Abstracts for Posters 1-8
Poster 1
Time-Dependent Relative Humidity in Nested Packaging
Vanessa M. Breslin, Brooke Tatarian, Maximilian Plajer, Maria J. Krisch, and Kenneth C. Waterman
FreeThink Technologies, Inc.
Presenting and Corresponding Author: Vanessa M Breslin, [email protected]
Purpose
The ability to model complex packaging options will expand the usefulness of the stability software ASAPprime®. Specifically, the ability to use single-layer packaging data to predict nested packaging moisture barrier behavior will provide a greater range of packaging options for modeling shelf life.
Methods
The study evaluated various packaging configurations consisting of a small HDPE bottle (75-cc) enclosed in a larger HDPE bottle (250-cc) and an LDPE bag enclosed in an HDPE bottle (250-cc) containing excipient, desiccant, or neither. The bottles were heat induction sealed and the bags were heat sealed before being exposed to a single controlled temperature and relative humidity condition (40°C/75% RH) to measure (using a data logger) the relative humidity (RH) in each compartment over 25 days. The experimental RH data for each nested packaging configuration was compared to the model predictions of RH as a function of time based on a two-compartment packaging model.
Results
The experimental RH data as a function of time are consistent with the two-compartment model predictions. As expected, the highly permeable LDPE bag inside of an HDPE bottle does not have a noticeable influence when predicting the RH change over time. Differences observed between some experimental data and model predictions are likely due to the data logger itself absorbing a significant amount of moisture.
Conclusion
A two-compartment packaging model that uses single-layer packaging data to predict the change in RH as a function of time in nested packaging shows reasonable agreement with the experimental data, with differences likely due to moisture absorption by the data loggers used in the measurements. Ongoing work will include incorporating moisture sorption of the data loggers for inclusion in the model prediction.
Poster 2
Targeted Delivery of siRNA to the Kidneys Using Mesoscale Lipid Nanoparticles
Anastasiia Vasylaki, Shakuntala Sookraj, Pratyusha Ghosh, Ryan Williams
Department of Biomedical Engineering, City College of New York
Presenting Author: Anastasiia Vasylaki, [email protected]
Corresponding Author: Ryan Williams, [email protected]
Purpose
Healthcare faces a significant challenge with the rising prevalence of acute and chronic kidney diseases, with limited treatment options. It has been demonstrated that nanoparticles in the mesoscale size range (300 – 400 nm) selectively target the proximal tubular epithelial cells in the kidneys. Lipid nanoparticles have proven successful in delivering nucleic acid therapeutics, however, kidney targeting of mesoscale nanoparticles has only been demonstrated with polymeric carriers. This research aims to develop a new lipid-based mesoscale nanoparticle formulation for delivering nucleic acid therapies as kidney disease treatment.
Methods
Mesoscale lipid nanoparticle (MLNP) formulation was prepared containing siRNA cargo, an ionizable lipid, a phospholipid, cholesterol, and a PEGylated lipid, using the nanoprecipitation method. MLNP size, PDI, and zeta potential were characterized via DLS. siRNA encapsulation efficiency was measured using RiboGreen assay. MLNP cytotoxicity to renal epithelial cells was tested using MTT assay. siRNA release from MLNPs was studied in PBS at 37°C. To assess LNP biodistribution, healthy C57/BI/6J mice were injected IV with 50 mg/kg of fluorescently labeled MLNPs. Different groups of mice were sacrificed at 1, 3, 5, and 7 days post-injection and organs were collected for fluorescent imaging.
Results
Five formulation modifications were tested to increase the size of lipid nanoparticles to the mesoscale range (300 – 400 nm). Increasing total lipid concentration showed the largest effect on size. To achieve stable PEGylation required for kidney targeting, the lipid composition of MLNPs has been optimized. Cell viability assays demonstrated that MLNP treatment does not affect the viability of renal epithelial cells. MLNP siRNA release profile showed sustained drug release over 72 h. In the biodistribution study, MLNPs primarily accumulated in the kidneys with a kidney/liver ratio of 2.18 after 24 h and >5.75 at the later time points.
Conclusion
In the current work, we have developed the first, to our knowledge, mesoscale lipid nanoparticle formulation optimized for targeted nucleic acid delivery to the kidneys. The MLNP formulation has demonstrated preliminary indications of safety and high selectivity for the kidneys. Additional in vitro and in vivo studies will be conducted to apply this drug delivery system for kidney disease treatment.
