/metabolismo , Proteína 1 Transportadora de Ânions Orgânicos/biossíntese , Proteína 1 Transportadora de Ânions Orgânicos/genética , Transportadores de Ânions Orgânicos/genética , Transportadores de Ânions Orgânicos Sódio-Independentes/biossíntese , Transportadores de Ânions Orgânicos Sódio-Independentes/genética , Proteínas de Transporte de Cátions Orgânicos/genética , Ligação Proteica , Ácido Úrico/sangue , Uricosúricos/efeitos adversosRESUMO
ACE910 is a humanized anti-factor IXa/X bispecific antibody mimicking the function of factor VIII (FVIII). We previously demonstrated in nonhuman primates that a single IV dose of ACE910 exerted hemostatic activity against hemophilic bleeds artificially induced in muscles and subcutis, and that a subcutaneous (SC) dose of ACE910 showed a 3-week half-life and nearly 100% bioavailability, offering support for effective prophylaxis for hemophilia A by user-friendly SC dosing. However, there was no direct evidence that such SC dosing of ACE910 would prevent spontaneous bleeds occurring in daily life. In this study, we newly established a long-term primate model of acquired hemophilia A by multiple IV injections of an anti-primate FVIII neutralizing antibody engineered in mouse-monkey chimeric form to reduce its antigenicity. The monkeys in the control group exhibited various spontaneous bleeding symptoms as well as continuous prolongation of activated partial thromboplastin time; notably, all exhibited joint bleeds, which are a hallmark of hemophilia. Weekly SC doses of ACE910 (initial 3.97 mg/kg followed by 1 mg/kg) significantly prevented these bleeding symptoms; notably, no joint bleeding symptoms were observed. ACE910 is expected to prevent spontaneous bleeds and joint damage in hemophilia A patients even with weekly SC dosing, although appropriate clinical investigation is required.
Assuntos
Anticorpos Biespecíficos/uso terapêutico , Anticorpos Monoclonais Humanizados/uso terapêutico , Fator IXa/imunologia , Fator X/imunologia , Hemofilia A/complicações , Hemorragia/complicações , Hemorragia/prevenção & controle , Animais , Anticorpos Biespecíficos/imunologia , Anticorpos Monoclonais Humanizados/imunologia , Linhagem Celular , Modelos Animais de Doenças , Hemorragia/patologia , Humanos , Articulações/efeitos dos fármacos , Articulações/patologia , Macaca fascicularis , Masculino , CamundongosRESUMO
Sodium-glucose cotransporter 2 (SGLT2) inhibitors showed a glucose lowering effect in type 2 diabetes patients through inducing renal glucose excretion. Detailed analysis of the mechanism of the glucosuric effect of SGLT2 inhibition, however, has been hampered by limitations of clinical study. Here, we investigated the mechanism of urinary glucose excretion using nonhuman primates with SGLT inhibitors tofogliflozin and phlorizin, both in vitro and in vivo. In cells overexpressing cynomolgus monkey SGLT2 (cSGLT2), both tofogliflozin and phlorizin competitively inhibited uptake of the substrate (α-methyl-d-glucopyranoside; AMG). Tofogliflozin was found to be a selective cSGLT2 inhibitor, inhibiting cSGLT2 more strongly than did phlorizin, with selectivity toward cSGLT2 1,000 times that toward cSGLT1; phlorizin was found to be a nonselective cSGLT1/2 inhibitor. In a glucose titration study in cynomolgus monkeys under conditions of controlled plasma drug concentration, both tofogliflozin and phlorizin increased fractional excretion of glucose (FEG) by up to 50% under hyperglycemic conditions. By fitting the titration curve using a newly introduced method that avoids variability in estimating the threshold of renal glucose excretion, we found that tofogliflozin and phlorizin lowered the threshold and extended the splay in a dose-dependent manner without significantly affecting the tubular transport maximum for glucose (TmG). Our results demonstrate the contribution of SGLT2 to renal glucose reabsorption (RGR) in cynomolgus monkeys and demonstrate that competitive inhibition of cSGLT2 exerts a glucosuric effect by mainly extending splay and lowering threshold without affecting TmG.
