Translational two-pore PBPK model to characterize whole-body disposition of different-size endogenous and exogenous proteins.

Two-pore physiologically based pharmacokinetic (PBPK) modeling has demonstrated its potential in describing the pharmacokinetics (PK) of different-size proteins. However, all existing two-pore models lack either diverse proteins for validation or interspecies extrapolation. To fill the gap, here we have developed and optimized a translational two-pore PBPK model that can characterize plasma and tissue disposition of different-size proteins in mice, rats, monkeys, and humans. Datasets used for model development include more than 15 types of proteins: IgG (150 kDa), F(ab)2 (100 kDa), minibody (80 kDa), Fc-containing proteins (205, 200, 110, 105, 92, 84, 81, 65, or 60 kDa), albumin conjugate (85.7 kDa), albumin (67 kDa), Fab (50 kDa), diabody (50 kDa), scFv (27 kDa), dAb2 (23.5 kDa), proteins with an albumin-binding domain (26, 23.5, 22, 16, 14, or 13 kDa), nanobody (13 kDa), and other proteins (110, 65, or 60 kDa). The PBPK model incorporates: (i) molecular weight (MW)-dependent extravasation through large and small pores via diffusion and filtration, (ii) MW-dependent renal filtration, (iii) endosomal FcRn-mediated protection from catabolism for IgG and albumin-related modalities, and (iv) competition for FcRn binding from endogenous IgG and albumin. The finalized model can well characterize PK of most of these proteins, with area under the curve predicted within two-fold error. The model also provides insights into contribution of renal filtration and lysosomal degradation towards total elimination of proteins, and contribution of paracellular convection/diffusion and transcytosis towards extravasation. The PBPK model presented here represents a cross-modality, cross-species platform that can be used for development of novel biologics.

Investigation of Antibody Pharmacokinetics in the Brain Following Intra-CNS Administration and Development of PBPK Model to Characterize the Data.

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.

Pharmacokinetics of AAV9 Mediated Trastuzumab Expression in Rat Brain Following Systemic and Local Administration.

INTRODUCTION: Recombinant adeno-associated viruses(rAAVs) are an attractive tool to ensure long-term expression monoclonal antibody(mAb) in the central nervous system(CNS). It is still unclear whether systemic injection or local CNS administration of AAV9 is more beneficial for the exposure of the expressed mAb in the brain. Hence, we compared the biodistribution and transgene expression following AAV9-Trastuzumab administration through different routes. METHODS AND RESULT: In-house generated AAV9-Trastuzumab vectors were administered at 5E+11 Vgs/rat through intravenous(IV), intracerebroventricular(ICV), intra-cisterna magna(ICM) and intrastriatal(IST) routes. Vector and trastuzumab blood/plasma concentrations were assessed at different time points up to the terminal time point of 21 days. Different brain regions in addition to the spinal cord, cerebrospinal fluid(CSF) and interstitial fluid(ISF), were also analyzed at the terminal time point. Our results show that vector biodistribution and Trastuzumab expression in the brain could the ranked as follows: IST>ICM>ICV>IV. Rapid clearance of vector was observed after administration via the ICM and ICV routes. The ICV route produced similar expression levels across different brain regions, while the ICM route had better expression in the hindbrain and spinal cord region. The IST route had higher expression in the forebrain region compared to the hindbrain region. A sharp decline in trastuzumab plasma concentration was observed across all routes of administration due to anti-trastuzumab antibody response. CONCLUSION: In this study we have characterized vector biodistribution and transgene mAb expression after AAV9 vector administration through different routes in rats. IST and ICM represent the best administration routes to deliver antibody genes to the brain.

Construction of nursing-sensitive quality indicator system for cardiac rehabilitation of patients undergoing percutaneous coronary intervention based on structure-process-outcome model.

