RESUMEN
Triple-negative breast cancer (TNBC) is the deadliest form of breast cancer with limited treatment options. The persistence of highly tumorigenic CD44-expressing subpopulation referred to as cancer stem cells (CSCs), endowed with the self-renewal capacity, has been associated with therapeutic resistance, hence clinical relapses. To mitigate these undesired events, targeted immunotherapies using antibody-photoconjugate (APC) or antibody-drug conjugate (ADC), were developed to specifically release cytotoxic payloads within targeted cells overexpressing cognate antigen receptors. Therefore, an αCD44(scFv)-SNAP-tag antibody fusion protein was engineered through genetic fusion of a single-chain antibody fragment (scFv) to a SNAPf-tag fusion protein, capable of self-conjugating with benzylguanine-modified light-sensitive near-infrared (NIR) phthalocyanine dye IRDye700DX (BG-IR700) or the small molecule toxin auristatin-F (BG-AURIF). Binding of the αCD44(scFv)-SNAPf-IR700 photoimmunoconjugate to antigen-positive cells was demonstrated by confocal microscopy and flow cytometry. By switching to NIR irradiation, CD44-expressing TNBC was selectively killed through induced phototoxic activities. Likewise, the αCD44(scFv)-SNAPf-AURIF immunoconjugate was able to selectively accumulate within targeted cells and significantly reduced cell viability through antimitotic activities at nano- to micromolar drug concentrations. This study provides an in vitro proof-of-concept for a future strategy to selectively destroy light-accessible superficial CD44-expressing TNBC tumors and their metastatic lesions which are inaccessible to therapeutic light.
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Aminobenzoatos , Receptores de Hialuranos , Inmunoconjugados , Oligopéptidos , Neoplasias de la Mama Triple Negativas , Humanos , Neoplasias de la Mama Triple Negativas/tratamiento farmacológico , Neoplasias de la Mama Triple Negativas/terapia , Neoplasias de la Mama Triple Negativas/patología , Receptores de Hialuranos/metabolismo , Inmunoconjugados/farmacología , Línea Celular Tumoral , Aminobenzoatos/farmacología , Aminobenzoatos/química , Femenino , Oligopéptidos/farmacología , Oligopéptidos/química , Anticuerpos de Cadena Única/farmacología , Inmunoterapia/métodos , Células Madre Neoplásicas/efectos de los fármacos , Células Madre Neoplásicas/metabolismoRESUMEN
PURPOSE: Triple-negative breast cancer (TNBC) is phenotypic of breast tumors lacking expression of the estrogen receptor (ER), the progesterone receptor (PgR), and the human epidermal growth factor receptor 2 (HER2). The paucity of well-defined molecular targets in TNBC, coupled with the increasing burden of breast cancer-related mortality, emphasizes the need to develop targeted diagnostics and therapeutics. While antibody-drug conjugates (ADCs) have emerged as revolutionary tools in the selective delivery of drugs to malignant cells, their widespread clinical use has been hampered by traditional strategies which often give rise to heterogeneous mixtures of ADC products. METHODS: Utilizing SNAP-tag technology as a cutting-edge site-specific conjugation method, a chondroitin sulfate proteoglycan 4 (CSPG4)-targeting ADC was engineered, encompassing a single-chain antibody fragment (scFv) conjugated to auristatin F (AURIF) via a click chemistry strategy. RESULTS: After showcasing the self-labeling potential of the SNAP-tag component, surface binding and internalization of the fluorescently labeled product were demonstrated on CSPG4-positive TNBC cell lines through confocal microscopy and flow cytometry. The cell-killing ability of the novel AURIF-based recombinant ADC was illustrated by the induction of a 50% reduction in cell viability at nanomolar to micromolar concentrations on target cell lines. CONCLUSION: This research underscores the applicability of SNAP-tag in the unambiguous generation of homogeneous and pharmaceutically relevant immunoconjugates that could potentially be instrumental in the management of a daunting disease like TNBC.