Assuntos
Compostos Benzidrílicos/farmacologia , Glucose/metabolismo , Glucosídeos/farmacologia , Macaca fascicularis/urina , Florizina/farmacologia , Inibidores do Transportador 2 de Sódio-Glicose , Transportador 2 de Glucose-Sódio/efeitos dos fármacos , Animais , Células COS/metabolismo , Células COS/patologia , Chlorocebus aethiops , DNA Complementar/genética , Relação Dose-Resposta a Droga , Técnicas In Vitro , Rim/metabolismo , Rim/patologia , Masculino , Metilglucosídeos/metabolismo , Modelos Animais , Transportador 1 de Glucose-Sódio/antagonistas & inibidores , Transportador 1 de Glucose-Sódio/efeitos dos fármacos , Transportador 1 de Glucose-Sódio/genética , Transportador 2 de Glucose-Sódio/genéticaRESUMO
BACKGROUND: Emicizumab, a factor (F) VIIIa-function mimetic bispecific antibody (BsAb) to FIXa and FX, has become an indispensable treatment option for people with hemophilia A (PwHA). However, a small proportion of PwHA still experience bleeds even under emicizumab prophylaxis, as observed in the long-term outcomes of clinical studies. A more potent BsAb may be desirable for such patients. OBJECTIVES: To identify a potent BsAb to FIXa and FX, NXT007, surpassing emicizumab by in vitro and in vivo evaluation. METHODS: New pairs of light chains for emicizumab's heavy chains were screened from phage libraries, and subsequent antibody optimization was performed. For in vitro evaluation, thrombin generation assays were performed with hemophilia A plasma. In vivo hemostatic activity was evaluated in a nonhuman primate model of acquired hemophilia A. RESULTS: NXT007 exhibited an in vitro thrombin generation activity comparable to the international standard activity of FVIII (100 IU/dL), much higher than emicizumab, when triggered by tissue factor. NXT007 also demonstrated a potent in vivo hemostatic activity at approximately 30-fold lower plasma concentrations than emicizumab's historical data. In terms of dose shift between NXT007 and emicizumab, the in vitro and in vivo results were concordant. Regarding pharmacokinetics, NXT007 showed lower in vivo clearance than those shown by typical monoclonal antibodies, suggesting that the Fc engineering to enhance FcRn binding worked well. CONCLUSION: NXT007, a potent BsAb, was successfully created. Nonclinical results suggest that NXT007 would have a potential to keep a nonhemophilic range of coagulation potential in PwHA or to realize more convenient dosing regimens than emicizumab.
Assuntos
Anticorpos Biespecíficos , Hemofilia A , Hemostáticos , Humanos , Hemostáticos/farmacologia , Hemostáticos/uso terapêutico , Trombina/metabolismo , Hemostasia , Coagulação Sanguínea , Fator VIIIRESUMO
To understand the risk of hypoglycemia associated with urinary glucose excretion (UGE) induced by sodium-glucose cotransporter (SGLT) inhibitors, it is necessary to know the relationship between the ratio of contribution of SGLT2 vs. SGLT1 to renal glucose reabsorption (RGR) and the glycemic levels in vivo. To examine the contributions of SGLT2 and SGLT1 in normal rats, we compared the RGR inhibition by tofogliflozin, a highly specific SGLT2 inhibitor, and phlorizin, an SGLT1 and SGLT2 (SGLT1/2) inhibitor, at plasma concentrations sufficient to completely inhibit rat SGLT2 (rSGLT2) while inhibiting rSGLT1 to different degrees. Under hyperglycemic conditions by glucose titration, tofogliflozin and phlorizin achieved ≥50% inhibition of RGR. Under hypoglycemic conditions by hyperinsulinemic clamp, RGR was reduced by 20-50% with phlorizin and by 1-5% with tofogliflozin, suggesting the smaller contribution of rSGLT2 to RGR under hypoglycemic conditions than under hyperglycemic conditions. Next, to evaluate the hypoglycemic potentials of SGLT1/2 inhibition, we measured the plasma glucose (PG) and endogenous glucose production (EGP) simultaneously after UGE induction by SGLT inhibitors. Tofogliflozin (400 ng/ml) induced UGE of about 2 mg·kg⻹·min⻹ and increased EGP by 1-2 mg·kg⻹·min⻹, resulting in PG in the normal range. Phlorizin (1,333 ng/ml) induced UGE of about 6 mg·kg⻹·min⻹ and increased EGP by about 4 mg·kg⻹·min⻹; this was more than with tofogliflozin, but the minimum PG was lower. These results suggest that the contribution of SGLT1 to RGR is greater under lower glycemic conditions than under hyperglycemic conditions and that SGLT2-selective inhibitors pose a lower risk of hypoglycemia than SGLT1/2 inhibitors.
