RESUMO
As the pharmaceutical industry places greater emphasis on pairing biological pathways with appropriate therapeutic intervention, an increase in the use of biologic drugs has emerged. With increasing complexity of biotherapeutics, absorption, distribution, metabolism, and excretion (ADME) studies have also become increasingly complex. The characterization of ADME properties is critical to tuning the pharmacokinetic profiles of next generation biologics (NGBs). The knowledge of the fate of a drug is essential for the enhancement of the design processes, elongation of exposure at the desired site of action, and achieving efficacy with minimum toxicity. In vivo proteolytic cleavage of biotherapeutics may lead to undesirable in vivo properties, such as rapid clearance, low bioavailability, and loss of pharmacodynamic effect. All of these may affect drug efficacy and/or generate safety concerns through increases in immunogenicity or off-target toxicity. The work herein describes the development of a robust, fully automated immunoaffinity purification (IA)-capillary electrophoresis-mass spectrometry (CE-MS) workflow. The reagents were carefully optimized to maximize isolation yields while minimizing the number of experimental steps to analytical results. The result is the development of a comprehensive integrated platform for the characterization of a wide range of biotherapeutics, including peptibodies, monoclonal antibodies, and bispecific antibodies. Empowered by this automated IA-CE-MS approach, implementing biotransformation studies at an early drug discovery stage can speed up the drug development process.
Assuntos
Produtos Biológicos , Eletroforese Capilar , Anticorpos Monoclonais , Descoberta de Drogas , Espectrometria de MassasRESUMO
There is an unmet medical need for nonopioid pain therapies in human populations; several pathways are under investigation for possible therapeutic intervention. Tetrahydrobiopterin (BH4) has received attention recently as a mediator of neuropathic pain. Recent reports have implicated sepiapterin reductase (SPR) in this pain pathway as a regulator of BH4 production. To evaluate the role of SPR inhibition on BH4 reduction, we developed analytical methods to monitor the relationship between the plasma concentration of test article and endogenous pterins and applied these in the rat spinal nerve ligation pain model. Sepiapterin is an endogenous substrate, which accumulates upon inhibition of SPR. In response to a potent inhibitor of SPR, plasma concentrations of sepiapterin increased proportionally with exposure. An indirect-effect pharmacokinetic/pharmacodynamic model was developed to describe the relationship between the plasma pharmacokinetics of test article and plasma sepiapterin levels in the rat, which was used to determine an in vivo SPR IC50 value. SPR inhibition and mechanical allodynia were assessed coordinately with pterin biomarkers in plasma and at the site of neuronal injury (i.e., dorsal root ganglion). Upon daily oral administration for 3 consecutive days, unbound plasma concentrations of test article exceeded the unbound in vivo rat SPR IC90 throughout the dose intervals, leading to a 60% reduction in BH4 in the dorsal root ganglion. Despite evidence for pharmacological modulation of the BH4 pathway, there was no significant effect on the tactile paw withdrawal threshold relative to vehicle-treated controls.
Assuntos
Oxirredutases do Álcool/antagonistas & inibidores , Oxirredutases do Álcool/metabolismo , Hiperalgesia/metabolismo , Neuralgia/metabolismo , Medição da Dor/métodos , Animais , Biopterinas/análogos & derivados , Biopterinas/antagonistas & inibidores , Biopterinas/metabolismo , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/uso terapêutico , Gânglios Espinais/efeitos dos fármacos , Gânglios Espinais/metabolismo , Humanos , Hiperalgesia/tratamento farmacológico , Masculino , Neuralgia/tratamento farmacológico , Medição da Dor/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Tato/efeitos dos fármacos , Tato/fisiologiaRESUMO
The identification of nonopioid alternatives to treat chronic pain has received a great deal of interest in recent years. Recently, the engineering of a series of Nav1.7 inhibitory peptide-antibody conjugates has been reported, and herein, the preclinical efforts to identify novel approaches to characterize the pharmacokinetic properties of the peptide conjugates are described. A cryopreserved plated mouse hepatocyte assay was designed to measure the depletion of the peptide-antibody conjugates from the media, with a correlation being observed between percentage remaining in the media and in vivo clearance (Pearson r = -0.5525). Physicochemical (charge and hydrophobicity), receptor-binding [neonatal Fc receptor (FcRn)], and in vivo pharmacokinetic data were generated and compared with the results from our in vitro hepatocyte assay, which was hypothesized to encompass all of the aforementioned properties. Correlations were observed among hydrophobicity; FcRn binding; depletion rates from the hepatocyte assay; and ultimately, in vivo clearance. Subsequent studies identified potential roles for the low-density lipoprotein and mannose/galactose receptors in the association of the Nav1.7 peptide conjugates with mouse hepatocytes, although in vivo studies suggested that FcRn was still the primary receptor involved in determining the pharmacokinetics of the peptide conjugates. Ultimately, the use of the cryopreserved hepatocyte assay along with FcRn binding and hydrophobic interaction chromatography provided an efficient and integrated approach to rapidly triage molecules for advancement while reducing the number of in vivo pharmacokinetic studies. SIGNIFICANCE STATEMENT: Although multiple in vitro and in silico tools are available in small-molecule drug discovery, pharmacokinetic characterization of protein therapeutics is still highly dependent upon the use of in vivo studies in preclinical species. The current work demonstrates the combined use of cryopreserved hepatocytes, hydrophobic interaction chromatography, and neonatal Fc receptor binding to characterize a series of Nav1.7 peptide-antibody conjugates prior to conducting in vivo studies, thus providing a means to rapidly evaluate novel protein therapeutic platforms while concomitantly reducing the number of in vivo studies conducted in preclinical species.