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Inmunoconjugados , Anticuerpos de Cadena Única , Neoplasias de la Mama Triple Negativas , Humanos , Inmunoconjugados/farmacología , Inmunoconjugados/química , Neoplasias de la Mama Triple Negativas/patología , Anticuerpos de Cadena Única/farmacología , Línea Celular Tumoral , Proteínas de la Membrana , Proteoglicanos Tipo Condroitín SulfatoRESUMEN
Antibody-drug conjugates (ADCs) are bifunctional molecules combining the targeting potential of monoclonal antibodies with the cancer-killing ability of cytotoxic drugs. This simple yet intelligently designed system directly addresses the lack of specificity encountered with conventional anti-cancer treatment regimes. However, despite their initial success, the generation of clinically sustainable and effective ADCs has been plagued by poor tumor penetration, undefined chemical linkages, unpredictable pharmacokinetic profiles, and heterogeneous mixtures of products. To this end, we generated a SNAP-tag-based fusion protein targeting the epidermal growth factor receptor (EGFR)-a biomarker of aggressive and drug-resistant cancers. Here, we demonstrate the use of a novel click coupling strategy to engineer a benzylguanine (BG)-linker-auristatin F (AuriF) piece that can be covalently tethered to the EGFR-targeting SNAP-tag-based fusion protein in an irreversible 1:1 stoichiometric reaction to form a homogeneous product. Furthermore, using these recombinant ADCs to target EGFR-overexpressing tumor cells, we provide a proof-of-principle for generating biologically active antimitotic therapeutic proteins capable of inducing cell death in a dose-dependent manner, thus alleviating some of the challenges of early ADC development.
RESUMEN
Monoclonal antibodies (mAbs) deliver great benefits to patients with chronic and/or severe diseases thanks to their strong specificity to the therapeutic target. As a result of this specificity, non-human primates (NHP) are often the only preclinical species in which therapeutic antibodies cross-react with the target. Here, we highlight the value and limitations that NHP studies bring to the design of safe and efficient early clinical trials. Indeed, data generated in NHPs are integrated with in vitro information to predict the concentration/effect relationship in human, and therefore the doses to be tested in first-in-human trials. The similarities and differences in the systems defining the pharmacokinetics and pharmacodynamics (PKPD) of mAbs in NHP and human define the nature and the potential of the preclinical investigations performed in NHPs. Examples have been collated where the use of NHP was either pivotal to the design of the first-in-human trial or, inversely, led to the termination of a project prior to clinical development. The potential impact of immunogenicity on the results generated in NHPs is discussed. Strategies to optimize the use of NHPs for PKPD purposes include the addition of PD endpoints in safety assessment studies and the potential re-use of NHPs after non-terminal studies or cassette dosing several therapeutic agents of interest. Efforts are also made to reduce the use of NHPs in the industry through the use of in vitro systems, alternative in vivo models, and in silico approaches. In the case of prediction of ocular PK, the body of evidence gathered over the last two decades renders the use of NHPs obsolete. Expert perspectives, advantages, and pitfalls with these alternative approaches are shared in this review.
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Productos Biológicos , Animales , Humanos , Productos Biológicos/farmacología , Primates , Anticuerpos MonoclonalesRESUMEN
The prediction of monoclonal antibody (mAb) disposition within solid tumors for individual patients is difficult due to inter-patient variability in tumor physiology. Improved a priori prediction of mAb pharmacokinetics in tumors may facilitate the development of patient-specific dosing protocols and facilitate improved selection of patients for treatment with anti-cancer mAb. Here, we report the use of dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI), with tumor penetration of the contrast agent gadobutrol used as a surrogate, to improve physiologically based pharmacokinetic model (PBPK) predictions of cetuximab pharmacokinetics in epidermal growth factor receptor (EGFR) positive xenografts. In the initial investigations, mice bearing Panc-1, NCI-N87, and LS174T xenografts underwent DCE-MRI imaging with the contrast agent gadobutrol, followed by intravenous dosing of an 125Iodine-labeled, non-binding mAb (8C2). Tumor concentrations of 8C2 were determined following the euthanasia of mice (3 h-6 days after 8C2 dosing). Potential predictor relationships between DCE-MRI kinetic parameters and 8C2 PBPK parameters were evaluated through covariate modeling. The addition of the DCE-MRI parameter Ktrans alone or Ktrans in combination with the DCE-MRI parameter Vp on the PBPK parameters for tumor blood flow (QTU) and tumor vasculature permeability (σTUV) led to the most significant improvement in the characterization of 8C2 pharmacokinetics in individual tumors. To test the utility of the DCE-MRI covariates on a priori prediction of the disposition of mAb with high-affinity tumor binding, a second group of tumor-bearing mice underwent DCE-MRI imaging with gadobutrol, followed by the administration of 125Iodine-labeled cetuximab (a high-affinity anti-EGFR mAb). The MRI-PBPK covariate relationships, which were established with the untargeted antibody 8C2, were implemented into the PBPK model with considerations for EGFR expression and cetuximab-EGFR interaction to predict the disposition of cetuximab in individual tumors (a priori). The incorporation of the Ktrans MRI parameter as a covariate on the PBPK parameters QTU and σTUV decreased the PBPK model prediction error for cetuximab tumor pharmacokinetics from 223.71 to 65.02%. DCE-MRI may be a useful clinical tool in improving the prediction of antibody pharmacokinetics in solid tumors. Further studies are warranted to evaluate the utility of the DCE-MRI approach to additional mAbs and additional drug modalities.
