Pharmaceutics

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Association of Gemcitabine and Paclitaxel to Drug-combination Nanoparticles Leads to Long-acting Pharmacokinetics, Enhances Plasma and Pancreatic Drug Exposure Relating to Tumor Regression: Interventional Studies in an Orthotopic Pancreatic Tumor Model
(2026-04-20) Wright, Catherine Sophaline; Ho, Rodney JY
Pancreatic cancer is one of the deadliest forms of cancer for which there is no cure available. Current first-line treatment regimens of drugs have made progress in improving the overall prognosis of pancreatic cancer but are ultimately limited by systemic toxicity from off-target drug exposure due to their inability to effectively localize to the tumor. Equipped with a reliable mouse model of pancreatic cancer and a novel technology called DcNP that enables producing a combination drug product containing two disparately water-soluble gemcitabine (G) and insoluble paclitaxel (T), this thesis research has evaluated the ability of GT-in-DcNP to enhance pancreatic tumor exposure and impact tumor progression. We found that a single dose of GT-in-DcNP administered intraperitoneally (IP) can lead to prolonged tumor regression that cannot be accomplished with GT in a free soluble formulation nor with GT-in-DcNP dosed intravenously. Future investigation on the impact of multiple IP doses of GT-in-DcNP may provide a better understanding of the ability of this route, dosing frequency, and additional optimization of dosage formulation to potentially eliminate pancreatic cancer. With additional refinement, validation, and safety studies, the findings of this research may serve as a targeted drug-combination strategy for consideration of further development as a product intended for the treatment of people living with pancreatic cancer.
Transcriptomic Profiling of SLC and ABC Transporters in the Human Term Placenta
(2026-04-20) Jung, Elijah M; Wang, Joanne
Solute carriers (SLC) and ATP-binding cassette (ABC) transporters are essential for placental solute exchange and fetal protection, yet their transcriptomic profiles in the human placenta remain poorly characterized. Moreover, although fetal sex influences placental development and function, its impact on transporter expression is yet to be determined. Using RNA sequencing, we profiled SLC and ABC transporter expression in two anatomical regions of healthy term human placentas with balanced fetal sex representation. In the intervillous region, 276 SLCs were detected, with the majority (60%) expressed at low levels (CPM < 25). The remaining 110 SLCs (40%) were expressed at higher levels (CPM 25–1650). Four transporters—SLC2A1 (GLUT1), SLC44A2 (CTL2), SLC38A2 (SNAT2), and SLC38A1 (SNAT1)—showed the highest expression (CPM > 500). Functional annotation of the top 110 SLCs revealed that transporters for metals, amino acids, and inorganic ions with each respectively accounting for 9% of the 110 annotated SLC genes. Other well-represented SLCs included xenobiotic, vitamin, sugar, and neurotransmitter transporters. Mitochondrial, lysosomal, and ER/Golgi transporters together accounted for approximately 37%, while 11% were orphan transporters with no known substrates. Thirty-seven ABC transcripts were detected in the intervillous region, primarily involved in xenobiotic and lipid transport. Compared with the intervillous region, 1,676 genes—including 32 SLCs and 2 ABC transporters—were differentially expressed in the decidual region. Although 79 genes in the intervillous region and 117 genes in the decidual region exhibited sexually dimorphic expression, none were SLC transporters. Notably, placentas from male fetuses showed significantly higher expression of ABCB2 (P-gp), a finding further confirmed by qPCR and western blot analysis. Together, these findings provide a comprehensive transcriptomic map of SLC and ABC transporters in the term human placenta and suggested potential impact of fetal sex on ABCB2 and xenobiotic protection.
Drug Combination Nanoparticles Carrying Gemcitabine and Paclitaxel: Formation Mechanism Influencing Pharmacokinetics, Drug Metabolism, and Efficacy in Triple-Negative Breast Cancer Mouse Models
(2026-04-20) Xu, Xiaolin; Ho, Rodney RJYH
Triple-negative breast cancer (TNBC) is one of the most aggressive subtypes of breast cancer and remains a major clinical challenge due to the absence of hormone receptors and human epidermal growth factor receptor 2 (HER2), rapid disease progression, and limited treatment options. Conventional combination chemotherapeutics are effective but limited by poor drug solubility, rapid systemic clearance, and a lack of synchronized delivery. Addressing these challenges requires an advanced drug delivery system capable of synchronizing the delivery of chemotherapeutics with diverse physicochemical properties to tumors in a long-acting and in vivo stable manner. To address this need, our research team developed a drug combination nanoparticle (DcNP) that enable the co-assembly of physicochemical diverse drugs, such as hydrophilic gemcitabine (G) and hydrophobic paclitaxel (T), into a nanoparticle, referred to as GT-in-DcNP. We previously demonstrated that one GT-in-DcNP composition achieves long-acting pharmacokinetics and synchronized delivery of both drugs in mouse models. However, the impact of the formation mechanism on in vivo pharmacokinetics (PK), metabolic protection of gemcitabine, and therapeutic efficacy across different GT-in-DcNP compositions remains unclear. To address these questions, various GT-in-DcNP compositions were designed and first characterized in vitro, where they exhibited comparable physicochemical properties. These formulations were subsequently evaluated for in vivo pharmacokinetics and therapeutic efficacy in both early- and late-stage TNBC mouse models. Mechanistic studies revealed that key preparation processes, such as controlled solvent removal, are essential for enabling short-acting gemcitabine to transition into a long-acting form in vivo. Using subcutaneous administration, a selected GT-in-DcNP composition effectively targeted lymphatic-vessel-rich 4T1 primary tumors in the mammary fat pads. GT-in-DcNP significantly increased tumor accumulation of both drugs, approximately 10-fold higher than that of the equivalent free-drug combination, resulting in primary tumor regression and restoration of mammary fat pad tissue. For systemic drug exposure, intravenous administration was employed to treat lung-metastatic tumor nodules in a 4T1-bearing mouse model. The PK behavior of multiple GT-in-DcNP compositions was first evaluated in healthy mice, confirming long-acting PK for both drugs across all formulations. In parallel, DcNP was shown to protect gemcitabine from rapid metabolism by cytidine deaminase, which likely contributes to its prolonged systemic exposure. This protective effect also enabled synchronized delivery of both drugs, with approximately 98% in vivo association efficiency of gemcitabine across compositions. Taken together, this dissertation elucidates the critical role of formulation mechanisms in enabling long-acting and synchronized delivery of physicochemical diverse drugs at various ratios using the DcNP platform, as demonstrated by GT-in-DcNP.