Assuntos
Dor Crônica/tratamento farmacológico , Antígenos de Histocompatibilidade Classe I/metabolismo , Imunoconjugados/farmacocinética , Canal de Sódio Disparado por Voltagem NAV1.7/metabolismo , Receptores Fc/metabolismo , Bloqueadores do Canal de Sódio Disparado por Voltagem/farmacocinética , Administração Intravenosa , Animais , Anticorpos Monoclonais/administração & dosagem , Anticorpos Monoclonais/farmacocinética , Criopreservação , Avaliação Pré-Clínica de Medicamentos/métodos , Hepatócitos , Antígenos de Histocompatibilidade Classe I/genética , Imunoconjugados/administração & dosagem , Macaca fascicularis , Masculino , Taxa de Depuração Metabólica , Camundongos , Camundongos Knockout , Peptídeos/administração & dosagem , Peptídeos/farmacocinética , Receptores Fc/genética , Distribuição Tecidual , Bloqueadores do Canal de Sódio Disparado por Voltagem/administração & dosagemRESUMO
Protein engineering is at an all-time high in biopharmaceutics. As a result, absorption, distribution, metabolism and excretion (ADME) of proteins has become more important to understand in the context of engineering strategies to optimize therapeutic properties of potential lead constructs. Immunoaffinity capture coupled with a newly developed capillary electrophoresis - mass spectrometry (CE-MS) system was used to characterize intact protein mass analysis of a wild type Fc-FGF21 construct and a sequence re-engineered Fc-FGF21 construct from an in vivo study. A number of truncated forms were observed and the time courses of the various proteolytic products were identified and compared between the two constructs. The abundances of the intact and truncated forms were used to provide the basis to semi-quantify ADME properties of the two protein forms. The use of this immunoaffinity capture followed by CE-MS based intact mass analysis workflow provided a qualitative and quantitative analysis of the pharmacokinetic profiles of the two proteins. The platform presented here holds great potential in characterization of the ADME properties of proteins.
Assuntos
Eletroforese Capilar/métodos , Espectrometria de Massas , Proteínas Recombinantes de Fusão/química , Animais , Cromatografia de Afinidade , Fatores de Crescimento de Fibroblastos/genética , Fatores de Crescimento de Fibroblastos/metabolismo , Meia-Vida , Fragmentos Fc das Imunoglobulinas/genética , Fragmentos Fc das Imunoglobulinas/metabolismo , Camundongos , Estabilidade Proteica , Proteínas Recombinantes de Fusão/imunologia , Proteínas Recombinantes de Fusão/farmacocinéticaRESUMO
AMG 416 (etelcalcetide) is a novel synthetic peptide agonist of the calcium-sensing receptor composed of a linear chain of seven d-amino acids (referred to as the d-amino acid backbone) with a d-cysteine linked to an l-cysteine via a disulfide bond. AMG 416 contains four basic d-arginine residues and is a +4 charged peptide at physiologic pH with a mol. wt. of 1048.3 Da. The pharmacokinetics (PK), disposition, and potential of AMG 416 to cause drug-drug interaction were investigated in nonclinical studies with two single (14)C-labels placed either at a potentially metabolically labile acetyl position or on the d-alanine next to d-cysteine in the interior of the d-amino acid backbone. After i.v. dosing, the PK and disposition of AMG 416 were similar in male and female rats. Radioactivity rapidly distributed to most tissues in rats with intact kidneys, and renal elimination was the predominant clearance pathway. No strain-dependent differences were observed. In bilaterally nephrectomized rats, minimal radioactivity (1.2%) was excreted via nonrenal pathways. Biotransformation occurred primarily via disulfide exchange with endogenous thiol-containing molecules in whole blood rather than metabolism by enzymes, such as proteases or cytochrome P450s; the d-amino acid backbone remained unaltered. A substantial proportion of the plasma radioactivity was covalently conjugated to albumin. AMG 416 presents a low risk for P450 or transporter-mediated drug-drug interactions because it showed no interactions in vitro. These studies demonstrated a (14)C label on either the acetyl or the d-alanine in the d-amino acid backbone would be appropriate for clinical studies.