Poster 3
PKPD Modeling and Quantitative Analysis of Immunotherapy Drug Pembrolizumab
Reyana Celebi and Marcel Musteata
Albany College of Pharmacy and Health Sciences
Presenting Author: Reyana Celebi, [email protected]
Purpose
This study develops an in-vitro fluidic pharmacokinetic system to assess pembrolizumab’s clearance and effect on cell growth as an alternative to animal models. Murine blood circulation dynamics are simulated within the fluidic system and integrate LC-MS/MS and SPME probes for drug quantification. PKAnalix software allows determination of clearance and elimination characteristics. This model supports and enables precise monitoring of drug metabolism and dose-dependent clearance changes such as target-mediated drug disposition. These approaches enhance pharmacokinetic monitoring of monoclonal antibodies as well as enhance immunotherapy dosing and translational research in oncology.
Methods
The fluidic apparatus consists of a mechanical arrangement of pumps and tubing that mimic the circulatory system of a mouse. Tubing carries drug solution from the injection point to the mixing chamber, then through the system circulation to mimic blood flow. Other features entail adjustable flow rates and a port for elimination and sample collection. With this system, drug concentration changes are monitored over time. Clearance kinetics were assessed in optimization trials with methyl blue injections.
Pembrolizumab samples collected over a time course were analyzed and quantified using LC-MS/MS with a Shimadzu LC system and AB Sciex QTRAP 5500 mass spectrometer, employing a Phenomenex Kinetex C18 column. A closed-loop fluidic system was used to evaluate pembrolizumab stability and adsorption within the system, which was quantified by LC-MS. SPME is a common microsampling method of analysis that was validated in previous studies.
Results
Data from optimization and validation trials showed calculated clearance values aligned with the elimination pump flow rate. Data plots by PKAnalix visually presented a lack of outliers and an expected profile of a multiple-dose regimen, in an IV Bolus, one compartment model. The system was validated to show the elimination pump provided the calculated clearance of methyl blue in the fluidic system. PEG minimized adsorption while maintaining signal intensity. Closed-loop trials confirmed pembrolizumab’s stability over time, validating the system’s ability to maintain drug integrity for multiple days.
Conclusion
This study successfully optimized a fluidic pharmacokinetic apparatus and system for creating realistic pharmacokinetic profiles, optimized quantification of pembrolizumab by LC-MS/MS, was validated using PKAnalix, and explored quantification of pembrolizumab by SPME. These findings support the fluidic pharmacokinetic generator apparatus and LC-MS/MS quantification as valuable tools for simulating monoclonal antibody pharmacokinetics and exploring its effect on cells.
Poster 4
Albumin is a critical factor contributing to plasma per- and polyfluoroalkyl substances (PFAS) concentration, accumulation, and retention in vivo
Olga Skende(1), Emily Kaye(1),Simon Vojta(2), Jitka Becanova(2), Rainer Lohmann(2), Fabian Fischer(1), Angela L. Slitt(1)
(1) Department of Biomedical and Pharmaceutical Sciences, University of Rhode Island (2) Graduate School of Oceanography, University of Rhode Island
Presenting Author: Olga Skende, [email protected]
Corresponding Author: Angela L. Slitt, [email protected]
Purpose
Per- and polyfluoroalkyl substances (PFAS) make up a class of synthetic molecules composed of chain-linked carbon- fluorine bonds. There are 10,000+ PFAS, with perfluorooctane sulfonate (PFOS) and perfluorohexane sulfonate (PFHxS) being some most frequently detected PFAS in serum of the United States general population. Newer PFAS, such as perfluorobutane sulfonic acid (PFBS) and 6:2-fluorotelomersulfonic acid (6:2 FTS), are now also detected in human serum and milk. As a class of chemicals, PFAS undergo little to no biotransformation and are slowly excreted. PFOS and PFHxS exhibit a strong binding affinity for serum albumin, which is hypothesized to be the predominant mechanism that dictates their slow elimination from the body. It is thought that albumin binding retains PFAS in the circulation, and prevents PFAS from undergoing renal excretion. However, to date, albumin binding as a critical factor for PFAS elimination has only been studied and modeled using in vitro assays. Thus, it was hypothesized that mice deficient in albumin would have reduced plasma PFAS concentrations due to decreased PFAS retention in plasma by albumin.
Methods
C57BL/6J mice expressing (Alb+/+) or lacking (Alb-/-) albumin were administered a single oral dose of a PFAS cocktail consisting of 0.1mg/kg PFOS, PFHxS, PFBS, and 6:2FTS at equal ratios (10 ml/kg, n=4-6/group). Plasma was collected via the submandibular vein over the course of 12 weeks at 1, 4, 24, and 72 hours, followed by 1, 2, 4, 6, 8, and 10 weeks. At 12 weeks post PFAS dosing, plasma and tissues were collected.
Results
Samples were processed using QuEtCheERS extraction material and prepared for LC/MS analysis. Plasma PFOS, PFHxS, PFBS, and 6:2FTS concentrations were markedly lower in Alb-/- mice as compared to Alb+/+ 1 hour, 4 hours, 24 hours, 72 hours, 1 week, 2 weeks, and 4 weeks after PFAS administration. Sample analysis for remaining time points and tissues is ongoing.
