RESUMEN
Despite the promising potential of direct central nervous system (CNS) antibody administration to enhance brain exposure, there remains a significant gap in understanding the disposition of antibodies following different intra-CNS injection routes. To bridge this knowledge gap, this study quantitatively investigated the brain pharmacokinetics (PK) of antibodies following intra-CNS administration. The microdialysis samples from the striatum (ST), cerebrospinal fluid (CSF) samples through cisterna magna (CM) puncture, plasma, and brain homogenate samples were collected to characterize the pharmacokinetics (PK) profiles of a non-targeting antibody, trastuzumab, following intracerebroventricular (ICV), intracisternal (ICM), and intrastriatal (IST) administration. For a comprehensive analysis, these intra-CNS injection datasets were juxtaposed against our previously acquired intravenous (IV) injection data obtained under analogous experimental conditions. Our findings highlighted that direct CSF injections, either through ICV or ICM, resulted in ~ 5-6-fold higher interstitial fluid (ISF) drug exposure than IV administration. Additionally, the low bioavailability observed following IST administration indicates the existence of a local degradation process for antibody elimination in the brain ISF along with the ISF bulk flow. The study further refined a physiologically based pharmacokinetic (PBPK) model based on new observations by adding the perivascular compartments, oscillated CSF flow, and the nonspecific uptake and degradation of antibodies by brain parenchymal cells. The updated model can well characterize the antibody PK following systemic and intra-CNS administration. Thus, our research offers quantitative insight into antibody brain disposition pathways and paves the way for determining optimal dosing and administration strategies for antibodies targeting CNS disorders.
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Anticuerpos , Encéfalo , Sistema Nervioso Central , Disponibilidad Biológica , Administración IntravenosaRESUMEN
Antibody-based therapeutics have recently gained keen attention for the treatment of pulmonary indications. However, systemically administered antibody exposure in the lungs needs to be better understood and remains a topic of interest. In this study, we evaluated the exposure of two different uPAR (urokinase-type plasminogen activator receptor) targeting full-length monoclonal IgGs in plasma and lung epithelial lining fluid (ELF) of mice after IP and IV administration. Antibody AK17 exhibited linear pharmacokinetics (PK) in plasma and ELF at 3 and 30 mg/kg single IV dose. The average plasma and ELF half-lives for AK17 and AK21 ranged between ~321-411 h and ~230-345 h, respectively, indicating sustained systemic and lung exposure of antibodies. The average ELF to the plasma concentration ratio of antibodies was ~0.01 and ~0.03 with IP and IV dosing, respectively, over 2 weeks post single dose. We simultaneously characterized plasma and ELF PK of antibody in mice by developing a minimal lung PBPK model for antibody. This model reasonably captured the plasma and ELF PK data while estimating three parameters. The model accounts for the convective transport of antibody into the tissues via blood and lymph flow. FcRn-mediated transcytosis was incorporated into the model for antibody distribution across the lung epithelial barrier. This model serves as a platform to predict the pulmonary PK of systemically administered antibodies and to support optimal dose selection for desired exposure in the lungs as the site of action.
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Pulmón , Receptores del Activador de Plasminógeno Tipo Uroquinasa , Ratones , Animales , Anticuerpos Monoclonales , AntibacterianosRESUMEN
Here, we have investigated the effect of size of protein therapeutics on brain pharmacokinetics (PK) following systemic administration in rats. All tested proteins were derived from trastuzumab that do not bind to any targets in rats. PK data generated with F(ab)2 (100 kDa), Fab (50 kDa), and scFv (27 kDa) fragments of trastuzumab, along with published PK data for FcRn non-binding and wild-type trastuzumab (150 kDa), were used to establish a relationship between the protein size and brain exposure. A large-pore microdialysis system was used to measure the PK of proteins in the plasma, the interstitial fluid (ISF) at the striatum (ST), and the cerebrospinal fluid (CSF) at the lateral ventricle (LV) and cisterna magna (CM). Concentrations of all the proteins in plasma, brain homogenate, ISF, and CSF were measured using ELISA. When evaluating the effect of protein size in the absence of FcRn binding, we found a bell-shaped relationship between the size and ISF/plasma AUC ratio, where 100 kDa F(ab)2 demonstrated the highest exposure. A similar bell-shaped relationship was observed for the brain homogenate/plasma AUC ratio, with a peak at 50 kDa. The CSF/plasma AUC ratio at LV increased monotonously with a decrease in the size of proteins. We observed that the exposure of protein therapeutics in different regions of the brain could be significantly different and there could be optimal sizes of protein therapeutics to accomplish maximum/selective exposure in selected brain regions following systemic administration.
