Sessions covered a new method for crossing the BBB, virtual population approaches, RNA nanoparticles, and much more.
By Cathy Yarbrough
Approximately 5,000 pharmaceutical scientists from 56 countries attended the 2019 American Association of Pharmaceutical Scientists PharmSci 360, November 3 to 6 in San Antonio. The topics of the meeting’s 150+ scientific sessions ranged from preclinical development to manufacturing and bioprocessing. The following text briefly describes some of the presentations.
Crossing the BBB
Many investigational drugs for brain cancer and neurological disorders such as Parkinson disease have failed not because they were ineffective or unsafe but because therapeutically relevant doses of the drugs could not cross the formidable blood-brain barrier (BBB).
In his keynote presentation, Non-invasive Drug and Gene Delivery to the Brain, Justin Hanes, Ph.D., Lewis J. Ort Professor at Johns Hopkins University (JHU), described how he and his colleagues developed a noninvasive method for temporarily opening the BBB to deliver both drug- and nucleic acid-loaded nanoparticles to specific brain regions.
The noninvasive method is the activation of circulating ultrasound contrast agent microbubbles (MB) by magnetic resonance (MR)-guided ultrasound (FUS). In a series of experiments with rodent and human brain tissue, Hanes and colleagues determined that MR-guided MB+FUS enables nanoparticles to cross the BBB.
However, in the initial experiments, the nanoparticles failed to disperse after entering the brain, said Hanes, also director of JHU’s Center for Nanomedicine. He and his colleagues determined that the nanoparticles were stuck in the complex adhesive, steric microenvironment of the brain’s extracellular space (ECS), which comprises 15 to 20 percent of total brain volume.
Fortunately, the researchers had solved a similar problem in previous studies with mucus-penetrating particles (MPPs) for drug and gene delivery to mucosal tissues of the lung airways. In those studies, the MPPs did not disperse throughout the mucosal surfaces. The researchers improved the dispersal of MPPs by coating them with an exceptionally dense layer of polyethylene glycol (PEG). PEG coating provided a nonadhesive surface, which enabled the MPNs to distribute uniformly.
Exceptionally dense PEG coating also proved effective with nanoparticles, Hanes said. PEG-coated nanoparticles as large as 114 nm in size avoided adhesive trapping in ECS and freely and rapidly diffused through the ECS and the parenchyma. Hanes said that experiments showed that PEG density and the diffusivity of the brain-penetrating nanoparticles (BPNs) were correlated.
The research conducted by Hanes and his colleagues was the first study to demonstrate that MR image-guided FUS+MB enabled PEG-coated nanoparticles to penetrate within the brain microenvironment. The research also determined that MR image-guided FUS+MB achieved sub-mm targeting of BPNs ranging from 40 to 65 nm in diameter. In addition, BPNs were shown to target both the vessel endothelium and the brain parenchyma without any uptake in nontargeted regions.
To reach targeted brain regions, BPNs must freely diffuse through ECS pores. A previous study by another lab suggested that BPNs had to be much smaller than 40 nm to penetrate the ECS. However, Hanes and his colleagues found that 30 percent of ECS pores in rodent and human brain tissue were larger than 100 nm and therefore accommodated nanoparticles larger than 40 nm.
In a series of subsequent experiments with rodent models, Hanes and his colleagues determined that MR-guided FUS+MB enabled the targeted and safe delivery of drug- and DNA-loaded BPNs to treat brain tumors and Parkinson disease. Cisplatin-loaded BPN efficiently penetrated the healthy brain parenchyma and brain tumor tissue ex vivo. In one experiment, 80 percent of rats with established brain tumors were cured by BPNs loaded with standard chemotherapy drugs, Hanes said.
Hanes and his colleagues also investigated the use of MR-guided FUS+MB to deliver nanoparticles loaded with the gene for GDNF (glial cell-derived neurotrophic factor) in a rodent model of Parkinson disease. A sustained high level of GDNF expression occurred in rodents treated with GDNF-BPNs. A single dose of GDNF-BPNs restored the density of dopaminergic neurons and motor neurons in the rodent model. In addition, dopamine levels and motor neuron function were restored.
Hanes’ collaborators in these studies include Richard J. Price, Ph.D., professor of Biomedical Engineering at the University of Virginia, and Jung Soo Suk, Ph.D., assistant professor of Ophthalmology at JHU.
Virtual Pop For QSP
Yougan Cheng, Ph.D.’s presentation, Virtual Population Approaches using Prior Clinical Data for Immuno-oncology Development, was one of seven PharmSci 360 symposia on the emerging field of quantitative systems pharmacology (QSP), a complex computational framework that can provide mechanistic insights about a biological system and the effects of drug treatment on system behavior.