BACKGROUND: Coronary heart disease (CHD) is a cardiovascular disease with high mortality. At present, percutaneous coronary intervention (PCI) is considered as the main effective treatment for CHD due to less trauma, shorter course of treatment, and better curative effect. However, PCI alone is not a permanent cure, so cardiac rehabilitation (CR) is needed for a supplement. Nowadays, the evaluation of the nursing-sensitive quality of CR after PCI focuses on the outcomes of patients, lacks a complete evaluation indicator system, and is prone to problems such as nursing management imbalance. OBJECTIVE: A scientific, sensitive, comprehensive and practical nursing-sensitive quality indicator system based on the structure-process-outcome model was constructed to provide a reference for evaluating nursing-sensitive quality of CR after PCI. METHODS: Firstly, through literature analysis and semi-structured interview, the indicator system was collected, screened and determined. Then, the framework of the indicator system was established, and the draft of nursing-sensitive quality indicator system of CR after PCI was formed. Subsequently, the nursing-sensitive quality indicator system of CR after PCI was initially established using Delphi method. Finally, the specific weight was determined by analytic hierarchy process (AHP), and the nursing-sensitive quality indicator system of CR after PCI was established and perfected. RESULTS: Two rounds of expert consultations were separately given 15 questionnaires, and all these questionnaires were returned, with a questionnaire response rate of 100%. Such result indicated that experts were highly motivated. Besides, the authoritative coefficients for two rounds of expert consultations were 0.865 and 0.888, and the coordination coefficients were 0.491 and 0.522, respectively. Hence, the experts' authority and coordination were high and the results were reliable. After the second round of expert consultation, the nursing-sensitive quality indicator system of CR after PCI was established, eventually. This system consisted of 3 first-level indicators (structural indicator, process indicator and outcome indicator), 11 s-level indicators and 29 third-level indicators. CONCLUSION: A relatively complete and reliable nursing-sensitive quality indicator system of CR after PCI has been established in this study. Such system is scientific and reliable and can provide a reference for the evaluation of clinical teaching quality of CR after PCI.

Expansion of platform physiologically-based pharmacokinetic model for monoclonal antibodies towards different preclinical species: cats, sheep, and dogs.

Monoclonal antibodies (mAbs) are becoming an important therapeutic option in veterinary medicine, and understanding the pharmacokinetic (PK) of mAbs in higher-order animal species is also important for human drug development. To better understand the PK of mAbs in these animals, here we have expanded a platform physiological-based pharmacokinetic (PBPK) model to characterize the disposition of mAbs in three different preclinical species: cats, sheep, and dogs. We obtained PK data for mAbs and physiological parameters for the three different species from the literature. We were able to describe the PK of mAbs following intravenous (IV) or subcutaneous administration in cats, IV administration in sheep, and IV administration dogs reasonably well by fixing the physiological parameters and just estimating the parameters related to the binding of mAbs to the neonatal Fc receptor. The platform PBPK model presented here provides a quantitative tool to predict the plasma PK of mAbs in dogs, cats, and sheep. The model can also predict mAb PK in different tissues where the site of action might be located. As such, the mAb PBPK model presented here can facilitate the discovery, development, and preclinical-to-clinical translation of mAbs for veterinary and human medicine. The model can also be modified in the future to account for more detailed compartments for certain organs, different pathophysiology in the animals, and target-mediated drug disposition.

Assessment of AAV9 distribution and transduction in rats after administration through Intrastriatal, Intracisterna magna and Lumbar Intrathecal routes.

Challenges in obtaining efficient transduction of brain and spinal cord following systemic AAV delivery have led to alternative administration routes being used in clinical trials that directly infuse the virus into the CNS. However, data comparing different direct AAV injections into the brain remain limited making it difficult to choose optimal routes. Here we tested both AAV9-egfp and AAV9-fLuc delivery via intrastriatal (IST), intracisterna magna (ICM) and lumbar intrathecal (LIT) routes in adult rats and assessed vector distribution and transduction in brain, spinal cord and peripheral tissues. We find that IST infusion leads to robust transgene expression in the striatum, thalamus and cortex with lower peripheral tissue transduction and anti-AAV9 capsid titers compared to ICM or LIT. ICM delivery provided strong GFP and luciferase expression across more brain regions than the other routes and similar expression in the spinal cord to LIT injections, which itself largely failed to transduce the rat brain. Our data highlight the strengths and weaknesses of each direct CNS delivery route which will help with future clinical targeting.

PBPK model for antibody disposition in mouse brain: validation using large-pore microdialysis data.