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Encéfalo , Líquido Extracelular , Animales , Área Bajo la Curva , Encéfalo/metabolismo , Preparaciones Farmacéuticas/metabolismo , Ratas , Trastuzumab/farmacocinéticaRESUMEN
In this manuscript, we present a translational physiologically-based pharmacokinetic (PBPK) model to characterize receptor-mediated transcytosis (RMT) of anti-transferrin receptor (TfR) monoclonal antibodies (mAbs) in the central nervous system (CNS). The model accounts for the state-of-the-art knowledge of the brain's anatomy and physiology, and physiological parameters were fixed according to different species. By estimating a few parameters associated with the TfR concentration, the TfR turnover, and the internalization rate, the model simultaneously characterizes plasma, whole brain, interstitial fluid (ISF), and cerebrospinal fluid (CSF) PK of unbound and bound anti-TfR mAbs with different binding affinities in mice, rats, and monkeys obtained from various literature sources within a threefold prediction error. The final PBPK model was validated using external anti-TfR mAb PK data in mice and monkeys with different affinities and doses. The simulation reasonably predicted plasma and brain PK of monovalent/bivalent anti-TfR mAbs within a threefold prediction error and characterized a bell-shaped relationship between the brain ISF/plasma AUC ratio and the KD value. Although further refinements of the PBPK model and clinical validation are required, this PBPK model may provide physiologically-based translation of CNS disposition of anti-TfR mAbs by accounting for the physiological difference of the endogenous RMT system among different species. The PBPK model may also guide selection of other endogenous receptors, lead optimization, and clinical development of novel CNS-targeted mAbs.
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Antineoplásicos Inmunológicos , Transcitosis , Animales , Anticuerpos Monoclonales/farmacocinética , Encéfalo/metabolismo , Haplorrinos/metabolismo , Ratones , Modelos Biológicos , RatasRESUMEN
The ocular pharmacokinetics (PK) of antibody-based therapies are infrequently studied in mice due to the technical difficulties in working with the small murine eye. This study is the first of its kind to quantitatively measure the PK of variously sized proteins in the plasma, cornea/ICB, vitreous humor, retina, and posterior cup (including choroid) of the mouse and to evaluate the relationship between molecular weight (MW) and antibody biodistribution coefficient (BC) to the eye. Proteins analyzed include trastuzumab (150 kDa), trastuzumab-vc-MMAE (T-vc-MMAE, 155 kDa), F(ab)2 (100 kDa), Fab (50 kDa), and scFv (27 kDa). As expected, ocular PK mirrored the systemic PK as plasma was the driving force for ocular exposure. For trastuzumab, T-vc-MMAE, F(ab)2, Fab, and scFv, respectively, the BCs in the cornea/ICB were 0.610%, 0.475%, 1.74%, 3.39%, and 13.7%; the BCs in the vitreous humor were 0.0198%, 0.0427%, 0.0934%, 0.234%, and 5.56%; the BCs for the retina were 0.539%, 0.230%, 0.704%, 2.44%, and 20.4%; the BCs for the posterior cup were 0.557%, 0.650%, 1.47%, 4.06%, and 13.9%. The relationship between BC and MW was best characterized by a log-log regression in which BC decreased as MW increased, with every doubling in MW leading to a decrease in BC by a factor of 3.44 × , 6.76 × , 4.74 × , and 3.43 × in cornea/ICB, vitreous humor, retina, and posterior cup, respectively. In analyzing the disposition of protein therapeutics to the eye, these findings enhance our understanding of the potential for ocular toxicity of systemically administered protein therapeutics and may aid in the discovery of systemically administered protein therapeutics for ocular disorders.