Assuntos
Compostos Benzidrílicos/efeitos adversos , Glucosídeos/efeitos adversos , Glicosúria/induzido quimicamente , Hipoglicemiantes/efeitos adversos , Rim/efeitos dos fármacos , Bloqueadores dos Canais de Sódio/efeitos adversos , Transportador 1 de Glucose-Sódio/antagonistas & inibidores , Inibidores do Transportador 2 de Sódio-Glicose , Absorção/efeitos dos fármacos , Animais , Compostos Benzidrílicos/administração & dosagem , Compostos Benzidrílicos/farmacocinética , Compostos Benzidrílicos/uso terapêutico , Glicemia/análise , Creatinina/metabolismo , Creatinina/urina , Relação Dose-Resposta a Droga , Gluconeogênese/efeitos dos fármacos , Glucosídeos/administração & dosagem , Glucosídeos/farmacocinética , Glucosídeos/uso terapêutico , Glicosúria/etiologia , Hiperglicemia/sangue , Hiperglicemia/tratamento farmacológico , Hiperglicemia/metabolismo , Hiperglicemia/urina , Hipoglicemia/sangue , Hipoglicemia/induzido quimicamente , Hipoglicemia/metabolismo , Hipoglicemia/urina , Hipoglicemiantes/administração & dosagem , Hipoglicemiantes/farmacocinética , Hipoglicemiantes/uso terapêutico , Rim/metabolismo , Masculino , Florizina/administração & dosagem , Florizina/efeitos adversos , Florizina/farmacocinética , Florizina/uso terapêutico , Ratos , Ratos Wistar , Bloqueadores dos Canais de Sódio/administração & dosagem , Bloqueadores dos Canais de Sódio/farmacocinética , Bloqueadores dos Canais de Sódio/uso terapêutico , Transportador 1 de Glucose-Sódio/metabolismo , Transportador 2 de Glucose-Sódio/metabolismoRESUMO
Sodium/glucose cotransporter 2 (SGLT2) is the predominant mediator of renal glucose reabsorption and is an emerging molecular target for the treatment of diabetes. We identified a novel potent and selective SGLT2 inhibitor, tofogliflozin (CSG452), and examined its efficacy and pharmacological properties as an antidiabetic drug. Tofogliflozin competitively inhibited SGLT2 in cells overexpressing SGLT2, and K(i) values for human, rat, and mouse SGLT2 inhibition were 2.9, 14.9, and 6.4 nM, respectively. The selectivity of tofogliflozin toward human SGLT2 versus human SGLT1, SGLT6, and sodium/myo-inositol transporter 1 was the highest among the tested SGLT2 inhibitors under clinical development. Furthermore, no interaction with tofogliflozin was observed in any of a battery of tests examining glucose-related physiological processes, such as glucose uptake, glucose oxidation, glycogen synthesis, hepatic glucose production, glucose-stimulated insulin secretion, and glucosidase reactions. A single oral gavage of tofogliflozin increased renal glucose clearance and lowered the blood glucose level in Zucker diabetic fatty rats. Tofogliflozin also improved postprandial glucose excursion in a meal tolerance test with GK rats. In db/db mice, 4-week tofogliflozin treatment reduced glycated hemoglobin and improved glucose tolerance in the oral glucose tolerance test 4 days after the final administration. No blood glucose reduction was observed in normoglycemic SD rats treated with tofogliflozin. These findings demonstrate that tofogliflozin inhibits SGLT2 in a specific manner, lowers blood glucose levels by increasing renal glucose clearance, and improves pathological conditions of type 2 diabetes with a low hypoglycemic potential.