Conclusion
This is the first study to prove that albumin is a critical factor contributing to plasma PFAS concentration, accumulation, and retention in vivo.
Poster 5
Optimizing Process Analytical Technology: Refractometer in Continuous Manufacturing of Lipid-Based Therapeutics
Luke Burroughs and Diane J. Burgess
University of Connecticut
Presenting Author: Luke Burroughs, [email protected]
Corresponding Author: Diane J. Burgess, [email protected]
Purpose
Continuous manufacturing (CM) of lipid-based therapeutics requires process analytical technology (PAT) to ensure critical quality attributes (CQAs) are maintained. Process refractometers offer inline verification of formulation concentrations, particularly for cryoprotectants. While refractive index (nD) is well-characterized for single-component systems, multi-component systems in lipid-based therapeutic manufacturing remain understudied. This research investigates the complex interactions of formulation and process parameters affecting refractive index in liposome manufacturing. By exploring these non-linear relationships, this work aims to enhance the application of refractometry as a PAT tool, ultimately improving the efficiency and reliability of CM for lipid-based therapeutics.
Methods
Measurements were conducted using Vaisala's PR-23 Series Process Refractometer at 589nm (nD), both statically and in-flow with the UConn CM platform. Formulations included monodispersed liposomes (50-200 d.nm) with varying charged lipid and PEG-lipid content. Single-component systems were first characterized for sucrose, ethanol, lipid concentration, liposome size, charged lipid percentage, temperature, flow rate, and pH. Subsequently, design of experiments (DoEs) were employed to evaluate different liposome formulations, incorporating various levels of sucrose and ethanol in binary and ternary systems. This approach enabled a comprehensive analysis of factors affecting refractive index in multi-component liposome manufacturing scenarios.
Results
Single component systems showed varying impacts on refractive index, with sucrose exerting the greatest influence, followed by ethanol, temperature, lipid concentration, pH, liposome size, flow rate, and charged lipid percentage. A three-factor, three-level design of experiments (DoE) involving sucrose, ethanol, and liposome concentrations revealed slight deviations from predictions based on single component system additive effects. The interplay of multiple formulation and process parameters on the refractive index highlights the need for formulation-specific field calibrations to achieve better accuracy, as opposed to relying on single component calibration curves.
Conclusion
Process refractometers offer valuable PAT capabilities for liposomal CM, enabling detection of cryoprotectant concentrations and other formulation/process variations. However, accurate concentration measurements necessitate field calibrations incorporating all formulation components under normal process flow conditions. Significant refractive index changes during production may result from multiple interacting factors, underscoring the need for careful process design when implementing control loops. This approach ensures robust inline monitoring and enhances the reliability of CM processes for lipid-based therapeutics.
Poster 6
Evaluation of Cluster of differentiation 36 (CD36) as a critical factor in perfluorooctanesulfonic acid (PFOS)-induced adverse liver outcomes
Jingmei Zeng, Juliana Agudelo Areiza, Olga Skende, Angela L. Slitt
University of Rhode Island
Presenting Author: Jingmei Zeng, [email protected]
Purpose
Perfluorooctanesulfonic acid (PFOS) is a synthetic perfluoroalkyl substance widely present in the environment and known to adversely affect human health. PFOS causes liver enlargement, induces steatosis, and causes cytotoxicity. CD36 is a membrane glycoprotein that facilitates the import of long-chain fatty acids into cells and contributes to lipid accumulation within the liver. The PFOS structure closely resembles a long-chain fatty acid, suggesting that CD36 may facilitate PFOS uptake into the liver. In vitro and modeling studies point to CD36 having PFOS binding sites. Therefore, it was hypothesized that CD36 is a critical factor for PFOS uptake and adverse liver effects. To investigate this, a Cd36-deficient mouse model was used to evaluate the role of CD36 in PFOS distribution and liver effects.
Methods
Wild-type mice (WT, C57BL6/J) and mice with Cd36 globally deleted (Cd36-/-, B6.129S1-Cd36tm1Mfe/J) between the ages of 7-9 weeks were administered vehicle (0.5% Tween20 in PBS) or PFOS (10 mg/kg in 0.5% Tween20 in PBS) by oral garage for seven days. Liver, brain, kidneys, lungs, and plasma were collected during necropsy on the eighth day. Lipids extracted from the liver and plasma were assessed using triglyceride, cholesterol, and ALT reagent kits. Samples were read by a SpectraMax plate reader.
Results
PFOS administration increased the liver-to-body weight ratio by 66% in WT mice and 72% in Cd36-/- mice. It also increased total liver lipids concentration similarly in WT and Cd36-/- mice by ~97%. Lastly, PFOS administration elevated liver triglyceride concentration by 83.7% in WT mice and 25% increase in Cd36-/- mice.