Regulation of Human Renal Drug Transporters by Inflammatory Cytokines and Pregnancy-related Hormones in Primary Proximal Tubular Epithelial Cells
(2026-02-05) Tsang, Yik Pui; Kelly, Edward J
Systemic inflammation and pregnancy both alter drug pharmacokinetics by changing physiology and modulating drug-metabolizing enzymes and transporters in eliminating organs. Regulation of hepatic enzymes in these states is relatively well described. However, the magnitude, direction, and mechanisms of renal transporter regulation remain poorly defined. This is despite clinical data suggesting that tubular secretory clearance can change independently of glomerular filtration rate and protein binding. Inflammation is characterized by elevated circulating pro-inflammatory cytokines, whereas pregnancy is accompanied by a distinct endocrine milieu with increased concentrations of multiple pregnancy-related hormones (PRHs). These soluble mediators are well known to regulate hepatic drug metabolizing enzymes and transporters (DMETs) and therefore represent plausible mechanistic drivers of renal transporter regulation. But mechanistic human data are lacking on whether and how inflammatory cytokines and pregnancy-related hormones (PRHs) regulate the major renal uptake and efflux transporters expressed in proximal tubular epithelial cells (PTECs). Published in vitro studies are further limited by non-selective transporter probes and by PTEC models that rapidly lose the expression and activity of key transporters. The overarching goal of this dissertation was to define how inflammatory cytokines and PRHs regulate human renal drug transporters in vitro, and to provide experimentally anchored parameters that can be incorporated into physiologically based pharmacokinetic (PBPK) models of inflammation, pregnancy, and their co-occurrence. The central hypothesis was that clinically relevant cytokine and hormone exposures cause coordinated, transporter-specific changes in mRNA expression and functional activity in human PTECs. To test this hypothesis, I pursued three specific aims: (1) establish an in vitro human PTEC system to quantify the mRNA expression and activity of individual renal transporters, including identification of selective substrate or substrate-inhibitor pairs for major uptake transporters; (2) quantify the effects of pro- and anti-inflammatory cytokines on renal transporter expression and activity; and (3) quantify the effects of trimester-matched PRH cocktails on renal transporter expression and activity. In Aim 1a, I systematically evaluated the selectivity of six candidate substrates (cidofovir, nicotinic acid, glycochenodeoxycholic acid sulfate [GCDCA-S], levocetirizine, ergothioneine, and atenolol) for the major renal uptake transporters (OAT1–4, OCT2, OCTN1/2) and for renal efflux transporters (P-gp, BCRP, MRPs, MATE1/2-K) using transporter-overexpressing HEK293 and MDCK-II cells and Sf9 membrane vesicles. Cidofovir, levocetirizine, and ergothioneine were confirmed as highly selective substrates of OAT1, OAT4, and OCTN1, respectively. Nicotinic acid was transported primarily by OAT2 but also by OAT1 and OAT3. GCDCA-S was transported by OAT3 and MRP2, and atenolol was transported by OCT2 and MATE1/2-K. Selective inhibitor combinations (e.g., nicotinic acid + quercetin for OAT2, GCDCA-S + cyclosporine A for OAT3, atenolol + mitoxantrone for OCT2) were identified to disentangle overlapping transport and establish selectivity towards OAT2, OAT3, and OCT2. These data provided a practical panel of substrates and substrate-inhibitor pairs that enables uptake transporter-specific activity measurements in primary human PTECs. In Aim 1b, I optimized an isolation and culture workflow for freshly isolated human PTECs seeded on Matrigel-coated Transwell inserts. This system preserved proximal tubule phenotype and maintained measurable OAT1–4, OCT2, and OCTN1 mRNA expression and activity over a defined 5–7-day assay window, overcoming the rapid dedifferentiation and loss of OAT activity observed previously in conventional flat-plate cultures. The combination of the selective substrates from Aim 1a and the optimized Transwell system provided a human-relevant platform for studying cytokine- and hormone-mediated regulation of renal transporters. In Aim 2, I first used plated PTECs to quantify how pathophysiologic concentrations of IL-6, IL-1β, TNF-α, and IFN-γ, tested individually and as a cocktail, altered renal transporter mRNA expression. Exposure to the cytokine cocktail for 48 h significantly downregulated mRNA expression of OCT2, OATP4C1, OAT4, MATE2-K, P-gp, and MRP2 and upregulated OCTN1 and MRP3, with IL-1β emerging as the main perpetrator. Building on these findings, I then used the Transwell human PTEC system to quantify both the mRNA expression of renal DMETs and the activity of uptake transporters (OAT1–4, OCT2, and OCTN1). IL-1β at 0.1 to 1 ng/mL robustly downregulated OAT1–3, OATP4C1, OCT2, OAT4, MATE2-K, MRP2, PEPT2, and endocytic receptors (cubilin, megalin), while inducing OCTN1 and MRP1/3. TNF-α reproduced the effects of IL-1β but only on OAT1–3. Functional assays showed concordant IL-1β-driven decreases in OAT1–3, OAT4, and OCT2 activity and increased OCTN1 activity. Mechanistic