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Ojo/metabolismo , Inmunoconjugados/farmacocinética , Fragmentos Fab de Inmunoglobulinas/metabolismo , Oligopéptidos/farmacocinética , Trastuzumab/farmacocinética , Animales , Inmunoconjugados/administración & dosificación , Inmunoconjugados/química , Fragmentos Fab de Inmunoglobulinas/administración & dosificación , Fragmentos de Inmunoglobulinas/administración & dosificación , Fragmentos de Inmunoglobulinas/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Desnudos , Peso Molecular , Oligopéptidos/administración & dosificación , Oligopéptidos/química , Distribución Tisular , Trastuzumab/administración & dosificación , Trastuzumab/químicaRESUMEN
Receptor-mediated transcytosis (RMT) is used to enhance the delivery of monoclonal antibodies (mAb) into the central nervous system (CNS). While the binding to endogenous receptors on the brain capillary endothelial cells (BCECs) may facilitate the uptake of mAbs in the brain, a strong affinity for the receptor may hinder the efficiency of transcytosis. To quantitatively investigate the effect of binding affinity on the pharmacokinetics (PK) of anti-transferrin receptor (TfR) mAbs in different regions of the rat brain, we conducted a microdialysis study to directly measure the concentration of free mAbs at different sites of interest. Our results confirmed that bivalent anti-TfR mAb with an optimal dissociation constant (KD) value (76 nM) among four affinity variants can have up to 10-fold higher transcytosed free mAb exposure in the brain interstitial fluid (bISF) compared to lower and higher affinity mAbs (5 and 174 nM). This bell-shaped relationship between KD values and the increased brain exposure of mAbs was also visible when using whole-brain PK data. However, we found that mAb concentrations in postvascular brain supernatant (obtained after capillary depletion) were almost always higher than the concentrations measured in bISF using microdialysis. We also observed that the increase in mAb area under the concentration curve in CSF compartments was less significant, which highlights the challenge in using CSF measurement as a surrogate for estimating the efficiency of RMT delivery. Our results also suggest that the determination of mAb concentrations in the brain using microdialysis may be necessary to accurately measure the PK of CNS-targeted antibodies at the site-of-actions in the brain.
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Anticuerpos Monoclonales/farmacocinética , Afinidad de Anticuerpos/inmunología , Encéfalo/metabolismo , Microdiálisis/métodos , Receptores de Transferrina/inmunología , Animales , Anticuerpos Monoclonales/líquido cefalorraquídeo , Anticuerpos Monoclonales/inmunología , Antineoplásicos Inmunológicos/administración & dosificación , Antineoplásicos Inmunológicos/sangre , Área Bajo la Curva , Transporte Biológico , Barrera Hematoencefálica/metabolismo , Encéfalo/citología , Células CHO , Cricetinae , Cricetulus , Células Endoteliales/metabolismo , Humanos , Masculino , Ratas Sprague-Dawley , Transcitosis , Trastuzumab/administración & dosificación , Trastuzumab/sangreRESUMEN
While protein therapeutics are one of the most successful class of drug molecules, they are expensive and not suited for treating chronic disorders that require long-term dosing. Adeno-associated virus (AAV) mediated in vivo gene therapy represents a viable alternative, which can deliver the genes of protein therapeutics to produce long-term expression of proteins in target tissues. Ongoing clinical trials and recent regulatory approvals demonstrate great interest in these therapeutics, however, there is a lack of understanding regarding their cellular disposition, whole-body disposition, dose-exposure relationship, exposure-response relationship, and how product quality and immunogenicity affects these important properties. In addition, there is a lack of quantitative studies to support the development of pharmacokinetic-pharmacodynamic models, which can support the discovery, development, and clinical translation of this delivery system. In this review, we have provided a state-of-the-art overview of current progress and limitations related to AAV mediated delivery of protein therapeutic genes, along with our perspective on the steps that need to be taken to improve clinical translation of this therapeutic modality.