Assuntos
Compostos Benzidrílicos/farmacologia , Glicemia/metabolismo , Diabetes Mellitus Experimental/tratamento farmacológico , Diabetes Mellitus Tipo 2/tratamento farmacológico , Glucosídeos/farmacologia , Hemoglobinas Glicadas/metabolismo , Inibidores do Transportador 2 de Sódio-Glicose , Animais , Diabetes Mellitus Tipo 2/sangue , Relação Dose-Resposta a Droga , Intolerância à Glucose/tratamento farmacológico , Teste de Tolerância a Glucose , Humanos , Hiperglicemia/sangue , Hiperglicemia/tratamento farmacológico , Rim/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos , Ratos , Ratos Sprague-Dawley , Ratos ZuckerRESUMO
Myostatin, a member of the transforming growth factor-ß superfamily, is an attractive target for muscle disease therapy because of its role as a negative regulator of muscle growth and strength. Here, we describe a novel antibody therapeutic approach that maximizes the potential of myostatin-targeted therapy. We generated an antibody, GYM329, that specifically binds the latent form of myostatin and inhibits its activation. Additionally, via "sweeping antibody technology", GYM329 reduces or "sweeps" myostatin in the muscle and plasma. Compared with conventional anti-myostatin agents, GYM329 and its surrogate antibody exhibit superior muscle strength-improvement effects in three different mouse disease models. We also demonstrate that the superior efficacy of GYM329 is due to its myostatin specificity and sweeping capability. Furthermore, we show that a GYM329 surrogate increases muscle mass in normal cynomolgus monkeys without any obvious toxicity. Our findings indicate the potential of GYM329 to improve muscle strength in patients with muscular disorders.
Assuntos
Anticorpos Monoclonais/farmacologia , Força Muscular/efeitos dos fármacos , Doenças Musculares/fisiopatologia , Miostatina/imunologia , Animais , Proteínas Morfogenéticas Ósseas/metabolismo , Modelos Animais de Doenças , Feminino , Fatores de Diferenciação de Crescimento/metabolismo , Macaca fascicularis , Masculino , Camundongos Endogâmicos C57BL , Músculo Esquelético/patologia , Músculo Esquelético/fisiopatologia , Atrofia Muscular/patologia , Atrofia Muscular/fisiopatologia , Tamanho do Órgão , Transdução de SinaisRESUMO
Agonistic antibodies targeting CD137 have been clinically unsuccessful due to systemic toxicity. Because conferring tumor selectivity through tumor-associated antigen limits its clinical use to cancers that highly express such antigens, we exploited extracellular adenosine triphosphate (exATP), which is a hallmark of the tumor microenvironment and highly elevated in solid tumors, as a broadly tumor-selective switch. We generated a novel anti-CD137 switch antibody, STA551, which exerts agonistic activity only in the presence of exATP. STA551 demonstrated potent and broad antitumor efficacy against all mouse and human tumors tested and a wide therapeutic window without systemic immune activation in mice. STA551 was well tolerated even at 150 mg/kg/week in cynomolgus monkeys. These results provide a strong rationale for the clinical testing of STA551 against a broad variety of cancers regardless of antigen expression, and for the further application of this novel platform to other targets in cancer therapy. SIGNIFICANCE: Reported CD137 agonists suffer from either systemic toxicity or limited efficacy against antigen-specific cancers. STA551, an antibody designed to agonize CD137 only in the presence of extracellular ATP, inhibited tumor growth in a broad variety of cancer models without any systemic toxicity or dependence on antigen expression.See related commentary by Keenan and Fong, p. 20.This article is highlighted in the In This Issue feature, p. 1.
Assuntos
Trifosfato de Adenosina , Neoplasias , Animais , Anticorpos Monoclonais/farmacologia , Antígenos de Neoplasias , Imunoterapia , Camundongos , Neoplasias/tratamento farmacológico , Microambiente Tumoral , Membro 9 da Superfamília de Receptores de Fatores de Necrose TumoralRESUMO
Prolonged signaling at the parathyroid hormone receptor 1 (PTHR1) correlates with the capacity of a ligand to bind to a G protein-independent receptor conformation (R0). As long-acting PTH (LA-PTH) ligands hold interest as potential treatments for hypoparathyroidism (HP), we explored the structural basis in the ligand for stable R0 binding and prolonged cAMP signaling. A series of PTH/PTHrP hybrid analogs were synthesized and tested for actions in vitro and in vivo. Of the series, [Ala1,3,12,Gln10,Arg11,Trp14]-PTH(1-14)/PTHrP(15-36) (M-PTH/PTHrP) bound with high affinity to R0, induced prolonged cAMP responses in UMR106 rat osteoblast-derived cells, and induced the most prolonged increases in serum calcium (sCa) in normal rats. Daily s.c. injection of M-PTH/PTHrP into thyroparathyroidectomized (TPTX) rats, a model of HP, normalized sCa without raising urine Ca. In contrast, oral alfacalcidol, a widely used treatment for HP, normalized sCa, but induced frank hypercalciuria. M-PTH/PTHrP exhibited low solubility in aqueous solutions of neutral pH; however, replacement of Leu18, Phe22, and His26 with the less hydrophobic residues, Ala, Ala, and Lys, at those respective positions markedly improved solubility while maintaining bioactivity. Indeed, we recently showed that the resultant analog [Ala18,22,Lys26]-M-PTH/PTHrP or LA-PTH, effectively normalizes sCa in TPTX rats and mediates prolonged actions in monkeys. These studies provide useful information for optimizing PTH and PTHrP ligand analogs for therapeutic development. © 2020 The Authors. JBMR Plus published by Wiley Periodicals, Inc. on behalf of American Society for Bone and Mineral Research.
