RESUMO
Migraine is a debilitating disease that affects almost 15% of the population worldwide and is the first cause of disability in people under 50 years of age, yet its etiology and pathophysiology remain incompletely understood. Recently, small molecules and therapeutic antibodies that block the calcitonin gene-related peptide (CGRP) signaling pathway have reduced migraine occurrence and aborted acute attacks of migraine in clinical trials and provided prevention in patients with episodic and chronic migraine. Heterogeneity is present within each diagnosis and patient's response to treatment, suggesting migraine as a final common pathway potentially activated by multiple mechanisms, e.g., not all migraine attacks respond to or are prevented by anti-CGRP pharmacological interventions. Consequently, other unique mechanisms central to migraine pathogenesis may present new targets for drug development. Pituitary adenylate cyclase-activating peptide (PACAP) is an attractive novel target for treatment of migraines. We generated a specific, high-affinity, neutralizing monoclonal antibody (ALD1910) with reactivity to both PACAP38 and PACAP27. In vitro, ALD1910 effectively antagonizes PACAP38 signaling through the pituitary adenylate cyclase-activating peptide type I receptor, vasoactive intestinal peptide receptor 1, and vasoactive intestinal peptide receptor 2. ALD1910 recognizes a nonlinear epitope within PACAP and blocks its binding to the cell surface. To test ALD1910 antagonistic properties directed against endogenous PACAP, we developed an umbellulone-induced rat model of neurogenic vasodilation and parasympathetic lacrimation. In vivo, this model demonstrates that the antagonistic activity of ALD1910 is dose-dependent, retaining efficacy at doses as low as 0.3 mg/kg. These results indicate that ALD1910 represents a potential therapeutic antibody to address PACAP-mediated migraine.
Assuntos
Anticorpos Monoclonais Humanizados/imunologia , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/imunologia , Animais , Especificidade de Anticorpos , Relação Dose-Resposta Imunológica , Epitopos/imunologia , Humanos , Cinética , Masculino , Transtornos de Enxaqueca/imunologia , Transtornos de Enxaqueca/prevenção & controle , Células PC12 , Ratos , Ratos Sprague-DawleyRESUMO
The pharmacokinetic (PK) behavior of monoclonal antibodies (mAbs) is influenced by target-mediated drug disposition, off-target effects, antidrug antibody-mediated clearance, and interaction with fragment-crystallizable domain (Fc) receptors such as neonatal Fc receptor. All of these interactions hold the potential to impact mAb biodistribution. Near infrared (NIR) fluorescent probes offer an approach complementary to radionuclides to characterize drug disposition. Notably, the use of FDA-approved IRDye800 (IR800; LI-COR, Lincoln, NE) as a protein-labeling agent in preclinical work holds the potential for quantitative tissue analysis. Here, we tested the utility of the IR800 dye as a quantitative mAb tracer during pharmacokinetic analysis in both plasma and tissues using a model mouse monoclonal IgG1 (8C2) labeled with ≤1.5 molecules of IR800. The plasma PK parameters derived from a mixture of IR800-8C2 and 8C2 dosed intravenously to C57BL/6 mice at 8 mg/kg exhibited a large discrepancy in exposure depending on the method of quantitation [CLplasma = 8.4 ml/d per kilogram (NIR fluorescence detection) versus 2.5 ml/d per kilogram (enzyme-linked immunosorbent assay)]. The disagreement between measurements suggests that the PK of 8C2 is altered by addition of the IR800 dye. Additionally, direct fluorescence analysis of homogenized tissues revealed several large differences in IR800-8C2 tissue uptake when compared with a previously published study using [(125)I]8C2, most notably an over 4-fold increase in liver concentration. Finally, the utility of IR800 in combination with whole body imaging was examined by comparison of IR800-8C2 levels observed in animal sagittal cross-sections to those measured in homogenized tissues. Our results represent the first PK analysis in both mouse plasma and tissues of an IR800-mAb conjugate and suggest that mAb disposition is significantly altered by IR800 conjugation to 8C2.