experiments with ERK, p38MAPK, JNK, and NF-κB inhibitors demonstrated that IL-1β suppresses OAT1/3 via JNK, OAT2 via p38MAPK, and induces OCTN1 via NF-κB. IL-6 classic and trans-signaling did not reproduce IL-1β-driven changes in transporter mRNA, despite IL-6 being a potent regulator for many hepatic DMET genes. These mechanistic, exposure-verified data can be used to inform PBPK models to predict renal secretory clearance and pathway-mediated drug interactions during inflammation. In Aim 3, I investigated whether PRHs drive the increased renal secretory clearance observed in pregnancy. Primary PTECs from three premenopausal female donors were exposed for 72 h to trimester-matched PRH cocktails (estrogens, progesterone, cortisol, testosterone, oxytocin, GH, and PGH) at physiologic (1×) or supraphysiologic (10×) concentrations, with medium refreshed every 24 h. At 1×, PRHs produced no significant changes in renal transporter, DME, or endocytic receptor mRNA expression or in uptake transporter activity, except for consistent downregulation of PEPT2. At 10×, selective mRNA changes emerged (e.g., induction of OAT1, OAT4, and MRP3; suppression of OCT2, OATP4C1, MATE2-K, MRP2, MRP4, and PEPT2), but these did not translate into measurable changes in OAT1–3, OAT4, OCT2, or OCTN1 activity. These findings argue against physiologic PRH concentrations as the primary driver of increased OAT-mediated secretion during pregnancy and point instead toward alternative mechanisms such as flow-dependent mechanotransduction and regulation by other hormones (e.g., prolactin, hCG). Together, this dissertation research delivers a validated workflow for quantifying individual renal transporter activities in human PTECs, a robust Transwell system that preserves proximal tubule transporter phenotype, and mechanistic, exposure-verified data of how inflammatory cytokines, but not physiologic PRHs, regulate renal drug transporters. These data can be integrated into PBPK models to predict inflammation-mediated changes in renal secretory clearance, and to refine dose selection for vulnerable populations experiencing acute or chronic inflammation, with or without pregnancy.
Characterization of Understudied Risk Factors in Chronic Kidney Disease
(2026-02-05) Mahadeo, Anish; Kelly, Edward J
Chronic kidney disease (CKD) is a major global health concern and is increasingly recognized as a leading cause of mortality. Furthermore, environmental causes of CKD are being acknowledged as this contributes to CKD of unknown etiology (CKDu), which is becoming an increasing burden in developing countries globally. A key pathophysiology reflects tubulointerstitial pathways in which the proximal tubule fails to complete adaptive repair after repeated subclinical acute kidney injury (AKI). The current hypothesis is that CKDu manifests hot, agricultural regions, where recurrent heat/dehydration interacts with local environmental exposures (water chemistry, metals, agrochemicals, mycotoxins). This dissertation investigates the contribution of one proposed CKDu driver, ochratoxin A (OTA). It will also explore the kidney injury mechanisms of post-translationally modified albumin using human-relevant in-vitro systems. This latter set of experiments sheds light on CKD more generally.Chapter 2 defines OTA’s mechanistic toxicology in human proximal tubule cells. RNA-seq, live/confocal imaging, and bioenergetic flux analyses show that OTA disrupts mitochondrial network homeostasis (fragmentation and hyperfusion), elevates mitochondrial ROS, and depresses oxidative phosphorylation and ATP generation at low-to-moderate, human-relevant concentrations. Cells exposed to OTA shift to a quiescent metabolic phenotype without compensatory glycolysis, consistent with a senescent-like state. Together, these data position mitochondrial dysfunction and blunted cytoprotective signaling as proximal events in OTA-mediated injury. Chapter 3 resolves renal accumulation mechanisms of OTA. A human transporter screen and kinetics identify high-affinity basolateral uptake via OAT1/3 and apical reabsorption via OAT4 and OATP1A2 with limited, non-saturable efflux via BCRP. Natural-product interaction studies show luteolin inhibits and caffeic acid activates BCRP-mediated efflux at physiologically relevant intestinal concentrations, suggesting potential modulation of intestinal absorption. Next, chapter 4 tests whether post-translationally modified albumin, a sustained proteinuric exposure in kidney and diabetic disease, amplifies proximal tubule injury mechanisms. In a 3D culture system, carbamylated albumin and to a lesser extent glycated albumin induces a robust, AKI-like program: TGF-β/SMURF2 activation, membrane/transport remodeling, cytokine release, and suppression of metallothionein-1 paralogs, aligning with Kidney Precision Medicine Project AKI transcriptomes. Overall, this work shines a light on understudied drivers of CKD, providing a mechanistic, bottom-up analysis and comparison of potential nephrotoxins to clinically relevant disease signatures that have been previously established. This work highlights the importance of mechanistic understanding of CKD risk factors that can be used to employ future mitigation and treatment of disease.