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Anticuerpos Monoclonales/inmunología , Medios de Contraste/química , Imagen por Resonancia Magnética , Neoplasias/inmunología , Animales , Anticuerpos Monoclonales/farmacocinética , Anticuerpos Monoclonales/uso terapéutico , Línea Celular Tumoral , Cetuximab/inmunología , Cetuximab/farmacocinética , Humanos , Masculino , Ratones , Modelos Biológicos , Neoplasias/sangre , Neoplasias/diagnóstico por imagen , Neoplasias/tratamiento farmacológicoRESUMEN
Harnessing the immunomodulatory activity of cytokines is a focus of therapies targeting inflammatory disease. The interleukin (IL)-1 superfamily contains pro-inflammatory and anti-inflammatory members that help orchestrate the immune response in adaptive and innate immunity. Of these molecules, IL-37 has robust anti-inflammatory activity across a range of disease models through inhibition of pro-inflammatory signaling cascades downstream of tumor necrosis factor, IL-1, and toll-like receptor pathways. We find that IL-37 is unstable with a poor pharmacokinetic and manufacturing profile. Here, we present the engineering of IL-37 from an unstable cytokine into an anti-inflammatory molecule with an excellent therapeutic likeness. We overcame these shortcomings through site-directed mutagenesis, the addition of a non-native disulfide bond, and the engineering of IL-37 as an Fc-fusion protein. Our results provide a platform for preclinical testing of IL-37 Fc-fusion proteins. The engineering approaches undertaken herein will apply to the conversion of similar potent yet short-acting cytokines into therapeutics.
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Antiinflamatorios , Citocinas , Citocinas/metabolismo , Inmunidad Innata , Inmunomodulación , Ingeniería de ProteínasRESUMEN
We present a novel approach for first-in-human (FIH) dose selection of the CD20xCD3 bispecific antibody, glofitamab, based on pharmacokinetic/pharmacodynamic (PKPD) assessment in cynomolgus monkeys to select a high, safe starting dose, with cytokine release (CR) as the PD endpoint. Glofitamab pharmacokinetics were studied in mice and cynomolgus monkeys; PKPD of IL-6, TNF-α and interferon-γ release following glofitamab, with/without obinutuzumab pretreatment (Gpt) was studied in cynomolgus monkeys. Potency differences for CR between cynomolgus monkeys and humans were determined by glofitamab incubation in whole blood of both species. The PKPD model for CR was translated to humans to project a starting dose that did not induce CR exceeding a clinically-predefined threshold. In cynomolgus monkeys, glofitamab showed a species-specific atypical high clearance, with and without B-cell debulking by Gpt. CR was related to glofitamab serum levels and B-cell counts. B-cell reduction by Gpt led to a marked decrease in CR. FIH starting dose (5 µg) was selected based on IL-6 release considering the markedly higher glofitamab in vitro potency in human vs monkey blood. This is a novel PKPD-based approach for selection of FIH starting dose for a CD20xCD3 bispecific antibody in B-cell lymphoma, evidenced in the glofitamab study, NP30179 (NCT03075696).