Assuntos
Calcimiméticos/farmacocinética , Peptídeos/farmacocinética , Receptores de Detecção de Cálcio/agonistas , Administração Intravenosa , Animais , Biotransformação , Calcimiméticos/administração & dosagem , Calcimiméticos/sangue , Calcimiméticos/toxicidade , Sistema Enzimático do Citocromo P-450/metabolismo , Cães , Interações Medicamentosas , Feminino , Células HEK293 , Humanos , Rim/metabolismo , Fígado/metabolismo , Masculino , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Estrutura Molecular , Peptídeos/administração & dosagem , Peptídeos/sangue , Peptídeos/toxicidade , Ligação Proteica , Ratos Endogâmicos BN , Receptores de Detecção de Cálcio/química , Receptores de Detecção de Cálcio/metabolismo , Eliminação Renal , Medição de Risco , Albumina Sérica/metabolismo , Relação Estrutura-Atividade , Distribuição Tecidual , TransfecçãoRESUMO
Past studies have demonstrated higher clearance for monoclonal antibodies possessing increased rates of non-specific endocytosis. However, this metric is oftentimes evaluated indirectly using biophysical techniques or cell surface binding studies that may not provide insight into the specific rates of cellular turnover. Furthermore, few examples evaluating non-specific endocytosis have been reported for a therapeutic antibody that reached clinical assessment. In the current report, we evaluated a therapeutic human immunoglobulin G2 monoclonal antibody targeted against the interleukin-4 receptor alpha chain (IL-4Rα) that exhibited elevated target independent clearance in previous Phase 1 and 2 studies. We confirmed high non-specific clearance of the anti-IL-4Rα antibody as compared to a reference antibody during pharmacokinetic assessments in wild type mice where target-mediated disposition was absent. We then developed a cell-based method capable of measuring cellular protein endocytosis and demonstrated the anti-IL-4Rα antibody exhibited marked non-specific uptake relative to the reference compound. Antibody homology modeling identified the anti-IL-4Rα antibody possessed positive charge patches whose removal via targeted mutations substantially reduced its non-specific endocytosis. We then expanded the scope of the study by evaluating panels of both preclinical and clinically relevant monoclonal antibodies and demonstrate those with the highest rates of non-specific uptake in vitro exhibited elevated target independent clearance, low subcutaneous bioavailability, or both. Our results support the observation that high non-specific endocytosis is a negative attribute in monoclonal antibody development and demonstrate the utility of a generic cell-based screen as a quantitative tool to measure non-specific endocytosis of protein therapeutics at the single-cell level.
RESUMO
In vivo clearance mechanisms of therapeutic monoclonal antibodies (mAbs) encompass both target-mediated and target-independent processes. Two distinct determinants of overall mAb clearance largely separate of target-mediated influences are non-specific cellular endocytosis and subsequent pH-dependent mAb recycling mediated by the neonatal Fc receptor (FcRn), where inter-mAb variability in the efficiency of both processes is observed. Here, we implemented a functional cell-based FcRn recycling assay via Madin-Darby canine kidney type II cells stably co-transfected with human FcRn and its light chain ß2-microglobulin. Next, a series of pH-dependent internalization studies using a model antibody demonstrated proper function of the human FcRn complex. We then applied our cellular assays to assess the contribution of both FcRn and non-specific interactions in the cellular turnover for a panel of 8 clinically relevant mAbs exhibiting variable human pharmacokinetic behavior. Our results demonstrate that the interplay of non-specific endocytosis rates, pH-dependent non-specific interactions, and engagement with FcRn all contribute to the overall recycling efficiency of therapeutic monoclonal antibodies. The predictive capacity of our assay approach was highlighted by successful identification of all mAbs within our panel possessing clearance in humans greater than 5 mL/day/kg. These results demonstrate that a combination of cell-based in vitro assays can properly resolve individual mechanisms underlying the overall in vivo recycling efficiency and non-target mediated clearance of therapeutic mAbs.