A Systems Approach to Vitamin A Homeostasis: Integrating Proteomics, Physiology, and Hormonal Modulation of Retinol, RBP4, and TTR
(2025-10-02) Yadav, Aprajita S; Isoherranen, Nina
Vitamin A (retinol) is an essential lipophilic micronutrient that plays critical roles in vision, immune function, development, and metabolic homeostasis. In circulation, retinol is bound to retinol binding protein 4 (RBP4), which in turn forms a complex with transthyretin (TTR). Formation of this higher molecular weight complex prevents glomerular filtration of RBP4. In the absence of nutritional deficiency or disease, circulating concentrations of retinol, RBP4, and TTR are thought to be relatively stable within an individual. The underlying sources of inter-individual variability in circulating retinol, RBP4, and TTR concentrations are not completely understood. Moreover, retinol, RBP4, and TTR are rarely quantified together in the same individual, limiting insight into how their concentrations or binding states may differ by individual or disease status, particularly in obesity and diabetes. This dissertation integrates proteomic quantification, clinical data, and kinetic modeling to investigate the sources of inter-individual variability in circulating retinol, RBP4, and TTR concentrations. To enable accurate quantitation, a targeted LC-MS/MS assay was developed and validated for absolute measurement of RBP4 and TTR in serum and plasma. The method allows for concurrent absolute quantification of RBP4 and TTR concentrations by using well characterized protein standards, robust surrogate peptides, stable isotope-labeled peptides for internal standards, and a process control to monitor digestion efficiency. Method accuracy across different biological matrices was confirmed with parallelism and matrix effect experiments. The assay was applied to samples from both healthy individuals and patients with diabetic kidney disease. Measured intra-individual variability in RBP4 and TTR concentrations was comparable to assay variance (6 – 8.5%), highlighting the low intra-individual variability of RBP4 and TTR concentrations in circulation. Compared to ELISA-based RBP4 and TTR measurements in samples from individuals with diabetic kidney disease, our LC-MS/MS assay showed overall agreement. Measurement of absolute concentrations of both RBP4 and TTR, combined with measurement of retinol concentrations, in serum enabled development and application of a kinetic binding model to predict complex formation between RBP4, TTR, and retinol using experimentally defined dissociation constants. The validated method was then applied to a carefully selected cohort of metabolically healthy individuals with a range of BMI (21–56 kg/m2), none of whom were diagnosed with diabetes, hepatic, or renal impairment. Prior studies have reported conflicting associations between serum RBP4 and BMI: some identified positive associations, while others reported no correlation. These discrepancies have been attributed to differences in study populations, including sex distribution, disease status, and critically, the analytical methods used to quantify RBP4. In the cohort studied here, serum RBP4 and TTR concentrations did not correlate with BMI. Instead, sex and age emerged as significant predictors of both serum TTR (p=0.02) and RBP4 (p=0.009) concentrations. A modest negative association between BMI and total serum retinol was observed (p=0.02). Using the developed kinetic binding model parameterized with experimentally derived dissociation constants, the majority of total RBP4 was predicted to be complexed with TTR, primarily as the ternary complex (retinol:RBP4:TTR). The model predicts that approximately 24% of total circulating RBP4 is unbound to retinol but remains complexed with TTR, highlighting RBP4:TTR as a distinct and previously underappreciated binding species. These findings reinforce the role of TTR in increasing RBP4 half-life by preventing glomerular filtration of RBP4. Results from the kinetic model further suggested that the sex differences in total RBP4 concentrations may be driven by differences in TTR concentrations between men and women. To explore hormonal regulation of sex differences, we examined the effects of estradiol and cortisol on retinoid and binding protein concentrations in two clinical studies. In postmenopausal women administered oral estradiol, linear mixed-effects modeling identified an inverse association between serum estradiol and RBP4 concentrations. In premenopausal women, oral hydrocortisone administration increased serum 13-cis-retinoic acid (13cisRA) concentrations, which were associated with cortisol exposure. These findings provide new clinical evidence that estradiol and cortisol may modulate circulating retinoids and their binding proteins, though further investigation is warranted. Collectively, this dissertation demonstrates the value of integrating quantitative proteomics with kinetic modeling to further our understanding of vitamin A homeostasis. These results highlight the importance of sex, age, and hormone exposure in modulating circulating retinoids and binding proteins and underscore how comorbidities such as hepatic or renal impairment can complicate interpretation of retinoid, RBP4, and TTR measurements. These findings also have implications for the utility of RBP4 as a biomarker, suggesting that concurrent measurement of retinol and TTR and inclusion of sex as a covariate should be considered in clinical studies investigating diagnostic and prognostic potential of RBP4 serum concentrations in disease states. Further studies on the relationships between retinol, RBP4, and TTR concentrations in populations with metabolic, hepatic, or renal disease are warranted. Together, this work contributes to a more clinically relevant understanding of vitamin A homeostasis.