RESUMO
1. The aim of the present study was to evaluate the effects of mitemcinal (GM-611), an orally active motilin receptor agonist, on delayed gastric emptying in a canine model of diabetic gastroparesis and to compare these effects with those of cisapride. 2. Moderate hyperglycaemia was induced by a single intravenous injection of a mixture of streptozotocin (30 mg/kg) and alloxan (50 mg/kg). Dogs that maintained moderate hyperglycaemia (fasting plasma glucose 200-300 mg/dL) without insulin treatment were selected and gastric emptying in these dogs was determined by the paracetamol method. 3. One year after the onset of diabetes, there was no difference in the gastric emptying of normal and diabetic dogs. However, after 5 years, the diabetic dogs showed delayed gastric emptying. The motor nerve conduction velocity of the tibial nerve was significantly lower in diabetic dogs compared with normal dogs at both time points. 4. Histopathological examination at the end of the study showed that there were fewer nerve fibres in both dorsal vagal and tibial nerves of diabetic dogs compared with normal dogs. The onset of delayed gastric emptying is thought to have occurred gradually, in parallel with abnormal autonomic nerve function induced by the long period of moderate hyperglycaemia. 5. Oral administration of mitemcinal (0.125, 0.25 or 0.5 mg/kg) dose-dependently accelerated delayed gastric emptying, significant at 0.5 mg/kg, in diabetic dogs, whereas cisapride (1, 3 or 10 mg/kg) had no significant effect. These results add to the existing evidence that mitemcinal is likely to be useful for treating diabetic gastroparesis.
Assuntos
Diabetes Mellitus Experimental/tratamento farmacológico , Eritromicina/análogos & derivados , Esvaziamento Gástrico/efeitos dos fármacos , Gastroparesia/tratamento farmacológico , Receptores dos Hormônios Gastrointestinais/agonistas , Receptores de Neuropeptídeos/agonistas , Administração Oral , Animais , Diabetes Mellitus Experimental/complicações , Diabetes Mellitus Experimental/fisiopatologia , Modelos Animais de Doenças , Cães , Eritromicina/administração & dosagem , Feminino , Esvaziamento Gástrico/fisiologia , Gastroparesia/complicações , Gastroparesia/fisiopatologia , Receptores dos Hormônios Gastrointestinais/fisiologia , Receptores de Neuropeptídeos/fisiologiaRESUMO
Emicizumab is a humanized bispecific antibody that binds simultaneously to factor (F) IXa and FX replacing the cofactor function of FVIIIa. Because emicizumab recognizes FIX/FIXa and FX/FXa, a question may arise whether emicizumab competes with antithrombin (AT) and/or tissue factor pathway inhibitor (TFPI), thereby enhancing overall hemostatic potential by blocking their antihemostatic effects. To address this question, we performed enzymatic assays using purified coagulation factors to confirm whether emicizumab interferes with the action of AT on FIXa or FXa, or with the action of TFPI on FXa. In those assays, we found no interference of emicizumab on the actions of AT and TFPI. We next assessed emicizumab's influences on the anticoagulation actions of AT or TFPI in thrombin generation assays triggered with FXIa or tissue factor (TF) in AT-depleted or TFPI-depleted plasma supplemented with AT or TFPI in vitro. In those assays, we employed anti-FIXa and anti-FX monospecific one-armed antibodies derived from emicizumab instead of emicizumab itself so as to prevent emicizumab's FVIIIa cofactor activity from boosting thrombin generation. Consequently, we found that neither anti-FIXa, anti-FX monospecific antibody, nor the mixture of the two interfered with the anticoagulation actions of AT or TFPI in plasma. Although emicizumab can bind to FIXa and FXa, our results showed no interference of emicizumab with the action of AT or TFPI on FIXa or FXa. This indicates that the presence of emicizumab is irrelevant to the action of AT and TFPI, and thus should not alter the coagulant/anticoagulant balance related to AT and TFPI.