RESUMO
Targeting antigens with antibodies exhibiting pH/Ca2+-dependent binding against an antigen is an attractive strategy to mitigate target-mediated disposition and antigen buffering. Studies have reported improved serum exposure of antibodies exhibiting pH/Ca2+-binding against membrane-bound receptors. Asialoglycoprotein receptor 1 (ASGR1) is a membrane-bound receptor primarily localized in hepatocytes. With a high expression level of approximately one million receptors per cell, high turnover, and rapid recycling, targeting this receptor with a conventional antibody is a challenge. In this study, we identified an antibody exhibiting pH/Ca2+-dependent binding to ASGR1 and generated antibody variants with increased binding to neonatal crystallizable fragment receptor (FcRn). Serum exposures of the generated anti-ASGR1 antibodies were analyzed in transgenic mice expressing human FcRn. Contrary to published reports of increased serum exposure of pH/Ca2+-dependent antibodies, the pH/Ca2+-dependent anti-ASGR1 antibody had rapid serum clearance in comparison to a conventional anti-ASGR1 antibody. We conducted sub-cellular trafficking studies of the anti-ASGR1 antibodies along with receptor quantification analysis for mechanistic understanding of the rapid serum clearance of pH/Ca2+-dependent anti-ASGR1 antibody. The findings from our study provide valuable insights in identifying the antigens, especially membrane bound, that may benefit from targeting with pH/Ca2+-dependent antibodies to obtain increased serum exposure.
Assuntos
Receptor de Asialoglicoproteína , Antígenos de Histocompatibilidade Classe I , Camundongos Transgênicos , Receptores Fc , Animais , Humanos , Receptor de Asialoglicoproteína/imunologia , Receptor de Asialoglicoproteína/metabolismo , Camundongos , Receptores Fc/imunologia , Receptores Fc/genética , Receptores Fc/metabolismo , Antígenos de Histocompatibilidade Classe I/imunologia , Antígenos de Histocompatibilidade Classe I/genética , Concentração de Íons de Hidrogênio , Anticorpos Monoclonais/imunologia , Cálcio/metabolismoRESUMO
In this investigation, we tested the hypothesis that a physiologically based pharmacokinetic (PBPK) model incorporating measured in vitro metrics of off-target binding can largely explain the inter-antibody variability in monoclonal antibody (mAb) pharmacokinetics (PK). A diverse panel of 83 mAbs was evaluated for PK in wild-type mice and subjected to 10 in vitro assays to measure major physiochemical attributes. After excluding for target-mediated elimination and immunogenicity, 56 of the remaining mAbs with an eight-fold variability in the area under the curve (AUC0-672h: 1.74 × 106 -1.38 × 107 ngâh/mL) and 10-fold difference in clearance (2.55-26.4 mL/day/kg) formed the training set for this investigation. Using a PBPK framework, mAb-dependent coefficients F1 and F2 modulating pinocytosis rate and convective transport, respectively, were estimated for each mAb with mostly good precision (coefficient of variation (CV%) <30%). F1 was estimated to be the mean and standard deviation of 0.961 ± 0.593, and F2 was estimated to be 2.13 ± 2.62. Using principal component analysis to correlate the regressed values of F1/F2 versus the multidimensional dataset composed of our panel of in vitro assays, we found that heparin chromatography retention time emerged as the predictive covariate to the mAb-specific F1, whereas F2 variability cannot be well explained by these assays. A sigmoidal relationship between F1 and the identified covariate was incorporated within the PBPK framework. A sensitivity analysis suggested plasma concentrations to be most sensitive to F1 when F1 > 1. The predictive utility of the developed PBPK model was evaluated against a separate panel of 14 mAbs biased toward high clearance, among which area under the curve of PK data of 12 mAbs was predicted within 2.5-fold error, and the positive and negative predictive values for clearance prediction were 85% and 100%, respectively. MAb heparin chromatography assay output allowed a priori identification of mAb candidates with unfavorable PK.