Use of a Genetically Modified Cell Culture Model to Investigate Human Breast Cancer Resistance Protein-mediated Milk Secretion of Drugs
(2025-10-02) Lee, Ahram; Wang, Joanne
Over 90% of breastfeeding women use at least one medication, which can expose their infants to drugs and potential toxicity. The Breast Cancer Resistance Protein (BCRP) transporter, encoded by the ABCG2 gene, is extensively studied in lactation. BCRP protein expression is elevated in lactating mammary epithelial cells (MECs). It is localized on the apical membrane of the MEC, where it plays a pivotal role in the secretion of endogenous and exogenous compounds into breast milk. BCRP actively transports nutrients such as riboflavin into milk, supporting infant development. However, BCRP can actively transport drugs and other xenobiotics into breast milk, increasing the risk of infant drug exposure and toxicity. Currently, there is no validated in vitro human mammary epithelial cell (hMEC) model to assess BCRP-mediated drug secretion during lactation. The goal of this study is to validate the MDCK-hBCRPcMDR1KO cell line as an in vitro model for evaluating BCRP-mediated drug transport into breast milk under physiologically relevant pH conditions. The MDCK-hBCRPcMDR1KO cell line that was engineered to overexpress human BCRP while lacking endogenous canine P-glycoprotein (P-gp), allows us to isolate BCRP-mediated transport without confounding effects from canine P-gp, which shares overlapping substrate specificity with BCRP. that eliminated substrate overlaps, We conducted bidirectional transport assays with cimetidine at apical pH 7.0 (human breast milk), pH 6.5 (intestine), and pH 7.4 (plasma). There was an approximately 2-fold increase in the efflux ratio compared to the MDCKcMDR1KO (control) cells, and the B-to-A transport of cimetidine was inhibited by the BCRP inhibitor KO143. Cimetidine transport was comparable across the three pHs. Our data suggest that, in contrast to membrane vesicles that showed increased BCRP activity under acidic conditions, pH had little impact on cimetidine transport in the monolayer model. These findings highlight that the MDCK-hBCRPcMDR1KO in vitro system is a useful system to study BCRP-mediated drug transport and should be further investigated as a versatile platform to evaluate BCRP-mediated drug secretion and drug-nutrient interaction at the blood-milk barrier.
Development of a lipid-based modular drug-RNA co-delivery nanoparticle approach for breast cancer therapy
(2025-08-01) Chen, Zhiqian; Ho, Rodney RH; Mu, Qingxin QM
Triple-negative breast cancer (TNBC) is a form of breast cancer characterized by the low expression of specific cell receptors, leading to the ineffectiveness of treatments targeting those receptors. While mono-chemotherapy was ineffective against TNBC, combined regimens of different chemotherapy drugs were selected as standard treatments against TNBC but still resulted low efficacy against metastatic breast cancer (MBC). Therapeutic nucleic acids, an alternative treatment, showed significant targeting ability and can overcome drug resistance. However, its stability problem and biological barriers made an appropriate carrier necessary. Therefore, drug-gene co-delivered nanoparticles have been explored to achieve synergistic therapeutic effects by combing the targeting ability of genes and the potency of drugs. Lipid-based nanoparticle, which showed good endosomal escape capability, has been explored as a carrier to co-deliver nucleic acid molecules and chemotherapy drugs to further enhance the multi-targeting ability of treatment and overcome biological barriers and drug resistance. In our previous studies, a lipid-based nanoparticle structure GT-in-DcNP enabled the co-delivery of two chemotherapy drugs and showed high efficacy and safety against TNBC. However, GT-in-DcNP structure was not designed for nucleic acid loading. To enable the loading of RNA molecules on GT-in-DcNP nanoparticle structure, layer-by-layer (LbL) approach was used. With a nanoparticle core, a layer of charged polymers, such as negatively charged RNA polymer or positively charged chitosan (Chi) polymer, could be coated on nanoparticle core through electrostatic interactions between opposite charges. On the outside of first polymer layer, another layer of polymers with opposite charges could be coated, further enhancing the biocompatibility, stability, or targeting according to the specific polymer coated. More than one layer of therapeutic agents could be loaded in LbL structure, which increased the loading capacity of therapeutic agents in lipid-based nanoparticles. This thesis research leverages on the previously developed GT-DCNP. The overarching goal of this study is developing a lipid-nanoparticle platform formulation approach to enable the RNA molecule loading with a LbL approach to enhance therapeutic efficacy against TNBC.In this study, DG-in-LNP core with different formulations were designed, prepared, and characterized. The LbL coating was carried out by electrostatic interactions between positively charged DG-in-LNP core, negatively charged nucleic acid molecules, and another layer of cationic polymer Chi. We validated that the addition of a cationic lipid (DOTAP) into DG-in-LNP core formulation was positively associated with nanoparticle average size and zeta potential, overturning the nanoparticle to positively charged structure and enabling the loading of negatively charged RNA molecules on its surface. Meanwhile, we added cholesterol into nanoparticle to stabilize its lipid structure. We also studied the impact of rehydration solvent and PTX loading on nanoparticle stability and surface charge. The formulation with the greatest stability and appropriate properties, including the size of 111.3 nm and zeta potential +15.6 mV, was used for further loading of RNA and Chi. In metastatic breast cancer cell lines, including 4T1 and MDA-MB-231, we found that the transfection efficiency of mRNA loaded in nanoparticle was low. Compared to nanoparticles formulated with mRNA, the siRNA nanoparticles had much smaller size and a more stable structure: The size of siRNA nanoparticle loaded with Chi was maintained under 150 nm, while the size of mRNA nanoparticle has exceeded 1000 nm. In MDA-MB-231 expressing luciferase reporter gene, siRNA nanoparticles with Chi loading and without were evaluated. We discovered that the loading of PTX in nanoparticle led to 20% to 40% higher luminescence signal. However, the increasing loading of Chi eliminated the difference in bioluminescence at the highest Chi loading ratio and overcome the disruption of siRNA delivery and transfection by PTX. Meanwhile, Chi also mitigated the toxicity induced by nanoparticle, enhancing the biocompatibility of nanoparticles. Nanoparticles loaded with PTX showed similar cytotoxicity as free PTX form, validating the successful release of PTX into cells. Overall, this study provides a novel approach that enables the loading of RNA molecules on stable lipid-based nanoparticle structure while enhancing the stability and targeting ability of nanoparticle.