RESUMO
The liver plays a major role in whole-body energy homeostasis by releasing secretory factors, termed hepatokines. To identify novel target genes associated with insulin resistance, we performed a comprehensive analysis of gene expression profiles using a DNA chip method in liver biopsy samples from humans with varying degrees of insulin resistance. Inhibin ßE (INHBE) was identified as a novel putative hepatokine with hepatic gene expression that positively correlated with insulin resistance and body mass index in humans. Quantitative real time-PCR analysis also showed an increase in INHBE gene expression in independent liver samples from insulin-resistant human subjects. Additionally, Inhbe gene expression increased in the livers of db/db mice, a rodent model of type 2 diabetes. To preliminarily screen the role of Inhbe in vivo in whole-body energy metabolic status, hepatic mRNA was knocked down with siRNA for Inhbe (siINHBE) in db/db mice. Treatment with siINHBE suppressed body weight gain during the two-week experimental period, which was attributable to diminished fat rather than lean mass. Additionally, treatment with siINHBE decreased the respiratory quotient and increased plasma total ketone bodies compared with treatment with non-targeting siRNA, both of which suggest enhanced whole-body fat utilization. Our study suggests that INHBE functions as a possible hepatokine to alter the whole-body metabolic status under obese insulin-resistant conditions.
Assuntos
Perfilação da Expressão Gênica , Subunidades beta de Inibinas/genética , Resistência à Insulina/genética , Fígado/patologia , Tecido Adiposo/citologia , Animais , Biópsia , Peso Corporal , Feminino , Humanos , Subunidades beta de Inibinas/deficiência , Subunidades beta de Inibinas/metabolismo , Fígado/metabolismo , Masculino , Camundongos , Pessoa de Meia-Idade , Fenótipo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/genéticaRESUMO
Modulating the complement system is a promising strategy in drug discovery for disorders with uncontrolled complement activation. Although some of these disorders can be effectively treated with an antibody that inhibits complement C5, the high plasma concentration of C5 requires a huge dosage and frequent intravenous administration. Moreover, a conventional anti-C5 antibody can cause C5 to accumulate in plasma by reducing C5 clearance when C5 forms an immune complex (IC) with the antibody, which can be salvaged from endosomal vesicles by neonatal Fc receptor (FcRn)-mediated recycling. In order to neutralize the increased C5, an even higher dosage of the antibody would be required. This antigen accumulation can be suppressed by giving the antibody a pH-dependent C5-binding property so that C5 is released from the antibody in the acidic endosome and then trafficked to the lysosome for degradation, while the C5-free antibody returns back to plasma. We recently demonstrated that a pH-dependent C5-binding antibody, SKY59, exhibited long-lasting neutralization of C5 in cynomolgus monkeys, showing potential for subcutaneous delivery or less frequent administration. Here we report the details of the antibody engineering involved in generating SKY59, from humanizing a rabbit antibody to improving the C5-binding property. Moreover, because the pH-dependent C5-binding antibodies that we first generated still accumulated C5, we hypothesized that the surface charges of the ICs partially contributed to a slow uptake rate of the C5-antibody ICs. This idea motivated us to engineer the surface charges of the antibody. Our surface-charge engineered antibody consequently exhibited a high capacity to sweep C5 and suppressed the C5 accumulation in vivo by accelerating the cycle of sweeping: uptake of ICs into cells, release of C5 from the antibody in endosomes, and salvage of the antigen-free antibody. Thus, our engineered anti-C5 antibody, SKY59, is expected to provide significant benefits for patients with complement-mediated disorders.