Assuntos
Anticorpos Monoclonais , Modelos Biológicos , Camundongos , Animais , Pinocitose , Bioensaio , HeparinaRESUMO
Interleukin-2 (IL-2), along with T-cell receptor (TCR) signaling, are required to control regulatory T cell (Treg) homeostasis and function in vivo. Due to the heightened sensitivity to IL-2, Tregs retain the ability to respond to low-dose or attenuated forms of IL-2, as currently being developed for clinical use to treat inflammatory diseases. While attenuated IL-2 increases Treg selectivity, the question remains as to whether a weakened IL-2 signal sufficiently enhances Treg suppressive function(s) toward disease modification. To understand this question, we characterized the in vivo activity and transcriptomic profiles of two different attenuated IL-2 muteins in comparison with wildtype (WT) IL-2. Our study showed that, in addition to favoring Tregs, the attenuated muteins induced disproportionately robust effects on Treg activation and conversion to effector Treg (eTreg) phenotype. Our data furthermore suggested that Tregs activated by attenuated IL-2 muteins showed reduced dependence on TCR signal, at least in part due to the enhanced ability of IL-2 muteins to amplify the TCR signal in vivo. These results point to a new paradigm wherein IL-2 influences Tregs' sensitivity to antigenic signal, and that the combination effect may be leveraged for therapeutic use of attenuated IL-2 muteins.
Assuntos
Interleucina-2 , Receptores de Antígenos de Linfócitos T , Linfócitos T Reguladores , Homeostase , Interleucina-2/genética , Interleucina-2/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo , Transdução de Sinais , HumanosRESUMO
PURPOSE: To investigate the mechanisms of clearance of AMG 386, an investigational recombinant peptide-Fc fusion protein (peptibody) that blocks tumor angiogenesis by neutralizing the interaction between angiopoietin-1 and -2 and the Tie2 receptor. METHODS: The role of the neonatal Fc receptor (FcRn) in AMG 386 clearance was assessed in wild-type and FcRn-knockout mice; the roles of the spleen and kidneys were assessed in splenectomized and 5/6th nephrectomized rats, respectively, compared with sham-operated rats. Animals were administered AMG 386 as a single intravenous dose of 3 or 10 mg/kg. Blood samples for pharmacokinetic analysis were collected periodically throughout a 504-hour postdose period. RESULTS: Compared with wild-type mice, AMG 386 clearance in FcRn-knockout mice was 18-fold faster at the 3-mg/kg dose (FcRn knockout, 13.2 mL/h/kg; wild-type, 0.728 mL/h/kg) and 14-fold faster at the 10-mg/kg dose (FcRn knockout, 10.7 mL/h/kg; wild-type, 0.777 mL/h/kg). Clearance in nephrectomized rats was slower than in sham-operated rats at both the 3-mg/kg dose (nephrectomized, 1.23 mL/h/kg; sham-operated, 1.75 mL/h/kg) and the 10-mg/kg dose (nephrectomized, 1.14 mL/h/kg; sham-operated, 1.65 mL/h/kg). Splenectomy had no apparent effect on the pharmacokinetics of AMG 386. CONCLUSIONS: The FcRn is integral to maintaining circulating levels of AMG 386 in mice. Renal clearance contributed approximately 30% to total AMG 386 clearance in rats.
Assuntos
Angiopoietina-1/antagonistas & inibidores , Angiopoietina-2/antagonistas & inibidores , Proteínas Recombinantes de Fusão/farmacocinética , Angiopoietina-1/metabolismo , Angiopoietina-2/metabolismo , Animais , Anticorpos Monoclonais/farmacocinética , Anticorpos Monoclonais/farmacologia , Antígenos de Histocompatibilidade Classe I/genética , Antígenos de Histocompatibilidade Classe I/metabolismo , Humanos , Rim/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neovascularização Patológica/tratamento farmacológico , Neovascularização Patológica/genética , Neovascularização Patológica/metabolismo , Ratos , Ratos Sprague-Dawley , Receptor TIE-2/antagonistas & inibidores , Receptor TIE-2/metabolismo , Receptores Fc/genética , Receptores Fc/metabolismo , Proteínas Recombinantes de Fusão/farmacologia , Baço/metabolismoRESUMO
The aim of this research was to develop a reliable non-radiometric method to measure the residual blood in tissue without the need for perfusion or radiometric measurements in biodistribution studies. It was found that the perfusion method not only was ineffective in removing blood from tissue, but also introduced additional variability in the determination of tissue drug exposure and was not reproducible across studies. In addition, the use of hemoglobin as an endogenous protein and biomarker for tissue blood content was studied and it was found that hemoglobin measurement in tissue was not a reliable and effective approach for determination of residual blood level in tissue. To evaluate an alternative method for addressing the tissue blood level in biodistribution studies, animals were dosed with a Residual Blood Determinant Reagent (RBDR) 5 min prior to tissue harvesting. The level of RBDR, an exogenous protein, was measured in whole blood homogenate and in tissue lysate. Based on the level of the RBDR, the vascular blood volume (VBV) in tissue was calculated and then the tissue exposures were corrected based on the blood volumes. The tissue VBVs measured by the RBDR method were comparable with the literature values obtained by radiometric measurements.
