Quantitative Pharmacology Research Articles

A Critical Neurodevelopmental Role for L-Type Voltage-Gated Calcium Channels in Neurite Extension and Radial Migration.

Despite many association studies linking gene polymorphisms and mutations of L-type voltage-gated Ca2+ channels (VGCCs) in neurodevelopmental disorders such as autism and schizophrenia, the roles of specific L-type VGCC during brain development remain unclear. Calcium signaling has been shown to be essential for neurodevelopmental processes such as sculpting of neurites, functional wiring, and fine tuning of growing networks. To investigate this relationship, we performed submembraneous calcium imaging using a membrane-tethered genetically encoded calcium indicator (GECI) Lck-G-CaMP7. We successfully recorded spontaneous regenerative calcium transients (SRCaTs) in developing mouse excitatory cortical neurons prepared from both sexes before synapse formation. SRCaTs originated locally in immature neurites independently of somatic calcium rises and were significantly more elevated in the axons than in dendrites. SRCaTs were not blocked by tetrodoxin, a Na+ channel blocker, but were strongly inhibited by hyperpolarization, suggesting a voltage-dependent source. Pharmacological and genetic manipulations revealed the critical importance of the Cav1.2 (CACNA1C) pore-forming subunit of L-type VGCCs, which were indeed expressed in immature mouse brains. Consistently, knocking out Cav1.2 resulted in significant alterations of neurite outgrowth. Furthermore, expression of a gain-of-function Cav1.2 mutant found in Timothy syndrome, an autosomal dominant multisystem disorder exhibiting syndromic autism, resulted in impaired radial migration of layer 2/3 excitatory neurons, whereas postnatal abrogation of Cav1.2 enhancement could rescue cortical malformation. Together, these lines of evidence suggest a critical role for spontaneous opening of L-type VGCCs in neural development and corticogenesis and indicate that L-type VGCCs might constitute a perinatal therapeutic target for neuropsychiatric calciochannelopathies.SIGNIFICANCE STATEMENT Despite many association studies linking gene polymorphisms and mutations of L-type voltage-gated Ca2+ channels (VGCCs) in neurodevelopmental disorders such as autism and schizophrenia, the roles of specific L-type VGCCs during brain development remain unclear. We here combined the latest Ca2+ indicator technology, quantitative pharmacology, and in utero electroporation and found a hitherto unsuspected role for L-type VGCCs in determining the Ca2+ signaling landscape of mouse immature neurons. We found that malfunctional L-type VGCCs in immature neurons before birth might cause errors in neuritic growth and cortical migration. Interestingly, the retarded corticogenesis phenotype was rescued by postnatal correction of L-type VGCC signal aberration. These findings suggest that L-type VGCCs might constitute a perinatal therapeutic target for neurodevelopment-associated psychiatric disorders.

Emerging roles for clinical pharmacometrics in cancer precision medicine.

Although significant progress has been made in cancer research, there exist unmet needs in patient care as reflected by the 'Cancer Moonshot' goals. This review appreciates the potential utility of quantitative pharmacology in cancer precision medicine. Precision oncology has received federal funding largely due to 'The Precision Medicine Initiative'. Precision medicine takes into account the inter-individual variability, and allows for tailoring the right medication or the right dose of drug to the best subpopulation of patients who will likely respond to the intervention, thus enhancing therapeutic success and reducing "financial toxicity" to patients, families and caregivers. The National Cancer Institute (NCI) committed US$ 70 million from its fiscal year 2016 budget to advance precision oncology research. Through the 'Critical Path Initiative', pharmacometrics has gained an important role in drug development; however, it is yet to find widespread clinical applicability. Stakeholders including clinicians and pharmacometricians need to work in concert to ensure that benefits of model-based approaches are harnessed to personalize cancer care to the individual needs of the patient via better dosing strategies, companion diagnostics, and predictive biomarkers. In medical oncology, where immediate patient care is the clinician's primary concern, pharmacometric approaches can be tailored to build models that rely on patient data already digitally available in the Electronic Health Record (EHR) to facilitate quick collaboration and avoid additional funding needs. Taken together, we offer a roadmap for the future of precision oncology which is fraught with both challenges and opportunities for pharmacometricians and clinicians alike.