(-)-Δ9-tetrahydrocannabinol and pregnancy: transporter-mediated tissue distribution and drug interactions
(2025-08-01) Chen, Xin; Unadkat, Jashvant D
As cannabis use during pregnancy increases, it is important to understand the mechanisms and extent of placental transfer of (-)-trans-Δ9-tetrahydrocannabinol (THC), the primary intoxicating constitute of cannabis, along with its circulating major metabolites. In a nonhuman primate study, the fetal plasma exposure to THC is about 30% of the maternal exposure. Similarly, in a human study of daily cannabis smokers, the average THC umbilical vein-to-maternal serum concentration ratio at delivery was 0.26 Â± 0.10 (n = 3). This reduced fetal exposure to THC in nonhuman primates and humans is likely attributed to the placenta’s ability to limit fetal exposure to xenobiotics. However, the exact mechanisms behind this protection are not fully understood. Since many transporters on both the maternal and fetal side of the syncytiotrophoblast are expressed in the placenta, we hypothesized that the reduced fetal exposure to THC could be attributed to apical (maternal-facing) efflux transporters [i.e., P-glycoprotein (P-gp) and/or breast cancer resistance protein (BCRP)], or basal (fetal-facing) uptake transporters [i.e., organic anion transporting polypeptide 2B1 (OATP2B1), organic cation transporter (OCT3), and/or organic anion transporter 4 (OAT4)], or both. Therefore, we evaluated, in vitro and in vivo, whether THC and its major metabolites, 11-hydroxy-THC (11-OH-THC), 11-nor-9-carboxy-THC (THC-COOH) are substrates of key placental efflux and uptake transporters at their pharmacologically relevant concentrations (Chapters 2, 3, and 4). Cannabinoid-drug interactions may occur if the cannabinoid, at its pharmacologically relevant plasma concentrations, is a significant inhibitor of the transporter involved in the drug’s tissue distribution or clearance. Therefore, we also investigated if THC and its major metabolites, 11-OH-THC and THC-COOH, are inhibitors of key placental/hepatic efflux and uptake transporters at their pharmacologically relevant concentrations (Chapters 2 and 4). To test the above hypotheses, we examined in Chapter 2, if THC and its major metabolites interact with key placental efflux transporters using cell lines that overexpress human P-gp or BCRP. At pharmacologically relevant concentrations, neither THC nor 11-OH-THC were substrates or inhibitors of P-gp or BCRP. THC-COOH, however, showed weak substrate and inhibitory activity for BCRP but not P-gp. Therefore, the placental efflux transporters P-gp and BCRP are unlikely to cause the reduced fetal-to-maternal exposure ratio of THC observed in humans or non-human primates. Moreover, THC and its metabolites are unlikely to produce cannabinoid-drug interactions at pharmacologically relevant concentrations. These findings contrast with earlier rodent studies which suggest that THC is a substrate of P-gp and Bcrp, suggesting species-specific differences in transport of THC. THC and its major metabolites are highly lipophilic with extensive nonspecific binding. Therefore, the above studies may be confounded by our inability to detect cannabinoid transport in the background of high non-specific binding. Others, using P-gp knock-out mice, have found that THC is a substrate of P-gp when it is administered orally. Therefore, in Chapter 3, we investigated maternal-fetal THC distribution and disposition in P-gp and/or Bcrp knockout pregnant mice. However, no significant changes in fetal-to-maternal area under the plasma concentration-time (AUC) ratios of THC or its metabolites were observed among all genotypes. Surprisingly, P-gp-deficient pregnant mice had significantly lower maternal brain/maternal plasma AUC ratios of THC compared to the wild type pregnant mice, suggesting an interaction of P-gp knock-out with other unknown transporters or brain fatty acid binding proteins (FABPs) to which THC binds. Since the above studies indicated that placental efflux transporters cannot explain the reduced fetal exposure to THC observed in human and non-human primates, in Chapter 4, we investigated if placental basal uptake transporters (i.e., OATP2B1, OCT3, OAT4) could be responsible for these observations. Given that THC and its metabolites are cleared by the liver, we also investigated if THC and its metabolites were substrates or inhibitors of the hepatic uptake transporters [OATP1B1, OATP1B3, OCT1, OAT4, sodium taurocholate cotransporter protein (NTCP)]. None of the cannabinoids interacted with these transporters, except for hepatic OCT1, which transported both THC and THC-COOH at their pharmacologically relevant concentrations. However, at these concentrations, they were not inhibitors of OCT1. Therefore, OATP2B1, OCT3, and OAT4 are also unlikely to be responsible for the reduced fetal exposure to THC. Also, this suggests that co-administration of OCT1 inhibitors with THC or THC-COOH could reduce the in vivo hepatic distribution of these cannabinoids provided OCT1 plays a significant role (vis-Ã -vis passive diffusion) in their distribution. In summary, our research indicates that the major placental efflux and uptake transporters, in humans or mice, are not responsible for limiting the observed fetal THC exposure in human and nonhuman primates, pointing to other placental transporters or alternative mechanisms. The identification of hepatic OCT1 as a transporter for THC and THC-COOH suggests possible OCT1-based drug interactions in their in vivo disposition. These findings lay the groundwork for future studies on cannabinoid pharmacokinetics, including during pregnancy, and highlight the importance of considering species differences when extrapolating from preclinical data to humans.