Assuntos
Volume Sanguíneo , Hemoglobinas , Animais , Distribuição Tecidual , Hemoglobinas/metabolismoRESUMO
Aim: To develop a method for the quantitation of effector functionless mouse surrogate IgG1 drug molecules in mouse matrices. Materials & methods: A panel of antibodies that bound specifically to N297G mutation-containing mouse IgG molecules was generated in rats. The panel was screened to identify an antibody that could be used as both the capture and detection reagent in an electrochemiluminescent immunoassay. Results & conclusion: The quantitative assay developed with the N297G-specific antibody passed acceptance criteria across multiple IgG1 fragment crystallizable (Fc)-containing protein formats and provides accurate quantitation of the total levels of mouse surrogate protein Fc present in in vivo mouse serum samples. These results are useful in understanding drug integrity and the development of precise pharmacokinetic/pharmacodynamic relationships.
Assuntos
Anticorpos Monoclonais , Imunoglobulina G , Animais , Imunoensaio/métodos , Imunoglobulina G/sangue , Imunoglobulina G/metabolismo , Testes Imunológicos , Camundongos , Ratos , SoroRESUMO
The decision to pursue a monoclonal antibody (mAb) as a therapeutic for disease intervention requires the assessment of many factors, such as target-biology, including the total target burden and its accessibility at the intended site of action, as well as mAb-specific properties like binding affinity and the pharmacokinetics in serum and tissue. Interleukin-36 receptor (IL-36 R) is a member of the IL-1 family cytokine receptors and an attractive target to treat numerous epithelial-mediated inflammatory conditions, including psoriatic and rheumatoid arthritis, asthma, and chronic obstructive pulmonary disease. However, information concerning the expression profile of IL-36 R at the protein level is minimal, so the feasibility of developing a therapeutic mAb against this target is uncertain. Here, we present a characterization of the properties associated with absorption, distribution, metabolism, and excretion of a high-affinity IL-36 R-targeted surrogate rat (IgG2a) mAb antagonist in preclinical mouse models. The presence of IL-36 R in the periphery was confirmed unequivocally as the driver of non-linear pharmacokinetics in blood/serum, although a predominant site of tissue accumulation was not observed based upon the kinetics of radiotracer. Additionally, the contribution of IL-36 R-mediated catabolism of mAb in kidney was tested in a 5/6 nephrectomized mouse model where minimal effects on serum pharmacokinetics were observed, although analysis of functional mAb in urine suggests that target can influence the amount of mAb excreted. Our data highlight an interesting case of target-mediated drug disposition (TMDD) where low, yet broadly expressed levels of membrane-bound target result in a cumulative effect to drive TMDD behavior typical of a large, saturable target sink. The potential differences between our mouse model and IL-36 R target profile in humans are also presented.
Assuntos
Algoritmos , Anticorpos Monoclonais/farmacocinética , Modelos Biológicos , Receptores de Interleucina/antagonistas & inibidores , Animais , Anticorpos Monoclonais/sangue , Anticorpos Monoclonais/imunologia , Afinidade de Anticorpos/imunologia , Feminino , Humanos , Cinética , Masculino , Camundongos Endogâmicos C57BL , Receptores de Interleucina/imunologia , Distribuição TecidualRESUMO
In this study we compared the pharmacokinetic profile of four unrelated antibodies, which do not bind to mammalian antigens, in IgG1 and IgG2 frameworks in both rats and non-human primates (NHP). This allowed for extensive cross comparison of the impact of antibody isotype, complementarity determining regions (CDR) and model species on pharmacokinetics without the confounding influence of antigen binding in the hosts. While antibody isotype had no significant impact on the pharmacokinetics, the CDRs do alter the profile, and there is an inverse correlation between the neonatal Fc receptor (FcRn) affinity and pharmacokinetic performance. Faster clearance rates were also associated with higher isoelectric points; however, although this panel of antibodies all possess basic isoelectric points, ranging from 8.44 to 9.18, they also have exceptional in vivo half-lives, averaging 369 hours, and low clearance rates, averaging 0.18 ml/h/kg in NHPs. This pattern of pharmacokinetic characteristics was conserved between rats and NHPs.