Getting Innovative Therapies Faster to Patients at the Right Dose: Impact of Quantitative Pharmacology Towards First Registration and Expanding Therapeutic Use.

Quantitative pharmacology (QP) applications in translational medicine, drug‐development, and therapeutic use were crowd‐sourced by the ASCPT Impact and Influence initiative. Highlighted QP case studies demonstrated faster access to innovative therapies for patients through 1) rational dose selection for pivotal trials; 2) reduced trial‐burden for vulnerable populations; or 3) simplified posology. Critical success factors were proactive stakeholder engagement, alignment on the value of model‐informed approaches, and utilizing foundational clinical pharmacology understanding of the therapy.

PK/PD modeling in the perinatal period

Perinatal period represents a continuous and dynamically changing state of growth and development. Marked differences in the pharmacokinetic (PK) and pharmacodynamic (PD) behavior of many drugs are reported between newborns and adults due to the developmental changes in physiological parameters during perinatal period. The lack of clinical trial data directly results in the variation and uncertainty of drug therapy in clinical practice. There has been a growing interest in exploring innovative methodology to optimize drug therapy in this vulnerable population. In this context, PK-PD modeling presents a promising and valuable approach. PK-PD modeling is the science of quantitative pharmacology. In application to pediatrics, it involves primarily developmental PK and PD modeling and simulation, which can combine information from many diverse sources as drug characteristics, developmental clinical pharmacology, physiological changes during childhood, pediatric disease and statistics. This presentation will use cases studies to demonstrate how to apply PK-PD modeling in optimization of clinical trial design and personalized drug therapy in perinatal period.

Reevaluating the role of megalin in renal vitamin D homeostasis using a human cell-derived microphysiological system

The role of megalin in the regulation of renal vitamin D homeostasis has previously been evaluated in megalin-knockout mice and rat proximal tubule epithelial cells. We revisited these hypotheses that were previously tested solely in rodent models, this time using a 3-dimensional proximal tubule microphysiological system incorporating primary human proximal tubule epithelial cells. Using this human cell-derived model, we confirmed that 25OHD3 is transported into the human proximal tubule epithelium via megalin-mediated endocytosis while bound to vitamin D binding protein. Building upon these findings, we then evaluated the role of megalin in modulating the cellular uptake and biological activity of 1α,25(OH)2D3. Inhibition of megalin function decreased the 1α,25(OH)2D3-mediated induction of both cytochrome P450 24A1 protein levels and 24-hydroxylation activity following perfusion with vitamin D binding protein and 1α,25(OH)2D3. The potential for reciprocal effects from 1α,25(OH)2D3 on megalin expression were also tested. Contrary to previously published observations from rat proximal tubule epithelial cells, 1α,25(OH)2D3 did not induce megalin gene expression, thus highlighting the potential for meaningful interspecies differences in the homeostatic regulation of megalin in rodents and humans. These findings challenge a recently promoted hypothesis, predicated on the rodent cell data, that attempts to connect 1α,25(OH)2D3-mediated regulation of renal megalin expression and the pathology of chronic kidney disease in humans. In addition to providing specific insights related to the importance of renal megalin in vitamin D homeostasis, these results constitute a proof-of-concept that human-derived microphysiological systems are a suitable replacement for animal models for quantitative pharmacology and physiology research.

Quantitative pharmacology studies in microphysiological systems (MPS)

Quantitative pharmacology studies in microphysiological systems (MPS)

Assessing the Combined Antibacterial Effect of Isoniazid and Rifampin on Four Mycobacterium tuberculosis Strains Using In Vitro Experiments and Response-Surface Modeling.