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Anticuerpos Biespecíficos , Linfoma de Células B , Animales , Citocinas , Humanos , Interleucina-6 , Linfoma de Células B/tratamiento farmacológico , Linfoma de Células B/patología , Macaca fascicularis , RatonesRESUMEN
A growing body of evidence supports the important role of molecular charge on antibody pharmacokinetics (PK), yet a quantitative description of the effect of charge on systemic and tissue disposition of antibodies is still lacking. Consequently, we have systematically engineered complementarity-determining regions (CDRs) of trastuzumab to create a series of variants with an isoelectric point (pI) range of 6.3-8.9 and a variable region (Fv) charge range of -8.9 to +10.9 (at pH 5.5), and have investigated in vitro and in vivo disposition of these molecules. These monoclonal antibodies (mAbs) exhibited incrementally enhanced binding to cell surfaces and cellular uptake with increased positive charge in antigen-negative cells. After single intravenous dosing in mice, a bell-shaped relationship between systemic exposure and Fv charge was observed, with both extended negative and positive charge patches leading to more rapid nonspecific clearance. Whole-body PK experiments revealed that, although overall exposures of most variants in the tissues were very similar, positive charge of mAbs led to significantly enhanced tissue:plasma concentration ratios for most tissues. In well-perfused organs such as liver, spleen, and kidney, the positive charge variants show superior accumulation. In tissues with continuous capillaries such as fat, muscle, skin, and bone, plasma concentrations governed tissue exposures. The in vitro and in vivo disposition data presented here facilitate better understanding of the impact of charge modifications on antibody PK, and suggest that alteration in the charge may help to improve tissue:plasma concentration ratios for mAbs in certain tissues. The data presented here also paves the way for the development of physiologically based pharmacokinetic models of mAbs that incorporate charge variations.
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Anticuerpos Monoclonales , Antineoplásicos Inmunológicos , Animales , Antígenos , Regiones Determinantes de Complementariedad , Punto Isoeléctrico , RatonesRESUMEN
New challenges and other topics in non-clinical safety testing of biotherapeutics were presented and discussed at the nineth European BioSafe Annual General Membership meeting in November 2019. The session topics were selected by European BioSafe organization committee members based on recent company achievements, agency interactions and new data obtained in the non-clinical safety testing of biotherapeutics, for which data sharing would be of interest and considered as valuable information. The presented session topics ranged from strategies of in vitro testing, immunogenicity prediction, bioimaging, and developmental and reproductive toxicology (DART) assessments to first-in-human (FIH) dose prediction and bioanalytical challenges, reflecting the entire space of different areas of expertise and different molecular modalities. During the 9th meeting of the European BioSafe members, the following topics were presented and discussed in 6 main sessions (with 3 or 4 presentations per session) and in three small group breakout sessions: 1) DART assessment with biotherapeutics: what did we learn and where to go?; 2) Non-animal testing strategies; 3) Seeing is believing: new frontiers in imaging; 4) Predicting immunogenicity during early drug development: hope or despair?; 5) Challenges in FIH dose projections; and 6) Non-canonical biologics formats: challenges in bioanalytics, PKPD and biotransformation for complex biologics formats. Small group breakout sessions were organized for team discussion about 3 specific topics: 1) Testing of cellular immune function in vitro and in vivo; 2) MABEL approach (toxicology and pharmacokinetic perspective); and 3) mRNA treatments. This workshop report presents the sessions and discussions at the meeting.
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Evaluación Preclínica de Medicamentos/métodos , HumanosRESUMEN
Safety assessment of biological drugs has its challenges due to the multiple new different modalities, for example, antibody-drug conjugates, bispecifics, nanobodies, fusion proteins and advanced therapy medicinal products (ATMPs), their different pharmacokinetic and pharmacodynamic properties, and their ability to trigger immunogenicity and toxicity. In the public and in the pharmaceutical industry, there is a strong and general desire to reduce the number of animals used in research and development of drugs and in particular reducing the use of nonhuman primates. Important discussions and activities are ongoing investigating the smarter designs of early research and dose range finding studies, reuse of animals, and replacing animal experiments with in vitro studies. Other important challenges include absence of a relevant species and design of studies and developing genetically modified animals for special investigative toxicology studies. Then, the learnings and challenges from the development of the first ATMPs are available providing valuable insights in the development path for these new potentially transformative treatments. Finally, development of strategies for assessment of immunogenicity and prediction of translation of immunogenicity and associated findings to the clinic. On this, the eighth meeting for the European BioSafe members, these challenges served as the basis for the presentations and discussions during the meeting. This article serves as the workshop report reviewing the presentations and discussions at the meeting.