Assuntos
Anticorpos/metabolismo , Imunoglobulina G/metabolismo , Animais , Anticorpos Monoclonais/imunologia , Células CHO , Regiões Determinantes de Complementaridade/imunologia , Cricetinae , Cricetulus , Antígenos de Histocompatibilidade Classe I/imunologia , Humanos , Masculino , Camundongos , Farmacocinética , Primatas/imunologia , Ligação Proteica , Ratos , Ratos Sprague-Dawley , Receptores Fc/imunologiaRESUMO
Drug discovery research on new pain targets with human genetic validation, including the voltage-gated sodium channel NaV1.7, is being pursued to address the unmet medical need with respect to chronic pain and the rising opioid epidemic. As part of early research efforts on this front, we have previously developed NaV1.7 inhibitory peptide-antibody conjugates with tarantula venom-derived GpTx-1 toxin peptides with an extended half-life (80 h) in rodents but only moderate in vitro activity (hNaV1.7 IC50 = 250 nM) and without in vivo activity. We identified the more potent peptide JzTx-V from our natural peptide collection and improved its selectivity against other sodium channel isoforms through positional analogueing. Here we report utilization of the JzTx-V scaffold in a peptide-antibody conjugate and architectural variations in the linker, peptide loading, and antibody attachment site. We found conjugates with 100-fold improved in vitro potency relative to those of complementary GpTx-1 analogues, but pharmacokinetic and bioimaging analyses of these JzTx-V conjugates revealed a shorter than expected plasma half-life in vivo with accumulation in the liver. In an attempt to increase circulatory serum levels, we sought the reduction of the net +6 charge of the JzTx-V scaffold while retaining a desirable NaV in vitro activity profile. The conjugate of a JzTx-V peptide analogue with a +2 formal charge maintained NaV1.7 potency with 18-fold improved plasma exposure in rodents. Balancing the loss of peptide and conjugate potency associated with the reduction of net charge necessary for improved target exposure resulted in a compound with moderate activity in a NaV1.7-dependent pharmacodynamic model but requires further optimization to identify a conjugate that can fully engage NaV1.7 in vivo.
Assuntos
Imunoconjugados , Canal de Sódio Disparado por Voltagem NAV1.7/metabolismo , Peptídeos/química , Venenos de Aranha/química , Bloqueadores do Canal de Sódio Disparado por Voltagem , Animais , Anticorpos/química , Descoberta de Drogas , Humanos , Imunoconjugados/química , Imunoconjugados/farmacocinética , Masculino , Camundongos , Terapia de Alvo Molecular , Canal de Sódio Disparado por Voltagem NAV1.7/imunologia , Peptídeos/farmacocinética , Venenos de Aranha/farmacocinética , Bloqueadores do Canal de Sódio Disparado por Voltagem/química , Bloqueadores do Canal de Sódio Disparado por Voltagem/farmacocinéticaRESUMO
The voltage-gated sodium channel NaV1.7 is a genetically validated pain target under investigation for the development of analgesics. A therapeutic with a less frequent dosing regimen would be of value for treating chronic pain; however functional NaV1.7 targeting antibodies are not known. In this report, we describe NaV1.7 inhibitory peptide-antibody conjugates as an alternate construct for potential prolonged channel blockade through chemical derivatization of engineered antibodies. We previously identified NaV1.7 inhibitory peptide GpTx-1 from tarantula venom and optimized its potency and selectivity. Tethering GpTx-1 peptides to antibodies bifunctionally couples FcRn-based antibody recycling attributes to the NaV1.7 targeting function of the peptide warhead. Herein, we conjugated a GpTx-1 peptide to specific engineered cysteines in a carrier anti-2,4-dinitrophenol monoclonal antibody using polyethylene glycol linkers. The reactivity of 13 potential cysteine conjugation sites in the antibody scaffold was tuned using a model alkylating agent. Subsequent reactions with the peptide identified cysteine locations with the highest conversion to desired conjugates, which blocked NaV1.7 currents in whole cell electrophysiology. Variations in attachment site, linker, and peptide loading established design parameters for potency optimization. Antibody conjugation led to in vivo half-life extension by 130-fold relative to a nonconjugated GpTx-1 peptide and differential biodistribution to nerve fibers in wild-type but not NaV1.7 knockout mice. This study describes the optimization and application of antibody derivatization technology to functionally inhibit NaV1.7 in engineered and neuronal cells.