Conclusion
Overall, these preliminary findings suggest that lack of Cd36 has a modest effect on PFOS-induced liver alterations. Ongoing studies will evaluate whether lack of Cd36 modified liver PFOS concentration.
Poster 7
Investigating the Cellular Internalization and Endosomal Escape of Polymeric Mesoscale Nanoparticles
Poojaa Jayanthi Venugopal, Adnan Arnaout, Ryan Williams
City College of New York
Presenting Author: Poojaa Jayanthi Venugopal, [email protected]
Purpose
Polymeric mesoscale nanoparticles (MNPs) are 300-400 nm particles designed for targeted drug delivery. Successful therapeutic delivery requires that these nanoparticles be efficiently internalized by cells and escape from endosomes to release their cargo into the cytoplasm. Various internalization pathways, including clathrin-mediated, caveolae-mediated, and macropinocytosis, have been observed in nanoparticle uptake. However, the dominant mechanism for MNP internalization remains unclear. Additionally, endosomal escape is a major bottleneck in nanoparticle-mediated drug delivery, as less than 2% of internalized nanoparticles successfully reach the cytoplasm. This study aims to identify the key internalization pathways utilized by MNPs and assess their ability to escape the endosomal compartments.
Methods
PLGA-PEG MNPs loaded with siGLO (fluorescent siRNA) were synthesized and characterized for size, polydispersity, and zeta potential using Dynamic Light Scattering (DLS). To investigate cellular internalization mechanisms, various specific and non-specific endocytosis inhibitors were used, including clathrin, caveolin, and macropinocytosis inhibitors. Additionally, endosomal escape was analyzed by treating kidney-relevant cells with early endosome inhibitors (EEA1, Rab5) and late endosome inhibitors (Rab7, Lamp1). Fluorescence imaging and quantitative analysis were used to assess nanoparticle uptake and trafficking.
Results
Treatment with endocytosis inhibitors led to a significant reduction in MNP fluorescence signal, with the most substantial decrease observed in cells treated with clathrin inhibitors, indicating that clathrin-mediated endocytosis is the dominant internalization pathway for these nanoparticles. Ongoing studies are focused on quantifying endosomal escape efficiency and determining the proportion of nanoparticles successfully reaching the cytoplasm.
Conclusion
This study provides critical insights into the mechanisms of MNP internalization and endosomal trafficking. Understanding these processes is essential for optimizing nanoparticle design to improve intracellular delivery efficiency. Future work will focus on strategies to enhance endosomal escape for more effective therapeutic applications.
Poster 8
In Vitro Lipolysis Model to Predict Food Effect of Poorly Water-Soluble Drugs Itraconazole, Rivaroxaban, and Ritonavir
Roshni P. Patel and James E. Polli
University of Maryland Baltimore
Presenting Author: Roshni P. Patel, [email protected]
Corresponding Author: James E. Polli, [email protected]
Purpose
Predicting the positive food effect of oral formulations due to food-mediated dissolution enhancement of lipophilic drugs is desirable. This study assesses the ability of in vitro lipolysis to predict a positive food effect.
Methods
Formulations of rivaroxaban and itraconazole were tested, where some but not all, exhibit a positive food effect in vivo. Amorphous solid dispersions (ASDs) of ritonavir, which show a negative food effect in vivo, were also studied. Fe-lipolysis and Fa-lipolysis media, mimicking fed and fasted intestinal conditions, were employed. Fe-lipolysis media included porcine bile extract, Trizma maleate, sodium chloride, L-α phosphatidylcholine, and sesame oil-incubated drug. Lipolysis was initiated with freshly prepared lipase suspension, and triglyceride hydrolysis was monitored via NaOH titration. Drug quantification in the aqueous phase was performed using UPLC. Fa-lipolysis was conducted similarly, with lower bile salt concentrations, no calcium chloride, and replacement of oil and pancreatic lipase with water.
Results
Results demonstrated frequent agreement between in vitro lipolysis predictions and in vivo human outcomes. For rivaroxaban, all eight formulations were correctly predicted: 2.5mg and 10mg strengths exhibited no food effect, while 20mg showed a positive food effect. For itraconazole, all four formulations were accurately predicted: Sporanox, Sempera, and generic capsules had a food effect, whereas Tolsura did not. For ritonavir, lipolysis predicted a positive food effect for API and Norvir products; however, in vivo, these formulations exhibit a negative food effect.
Conclusion
In vitro lipolysis demonstrated favorable predictability of positive food effects for lipophilic drug formulations, successfully predicting the dose-dependent food effect of rivaroxaban and the formulation-dependent positive food effect of itraconazole. However, it failed to capture the negative food effect of ritonavir ASDs, indicating a need for further refinement of the model. Despite this limitation, in vitro lipolysis shows promise as a predictive tool for positive food effects of poorly soluble drugs.