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Dependovirus/genética , Terapia Genética , Proteínas/genética , Humanos , Modelos Moleculares , Proteínas/química , Proteínas/farmacocinéticaRESUMEN
Crispness retention is a postharvest trait that fruit of the 'Honeycrisp' apple and some of its progeny possess. To investigate the molecular mechanisms of crispness retention, progeny individuals derived from a 'Honeycrisp' × MN1764 population with fruit that either retain crispness (named "Retain"), lose crispness (named "Lose"), or that are not crisp at harvest (named "Non-crisp") were selected for transcriptomic comparisons. Differentially expressed genes (DEGs) were identified using RNA-Seq, and the expression levels of the DEGs were validated using nCounter®. Functional annotation of the DEGs revealed distinct ripening behaviors between fruit of the "Retain" and "Non-crisp" individuals, characterized by opposing expression patterns of auxin- and ethylene-related genes. However, both types of genes were highly expressed in the fruit of "Lose" individuals and 'Honeycrisp', which led to the potential involvements of genes encoding auxin-conjugating enzyme (GH3), ubiquitin ligase (ETO), and jasmonate O-methyltransferase (JMT) in regulating fruit ripening. Cell wall-related genes also differentiated the phenotypic groups; greater numbers of cell wall synthesis genes were highly expressed in fruit of the "Retain" individuals and 'Honeycrisp' when compared with "Non-crisp" individuals and MN1764. On the other hand, the phenotypic differences between fruit of the "Retain" and "Lose" individuals could be attributed to the functioning of fewer cell wall-modifying genes. A cell wall-modifying gene, MdXTH, was consistently identified as differentially expressed in those fruit over two years in this study, so is a major candidate for crispness retention.
RESUMEN
In this study, we evaluated the effect of size on tumor disposition of protein therapeutics, including the plasma and tumor pharmacokinetics (PK) of trastuzumab (â¼150 kDa), FcRn-nonbinding trastuzumab (â¼150 kDa), F(ab)2 fragment of trastuzumab (â¼100 kDa), Fab fragment of trastuzumab (â¼50 kDa), and trastuzumab scFv (â¼27 kDa) in both antigen (i.e., HER2)-overexpressing (N87) and antigen-nonexpressing (MDA-MB-468) tumor-bearing mice. The observed data were used to develop the maximum tumor uptake versus molecular weight and tumor-to-plasma area under the curve (AUC) ratio versus molecular weight relationships. Comparison of the PK of different sizes of FcRn nonbinding molecules in target-expressing tumor showed that â¼100 kDa is an optimal size to achieve maximum tumor uptake and â¼50 kDa is an optimal size to achieve maximum tumor-to-plasma exposure ratio of protein therapeutics. The PK data were also used to validate a systems PK model for tumor disposition of different-sized protein therapeutics. The PK model was able to predict a priori the PK of all five molecules in both tumor types reasonably well (within 2- to 3-fold). In addition, the model captured the bell-shaped relationships observed between maximum tumor uptake and molecular weight and between tumor-to-plasma AUC ratio and molecular weight. Our results provide an unprecedented insight into the effect of size and target engagement on the tumor PK of protein therapeutics. Our results also provide further validation of the tumor disposition model, which can be used to support discovery, development, and preclinical-to-clinical translation of different sizes of protein therapeutics. SIGNIFICANCE STATEMENT: This article highlights the importance of molecular size and target engagement on the tumor disposition of protein therapeutics. Our results suggest that â¼100 kDa is an optimal size to achieve maximum tumor uptake and â¼50 kDa is an optimal size to achieve maximum tumor-to-plasma exposure ratio for non-FcRn-binding targeted protein therapeutics. We also demonstrate that a systems pharmacokinetics model developed to characterize tumor disposition of protein therapeutics can predict a priori the disposition of different-sized protein therapeutics in target-expressing and target-nonexpressing solid tumors.