While isoniazid and rifampin have been the cornerstone of tuberculosis therapy caused by drug-susceptible Mycobacterium tuberculosis for more than 40 years, their combined action has never been thoroughly assessed by modern quantitative pharmacology approaches. The aims of this work were to perform in vitro experiments and mathematical modeling of the antibacterial effect of isoniazid and rifampin alone and in combination against various strains of Mycobacterium tuberculosis After MIC determination of H37Rv and three strains belonging to the Beijing, Euro-American, and Indo-Oceanic lineages, the antibacterial effects of isoniazid and rifampin alone and in combination were studied in static time-kill experiments. A sigmoidal maximum effect model (Hill equation) and a response-surface model were used to describe the effect of the drugs alone and in combination, respectively. The killing effect of isoniazid and rifampin alone were well described by the Hill equation. Rifampin displayed a more concentration-dependent effect than isoniazid around the MIC. The pharmacodynamics parameters of each drug (maximal effect, median effect concentration, and coefficient of sigmoidicity) were quite similar between the four strains. The response-surface model from Minto et al. fit data of combined effect very well with low bias and imprecision (C. F. Minto, T. W. Schnider, T. G. Short, K. M. Gregg, A. Gentilini, Anesthesiology 92:1603-1616, 2000, https://doi.org/10.1097/00000542-200006000-00017). Response-surface modeling showed that the combined action of isoniazid and rifampin was synergistic for the H37Rv, Beijing, and Euro-American strains but only additive for the Indo-Oceanic strain. This study can serve as a motivating example for preclinical evaluation of combined action of antituberculous drugs.

Highlights of This Issue

Prospective prediction and mechanistic understanding of drug penetration across human blood-brain barrier (BBB) are critical to rational drug development and therapy for brain cancer. By using a translational research strategy that leverages preclinical studies, pharmacokinetic modeling, and clinical trials, Li and colleagues developed an IVIVE-PBPK modeling approach for quantitatively and mechanistically understanding tumor penetration of AZD1775 in glioblastoma patients. The knowledge and tool generated are of enormous value to the field of quantitative pharmacology and also to the development of more effective therapies for brain cancer, where sufficient drug brain penetration is the prerequisite for efficacy.Mutation signatures of homologous recombination deficiency (HRD) have recently been shown to be prevalent in many cancer types. To examine their clinical implications, Zhao and colleagues performed whole-genome sequencing of 93 breast cancers, 33 of which were treated with platinum-based chemotherapy. They quantified 6 HRD-associated mutation signatures shown to robustly predict BRCA1/2 status. Their observational analysis found that these HRD signatures also were associated with improved outcomes and treatment durations on platinum-based chemotherapy. They demonstrate an approach for computing HRD signatures, which may aid in the clinical translation of this potential predictive biomarker.Philadelphia chromosome positive acute lymphoblastic leukemia (BCR-ABL+ B-ALL) has poor prognosis in both pediatric and adult patients with the disease-free survival of 25%–30% in children and less than 20% in adults. Antiapoptotic MCL-1 is essential for the survival of BCR-ABL+ leukemic cells and they are resistant to BH3-mimetic navitoclax. Budhraja and colleagues identified that dihydroartemisinin (DHA), an antimalarial drug, represses MCL-1 expression and sensitizes BCR-ABL+ B-ALL cells to synergistic killing by navitoclax in vitro in patient-derived xenografts and in mouse models. The data suggested that combination of DHA and navitoclax could have implications to improve therapeutic response in BCR-ABL+ B-ALL.The high prevalence of AR in ER+ breast cancer along with the preclinical and clinical efficacy of classic androgens provide rationale for developing a new generation of selective AR agonist for this indication. Yu and colleagues report that RAD140, an oral, tissue-selective androgen receptor modulator (SARM), significantly inhibits the growth of AR/ER+ breast cancer models, an effect further enhanced by the coadministration of CDK4/6 inhibitor. Importantly, RAD140 treatment led to substantial decrease in ESR1 mRNA, suggesting a distinct AR-mediated mechanism of action compared with the agents targeting ER protein. RAD140 is now being evaluated in recurrent ER+ breast cancer.