Poster 1
Time-Dependent Relative Humidity in Nested Packaging
Vanessa M. Breslin, Brooke Tatarian, Maximilian Plajer, Maria J. Krisch, and Kenneth C. Waterman
FreeThink Technologies, Inc.
Presenting and Corresponding Author: Vanessa M Breslin, [email protected]
Purpose
The ability to model complex packaging options will expand the usefulness of the stability software ASAPprime®. Specifically, the ability to use single-layer packaging data to predict nested packaging moisture barrier behavior will provide a greater range of packaging options for modeling shelf life.
Methods
The study evaluated various packaging configurations consisting of a small HDPE bottle (75-cc) enclosed in a larger HDPE bottle (250-cc) and an LDPE bag enclosed in an HDPE bottle (250-cc) containing excipient, desiccant, or neither. The bottles were heat induction sealed and the bags were heat sealed before being exposed to a single controlled temperature and relative humidity condition (40°C/75% RH) to measure (using a data logger) the relative humidity (RH) in each compartment over 25 days. The experimental RH data for each nested packaging configuration was compared to the model predictions of RH as a function of time based on a two-compartment packaging model.
Results
The experimental RH data as a function of time are consistent with the two-compartment model predictions. As expected, the highly permeable LDPE bag inside of an HDPE bottle does not have a noticeable influence when predicting the RH change over time. Differences observed between some experimental data and model predictions are likely due to the data logger itself absorbing a significant amount of moisture.
Conclusion
A two-compartment packaging model that uses single-layer packaging data to predict the change in RH as a function of time in nested packaging shows reasonable agreement with the experimental data, with differences likely due to moisture absorption by the data loggers used in the measurements. Ongoing work will include incorporating moisture sorption of the data loggers for inclusion in the model prediction.
Poster 2
Targeted Delivery of siRNA to the Kidneys Using Mesoscale Lipid Nanoparticles
Anastasiia Vasylaki, Shakuntala Sookraj, Pratyusha Ghosh, Ryan Williams
Department of Biomedical Engineering, City College of New York
Presenting Author: Anastasiia Vasylaki, [email protected]
Corresponding Author: Ryan Williams, [email protected]
Purpose
Healthcare faces a significant challenge with the rising prevalence of acute and chronic kidney diseases, with limited treatment options. It has been demonstrated that nanoparticles in the mesoscale size range (300 – 400 nm) selectively target the proximal tubular epithelial cells in the kidneys. Lipid nanoparticles have proven successful in delivering nucleic acid therapeutics, however, kidney targeting of mesoscale nanoparticles has only been demonstrated with polymeric carriers. This research aims to develop a new lipid-based mesoscale nanoparticle formulation for delivering nucleic acid therapies as kidney disease treatment.
Methods
Mesoscale lipid nanoparticle (MLNP) formulation was prepared containing siRNA cargo, an ionizable lipid, a phospholipid, cholesterol, and a PEGylated lipid, using the nanoprecipitation method. MLNP size, PDI, and zeta potential were characterized via DLS. siRNA encapsulation efficiency was measured using RiboGreen assay. MLNP cytotoxicity to renal epithelial cells was tested using MTT assay. siRNA release from MLNPs was studied in PBS at 37°C. To assess LNP biodistribution, healthy C57/BI/6J mice were injected IV with 50 mg/kg of fluorescently labeled MLNPs. Different groups of mice were sacrificed at 1, 3, 5, and 7 days post-injection and organs were collected for fluorescent imaging.
Results
Five formulation modifications were tested to increase the size of lipid nanoparticles to the mesoscale range (300 – 400 nm). Increasing total lipid concentration showed the largest effect on size. To achieve stable PEGylation required for kidney targeting, the lipid composition of MLNPs has been optimized. Cell viability assays demonstrated that MLNP treatment does not affect the viability of renal epithelial cells. MLNP siRNA release profile showed sustained drug release over 72 h. In the biodistribution study, MLNPs primarily accumulated in the kidneys with a kidney/liver ratio of 2.18 after 24 h and >5.75 at the later time points.
Conclusion
In the current work, we have developed the first, to our knowledge, mesoscale lipid nanoparticle formulation optimized for targeted nucleic acid delivery to the kidneys. The MLNP formulation has demonstrated preliminary indications of safety and high selectivity for the kidneys. Additional in vitro and in vivo studies will be conducted to apply this drug delivery system for kidney disease treatment.