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Antígenos de Histocompatibilidad Clase I/metabolismo , Neoplasias/tratamiento farmacológico , Receptor ErbB-2/antagonistas & inhibidores , Receptores Fc/metabolismo , Anticuerpos de Cadena Única/farmacología , Trastuzumab/farmacocinética , Animales , Área Bajo la Curva , Línea Celular Tumoral , Humanos , Masculino , Ratones , Modelos Biológicos , Peso Molecular , Neoplasias/sangre , Neoplasias/patología , Receptor ErbB-2/metabolismo , Anticuerpos de Cadena Única/administración & dosificación , Anticuerpos de Cadena Única/química , Distribución Tisular , Trastuzumab/administración & dosificación , Trastuzumab/química , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
In this manuscript, we have presented the development of a novel platform physiologically-based pharmacokinetic (PBPK) model to characterize brain disposition of mAbs in the mouse, rat, monkey and human. The model accounts for known anatomy and physiology of the brain, including the presence of distinct blood-brain barrier and blood-cerebrospinal fluid (CSF) barrier. CSF and interstitial fluid turnover, and FcRn mediated transport of mAbs are accounted for. The model was first used to characterize published and in-house pharmacokinetic (PK) data on the disposition of mAbs in rat brain, including the data on PK of mAb in different regions of brain determined using microdialysis. Majority of model parameters were fixed based on literature reported values, and only 3 parameters were estimated using rat data. The rat PBPK model was translated to mouse, monkey, and human, simply by changing the values of physiological parameters corresponding to each species. The translated PBPK models were validated by a priori predicting brain PK of mAbs in all three species, and comparing predicted exposures with observed data. The platform PBPK model was able to a priori predict all the validation PK profiles reasonably well (within threefold), without estimating any parameters. As such, the platform PBPK model presented here provides an unprecedented quantitative tool for prediction of mAb PK at the site-of-action in the brain, and preclinical-to-clinical translation of mAbs being developed against central nervous system (CNS) disorders. The proposed model can be further expanded to account for target engagement, disease pathophysiology, and novel mechanisms, to support discovery and development of novel CNS targeting mAbs.
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Anticuerpos Monoclonales/farmacocinética , Encéfalo/metabolismo , Modelos Biológicos , Investigación Biomédica Traslacional/métodos , Animales , Anticuerpos Monoclonales/administración & dosificación , Anticuerpos Monoclonales/sangre , Anticuerpos Monoclonales/líquido cefalorraquídeo , Haplorrinos , Humanos , Ratones , Especificidad de Órganos , Ratas , Especificidad de la Especie , Distribución TisularRESUMEN
Here, we present the first case-study where microdialysis is used to investigate the pharmacokinetics of antibody in different regions of rat brain. Endogenous IgG was used to understand antibody disposition at steady-state and exogenously administered trastuzumab was used to understand the disposition in a dynamic setting. Microdialysis samples from the striatum (ST), lateral ventricle (LV), and cisterna magna (CM) were collected, along with plasma and brain homogenate, to comprehensively understand brain pharmacokinetics of antibodies. Antibody concentrations in cerebrospinal fluid (CSF) were found to vary based on the site-of-collection, where CM concentrations were several-fold higher than LV. In addition, antibody concentrations in CSF (CM/LV) were found to not accurately represent the concentrations of antibody inside brain parenchyma (e.g., ST). Elimination of CSF from CM was found to be slower than LV, and the entry and exit of antibody from ST was also slower. Pharmacokinetics of exogenously administered antibody revealed that the entry of antibody into LV via the blood-CSF barrier may represent an early pathway for antibody entry into the brain. Plasma concentrations of antibody were 247-667, 104-184, 165-435, and 377-909 fold higher than the antibody concentrations in LV, CM, ST, and brain homogenate. It was found that the measurement of antibody pharmacokinetics in different regions of the brain using microdialysis provides an unprecedented insight into brain disposition of antibody. This insight can help in designing better molecules, dosing regimens, and route of administration, which can in turn improve the efficacy of antibodies for central nervous system disorders.