Quantitative contributions of processes by which polyanion drugs reduce intracellular bioavailability and transfection efficiency of cationic siRNA lipoplex

RNA Interference (RNAi) is a potentially useful tool to correct the detrimental effects of faulty genes; several RNAi are undergoing clinical evaluation in various diseases. The present study identified the relative contributions of three mechanisms by which polyanion drugs reduced the gene silencing activity of Lipoplex, a complex of small interfering RNA (siRNA) and cationic liposomes. The study used a siRNA against the chemoresistance gene survivin and two model polyanion drugs (suramin, heparin). Products of Lipoplex destabilization were separated, identified, and/or quantified using ultrafiltration, gel electrophoresis, and RT-qPCR (quantitative reverse transcription polymerase chain reaction). Cell binding and endocytosis of fluorescence-labeled Lipoplex and the amount of siRNA at its site of action RISC (RNA-induced silencing complex) were evaluated using endocytosis markers, confocal microscopy, quantitative image analysis, immunoprecipitation, and RT-qPCR. The results show suramin and heparin exerted multiple concentration-dependent effects. First, these agents altered several Lipoplex properties (i.e., reduced particle size, changed surface charge, modified composition of protein biocorona). Second, both caused Lipoplex destabilization to release double- and single-strand siRNA and/or smaller siRNA-lipid complexes with reduced siRNA cargo. Third, both prevented the cell surface binding and internalization of Lipoplex, diminished the siRNA concentration in RISC, and retarded the mRNA knockdown. Suramin and heparin yielded qualitatively and quantitatively different results. Analysis of the experimental results of suramin using quantitative pharmacology (QP) modeling indicated the major cause of gene silencing activity loss depended on drug concentration, changing from inhibition of endocytosis at lower concentration (accounting for 60% loss at ~9μM) to inhibition of cell surface binding and loss of siRNA cargo at higher concentrations (accounting for 64% and 27%, respectively, at 70μM). In summary, the present study demonstrates the complex and dynamic interactions between polyanions and Lipoplex, and the use of QP modeling to delineate the contributions of three mechanisms to the eventual loss of gene silencing activity.

Exosome is a mechanism of intercellular drug transfer: Application of quantitative pharmacology

PurposeExosomes are small membrane vesicles (30–100nm in diameter) secreted by cells into extracellular space. The present study evaluated the effect of chemotherapeutic agents on exosome production and/or release, and quantified the contribution of exosomes to intercellular drug transfer and pharmacodynamics. MethodsHuman cancer cells (breast MCF7, breast-to-lung metastatic LM2, ovarian A2780 and OVCAR4) were treated with paclitaxel (PTX, 2–1000nM) or doxorubicin (DOX, 20–1000nM) for 24–48h. Exosomes were isolated from the culture medium of drug-treated donor cells (Donor cells) using ultra-centrifugation, and analyzed for acetylcholinesterase activity, total proteins, drug concentrations, and biological effects (cytotoxicity and anti-migration) on drug-naïve recipient cells (Recipient cells). These results were used to develop computational predictive quantitative pharmacology models. ResultsCells in exponential growth phase released ~220 exosomes/cell in culture medium. PTX and DOX significantly promoted exosome production and/or release in a dose- and time-dependent manner, with greater effects in ovarian cancer cells than in breast cancer cells. Exosomes isolated from Donor cells contained appreciable drug levels (2–7pmole/106 cells after 24h treatment with 100–1000nM PTX), and caused cytotoxicity and inhibited migration of Recipient cells. Quantitative pharmacology models that integrated cellular PTX pharmacokinetics with PTX pharmacodynamics successfully predicted effects of exosomes on intercellular drug transfer, cytotoxicity of PTX on Donor cells and cytotoxicity of PTX-containing exosomes on Recipient cells. Additional model simulations indicate that within clinically achievable PTX concentrations, the contribution of exosomes to active drug efflux increased with drug concentration and exceeded the p-glycoprotein efflux when the latter was saturated. ConclusionsOur results indicate (a) chemotherapeutic agents stimulate exosome production or release, and (b) exosome is a mechanism of intercellular drug transfer that contributes to pharmacodynamics of neighboring cells.

Application of Quantitative Pharmacology Approaches in Bridging Pharmacokinetics and Pharmacodynamics of Domagrozumab From Adult Healthy Subjects to Pediatric Patients With Duchenne Muscular Disease.