Pharmacological profiling of conventional antiseizure medicines against seizures and kindling acquisition in female mice
(2025-08-01) Tieu, Taryn; Barker-Haliski, Melissa
Rationale: Historical ASM discovery has primarily relied on demonstration of efficacy in clinically validated acute seizure models evoked in male, wild-type rodents (e.g., maximal electroshock, subcutaneous pentylenetetrazol, or kindling). However, little work has been done to establish ASM response profiles in female rodents with acute or chronic seizures. Therefore, this thesis aimed to build on our previously established female C57BL/6J studies (Lehmann & Barker-Haliski 2023) to fill this knowledge gap and to define the acute median effective dose (ED50) of 4 FDA-approved ASMs (levetiracetam (LEV), carbamazepine (CBZ), valproic acid (VPA), and phenobarbital (PB)) in female outbred CF-1 mice using the 6 Hz 32 mA model of focal seizures. Further, no compound has yet been found to prevent the development of epilepsy so we propose a moderate-throughput anti-epileptogenesis drug screening paradigm for early compound profiling via repeated administration of anticonvulsant doses of VPA to assess the potential to prevent the formation of chronic hyperexcitable neuronal network or modify the severity of behavioral comorbidities of epilepsy in male versus female mice using the corneal kindled mouse (CKM) model of chronic seizures. Methods: Female outbred CF-1 mice were used to quantify of an ED50 of candidate ASMs (n=8 mice/dose) in a 6 Hz 32 mA test. Motor impairment was assessed via performance on a fixed speed rotarod. To establish a moderate-throughput, early-stage disease modification screening paradigm, male (n=40) and female (n=45) CF-1 mice underwent 60 Hz corneal kindling, a model of chronic network hyperexcitability, for 3-4 weeks in the presence or absence of twice daily VPA (150 mg/kg, intraperitoneal – i.p.) or vehicle administration. Mice were monitored twice daily for the presence and severity of evoked behavioral seizures until all animals met the kindled seizure endpoint. Following the kindling acquisition period, all CKM were challenged in an open field test to quantify the extent to which ASM administration during the kindling process modified anxiety-like behavioral comorbidities of epilepsy. Results: The calculated ED50 [and 95% confidence intervals] in female CF-1 mice for VPA (100 mg/kg [60.5-139]), CBZ (14.0 mg/kg [8.77-23.1]), LEV (14.3 mg/kg [0.591-36.0]) and PB (18.3 mg/kg [6.8-30.4]) were consistent with our previously reported values in the 6 Hz test in inbred female C57BL/6J mice (Lehmann and Barker-Haliski 2023). Notably, CBZ was poorly tolerated in CF-1 female mice: 87.5% of female mice were impaired on a rotarod at the highest dose tested. No other motor impairment was noted. In both male and female corneal kindling acquisition studies, VPA prevented kindled seizure presentation. The number of CKM reaching the fully kindled state was lower in both female (2/10) and male (2/11) mice than in VEH-treated female (8/10) and male (14/15) mice (male: X2 value = 18.57, p < 0.001; female: X2 value = 12.77, p < 0.001). However, repeated VPA administration did not significantly impact CKM activity in the center of an open field versus the activity of VEH-treated mice, suggesting no disease modifying effect of VPA administration on the anxiety-like behavioral comorbidity of epilepsy. Conclusion: This study establishes the anticonvulsant properties of four prototypical ASMs in wild-type outbred female CF-1 mice to inform future preclinical epilepsy studies using novel investigational compounds. This work also establishes the feasibility of a disease-modification drug screening paradigm for epilepsy in male and female mice using the corneal kindled mouse model and open field test of anxiety-like behaviors, a known clinical comorbidity of epilepsy. Altogether, this work offers a strategy on which to rapidly assess the anticonvulsant and disease modifying potential of candidate investigational treatments for the symptomatic management, and possibly prevention, of epilepsy.
Development of KRAS-Targeting siRNA and Chemotherapy Co-Delivery Chitosan NPs for Enhanced Treatment of Pancreatic Cancer
(2025-08-01) Chien, Yu-Ting; Zhang, Miqin MZ
Pancreatic ductal adenocarcinoma (PDAC) remains one of the most lethal cancers due to its aggressive progression, late-stage diagnosis, and limited treatment options. These clinical challenges are compounded by intrinsic resistance to chemotherapy and the high prevalence of activating mutations in the Kirsten rat sarcoma viral oncogene homolog (KRAS). To address these barriers, we developed a chitosan-based nanoparticles platform for the co-delivery of paclitaxel (PTX) and small interfering RNA (siRNA) targeting a prevalent KRAS variant, G12D (CP-PTX-KRAS G12D siRNA) to pancreatic cancer cells. The CP-PTX conjugate was synthesized via carbodiimide-mediated coupling between pegylated chitosan and carboxyl-modified PTX, then complexed with siRNA through electrostatic interactions to form dual-functional nanoparticles.The optimized formulation exhibited a compact size (~26.5 nm) and a moderately negative zeta potential (−9.8 mV), indicating stable siRNA loading and favorable physicochemical properties for intracellular delivery. The platform was initially validated using PolyIC, a double-stranded RNA and firefly luciferase siRNA to optimize formulation parameters and assess delivery efficiency. Cytotoxicity assays in KRAS G12D-mutant PANC-1 cells revealed dose-dependent viability reductions with single-agent PTX and KRAS siRNA, exhibiting IC50 values of 0.5470 ÂµM and 0.2784 ÂµM, respectively. Co-treatment at fixed PTX:siRNA molar ratios (1:0.2, 1:0.5, 1:1, 1:2) significantly enhanced cytotoxicity compared to single agents. CP-PTX-siRNA nanoparticles achieved consistently lower total IC50 values than corresponding free drug combinations across all tested ratios. At the 1:0.2 ratio, the nanoparticles formulation showed a total IC50 of 0.0921 ÂµM (vs. 0.1328 ÂµM for free drug), with a CI value of 0.317, indicating strong synergy. Similar trends were observed for other ratios, with all CI values <1, supporting synergistic interactions between PTX and siRNA when co-delivered via nanoparticles. These findings demonstrate the potential of CP-PTX-siRNA nanoparticles as an effective co-delivery platform for chemo-gene therapy in KRAS-driven pancreatic cancer. This system offers a modular and biocompatible approach to overcoming drug resistance and genetic drivers of malignancy, supporting its further development for translational cancer therapy.