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Alternativas a las Pruebas en Animales/métodos , Anticuerpos Monoclonales/farmacocinética , Productos Biológicos/farmacocinética , Biomarcadores Farmacológicos , Congresos como Asunto , Evaluación Preclínica de Medicamentos/métodos , Animales , HumanosRESUMEN
T-cell bispecific antibodies (TCBs) crosslink tumor and T-cells to induce tumor cell killing. While TCBs are very potent, on-target off-tumor toxicity remains a challenge when selecting targets. Here, we describe a protease-activated anti-folate receptor 1 TCB (Prot-FOLR1-TCB) equipped with an anti-idiotypic anti-CD3 mask connected to the anti-CD3 Fab through a tumor protease-cleavable linker. The potency of this Prot- FOLR1-TCB is recovered following protease-cleavage of the linker releasing the anti-idiotypic anti-CD3 scFv. In vivo, the Prot-FOLR1-TCB mediates antitumor efficacy comparable to the parental FOLR1-TCB whereas a noncleavable control Prot-FOLR1-TCB is inactive. In contrast, killing of bronchial epithelial and renal cortical cells with low FOLR1 expression is prevented compared to the parental FOLR1-TCB. The findings are confirmed for mesothelin as alternative tumor antigen. Thus, masking the anti-CD3 Fab fragment with an anti-idiotypic mask and cleavage of the mask by tumor-specific proteases can be applied to enhance specificity and safety of TCBs.
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Anticuerpos Biespecíficos/inmunología , Anticuerpos Biespecíficos/metabolismo , Complejo CD3/inmunología , Receptor 1 de Folato/inmunología , Péptido Hidrolasas/metabolismo , Linfocitos T/inmunología , Animales , Anticuerpos Biespecíficos/química , Anticuerpos Biespecíficos/uso terapéutico , Línea Celular Tumoral , Proteínas Ligadas a GPI/inmunología , Humanos , Inmunoterapia , Activación de Linfocitos/efectos de los fármacos , Mesotelina , Ratones , Terapia Molecular Dirigida , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Minipigs have been proposed as animal model to study the subcutaneous (SC) absorption of monoclonal antibodies (mAb), because they are more translatable to humans than other species. However, the minipig SC tissue structure differs markedly depending on its location. This study explored different SC administration sites for mAb SC administration, to explore which site translates best to humans. The study assessed the SC absorption of tocilizumab (Actemra®) following administration at several injection sites: Inguinal area, flank, caudal to the ear, and interscapular area, in comparison with an IV group. After SC administration, tocilizumab absorption was most rapid from the inguinal administration site, and slowest after administration behind the ear, with absorption from the other sites in between. Tocilizumab bioavailability was 98.6, 88.3, 74.1, and 86.3% after administration in inguinal area, flank, behind the ear, and interscapular area, as determined by non-compartmental analysis. Fitting of a single first-order absorption rate constant by compartmental analysis was dissatisfactory. A combined fitting of all data was done assuming two different kinds of SC depots, one undergoing fast absorption, the other undergoing a slower absorption. The split between these absorption depots differed across administration sites, with absorption from "fast depot" in inguinal area > flank > interscapular area > behind the ear. Comparisons with clinical data show that tocilizumab PK after SC administration behind the ear translates best to humans, considering both bioavailability and rate of absorption. Whether this translation from minipigs to humans is prototypic for other mAb remains to be confirmed.
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Anticuerpos Monoclonales Humanizados/administración & dosificación , Modelos Animales , Absorción Cutánea , Porcinos Enanos , Animales , Anticuerpos Monoclonales Humanizados/farmacocinética , Humanos , Inyecciones Subcutáneas , Masculino , Modelos Biológicos , PorcinosRESUMEN
PURPOSE: The tumor microenvironment plays a key role in cancer development and progression and is involved in resistance to chemo- and immunotherapy. Cancer-associated fibroblast expressing fibroblast-activating protein α (FAPα) is one of the predominant stroma cell types and is involved in resistance to immunotherapy. EXPERIMENTAL DESIGN: We generated OMTX705, a novel antibody-drug conjugate from a humanized anti-FAP antibody linked to a new cytolysin. Here, we studied its antineoplastic activity in vitro and in preclinical mouse models alone and in combination with chemotherapy as well as immunotherapy in PD-1-resistant tumors. RESULTS: In Avatar models, OMTX705 showed a 100% tumor growth inhibition and prolonged tumor regressions as single agent and in combination with chemotherapy. Treatment rechallenge following treatment discontinuation induced additional tumor regression, suggesting lack of treatment resistance. In a mouse model with a humanized immune system resistant to PD-1 inhibition, OMTX705 increased tumor infiltration by CD8+ T cells, induced complete regressions, and delayed tumor recurrence. CONCLUSIONS: These data suggest that FAP targeting with OMTX705 represents a novel and potent strategy for cancer treatment, including tumors resistant to immunotherapy, and support its clinical development.