Domagrozumab, a monoclonal antibody that binds to myostatin, is being developed for Duchenne muscular dystrophy (DMD) boys following a first-in-human study in healthy adults. Literature reporting pharmacokinetic parameters of monoclonal antibodies suggested that body-weight- and body-surface-area-adjusted clearance and volume of distribution estimates between adults and children are similar for subjects older than 6 years. Population modeling identified a Michaelis-Menten binding kinetics model to optimally characterize the target mediated drug disposition profile of domagrozumab and identified body mass index on the volume of distribution as the only significant covariate. Model parameters were predicted with high-precision pharmacokinetics (clearance 1.01 × 10-4 L/[h·kg]; central volume of distribution 457 × 10-4 L/kg; maximum elimination rate 17.5 × 10-4 nmol/[h·kg], Km 10.6 nmol/L) and pharmacodynamics (myostatin turnover rate 457 × 10-4 h-1 ; complex removal rate 90 × 10-4 h-1 ; half-saturation constant 4.32 nmol/L) and were used to predict target coverage for dosage selection in the DMD population. Additionally, allometric approaches (estimated scaling exponents (standard error) for clearance and volume were 0.81 [0.01] and 0.98 [0.02], respectively) in conjunction with a separate analysis to obtain the population mean weight and standard deviation suggested that if dosed per body weight, an only 11% difference in clearance is expected between the heaviest and lightest patient, thus preventing the need for dose adjustment. In summary, quantitative approaches were instrumental in bridging and derisking the fast-track development of domagrozumab in DMD.

Clinical Pharmacology: a Discipline at the Nexus Between Translational Science and Precision Medicine: Commentary on “Enhancing Value of Clinical Pharmacodynamics in Oncology Drug Development: An Alliance Between Quantitative Pharmacology and Translational Science”

Clinical Pharmacology: a Discipline at the Nexus Between Translational Science and Precision Medicine: Commentary on “Enhancing Value of Clinical Pharmacodynamics in Oncology Drug Development: An Alliance Between Quantitative Pharmacology and Translational Science”

Quantitative Pharmacology of Intercellular Drug Shuttle by Exosomes

BackgroundExosomes are extracellular vesicles (30–100 nm) that contain bioactive molecules, secreted by most cell types and play a role in intercellular communications. Tumor cells exosomes may affect tumor progression, metastasis, and therapeutic efficacy and resistance. The current study used in vitro experiments and in silico studies to investigate the roles of exosomes in intercellular drug transfer and the quantitative relationship between exosomes and pharmacodynamics of chemotherapy drugs in solid tumor cells.MethodsEffects of paclitaxel (PTX) and doxorubicin (DOX) on exocytosis were studied in human breast (MCF7 and a lung metastatic subline of MDA‐MB‐231 cells, LM2) and ovarian (A2780 and OVCAR4) cancer cells. Inhibition of exocytosis was studied using omeprazole (proton pump inhibitor) and GW4869 (ceramide synthesis inhibitor). Time‐lapse laser confocal microscopy was used together with RFP‐transfected cells to monitor the release of exosomes into extracellular fluid. Extracellular exosomes were collected and quantified based on their acetylcholinesterase activity. Drug concentrations were measured using LC‐MS/MS. Pharmacodynamics of exosomes were measured in recipient cells, using the sulforhodamine B assay for anti‐proliferation and the wound‐healing assay for anti‐migration.ResultsAll four cells in exponential growth phase maintained similar baseline exosome level (~220 exosomes/cell). PTX (2–1000 nM) or DOX (10–1000 nM) treatment for 24 or 48 h stimulated exosome production/secretion, in dose‐ and time‐dependent manner, by up to 10‐fold. After 24 h treatment, the amount of PTX in exosomes was 5–10% of that in donor cells, and these PTX‐containing exosomes inhibited cell viability and migration in vitro. Pretreatment for 24 h with omeprazole (10 μM) or GW4869 (10 μg/mL) reduced the production/secretion of, and the drug amount in, exosomes by >20%, and enhanced the exosome cytotoxicity in donor cells. Our quantitative pharmacology model, comprising 6 intracellular and intercellular compartments and 11 rate constants, captured and characterized the effect of exocytosis on intercellular transfer of drug‐containing exosomes and PTX pharmacodynamics in donor and recipient cells with good fitness (CV <19%).ConclusionOur results indicate (a) chemotherapeutic agents stimulate exosome production/secretion, (b) exocytosis is a mechanism of intercellular drug transfer responsible for conferring biological activities on neighboring recipient cells, and (c) successful application of quantitative pharmacology to depict the spatial and temporal effects of exosomes on chemotherapy pharmacodynamics.Support or Funding InformationSupported in part by RO1CA163015 (GW, JA) from National Cancer Institute and RO1EB015253 (JA, GW) from National Institute of Biomedical Imaging and Bioengineering, DHHS and RSG‐16‐006‐01‐CCE (SW) from American Cancer Society.