Poster 3
PKPD Modeling and Quantitative Analysis of Immunotherapy Drug Pembrolizumab
Reyana Celebi and Marcel Musteata
Albany College of Pharmacy and Health Sciences
Presenting Author: Reyana Celebi, [email protected]
Purpose
This study develops an in-vitro fluidic pharmacokinetic system to assess pembrolizumab’s clearance and effect on cell growth as an alternative to animal models. Murine blood circulation dynamics are simulated within the fluidic system and integrate LC-MS/MS and SPME probes for drug quantification. PKAnalix software allows determination of clearance and elimination characteristics. This model supports and enables precise monitoring of drug metabolism and dose-dependent clearance changes such as target-mediated drug disposition. These approaches enhance pharmacokinetic monitoring of monoclonal antibodies as well as enhance immunotherapy dosing and translational research in oncology.
Methods
The fluidic apparatus consists of a mechanical arrangement of pumps and tubing that mimic the circulatory system of a mouse. Tubing carries drug solution from the injection point to the mixing chamber, then through the system circulation to mimic blood flow. Other features entail adjustable flow rates and a port for elimination and sample collection. With this system, drug concentration changes are monitored over time. Clearance kinetics were assessed in optimization trials with methyl blue injections.
Pembrolizumab samples collected over a time course were analyzed and quantified using LC-MS/MS with a Shimadzu LC system and AB Sciex QTRAP 5500 mass spectrometer, employing a Phenomenex Kinetex C18 column. A closed-loop fluidic system was used to evaluate pembrolizumab stability and adsorption within the system, which was quantified by LC-MS. SPME is a common microsampling method of analysis that was validated in previous studies.
Results
Data from optimization and validation trials showed calculated clearance values aligned with the elimination pump flow rate. Data plots by PKAnalix visually presented a lack of outliers and an expected profile of a multiple-dose regimen, in an IV Bolus, one compartment model. The system was validated to show the elimination pump provided the calculated clearance of methyl blue in the fluidic system. PEG minimized adsorption while maintaining signal intensity. Closed-loop trials confirmed pembrolizumab’s stability over time, validating the system’s ability to maintain drug integrity for multiple days.
Conclusion
This study successfully optimized a fluidic pharmacokinetic apparatus and system for creating realistic pharmacokinetic profiles, optimized quantification of pembrolizumab by LC-MS/MS, was validated using PKAnalix, and explored quantification of pembrolizumab by SPME. These findings support the fluidic pharmacokinetic generator apparatus and LC-MS/MS quantification as valuable tools for simulating monoclonal antibody pharmacokinetics and exploring its effect on cells.
Poster 4
Albumin is a critical factor contributing to plasma per- and polyfluoroalkyl substances (PFAS) concentration, accumulation, and retention in vivo
Olga Skende(1), Emily Kaye(1),Simon Vojta(2), Jitka Becanova(2), Rainer Lohmann(2), Fabian Fischer(1), Angela L. Slitt(1)
(1) Department of Biomedical and Pharmaceutical Sciences, University of Rhode Island (2) Graduate School of Oceanography, University of Rhode Island
Presenting Author: Olga Skende, [email protected]
Corresponding Author: Angela L. Slitt, [email protected]
Purpose
Per- and polyfluoroalkyl substances (PFAS) make up a class of synthetic molecules composed of chain-linked carbon- fluorine bonds. There are 10,000+ PFAS, with perfluorooctane sulfonate (PFOS) and perfluorohexane sulfonate (PFHxS) being some most frequently detected PFAS in serum of the United States general population. Newer PFAS, such as perfluorobutane sulfonic acid (PFBS) and 6:2-fluorotelomersulfonic acid (6:2 FTS), are now also detected in human serum and milk. As a class of chemicals, PFAS undergo little to no biotransformation and are slowly excreted. PFOS and PFHxS exhibit a strong binding affinity for serum albumin, which is hypothesized to be the predominant mechanism that dictates their slow elimination from the body. It is thought that albumin binding retains PFAS in the circulation, and prevents PFAS from undergoing renal excretion. However, to date, albumin binding as a critical factor for PFAS elimination has only been studied and modeled using in vitro assays. Thus, it was hypothesized that mice deficient in albumin would have reduced plasma PFAS concentrations due to decreased PFAS retention in plasma by albumin.
Methods
C57BL/6J mice expressing (Alb+/+) or lacking (Alb-/-) albumin were administered a single oral dose of a PFAS cocktail consisting of 0.1mg/kg PFOS, PFHxS, PFBS, and 6:2FTS at equal ratios (10 ml/kg, n=4-6/group). Plasma was collected via the submandibular vein over the course of 12 weeks at 1, 4, 24, and 72 hours, followed by 1, 2, 4, 6, 8, and 10 weeks. At 12 weeks post PFAS dosing, plasma and tissues were collected.
Results
Samples were processed using QuEtCheERS extraction material and prepared for LC/MS analysis. Plasma PFOS, PFHxS, PFBS, and 6:2FTS concentrations were markedly lower in Alb-/- mice as compared to Alb+/+ 1 hour, 4 hours, 24 hours, 72 hours, 1 week, 2 weeks, and 4 weeks after PFAS administration. Sample analysis for remaining time points and tissues is ongoing.