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Antineoplásicos/farmacología , Resistencia a Antineoplásicos/efectos de los fármacos , Inmunoconjugados/farmacología , Proteínas de la Membrana/antagonistas & inhibidores , Animales , Anticuerpos Monoclonales Humanizados/farmacología , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Endopeptidasas , Humanos , Inmunomodulación/efectos de los fármacos , Ratones , Microambiente Tumoral/efectos de los fármacos , Microambiente Tumoral/inmunología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The pharmacokinetic (PK) properties of therapeutic antibodies directly affect efficacy, dose and dose intervals, application route and tissue penetration. In indications where health-care providers and patients can choose between several efficacious and safe therapeutic options, convenience (determined by dosing interval or route of application), which is mainly driven by PK properties, can affect drug selection. Therapeutic antibodies can have greatly different PK even if they have identical Fc domains and show no target-mediated drug disposition. Biophysical properties like surface charge or hydrophobicity, and binding to surrogates for high abundant off-targets (e.g., baculovirus particles, Chinese hamster ovary cell membrane proteins) were proposed to be responsible for these differences. Here, we used heparin chromatography to separate a polyclonal mix of endogenous human IgGs (IVIG) into fractions that differ in their PK properties. Heparin was chosen as a surrogate for highly negatively charged glycocalyx components on endothelial cells, which are among the main contributors to nonspecific clearance. By directly correlating heparin retention time with clearance, we identified heparin chromatography as a tool to assess differences in unspecific cell-surface interaction and the likelihood for increased pinocytotic uptake and degradation. Building on these results, we combined predictors for FcRn-mediated recycling and cell-surface interaction. The combination of heparin and FcRn chromatography allow identification of antibodies with abnormal PK by mimicking the major root causes for fast, non-target-mediated, clearance of therapeutic, Fc-containing proteins.
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Cromatografía/métodos , Células Endoteliales/metabolismo , Inmunoglobulinas Intravenosas/química , Animales , Células CHO , Cricetulus , Heparina/química , Antígenos de Histocompatibilidad Clase I/metabolismo , Humanos , Inmunoglobulinas Intravenosas/metabolismo , Tasa de Depuración Metabólica , Pinocitosis , Unión Proteica , Proteolisis , Receptores Fc/metabolismoRESUMEN
For therapeutic proteins, the currently established standard development path generally does not foresee biotransformation studies by default because it is well known that the clearance of therapeutic proteins proceeds via degradation to small peptides and individual amino acids. In contrast to small molecules, there is no general need to identify enzymes involved in biotransformation because this information is not relevant for drug-drug interaction assessment and for understanding the clearance of a therapeutic protein. Nevertheless, there are good reasons to embark on biotransformation studies, especially for complex therapeutic proteins. Typical triggers are unexpected rapid clearance, species differences in clearance not following the typical allometric relationship, a mismatch in the pharmacokinetics/pharmacodynamics (PK/PD) relationship, and the need to understand observed differences between the results of multiple bioanalytical methods (e.g., total vs. target-binding competent antibody concentrations). Early on during compound optimization, knowledge on protein biotransformation may help to design more stable drug candidates with favorable in vivo PK properties. Understanding the biotransformation of a therapeutic protein may also support designing and understanding the bioanalytical assay and ultimately the PK/PD assessment. Especially in cases where biotransformation products are pharmacologically active, quantification and assessment of their contribution to the overall pharmacological effect can be important for establishing a PK/PD relationship and extrapolation to humans. With the increasing number of complex therapeutic protein formats, the need for understanding the biotransformation of therapeutic proteins becomes more urgent. This article provides an overview on biotransformation processes, proteases involved, strategic considerations, regulatory guidelines, literature examples for in vitro and in vivo biotransformation, and technical approaches to study protein biotransformation. SIGNIFICANCE STATEMENT: Understanding the biotransformation of complex therapeutic proteins can be crucial for establishing a pharmacokinetic/pharmacodynamic relationship. This article will highlight scientific, strategic, regulatory, and technological features of protein biotransformation.