Quantitative Pharmacology to Interrogate Mechanisms of Lipoplex Destabilization by Serum Proteins

Short interfering RNA (siRNA) represents an option to correct genetic lesions. Due to its instability and unfavorable transport properties (high MW, large size, negative charges), siRNA is commonly delivered as a complex with cationic liposomes or Lipoplex. Lipoplex, ~100–200 nm diameter, is multilamellar comprising alternating layers of lipids and siRNA (located on both the surface (siRNAsurface) and inner layers (siRNAinner)). This study investigated the kinetics of Lipoplex destabilization by serum proteins and the changes in destabilization products, as functions of siRNA and protein concentrations, and used quantitative pharmacology (QP) modeling to infer likely mechanisms.Experiments used five methods to separate, identify and quantify the products of Lipoplex destabilization by fetal bovine serum (FBS), i.e., ultrafiltration, centrifugation, confocal microscopy with quantitative image analysis, gel electrophoresis, and RT‐qPCR (quantitative reverse transcriptase polymerase chain reaction). The results show the products of Lipoplex destabilization depended on concentrations of both proteins and siRNA, with free or degraded siRNA as a major product (maximal release of 65%). Computational models were developed in MATLAB, are based on a system of ordinary differential equations solved with a variable‐step and variable‐order solver. Model parameters were determined by assigning initial estimates, followed by optimization using MATLAB fminsearch algorithm to obtain the best‐fit parameter values (i.e., lowest sum of squared errors (SSE) between model‐simulated and experimental results).We evaluated four models based in part on literature data on protein binding, Lipoplex structure and changes in protein‐Lipoplex interactions, and on our experimental results. Each model described a mechanism or a combination of mechanisms resulting in concentration‐dependent siRNA release. Two models, one depicting cooperative protein binding to Lipoplex and one depicting replacement of low affinity proteins from their Lipoplex binding sites by high affinity proteins, yielded unsatisfactory fit to the experimental results as indicated by the high SSE (5327–6835). In contrast, extending these models to include the additional feature of saturable or limited cationic lipid binding sites for which protein and siRNA compete yielded better fitting results (SSE of 4010 and 376).In summary, this study demonstrates the release of siRNA from Lipoplex due to interactions with serum proteins was dependent on the protein:siRNA molar concentration ratio (Ratio), and that the likely mechanism involves proteins competing and thereby releasing siRNAsurface from the cationic lipid binding sites, with accelerated siRNA release when high abundance low affinity proteins are replaced by low abundance high affinity proteins at higher Ratio, until all siRNAsurface is released.Support or Funding InformationSupported in part by research grants RO1 CA163015 (GW, JA) from National Cancer Institute and RO1EB015253 (JA, GW) from National Institute of Biomedical Imaging and Bioengineering, DHHS.

Polyanionic Drugs Reduce Intracellular Bioavailability and Transfection Efficiency of Cationic siRNA Lipoplex: Quantitative Pharmacology Studies