Assuntos
Imunoconjugados/farmacologia , Canal de Sódio Disparado por Voltagem NAV1.7/metabolismo , Peptídeos/farmacologia , Bloqueadores do Canal de Sódio Disparado por Voltagem/farmacologia , Animais , Células HEK293 , Humanos , Imunoconjugados/química , Imunoconjugados/farmacocinética , Masculino , Camundongos , Modelos Moleculares , Peptídeos/química , Peptídeos/farmacocinética , Distribuição Tecidual , Bloqueadores do Canal de Sódio Disparado por Voltagem/química , Bloqueadores do Canal de Sódio Disparado por Voltagem/farmacocinéticaRESUMO
BACKGROUND: AMG 517 or 1-aminobenzotriazole were quantified by LC-MS/MS from low blood/plasma volumes for rat pharmacokinetic (PK) characterization in order to qualify manual/automated dried blood spot (DBS) sampling and plasma separation capillary sampling. In addition, mouse serial automated blood sampling was compared with standard composite sampling. MATERIALS & METHODS: AMG 517 or 1-aminobenzotriazole was administered to rats or mice and multiple microsampling techniques were used to obtain blood or plasma. RESULTS: PK parameters derived from DBS and whole blood-obtained drug concentrations were within 7% for manual DBS and 20% for automated DBS. Plasma PK parameters derived from capillary or standard plasma-obtained drug concentrations differed by 6%. Plasma PK parameters obtained from serial automated blood sampling or manual composite sampling were within 20%. CONCLUSION: Collectively, these results suggest that the microsampling applications that were investigated are attractive approaches for quantifying drug candidates in low matrix volumes that can be successfully employed within discovery-stage rodent PK studies.
Assuntos
Benzotiazóis/sangue , Cromatografia Líquida de Alta Pressão/métodos , Pirimidinas/sangue , Espectrometria de Massas em Tandem/métodos , Triazóis/sangue , Animais , Benzotiazóis/farmacocinética , Coleta de Amostras Sanguíneas/métodos , Descoberta de Drogas , Masculino , Camundongos , Farmacocinética , Pirimidinas/farmacocinética , Ratos , Ratos Sprague-Dawley , Triazóis/farmacocinéticaRESUMO
BACKGROUND: Development of an alternative sampling method that uses small amounts of whole blood, such as dried blood spots (DBS), would be an advance in the quantitative assay field. Previously, we assessed the ability to quantitate therapeutic monoclonal antibodies present in DBS compared with a typical serum sample-based method, and concluded that measurements in DBS were reproducible and yielded methods that met requirements for precision, accuracy and sensitivity. The goal herein was to assess the measurement of therapeutic antibodies in DBS compared with serum and plasma in vivo. RESULTS: Comparison of DBS versus serum in Sprague-Dawley rats and DBS versus plasma in cynomolgus monkeys for measurement of antibody concentrations revealed a two- to three-fold difference in exposure between the samples. CONCLUSION: Overall, there was good correlation between DBS versus serum and DBS versus plasma, but there was a discrepancy in DBS exposures, presumably attributable to hematocrit and recovery effects.
Assuntos
Anticorpos Monoclonais/sangue , Anticorpos Monoclonais/uso terapêutico , Coleta de Amostras Sanguíneas , Teste em Amostras de Sangue Seco , Animais , Ensaio de Imunoadsorção Enzimática , Haplorrinos , Hematócrito , Humanos , Espectrometria de Massas , Ratos , Ratos Sprague-DawleyRESUMO
BACKGROUND: Dried blood spot (DBS) sampling in combination with LC-MS/MS has been used increasingly in drug discovery for quantitative analysis to support pharmacokinetic (PK) studies. In this study, we assessed the effect of blood-to-plasma (B:P) partitioning on the bioanalytical performance and PK data acquired by DBS for a compound AMG-1 with species and concentration-dependent B:P ratio. RESULTS: B:P partitioning did not adversely affect bioanalytical performance of DBS for AMG-1. For rat, (B:P ratio of 0.63), PK profiles from DBS and plasma methods were comparable. For dog, concentration-dependence of B:P ratio was observed both in vivo and in vitro. Additional studies demonstrated concentration-dependence of the compound's unbound fraction in plasma, which may contribute to the concentration-dependence of the B:P ratio. CONCLUSION: DBS is a promising sampling technique for preclinical pharmacokinetic studies. For compounds with high B:P ratio, caution needs to be applied for data comparison and interpretation between matrices.