Assuntos
Anticorpos Monoclonais/genética , Ativação do Complemento/efeitos dos fármacos , Complemento C5/antagonistas & inibidores , Engenharia de Proteínas/métodos , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/uso terapêutico , Afinidade de Anticorpos , Ativação do Complemento/imunologia , Complemento C5/imunologia , Complemento C5/isolamento & purificação , Simulação por Computador , Descoberta de Drogas/métodos , Endossomos/imunologia , Antígenos de Histocompatibilidade Classe I/genética , Antígenos de Histocompatibilidade Classe I/imunologia , Humanos , Concentração de Íons de Hidrogênio , Doenças do Sistema Imunitário/tratamento farmacológico , Doenças do Sistema Imunitário/imunologia , Macaca fascicularis , Camundongos , Camundongos Transgênicos , Mutagênese , Receptores Fc/genética , Receptores Fc/imunologia , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/isolamento & purificação , Fatores de TempoRESUMO
Emicizumab, a humanised bispecific antibody recognising factors (F) IX/IXa and X/Xa, can accelerate FIXa-catalysed FX activation by bridging FIXa and FX in a manner similar to FVIIIa. However, details of the emicizumab-antigen interactions have not been reported so far. In this study, we first showed by surface plasmon resonance analysis that emicizumab bound FIX, FIXa, FX, and FXa with moderate affinities (KD = 1.58, 1.52, 1.85, and 0.978 µM, respectively). We next showed by immunoblotting analysis that emicizumab recognised the antigens' epidermal growth factor (EGF)-like domains. We then performed KD-based simulation of equilibrium states in plasma for quantitatively predicting the ways that emicizumab would interact with the antigens. The simulation predicted that only a small part of plasma FIX, FX, and emicizumab would form antigen-bridging FIX-emicizumab-FX ternary complex, of which concentration would form a bell-shaped relationship with emicizumab concentration. The bell-shaped concentration dependency was reproduced by plasma thrombin generation assays, suggesting that the plasma concentration of the ternary complex would correlate with emicizumab's cofactor activity. The simulation also predicted that at 10.0-100 µg/ml of emicizumab-levels shown in a previous study to be clinically effective-the majority of plasma FIX, FX, and emicizumab would exist as monomers. In conclusion, emicizumab binds FIX/FIXa and FX/FXa with micromolar affinities at their EGF-like domains. The KD-based simulation predicted that the antigen-bridging ternary complex formed in circulating plasma would correlate with emicizumab's cofactor activity, and the majority of FIX and FX would be free and available for other coagulation reactions.
Assuntos
Anticorpos Biespecíficos/imunologia , Anticorpos Biespecíficos/farmacologia , Anticorpos Monoclonais Humanizados/imunologia , Anticorpos Monoclonais Humanizados/farmacologia , Fator VIIIa/imunologia , Anticorpos Biespecíficos/sangue , Anticorpos Monoclonais Humanizados/sangue , Especificidade de Anticorpos , Reações Antígeno-Anticorpo , Sítios de Ligação , Materiais Biomiméticos/farmacologia , Simulação por Computador , Fator IX/antagonistas & inibidores , Fator IX/imunologia , Fator IXa/antagonistas & inibidores , Fator IXa/imunologia , Fator X/antagonistas & inibidores , Fator X/imunologia , Fator Xa/imunologia , Inibidores do Fator Xa/sangue , Inibidores do Fator Xa/imunologia , Inibidores do Fator Xa/farmacologia , Humanos , Modelos ImunológicosRESUMO
Dysregulation of the complement system is linked to the pathogenesis of a variety of hematological disorders. Eculizumab, an anti-complement C5 monoclonal antibody, is the current standard of care for paroxysmal nocturnal hemoglobinuria (PNH) and atypical hemolytic uremic syndrome (aHUS). However, because of high levels of C5 in plasma, eculizumab has to be administered biweekly by intravenous infusion. By applying recycling technology through pH-dependent binding to C5, we generated a novel humanized antibody against C5, SKY59, which has long-lasting neutralization of C5. In cynomolgus monkeys, SKY59 suppressed C5 function and complement activity for a significantly longer duration compared to a conventional antibody. Furthermore, epitope mapping by X-ray crystal structure analysis showed that a histidine cluster located on C5 is crucial for the pH-dependent interaction with SKY59. This indicates that the recycling effect of SKY59 is driven by a novel mechanism of interaction with its antigen and is distinct from other known pH-dependent antibodies. Finally, SKY59 showed neutralizing effect on C5 variant p.Arg885His, while eculizumab does not inhibit complement activity in patients carrying this mutation. Collectively, these results suggest that SKY59 is a promising new anti-C5 agent for patients with PNH and other complement-mediated disorders.