RNA Interference (RNAi) is a potentially useful tool to correct the detrimental effects of faulty genes, and several such agents are undergoing clinical evaluation. The present study evaluated the effects of polyanionic drugs (suramin, heparin) on the in vitro stability, cellular processing, and biological activity of Lipoplex, a complex between small interfering RNA (siRNA) and cationic liposome (50% DOTAP, 30% Cholesterol, 19% DOPE, 1% DSPE‐PEG). The resulting Lipoplex had a diameter of 195 nm and surface charge of +33.9 mV. Two siRNA, against survivin and metadherin, were used. The destabilization products of Lipoplex were separated, identified, and quantified using ultrafiltration, gel electrophoresis, and RT‐qPCR (quantitative reverse transcription polymerase chain reaction). Binding and endocytosis of Lipoplex in cells, and the amount of siRNA located in its target site RISC (RNA‐induced silencing complex) were evaluated using endocytosis markers, confocal microscopy, quantitative image analysis, immunoprecipitation, and RT‐qPCR. The results show suramin and heparin exerted several effects that were dependent on their concentrations as well as Lipoplex concentration. These agents altered the Lipoplex physical properties (i.e., reduced particle size, changed surface charge from positive to negative, removed protein‐biocorona or modified its composition). These changes are associated with Lipoplex destabilization and release of lipids and siRNA (ds/ss siRNA) and formation smaller Lipoplex with reduced siRNA content. The Lipoplex destabilization products due to suramin and heparin were different from each other and from those caused by serum proteins, both qualitatively and quantitatively. In addition, suramin and heparin prevented the cell surface binding and internalization of Lipoplex, diminished the intracellular bioavailability of siRNA in RISC, and retarded the mRNA knockdown by Lipoplex. Quantitative pharmacology analysis using experimental results and computational simulations indicated that polyanion:siRNA molar concentration ratio (Ratio) determined the relative contribution of two mechanisms to the reduction of Lipoplex‐conferred gene silencing; the Lipoplex destabilization and loss of siRNA cargo had a greater contribution compared to the inhibition of Lipoplex cellular binding and endocytosis at lower Ratio (e.g., 17:1), whereas the latter had a greater contribution at higher Ratio (e.g., 128:1). In summary, interactions with polyanionic drugs destabilize Lipoplex result in different products for different drugs, and the three‐way interactions between polyanionic drugs, Lipoplex and cell membrane binding sites diminish the Lipoplex biological activity.Support or Funding InformationSupported in part by research grants, R01CA158300 (GW, JA) and R01CA163015 (GW, JA) from National Cancer Institute and R01EB015253 (JA, GW) from National Institute of Biomedical Imaging and Bioengineering, NIH, DHHS.

Quantitative Pharmacology of Internalization, Intracellular Trafficking and Gene Silencing of siRNA

RNA interference (RNAi) represents a promising approach to correct genetic lesions, and several RNAi candidates including short interfering RNA (siRNA) are in clinical evaluation. In cells, siRNA is loaded on RISC (RNA Induced Silencing Complex) and causes degradation of the targeted mRNA. Due to instability and immunogenicity, siRNA is delivered as a complex with cationic liposomes (Lipoplex, 100–200 nm diameter). Hence, siRNA pharmacodynamics is determined by multiple kinetic steps including cell binding, endocytosis, endosomal transport and recycling, cytoplasmic release from endosomes and Lipoplex, loading in cytoplasmic RISC, and degradation of mRNA and protein. The goal was to develop quantitative pharmacology models of the concentration/time dependent mRNA and protein knockdowns by Lipoplex.For model development, Lipoplex and siRNA transfer between extracellular and intracellular compartments or organelles, and degradation of mRNA/protein were described as first order processes, and the mathematical formulation was based on a system of ordinary differential equations solved numerically. Model parameters were identified by first assigning initial estimates, then fitting the model to the experimental data to yield the best‐fit parameters (i.e., lowest sum of squared errors and within experimental variations).For experimental results, human breast MCF7 cancer cells were incubated with fluorescence‐labeled Lipoplex, and Lipoplex cellular binding and endocytosis were monitored using live cell confocal microscopy or fluorescence microscopy together with markers for endosomal organelles (Rab5 for early endosomes and macropinosomes, Rab‐7 for late endosomes, LAMP1 for lysosomes). Images were acquired every 10 min for up to 12 h, and the fluorescence intensity (e.g., signals colocalizing with the markers) were converted to siRNA molecules using quantitative image analysis (ImageJ and MatLab software). The siRNA molecules on RISC (siRNARISC) were quantified using RISC immunoprecipitation followed by siRNA extraction and quantification with RT‐qPCR (quantitative reverse transcription polymerase chain reaction). The mRNA level was measured using RT‐qPCR and the protein level using Western blotting. The experimental results and computational modeling allowed the definition of two models: (i) the kinetic model described the steps from extracellular Lipoplex to siRNARISC and comprised 16 compartments and 18 rate constants. (ii) The pharmacodynamic model described the mRNA and protein knockdown by siRNARISC and comprised 3 compartments and 7 rate constants. The two models jointly yielded the concentration/time dependent gene silencing by Lipoplex. In conclusion, this study demonstrates the successful application of quantitative pharmacology to capture the kinetics of siRNA embedded in Lipoplex deliver to its target RISC and the resulting pharmacodynamics of gene silencing.Support or Funding InformationSupported in part by research grants RO1 CA163015 (GW, JA) from National Cancer Institute and RO1EB015253 (JA, GW) from National Institute of Biomedical Imaging and Bioengineering, DHHS.