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Preparaciones Farmacéuticas/metabolismo , Proteínas/farmacocinética , Bibliotecas de Moléculas Pequeñas/farmacocinética , Animales , Biotransformación , Interacciones Farmacológicas , Humanos , Preparaciones Farmacéuticas/administración & dosificación , Proteínas/administración & dosificación , Proteínas/farmacología , Bibliotecas de Moléculas Pequeñas/administración & dosificación , Bibliotecas de Moléculas Pequeñas/farmacologíaRESUMEN
The pharmacokinetics (PK) of the anti-CD20 monoclonal antibody obinutuzumab was assessed after single intravenous dosing to cynomolgus monkeys. In addition, the pharmacokinetic-pharmacodynamic (PKPD) relationship for B-cell depletion was characterized. The PKPD model was used to estimate the B-cell repopulation during the recovery phase of chronic toxicology studies, thereby supporting the study design, in particular planning the recovery phase duration. Marked immunogenicity against obinutuzumab was observed approximately 10 days after single dose, leading to an up to â¼30-fold increase in obinutuzumab clearance in the affected monkeys. Despite this accelerated clearance, the PK could be characterized, either by disregarding the clearance in noncompartmental PK analysis or by capturing it explicitly as an additional time-dependent clearance process in compartmental modeling. This latter step was crucial to model the PKPD of B-cells as an indirect response to obinutuzumab exposure, showing that-without immune response-the limiting factor is obinutuzumab elimination with concentrations below 0.02 µg/mL required for initiation of B-cell recovery. Overall, the results demonstrate that despite a marked anti-drug antibody response in the nonclinical animal species, the PK and PKPD of obinutuzumab could be characterized successfully by appropriately addressing the immune-modulated clearance pathway in data analysis and modeling.
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Anticuerpos Monoclonales Humanizados/farmacocinética , Anticuerpos Monoclonales/farmacocinética , Antígenos CD20/metabolismo , Animales , Linfocitos B/efectos de los fármacos , Linfocitos B/metabolismo , Macaca fascicularisRESUMEN
A selective, sensitive and rapid mice dried blood spot (DBS) method has been developed and validated for the simultaneous quantification of bendamustine (BM) and γ-hydroxy-bendamustine (HBM) as per regulatory guidelines using an LC-MS/MS. Quality control, calibration curve and study sample DBS cards were sonicated with 5% formic acid in water before extraction with ethyl acetate enriched with internal standard (I.S.). The organic layer was evaporated and residue was reconstituted in 0.1% formic acid in acetonitrile for LC-MS/MS analysis. Chromatographic resolution of both analytes (BM and HBM) and the I.S. (loperamide) was achieved on an Atlantis dC18 column using 0.2% formic acid:acetonitrile (25:75, v/v) as an eluant delivered at a constant flow-rate of 0.5â¯mL/min. The total chromatographic run time was 3.2â¯min. The MS/MS ion transitions monitored were m/z 358.0 â 228.0, 374.0 â 338.0 and 477.0 â 210.0 for BM, HBM and the I.S, respectively. The assay was linear in the range of 5.65-2544â¯ng/mL for both BM and HBM. The within-run and between-run accuracy and within-run and between-run precision were in the range of 0.96-1.00 and 1.36-9.94%, respectively for BM; 0.88-1.03 and 4.57-11.7%, respectively for HBM on mice DBS cards. Stability studies showed that both analytes were stable at room temperature for 7 days and at -80⯰C for 55 days on DBS cards. The validated DBS method has been applied to a pharmacokinetic study in mice.
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Clorhidrato de Bendamustina/análogos & derivados , Clorhidrato de Bendamustina/sangre , Clorhidrato de Bendamustina/farmacocinética , Pruebas con Sangre Seca/métodos , Animales , Calibración , Cromatografía Líquida de Alta Presión , Estabilidad de Medicamentos , Hematócrito , Humanos , Límite de Detección , Modelos Lineales , Masculino , Ratones , Ratones Endogámicos BALB C , Reproducibilidad de los Resultados , Espectrometría de Masas en Tándem , TemperaturaRESUMEN
Fomepizole is used as an antidote to treat methanol poisoning due to its selectivity towards alcohol dehydrogenase. In the present study, the goal is to develop a method to predict the fomepizole human plasma concentration versus time profile based on the preclinical pharmacokinetics using the assumption of superimposability on simulated time course profiles of animals and humans. Standard allometric equations with/without correction factors were also assimilated in the prediction. The volume of distribution at steady state (Vss) predicted by simple allometry (57.55 L) was very close to the reported value (42.17 L). However, clearance (CL) prediction by simple allometry was at least 3-fold higher to the reported value (33.86 mL/min); hence, multiple correction factors were used to predict the clearance. Both brain weight and maximum life span potential could predict the CL with 1.22- and 1.01-fold difference. Specifically, the predicted Vss and CL values via interspecies scaling were used in the prediction of series of human intravenous pharmacokinetic parameters, while the simulation of human oral profile was done by the use of absorption rate constant (Ka) from dog following the applicability of human bioavailability value scaled from dog data. In summary, the findings indicate that the utility of diverse allometry approaches to derive the human pharmacokinetics of fomepizole after intravenous/oral dosing.