Assuntos
Anticorpos Monoclonais/farmacocinética , Corantes Fluorescentes/química , Animais , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Espectroscopia de Luz Próxima ao Infravermelho , Distribuição TecidualRESUMO
To assess the potential for male-mediated drug transfer to their female partner and/or developing conceptus, vaginal uptake of a monoclonal antibody (mAb) biotherapeutic was assessed in cynomolgus monkeys. A human IgG2 mAb (IgG2X; bound human and cynomolgus monkey neonatal Fc-receptor, FcRn, with similar high affinity) was administered intravaginally (IvG; 100mg/dose) to 5 pregnant cynomolgus monkeys biweekly from gestation day (gd) 21 to gd133. In all maternal samples collected before gd119, IgG2X plasma concentrations were below the limit of quantification (BLQ; <25ng/mL). After dosing on gd119 and 133, maternal IgG2X plasma concentrations remained BLQ in 3/5 monkeys and were very low in 2/5 (up to 116ng/mL; â¼0.01% of the IvG dose). IgG2X was BLQ in all fetal plasma samples. These data indicate that male-mediated mAb drug transfer via seminal fluid does not present a health risk to the female partner and is not bioavailable to the developing conceptus.
Assuntos
Anticorpos Monoclonais/administração & dosagem , Anticorpos Monoclonais/farmacocinética , Sangue Fetal/metabolismo , Imunoglobulina G/administração & dosagem , Imunoglobulina G/farmacologia , Administração Intravaginal , Animais , Anticorpos Monoclonais/sangue , Feminino , Imunoglobulina G/sangue , Macaca fascicularis , Masculino , Exposição Materna , Troca Materno-Fetal , Gravidez , Sêmen/metabolismoRESUMO
Understanding species differences in the placental transfer of monoclonal antibodies is important to inform species selection for nonclinical safety assessment, interpret embryo-fetal changes observed in these studies, and extrapolate their human relevance. Data presented here for a fully human immunoglobulin G2 monoclonal antibody (IgG2X) revealed that, during organogenesis, in both the cynomolgus monkey (gestation day 35 [gd35]) and the rat (gd10) the extent of IgG2X placental transfer (approximately 0.5% maternal plasma concentration, MPC) was similar to the limited published human data for endogenous IgG. At this early gestational stage, IgG2X placental transfer was approximately 6-fold higher in the rabbit (gd10). By the end of organogenesis, rat embryonic plasma concentrations (gd16) exceeded those in the cynomolgus monkey (gd50) by approximately 3-fold. These data suggest that relative to the cynomolgus monkey, the rabbit (and to a lesser extent the rat) may overestimate potential harmful effects to the human embryo during this critical period of development. Beyond organogenesis, fetal IgG2X plasma concentrations increased approximately 10-fold early in the second trimester (gd50-70) in the cynomolgus monkey and remained relatively unchanged thereafter (at approximately 5% MPC). Late gestational assessment was precluded in rabbits due to immunogenicity, but in rats, fetal IgG2X plasma concentrations increased more than 6-fold from gd16 to gd21 (reaching approximately 15% MPC). In rats, maternal exposure consistent with that achieved by ICH S6(R1) high-dose selection criteria resulted in embryonic plasma concentrations, reaching pharmacologically relevant levels during organogenesis. Furthermore, dose proportional exposure in both mothers and embryos indicated that this was unlikely to occur at the lower therapeutic dose levels used in humans.
Assuntos
Anticorpos Monoclonais/farmacocinética , Troca Materno-Fetal , Organogênese/efeitos dos fármacos , Placenta/efeitos dos fármacos , Animais , Relação Dose-Resposta a Droga , Embrião de Mamíferos/efeitos dos fármacos , Feminino , Feto/efeitos dos fármacos , Feto/embriologia , Idade Gestacional , Imunoglobulina G/metabolismo , Macaca fascicularis , Exposição Materna , Placenta/metabolismo , Gravidez , Coelhos , Ratos , Ratos Sprague-DawleyRESUMO
Proprotein convertase subtilisin/kexin type 9 (PCSK9) regulates serum LDL cholesterol (LDL-C) by interacting with the LDL receptor (LDLR) and is an attractive therapeutic target for LDL-C lowering. We have generated a neutralizing anti-PCSK9 antibody, mAb1, that binds to an epitope on PCSK9 adjacent to the region required for LDLR interaction. In vitro, mAb1 inhibits PCSK9 binding to the LDLR and attenuates PCSK9-mediated reduction in LDLR protein levels, thereby increasing LDL uptake. A combination of mAb1 with a statin increases LDLR levels in HepG2 cells more than either treatment alone. In wild-type mice, mAb1 increases hepatic LDLR protein levels approximately 2-fold and lowers total serum cholesterol by up to 36%: this effect is not observed in LDLR(-/-) mice. In cynomolgus monkeys, a single injection of mAb1 reduces serum LDL-C by 80%, and a significant decrease is maintained for 10 days. We conclude that anti-PCSK9 antibodies may be effective therapeutics for treating hypercholesterolemia.