Conclusion
This is the first study to prove that albumin is a critical factor contributing to plasma PFAS concentration, accumulation, and retention in vivo.
Poster 5
Optimizing Process Analytical Technology: Refractometer in Continuous Manufacturing of Lipid-Based Therapeutics
Luke Burroughs and Diane J. Burgess
University of Connecticut
Presenting Author: Luke Burroughs, [email protected]
Corresponding Author: Diane J. Burgess, [email protected]
Purpose
Continuous manufacturing (CM) of lipid-based therapeutics requires process analytical technology (PAT) to ensure critical quality attributes (CQAs) are maintained. Process refractometers offer inline verification of formulation concentrations, particularly for cryoprotectants. While refractive index (nD) is well-characterized for single-component systems, multi-component systems in lipid-based therapeutic manufacturing remain understudied. This research investigates the complex interactions of formulation and process parameters affecting refractive index in liposome manufacturing. By exploring these non-linear relationships, this work aims to enhance the application of refractometry as a PAT tool, ultimately improving the efficiency and reliability of CM for lipid-based therapeutics.
Methods
Measurements were conducted using Vaisala's PR-23 Series Process Refractometer at 589nm (nD), both statically and in-flow with the UConn CM platform. Formulations included monodispersed liposomes (50-200 d.nm) with varying charged lipid and PEG-lipid content. Single-component systems were first characterized for sucrose, ethanol, lipid concentration, liposome size, charged lipid percentage, temperature, flow rate, and pH. Subsequently, design of experiments (DoEs) were employed to evaluate different liposome formulations, incorporating various levels of sucrose and ethanol in binary and ternary systems. This approach enabled a comprehensive analysis of factors affecting refractive index in multi-component liposome manufacturing scenarios.
Results
Single component systems showed varying impacts on refractive index, with sucrose exerting the greatest influence, followed by ethanol, temperature, lipid concentration, pH, liposome size, flow rate, and charged lipid percentage. A three-factor, three-level design of experiments (DoE) involving sucrose, ethanol, and liposome concentrations revealed slight deviations from predictions based on single component system additive effects. The interplay of multiple formulation and process parameters on the refractive index highlights the need for formulation-specific field calibrations to achieve better accuracy, as opposed to relying on single component calibration curves.
Conclusion
Process refractometers offer valuable PAT capabilities for liposomal CM, enabling detection of cryoprotectant concentrations and other formulation/process variations. However, accurate concentration measurements necessitate field calibrations incorporating all formulation components under normal process flow conditions. Significant refractive index changes during production may result from multiple interacting factors, underscoring the need for careful process design when implementing control loops. This approach ensures robust inline monitoring and enhances the reliability of CM processes for lipid-based therapeutics.
Poster 6
Evaluation of Cluster of differentiation 36 (CD36) as a critical factor in perfluorooctanesulfonic acid (PFOS)-induced adverse liver outcomes
Jingmei Zeng, Juliana Agudelo Areiza, Olga Skende, Angela L. Slitt
University of Rhode Island
Presenting Author: Jingmei Zeng, [email protected]
Purpose
Perfluorooctanesulfonic acid (PFOS) is a synthetic perfluoroalkyl substance widely present in the environment and known to adversely affect human health. PFOS causes liver enlargement, induces steatosis, and causes cytotoxicity. CD36 is a membrane glycoprotein that facilitates the import of long-chain fatty acids into cells and contributes to lipid accumulation within the liver. The PFOS structure closely resembles a long-chain fatty acid, suggesting that CD36 may facilitate PFOS uptake into the liver. In vitro and modeling studies point to CD36 having PFOS binding sites. Therefore, it was hypothesized that CD36 is a critical factor for PFOS uptake and adverse liver effects. To investigate this, a Cd36-deficient mouse model was used to evaluate the role of CD36 in PFOS distribution and liver effects.
Methods
Wild-type mice (WT, C57BL6/J) and mice with Cd36 globally deleted (Cd36-/-, B6.129S1-Cd36tm1Mfe/J) between the ages of 7-9 weeks were administered vehicle (0.5% Tween20 in PBS) or PFOS (10 mg/kg in 0.5% Tween20 in PBS) by oral garage for seven days. Liver, brain, kidneys, lungs, and plasma were collected during necropsy on the eighth day. Lipids extracted from the liver and plasma were assessed using triglyceride, cholesterol, and ALT reagent kits. Samples were read by a SpectraMax plate reader.
Results
PFOS administration increased the liver-to-body weight ratio by 66% in WT mice and 72% in Cd36-/- mice. It also increased total liver lipids concentration similarly in WT and Cd36-/- mice by ~97%. Lastly, PFOS administration elevated liver triglyceride concentration by 83.7% in WT mice and 25% increase in Cd36-/- mice.