The objective of this manuscript was to validate a physiologically-based pharmacokinetic (PBPK) model developed to characterize brain pharmacokinetics (PK) of monoclonal antibodies (mAbs) using novel large-pore microdialysis data generated in mice. To support this objective, brain, CSF, and ISF PK of a human anti-tetanus toxin (TeTx) antibody was measured in mice following intraperitoneal (IP) administration. This antibody has no binding in mice. In addition, our recently published mouse brain PK data generated following intravenous (IV) and IP administration of trastuzumab in mice, and other published PK data for brain disposition of antibody in mice, were used to evaluate the PBPK model. All the model parameters were obtained from literature or kept the same as in our previously published manuscript. The revised PBPK model was able to characterize the PK of antibodies in mice brain, CSF, and ISF reasonably well (i.e., within a three-fold error). However, a priori selected parameters led to underprediction of ISF PK during the initial phase of the profile. A local sensitivity analysis suggested that minor changes in several brain-related parameters can help overcome this discrepancy, where an increase in the convective flow of antibodies across BBB was found to be the most parsimonious way to capture all the PK profiles well. However, the presence of this pathway needs further validation. As such, here we have presented an improved PBPK model to characterize and predict the PK of mAbs in different regions of the mouse brain following systemic administration. This model can serve as a quantitative tool to facilitate the discovery, preclinical evaluation, and preclinical-to-clinical translation of novel antibodies targeted against CNS disorders.

Effect of the Size of Protein Therapeutics on Brain Pharmacokinetics Following Systematic Administration.

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.

Algorithmic multiscale analysis for the FcRn mediated regulation of antibody PK in human.

A demonstration is provided on how algorithmic asymptotic analysis of multi-scale pharmacokinetics (PK) systems can provide (1) system level understanding and (2) predictions on the response of the model when parameters vary. Being algorithmic, this type of analysis is not hindered by the size or complexity of the model and requires no input from the investigator. The algorithm identifies the constraints that are generated by the fast part of the model and the components of the slow part of the model that drive the system within these constraints. The demonstration is based on a typical monoclonal antibody PK model. It is shown that the findings produced by the traditional methodologies, which require significant input by the investigator, can be produced algorithmically and more accurately. Moreover, additional insights are provided by the algorithm, which cannot be obtained by the traditional methodologies; notably, the dual influence of certain reactions depending on whether their fast or slow component dominates. The analysis reveals that the importance of physiological processes in determining the systemic exposure of monoclonal antibodies (mAb) varies with time. The analysis also confirms that the rate of mAb uptake by the cells, the binding affinity of mAb to neonatal Fc receptor (FcRn), and the intracellular degradation rate of mAb are the most sensitive parameters in determining systemic exposure of mAbs. The algorithmic framework for analysis introduced and the resulting novel insights can be used to engineer antibodies with desired PK properties.

Towards a translational physiologically-based pharmacokinetic (PBPK) model for receptor-mediated transcytosis of anti-transferrin receptor monoclonal antibodies in the central nervous system.

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.

Current progress and limitations of AAV mediated delivery of protein therapeutic genes and the importance of developing quantitative pharmacokinetic/pharmacodynamic (PK/PD) models.

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.

Brain pharmacokinetics of anti-transferrin receptor antibody affinity variants in rats determined using microdialysis.

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.

Determination of ADC Cytotoxicity in Immortalized Human Cell Lines.

Cytotoxicity assays are a necessary first step to triage ADC molecules before moving them forward to relatively time-consuming and expensive in vivo studies. When cells are exposed to ADC molecules, antigen expressing cells can effectively take up those molecules and eventually die as a result of the released payload. This cytotoxic property of ADCs can be evaluated by measuring the percentage of living cells at the end of the incubation period. Tetrazolium colorimetric assay (MTT) is a widely used method that can be used to measure cell viability. Here we describe how to use an MTT assay to measure the cytotoxic effect of ADCs and calculate the corresponding IC50. Besides the cytotoxic behavior on antigen expressing cells, ADCs can also demonstrate bystander killing of antigen negative cells in the vicinity of antigen expressing cells. Here, we report how to use a co-culture experiment to evaluate the bystander effect of ADC with the help of fluorescent protein transfected antigen negative cells.

A translational platform PBPK model for antibody disposition in the brain.

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.