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Antídotos/farmacocinética , Fomepizol/farmacocinética , Administración Intravenosa , Animales , Antídotos/administración & dosificación , Disponibilidad Biológica , Fomepizol/administración & dosificación , Fomepizol/sangre , Humanos , Masculino , Ratones , Modelos Biológicos , Conejos , RatasRESUMEN
BACKGROUND: Manual skull drilling is an old but in modern neurosurgery still established procedure which can be applied quickly and universally in emergency situations. Electrical drilling requires more complex equipment and is usually reserved to the Operating Room (OR). It also seems desirable to apply an electrical drill for bedside usage but a suitable product does not exist so far. METHOD: Our experimental study using a manually and an electrically driven skull drill included a total of 40 holes drilled into synthetic biomechanical sheets. Half of the holes were produced with a prototype electrical drilling machine of the company Kaiser Technology and half of them with a traditional manual drill. Different drilling parameters such as the geometry of the borehole, the drilling forces and the drilling vibrations were captured during all experiments. RESULTS: The electrical drilling needed higher vertical force by the operators and a longer time to penetrate the sheet. A reason was the relatively lower rotational speed provided by this particular drill. When drilling electrically the vibrations were substantially less which in turn led to a more precise shape of the holes (revealed by observation via a microscope). CONCLUSIONS: The electrification of bedside drilling can in principle enable emergency craniostomies to be performed with greater ease and accuracy. The power of the electric drive, however, must be at least equivalent to the power of the traditional manual drill. Otherwise, the vertical forces exerted on the scull by the operator become inhibitive. The challenge is to combine cost-efficiency and re-sterilizability of an electrically driven drilling machine which at the same time is small and simple enough to qualify for emergency applications.
Asunto(s)
Electricidad , Diseño de Equipo , Procedimientos Neuroquirúrgicos/instrumentación , Instrumentos Quirúrgicos , HumanosRESUMEN
Sensitive and high-throughput measurement of biotherapeutics and biomarkers in plasma and tissues is critical for protein-drug development. Enrichment of target signature peptide (SP) after sample digestion permits sensitive LC-MS-based protein quantification and carries several prominent advantages over protein-level enrichment; however, developing high-quality antipeptide antibodies is challenging. Here we describe a novel, antibody-free, peptide-level-enrichment technique enabling high-throughput, sensitive, and robust quantification of proteins in biomatrices, by highly selective removal of matrix peptides and components via cation-exchange (CX) reversed-phase (RP) SPE with strategically regulated pH and ionic and organic strengths. Multiple-mechanism washing and elution achieved highly selective separation despite the low plate number of the SPE cartridge. We first investigated the adsorption-desorption behaviors of peptides on CX-RP sorbent and the coexisting, perplexing effects of pH, and ionic and organic strengths on the selectivity for SP enrichment, which has not been previously characterized. We demonstrated that the selectivity for separating target SPs from matrix peptides was closely associated with buffer pH relative to the pI of the SP, and pH values of pI - 2, pI, and pI + 2 respectively provided exceptional specificity for the ionic wash, the hydrophobic wash, and selective elution. Furthermore, desorption of peptides from the mixed-mode sorbent showed exponential and linear dependence, respectively, on organic-solvent percentage and salt percentage. On the basis of these findings, we established a streamlined procedure for rapid and robust method development. Quantification of biotherapeutics, targets, and biomarkers in plasma and tissues was used as the model system. Selective enrichment of target SPs was achieved along with elimination of 87-95% of matrix peptides, which improved the LOQ by 20-fold (e.g., 2 ng per gram of tissue). Application was demonstrated by sensitive quantification of time courses of mAb (T84.66) and target (CEA) in plasma and tumor tissues from a low-dose mouse PK study. For the first time, down-regulation of membrane-associated antigen following mAb treatment was observed. The CX-RP enrichment is robust, high-throughput, and universally applicable and thus is highly valuable for ultrasensitive, large-scale measurement of target protein in plasma and tissues.