Assuntos
Anticorpos Neutralizantes/imunologia , Complemento C5/antagonistas & inibidores , Complemento C5/imunologia , Animais , Anticorpos Neutralizantes/administração & dosagem , Anticorpos Neutralizantes/química , Complemento C5/química , Cristalografia por Raios X , Hemoglobinúria Paroxística/tratamento farmacológico , Humanos , Macaca fascicularis , Ligação Proteica , Conformação ProteicaRESUMO
Cancer care is being revolutionized by immunotherapies such as immune checkpoint inhibitors, engineered T cell transfer, and cell vaccines. The bispecific T cell-redirecting antibody (TRAB) is one such promising immunotherapy, which can redirect T cells to tumor cells by engaging CD3 on a T cell and an antigen on a tumor cell. Because T cells can be redirected to tumor cells regardless of the specificity of T cell receptors, TRAB is considered efficacious for less immunogenic tumors lacking enough neoantigens. Its clinical efficacy has been exemplified by blinatumomab, a bispecific T cell engager targeting CD19 and CD3, which has shown marked clinical responses against hematological malignancies. However, the success of TRAB in solid tumors has been hampered by the lack of a target molecule with sufficient tumor selectivity to avoid "on-target off-tumor" toxicity. Glypican 3 (GPC3) is a highly tumor-specific antigen that is expressed during fetal development but is strictly suppressed in normal adult tissues. We developed ERY974, a whole humanized immunoglobulin G-structured TRAB harboring a common light chain, which bispecifically binds to GPC3 and CD3. Using a mouse model with reconstituted human immune cells, we revealed that ERY974 is highly effective in killing various types of tumors that have GPC3 expression comparable to that in clinical tumors. ERY974 also induced a robust antitumor efficacy even against tumors with nonimmunogenic features, which are difficult to treat by inhibiting immune checkpoints such as PD-1 (programmed cell death protein-1) and CTLA-4 (cytotoxic T lymphocyte-associated protein-4). Immune monitoring revealed that ERY974 converted the poorly inflamed tumor microenvironment to a highly inflamed microenvironment. Toxicology studies in cynomolgus monkeys showed transient cytokine elevation, but this was manageable and reversible. No organ toxicity was evident. These data provide a rationale for clinical testing of ERY974 for the treatment of patients with GPC3-positive solid tumors.
Assuntos
Anticorpos Biespecíficos/uso terapêutico , Glipicanas/imunologia , Neoplasias/imunologia , Neoplasias/patologia , Linfócitos T/imunologia , Animais , Anticorpos Biespecíficos/administração & dosagem , Anticorpos Biespecíficos/farmacocinética , Antineoplásicos/farmacocinética , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Complexo CD3/metabolismo , Citocinas/metabolismo , Humanos , Imunocompetência/efeitos dos fármacos , Injeções Intravenosas , Ativação Linfocitária/efeitos dos fármacos , Ativação Linfocitária/imunologia , Macaca fascicularis , Camundongos Transgênicos , Esteroides/farmacologia , Esteroides/uso terapêutico , Linfócitos T/efeitos dos fármacosRESUMO
Hypoparathyroidism is a disease of chronic hypocalcemia and hyperphosphatemia due to a deficiency of parathyroid hormone (PTH). PTH and analogs of the hormone are of interest as potential therapies. Accordingly, we examined the pharmacological properties of a long-acting PTH analog, [Ala(1,3,12,18,22) , Gln(10) ,Arg(11) ,Trp(14) ,Lys(26) ]-PTH(1-14)/PTHrP(15-36) (LA-PTH) in thyroparathyroidectomized (TPTX) rats, a model of HP, as well as in normal monkeys. In TPTX rats, a single intravenous administration of LA-PTH at a dose of 0.9 nmol/kg increased serum calcium (sCa) and decreased serum phosphate (sPi) to near-normal levels for longer than 48 hours, whereas PTH(1-34) and PTH(1-84), each injected at a dose 80-fold higher than that used for LA-PTH, increased sCa and decreased sPi only modestly and transiently (<6 hours). LA-PTH also exhibited enhanced and prolonged efficacy versus PTH(1-34) and PTH(1-84) for elevating sCa when administered subcutaneously (s.c.) into monkeys. Daily s.c. administration of LA-PTH (1.8 nmol/kg) into TPTX rats for 28 days elevated sCa to near normal levels without causing hypercalciuria or increasing bone resorption markers, a desirable goal in the treatment of hypoparathyroidism. The results are supportive of further study of long-acting PTH analogs as potential therapies for patients with hypoparathyroidism. © 2016 American Society for Bone and Mineral Research.