Conclusion
Overall, these preliminary findings suggest that lack of Cd36 has a modest effect on PFOS-induced liver alterations. Ongoing studies will evaluate whether lack of Cd36 modified liver PFOS concentration.
Poster 7
Investigating the Cellular Internalization and Endosomal Escape of Polymeric Mesoscale Nanoparticles
Poojaa Jayanthi Venugopal, Adnan Arnaout, Ryan Williams
City College of New York
Presenting Author: Poojaa Jayanthi Venugopal, [email protected]
Purpose
Polymeric mesoscale nanoparticles (MNPs) are 300-400 nm particles designed for targeted drug delivery. Successful therapeutic delivery requires that these nanoparticles be efficiently internalized by cells and escape from endosomes to release their cargo into the cytoplasm. Various internalization pathways, including clathrin-mediated, caveolae-mediated, and macropinocytosis, have been observed in nanoparticle uptake. However, the dominant mechanism for MNP internalization remains unclear. Additionally, endosomal escape is a major bottleneck in nanoparticle-mediated drug delivery, as less than 2% of internalized nanoparticles successfully reach the cytoplasm. This study aims to identify the key internalization pathways utilized by MNPs and assess their ability to escape the endosomal compartments.
Methods
PLGA-PEG MNPs loaded with siGLO (fluorescent siRNA) were synthesized and characterized for size, polydispersity, and zeta potential using Dynamic Light Scattering (DLS). To investigate cellular internalization mechanisms, various specific and non-specific endocytosis inhibitors were used, including clathrin, caveolin, and macropinocytosis inhibitors. Additionally, endosomal escape was analyzed by treating kidney-relevant cells with early endosome inhibitors (EEA1, Rab5) and late endosome inhibitors (Rab7, Lamp1). Fluorescence imaging and quantitative analysis were used to assess nanoparticle uptake and trafficking.
Results
Treatment with endocytosis inhibitors led to a significant reduction in MNP fluorescence signal, with the most substantial decrease observed in cells treated with clathrin inhibitors, indicating that clathrin-mediated endocytosis is the dominant internalization pathway for these nanoparticles. Ongoing studies are focused on quantifying endosomal escape efficiency and determining the proportion of nanoparticles successfully reaching the cytoplasm.
Conclusion
This study provides critical insights into the mechanisms of MNP internalization and endosomal trafficking. Understanding these processes is essential for optimizing nanoparticle design to improve intracellular delivery efficiency. Future work will focus on strategies to enhance endosomal escape for more effective therapeutic applications.
Poster 8
In Vitro Lipolysis Model to Predict Food Effect of Poorly Water-Soluble Drugs Itraconazole, Rivaroxaban, and Ritonavir
Roshni P. Patel and James E. Polli
University of Maryland Baltimore
Presenting Author: Roshni P. Patel, [email protected]
Corresponding Author: James E. Polli, [email protected]
Purpose
Predicting the positive food effect of oral formulations due to food-mediated dissolution enhancement of lipophilic drugs is desirable. This study assesses the ability of in vitro lipolysis to predict a positive food effect.
Methods
Formulations of rivaroxaban and itraconazole were tested, where some but not all, exhibit a positive food effect in vivo. Amorphous solid dispersions (ASDs) of ritonavir, which show a negative food effect in vivo, were also studied. Fe-lipolysis and Fa-lipolysis media, mimicking fed and fasted intestinal conditions, were employed. Fe-lipolysis media included porcine bile extract, Trizma maleate, sodium chloride, L-α phosphatidylcholine, and sesame oil-incubated drug. Lipolysis was initiated with freshly prepared lipase suspension, and triglyceride hydrolysis was monitored via NaOH titration. Drug quantification in the aqueous phase was performed using UPLC. Fa-lipolysis was conducted similarly, with lower bile salt concentrations, no calcium chloride, and replacement of oil and pancreatic lipase with water.
Results
Results demonstrated frequent agreement between in vitro lipolysis predictions and in vivo human outcomes. For rivaroxaban, all eight formulations were correctly predicted: 2.5mg and 10mg strengths exhibited no food effect, while 20mg showed a positive food effect. For itraconazole, all four formulations were accurately predicted: Sporanox, Sempera, and generic capsules had a food effect, whereas Tolsura did not. For ritonavir, lipolysis predicted a positive food effect for API and Norvir products; however, in vivo, these formulations exhibit a negative food effect.
Conclusion
In vitro lipolysis demonstrated favorable predictability of positive food effects for lipophilic drug formulations, successfully predicting the dose-dependent food effect of rivaroxaban and the formulation-dependent positive food effect of itraconazole. However, it failed to capture the negative food effect of ritonavir ASDs, indicating a need for further refinement of the model. Despite this limitation, in vitro lipolysis shows promise as a predictive tool for positive food effects of poorly soluble drugs.