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Barrera Hematoencefálica/metabolismo , Encéfalo/metabolismo , Microdiálisis/métodos , Trastuzumab/farmacocinética , Animales , Antineoplásicos Inmunológicos/líquido cefalorraquídeo , Antineoplásicos Inmunológicos/farmacocinética , Cisterna Magna/metabolismo , Cuerpo Estriado/metabolismo , Inmunoglobulina G/metabolismo , Ventrículos Laterales/metabolismo , Masculino , Ratas Sprague-Dawley , Trastuzumab/líquido cefalorraquídeoRESUMEN
Loss of crispness in apple fruit during storage reduces the fruit's fresh sensation and consumer acceptance. Apple varieties that maintain crispness thus have higher potential for longer-term consumer appeal. To efficiently phenotype crispness, several instrumental methods have been tested, but variable results were obtained when different apple varieties were assayed. To extend these studies, we assessed the extent to which instrumental measurements correlate to and predict sensory crispness, with a focus on crispness maintenance. We used an apple breeding family derived from a cross between "Honeycrisp" and "MN1764," which segregates for crispness maintenance. Three types of instrumental measurements (puncture, snapping, and mechanical-acoustic tests) and sensory evaluation were performed on fruit at harvest and after 8 weeks of cold storage. Overall, 20 genotypes from the family and the 2 parents were characterized by 19 force and acoustic measures. In general, crispness was more related to force than to acoustic measures. Force linear distance and maximum force as measured by the mechanical-acoustic test were best correlated with sensory crispness and change in crispness, respectively. The correlations varied by apple genotype. The best multiple linear regression model to predict change in sensory crispness between harvest and storage of fruit of this breeding family incorporated both force and acoustic measures. PRACTICAL APPLICATIONS: This work compared the abilities of instrumental tests to predict sensory crispness maintenance of apple fruit. The use of an instrumental method that is highly correlated to sensory crispness evaluation can enhance the efficiency and reduce the cost of measuring crispness for breeding purposes. This study showed that sensory crispness and change in crispness after storage of an apple breeding family were reliably predicted with a combination of instrumental measurements and multiple variable analyses. The strategy potentially can be applied to other apple varieties for more accurate interpretation of crispness maintenance measured instrumentally.
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Almacenamiento de Alimentos , Tecnología de Alimentos , Malus/genética , Percepción del Gusto , Genoma de Planta , Humanos , FitomejoramientoRESUMEN
An ex vivo degenerative intervertebral disc (IVD) organ culture system was established for the screening of disc regeneration agents. Its application was demonstrated by a stem cell and growth factor-based therapeutic approach for the amelioration of IVD. An ex vivo culture system using chymopapain to partially digest nucleus proposus tissue was established to mimic human IVD degeneration. This system was then used for the evaluation of different therapeutic regimens including: mesenchymal stem cell derived from eGFP-transgenic porcine (MSC-GFP), platelet-rich plasma (PRP) and MSC-GFP/PRP combined treatment, and confirmed in in vivo animal model. Chondrogenic-specific gene products including Col II and aggrecan were found upregulated and chondrogenic matrix deposition increased, as evident by sustained fluorescent signals over 4 weeks, in the MSC-GFP implanted group. Previously, we demonstrated in vitro stage-specific chondrogenesis of MSC by chondrocytic commitment. These same molecules upregulated for chondrogenesis were also observed in MSC-GFP group. PRP that has been shown to promote nucleus pulposus (NP) regeneration also resulted in significant increased levels of mRNA involved in chondrogenesis and matrices accumulation. The ex vivo IVD regeneration results were repeated and supported by in vivo porcine degenerative system. Moreover, the disc height index (DHI) was significantly increased in both in vivo MSC-GFP and PRP regeneration groups. Unexpectedly, the MSC-GFP/PRP combined therapy demonstrated an inclination towards osteogenesis in ex vivo system. The ex vivo degenerative IVD culture system described in this study could serve as an alternative and more accessible model over large animal model. This system also provides a high-throughput platform for screening therapeutic agents for IVD regeneration.