Using quantitative pharmacology to overcome challenges in hematopoietic cell transplantation: Application towards primary immune deficiencies and inborn errors of metabolism

Using quantitative pharmacology to overcome challenges in hematopoietic cell transplantation: Application towards primary immune deficiencies and inborn errors of metabolism

Enhancing value of clinical pharmacodynamics in oncology drug development: An alliance between quantitative pharmacology and translational science.

Clinical pharmacodynamic evaluation is a key component of the "pharmacologic audit trail" in oncology drug development. We posit that its value can and should be greatly enhanced via application of a robust quantitative pharmacology framework informed by biologically mechanistic considerations. Herein, we illustrate examples of intersectional blindspots across the disciplines of quantitative pharmacology and translational science and offer a roadmap aimed at enhancing the caliber of clinical pharmacodynamic research in the development of oncology therapeutics.

Harnessing Novel Quantitative Pharmacology Approaches to Optimize the Treatment of Children With Tuberculosis

The World Health Organization (WHO) estimates that a million children each year develop tuberculosis [1], with models suggesting that only a third of these children are diagnosed and started on treatment [2]. Although the WHO-recommended treatment regimen for children with drug-susceptible tuberculosis is generally well tolerated and leads to good treatment outcomes, 6 months of daily treatment can be challenging for children and families. For multidrug-resistant tuberculosis (disease caused by Mycobacterium tuberculosis resistant to isoniazid and rifampicin), treatment regimens are long (often for >18 months) and are associated with significant toxicity. There is, therefore, a pressing case for new shorter, well-tolerated regimens that can treat both drug-susceptible and multidrug-resistant organisms.

Quantitative methods in electroencephalography to access therapeutic response.

Pharmacometrics or Quantitative Pharmacology aims to quantitatively analyze the interaction between drugs and patients whose tripod: pharmacokinetics, pharmacodynamics and disease monitoring to identify variability in drug response. Being the subject of central interest in the training of pharmacists, this work was out with a view to promoting this idea on methods to access the therapeutic response of drugs with central action. This paper discusses quantitative methods (Fast Fourier Transform, Magnitude Square Coherence, Conditional Entropy, Generalised Linear semi-canonical Correlation Analysis, Statistical Parametric Network and Mutual Information Function) used to evaluate the EEG signals obtained after administration regimen of drugs, the main findings and their clinical relevance, pointing it as a contribution to construction of different pharmaceutical practice. Peter Anderer et. al in 2000 showed the effect of 20mg of buspirone in 20 healthy subjects after 1, 2, 4, 6 and 8h after oral ingestion of the drug. The areas of increased power of the theta frequency occurred mainly in the temporo-occipital - parietal region. It has been shown by Sampaio et al., 2007 that the use of bromazepam, which allows the release of GABA (gamma amino butyric acid), an inhibitory neurotransmitter of the central nervous system could theoretically promote dissociation of cortical functional areas, a decrease of functional connectivity, a decrease of cognitive functions by means of smaller coherence (electrophysiological magnitude measured from the EEG by software) values. Ahmad Khodayari-Rostamabad et al. in 2015 talk that such a measure could be a useful clinical tool potentially to assess adverse effects of opioids and hence give rise to treatment guidelines. There was the relation between changes in pain intensity and brain sources (at maximum activity locations) during remifentanil infusion despite its potent analgesic effect. The statement of mathematical and computational aspects in the use of clinical data is frequent and elucidation of these aspects we use PhysioNet https://www.physionet.org/, Clinical Database online supported by the National Institutes of Health (National Institutes of Health of United States of America/NIH-USA) for the acquisition of EEG data and the Matlab program to do the simulations with the methods and thus create opportunities greater understanding.