Cheng, a principal scientist at Bristol-Myers Squibb, described the development of a suitable virtual population (VPop) for an in-house, multi-therapy QSP platform on the mechanistic interaction of key components of the cancer-immunity cycle and the synergy of immune-oncology (I-O) drug combinations for treating melanoma patients. The VPop was designed to quantitatively reproduce observed biomarker and response variations in real world melanoma patients, he said.
The QSP platform and VPop model was “built from scratch,” said Cheng, who weighted mechanistic variability and prevalence in order to capture population variability of numerous characteristics such as lesion size response at various time points during anti-CTLA4 and anti-PD-1 therapies.
The resulting QSP platform and VPop (QSP/VPop) integrated quantitative clinical anti-CTLA4 and anti-PD1 biomarker and efficacy information from clinical trials in first-line melanoma. The VPop captured clinical distributions simultaneously across multiple interventions as well as the overall clinical response rate. “One of the most important results is that the virtual population captures the overall response rate,” he said.
Cheng and his colleagues used QSP/VPop to predict the anticipated dose response of an investigational I-O drug for melanoma patients whose disease had progressed despite anti-PD-1 therapy. QSP/VPop also helped the scientists identify the role of biomarkers in patients’ response to the therapy.
The QSP/VPop accurately predicted second line anti-CTLA4 therapy response after progression on anti-PD1 therapy as well as patients’ response to anti-CTLA4 and anti-PD1 therapy combination, Cheng said. As a result, he and his colleagues were able to use the platform to predict the dose responses of a BMS investigational compound and formulate hypotheses for unexpected biomarker observations with the new therapy.
The platform’s usefulness is not limited to a single project. It has been and will be used to support other I-O programs, Cheng pointed out.
RNAi Delivery Exosomes
In his Rapid Fire presentation, RNA Nanoparticles Cooperated with Exosome as Efficient in vivo RNAi Delivery for Cancer Treatment, Zhefeng Li described the development of a novel drug delivery system that combines antibody-like RNA nanoparticles with exosomes to enable the targeted delivery of siRNA to cancer cells.
Li, a fifth year Ph.D. student in pharmaceutics and pharmacology at Ohio State University (OSU), and his collaborators, Fengmei Pi, Ph.D., and Zhen Zheng, Ph.D., evaluated the formation in mice models of prostate cancer, triple negative breast cancer, patient derived colon cancer, and cervical cancer. The research was conducted in the lab of Peixuan Guo, Ph.D., Sylvan G. Frank Endowed Chair of Pharmaceutics and Pharmacology at OSU.
To develop the formulation, Li and his colleagues first placed membrane-anchoring cholesterol molecules at the tails of arrow-shaped RNA nanoparticles. As a result, the outer surfaces of the exosomes displayed RNA aptamers or folate molecules, which increased cancer cell binding and uptake. Folate has a high affinity for the folate receptor protein that is commonly expressed on the surface of many human cancer cells.
In subsequent studies, the researchers determined that the decorated exosomes facilitated the specific delivery of siRNA to the cytosol of cancer cells by a direct fusion mechanism that avoided endosome trapping.
“Remarkably, the RNA payloads showed tumor suppression in all four cancer models,” said Li.
Prostate cancer growth was inhibited in mice models treated with survivin-loaded exosomes that were designed to bind to prostate-specific membrane antigen. “Similarly, exosomes displaying the epidermal growth-factor receptor inhibited breast cancer growth in the mice models,” Li said. Tumor growth was suppressed in mice models of patient-derived colorectal cancer treated with exosomes loaded with survivin siRNA and displaying folate. Tumor suppression was documented in the cervical cancer mice model treated with folate-displayed, ginger-derived exosomes loaded with survivin siRNA.
Biologics—Quo Vadis?
The history and future of biotech formulations and the challenges of developing these medicines were the focus of Hanns-Christian Mahler, Ph.D., FAAPS’s keynote presentation, Formulating Biologics—Quo vadis?
The formulations of the first wave of therapeutic proteins shared several characteristics, said Mahler, head of Drug Product Services at Lonza AG. Seventy percent of early biotech drugs were liquid dosage forms, while 30 percent were lyophilized formulations. While biologic formulations in the 1980s depended on a variety of buffer systems, the phosphate buffer was the most frequently utilized, he said.
Not all of the early biotech formulations included surfactants, which is an interesting finding, said Mahler. Many formulations contained a variety of very unconventional excipients, such as albumin, as well as excipients that were used in the manufacture of small molecule drugs.
In the early years of the development of therapeutic proteins, formulation scientists focused on the biochemical stability of the protein separately from how processing steps and container materials could affect the protein’s stability. In addition, formulation scientists tried to simulate a protein’s physiological environment.
Today’s therapeutic protein formulations also share several characteristics. The ratio of liquid to lyophilized dosage forms remains 70 to 30 percent. Mahler noted that monoclonal antibody (mAb) formulations have been commoditized as liquids. More novel proteins may be manufactured by lyophilization, he added.
Many current biologic formulations use a histidine buffer, and surfactants are part of most current formulations. Excipient choices are very limited, and qualifying new excipients has become more challenging, costly, and time-consuming, Mahler added.
“The art of drug product development of biologics has improved over the last decades by advancements in analytical methods, deeper understanding of protein/excipient interactions, and material and process sciences,” he said.
The increased development of diverse molecules including highly complex biologics that require targeted formulations was among Mahler’s predictions about the future of biotech formulations. He also said that pharmaceutical scientists will need to focus on improving the development of pharmaceutically advanced solutions such as gene therapy, cell therapy, and tissue engineering products.
Mahler predicted that more biologics will be delivered in parenteral drug/device combination products such as auto-injectors, prefilled syringes, and vial and transfer syringes.
He also predicted that more formulation scientists will adopt a more holistic, integrated approach to biologics development. “Developing adequate, integrated drug products—considering drug substance process, formulation, dosage form, primary packaging, drug product manufacture, and usability—is key to ensure product quality, competitiveness, and efficacy and safety for patients,” Mahler said.
The challenges in developing biotech formulations include ensuring container closure integrity and preventing interactions of formulation components and primary packaging. Such interactions can lead to the development of insoluble particulates. For example, in delamination, the glass in vials can leach glass particles that migrate into the formulation and affect the product’s quality and safety. “Water is a pretty aggressive solvent,” said Mahler.
Interaction between the container and formulation also can affect the product’s appearance. An example is the fogging that can occur in containers of lyophilized drugs. The interaction of process residuals also can produce reactions with vaporized hydrogen perioxide and tungsten.
Mechanistic Model For Dosing
In her Rapid Fire presentation, Charvi Nanavati, Ph.D., assistant director, Pharmacokinetics and Clinical Pharmacology at Ionis Pharmaceuticals, described the development of a mechanism-based pharmacokinetic/pharmacodynamic (PK/PD) population model to support clinical studies of the investigational therapy, ISIS 702843, for the treatment of non-transfusion dependent thalassemia (NTDT).
ISIS 702843 is an antisense oligonucleotide (ASO)/nucleic acid-based therapy that targets the transmembrane protease serine 6 (TMPRSS6) pathway for iron regulation. TMPRSS6 is a key negative regulator of hepcidin. Inhibiting TMPRSS6 can lead to increased production of hepcidin, which eventually can result in more effective red blood cell production, said Nanavati.
Nanavati briefly summarized the rationale for targeting this pathway as well as the clinical proof of concept for the ASO ISIS 702843.
In the phase 1 clinical trial, a dose-dependent pharmacokinetic and hepcidin profile with a reduction in transferrin saturation (TSAT) characterized the healthy volunteers who received four doses of ISIS 702843.
The mechanistic model, which was based on data from the phase 1 trial of ISIS 702843 with healthy volunteers, was developed to aid in recommending dosing strategies for the phase 2 study, Nanavati said. The model characterized the exposure-response relationship between plasma concentrations of the drug and hepcidin and TSAT, the two key biomarkers for NTDT.
The recommended dosing strategies for the phase 2 trial were based on model simulations that evaluated exposure-response relationships at different doses and dosing regimens. Nanavati said that the evaluation revealed that body weight was a significant covariate for central plasma clearance and central volume in the phase 1 trial. However, simulations with a virtual patient population treated with the recommended dosing regimen suggested that body weight would likely not have a clinical impact on hepcidin and TSAT responses in the phase 2 trial, she said.
Nanavati said that the current mechanistic model can serve as a platform upon which a more comprehensive model that includes additional data from patients as well as clinical endpoints can be built.
Her presentation was titled Mechanism-Based Population Pharmacokinetic/Pharmacodynamic (PK/PD) Modeling of ISIS 702843, a TMPRSS6 Targeting Antisense Oligonucleotide (ASO) to Guide Early Clinical Development.
A Disconnect Corrected
In his Rapid Fire presentation, Alexander Kozhich, Ph.D., senior principal scientist at Bristol-Myers Squibb, described how acoustic liquid handling technology can bridge the gap between blood microsampling and microfluidics in preclinical studies of novel biologics.
By bridging the gap, sample preparation and analysis of small volumes of blood samples from laboratory mice can be fully automated, said Kozhich, whose presentation was titled Microsampling, Acoustic Technology and Microfluidics Combination in Multidomain Biologics Bioanalysis.
Kozhich said that the complexity of a novel biologic requires that multiple pharmacokinetic (PK) and biomarker measurements be obtained so that an accurate description of the drug’s PK, pharmacodynamics (PD), and safety parameters can be generated. Such a description is based on multiple PK and biomarker measurements in samples from laboratory animals treated with the novel drug.
These detailed descriptions can be accomplished with composite sampling in which sample volumes are large enough to conduct multiple assays. However, serial sampling can generate much better data than can composite sampling, Kozhich said. For example, serial sampling provides more accurate data for PK/PD profiling and is less costly because it requires significantly fewer laboratory animals, less dosing material, and less biohazardous waste. However, with serial sampling, limitations on sample size prevent the conduct of multiple assays.
The oldest and best known sampling method is dry blood spot (DBS). While successfully used with small molecule compounds, DBS microsampling is not effective with biologics because of the irreversible denaturation of protein therapeutics during the drying step. The larger sample volume required for traditional ligand binding assays also could lessen DBS’s effectiveness, Kozhich said.
In studies with mice, measurements can be conducted with plasma or serum, but sample volume should be low enough to allow frequent serial sampling (10–20 μL) of the animals, Kozhich said. Frequent sampling would produce 4–8 μL of plasma. However, this volume is not large enough to allow accurate dispensing for bioanalysis either manually or by using traditional liquid handlers.
“So here we have a disconnect,” Kozhich said. Although microfluidics technology has drastically reduced sample volume requirements to less than 5 μL after minimum required dilution, the collection and handling of such small sample volumes can be challenging because researchers cannot accurately dilute and transfer the samples by using manual pipettes or automated liquid handlers.
The solution: using acoustic liquid transfer technology, which can dilute 25 nL of plasma/serum into 10 uL with high precision. “In essence, the acoustic liquid handler enabled us to prepare plasma/serum samples in the size needed for a microfluidics platform from a less than 10 uL sample,” he said.
Using capillary microsampling, acoustic liquid handling and microfluidic immunoassay, Kozhich and his colleagues obtained very small volumes of serum samples to measure several forms of mAb therapeutics and three biomarkers as well as assess anti-drug antibodies, he added.
All assays were performed with acceptable precision and accuracy for standards and quality control. The results were used to generate a comprehensive PK and PD dataset to enable informed decisions about the drug’s development, Kozhich added.
Qualifying Biomarkers
The Food and Drug Administration’s (FDA’s) biomarker qualification program was the focus of Christopher Leptak, M.D., Ph.D.’s keynote presentation at PharmSci 360. Leptak is director of FDA’s Office of New Drugs’ regulatory science program and the Center for Drug Evaluation and Research’s biomedical qualification (BQ) program.
FDA established the BQ program in response to the 21st Century Cures Act of 2016. In 2019, FDA issued the draft guidance Biomarker Qualification: Evidentiary Framework, which describes the type and level of scientific data that researchers must provide to obtain BQ for biomarkers.
The evidentiary framework is broadly applicable to all BQ submissions regardless of whether the biomarker is disease or treatment related. Leptak said that FDA-qualified pharmacodynamic/response biomarkers can become clinical trial endpoints and, for a very small subset, surrogate endpoints.
During August 2017 through October 2019, FDA’s BQ program approved two biomarkers, a clinical nephrotoxicity safety panel to aid in dose escalation studies and a monitoring biomarker for treatment initiation in malaria challenge model clinical trials.
Leptak described the formal three-stage process for reviewing submissions to the BQ program. The first stage is submission of a letter of intent (LOL). If FDA accepts the LOL, the next step is the submission of a proposal that addresses the benefit and risk of using the biomarker in drug development and its context of use (COU). If FDA accepts the proposal, the applicant can submit a full qualification package that contains the accumulated data supporting the qualification of the biomarker for the proposed COU.
“The specific COU for a biomarker drives the extent of evidence needed for BQ,” Leptak said. Within its stated COU, a qualified biomarker must have a specific interpretation and application in both drug development and regulatory review. Thus, once a biomarker is FDA qualified, it can be used under the same COU in multiple drug development programs, he pointed out.
A total of 261 LOLs, and eight full qualification packages have been submitted to the BQ program during the over-two-year period ending in October 2019. FDA staff also are reviewing eight transition summaries from the agency’s previous biomarker development program, which was initiated in 2007. The BQ program replaces the legacy program.
Leptak’s keynote presentation was titled Regulatory Perspective on Biomarker Qualification and Impact on Drug Development.
Cathy Yarbrough is a freelance science writer.
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