A Comparative Analysis of Nephrotoxicity Induced by Voclosporin and Cyclosporine A Using Kidney Microphysiological Systems
(2025-05-12) Aryeh, Kayenat; Kelly, Edward J
Calcineurin inhibitor (CNI)-induced nephrotoxicity presents a major obstacle to long-term kidney transplant survival and patient quality of life. While the classic CNI cyclosporin A (CsA) is effective in preventing graft rejection, it can cause substantial renal injury through complex mechanisms involving mitochondrial dysfunction, excessive reactive oxygen species (ROS), endoplasmic reticulum (ER) stress, and disrupted cell cycle regulation—processes exacerbated by the hypoxic environment of the kidney cortex where proximal tubule epithelial cells (PTECs) primarily localize. In contrast, the newer agent voclosporin (VOC) achieves immunosuppression with notably reduced nephrotoxic effects, yet the molecular basis for this difference remains unclear. We combined RNA sequencing with a physiologically relevant, three-dimensional kidney microphysiological system (MPS) to compare the transcriptomic and cellular responses of PTECs exposed to CsA and VOC. Our analyses revealed that CsA induces broad perturbations in cell cycle progression and proteostatic pathways, coupled with robust ER stress responses and enrichment of ferroptosis-related genes associated with oxidative damage. In contrast, VOC elicited subtler alterations in similar pathways, suggesting a more measured adaptive response that preserves mitochondrial integrity and maintains ATP and ROS homeostasis. These findings underscore the importance of delineating the distinct cellular and transcriptomic mechanisms induced by CsA and VOC, which will be critical for developing safer immunosuppressive regimens, improving graft longevity, and ultimately enhancing patient outcomes in solid organ transplantation.
Effect of Pro-inflammatory Cytokines on Intestinal Drug Transporters in Human Enteroid Monolayers
(2025-01-23) Wang, Kai; Thummel, Kenneth E.
Altered drug pharmacokinetics during inflammation or infections have been linked with elevated proinflammatory cytokines in the plasma. Although oral medication is the most common route of drug administration, data on the impact of proinflammatory cytokines on the expression and activity of intestinal drug transporters remains limited. Using a novel enteroid in vitro model, we investigated the effects of key proinflammatory cytokines (i.e., interleukin (IL)-1β, IL-6, tumor necrosis factor (TNF)-α, and interferon (IFN)-γ) on the mRNA expression of major intestinal transporters, and the activity of intestinal P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP). Differentiated enteroid monolayers were exposed to cytokines, individually or as a cocktail, at 0.1, 1, and 10 ng/mL, encompassing their pathophysiological plasma concentrations in various inflammatory or infectious diseases. After 48 hours of exposure to the cytokine cocktail, significant concentration-dependent downregulation of P-gp, BCRP, multidrug resistance proteins (MRP) 2/3, organic solute transporter-α/β, serotonin transporter, and organic anion transporter polypeptide 2B1 was observed, along with upregulation of MRP4. Among the cytokines tested, IL-1β exhibited the most pronounced effects. Subsequent TranswellÂ® assays showed a significant decrease in the efflux ratio of nitrofurantoin, a selective BCRP substrate, after 48 hours of incubation with 1 ng/mL of each cytokine or the 1 ng/mL cytokine cocktail. Interestingly, the efflux ratio of digoxin, a P-gp substrate, was not affected by cytokines. These findings demonstrate that proinflammatory cytokines can transcriptionally dysregulate intestinal drug transporters and downregulate the activity of intestinal BCRP in vitro, highlighting the potential impact of inflammation on oral drug pharmacokinetics and the importance of optimizing dosing regimens for patients with inflammatory conditions.
Development and Characterization of a Human Intestinal Organoid Monolayer Model to Predict Oral Drug Disposition
(2025-01-23) Arian, Christopher Michael; Thummel, Kenneth E
The intestine plays an important role in governing the disposition of orally administered medication, serving important gate-keeping functions that can profoundly impact the systemic blood exposure of such medications. While currently used in vitro systems have substantially enhanced our knowledge of intestinal physiology as it relates to drug disposition, they suffer from limitations which often hinders their ability to broadly and accurately predict the fate of orally administered drugs. The use of complex in vitro systems, such as the intestinal microphysiological system (MPS) and adult human stem cell-derived intestinal organoids (enteroids), have profoundly expanded our understanding of intestinal development, physiology, and pathology. However, research employing these models for use in the field of pharmaceutical sciences remains relatively sparse. The projects described in this dissertation proposal seek to develop and characterize an intestinal MPS and a long-term cultured enteroid monolayer model for use in pharmaceutical sciences research.Chapter 2 describes the development and characterization of an intestinal MPS model cultured with LS180 cells, an immortalized colorectal adenocarcinoma cell line, and primary human umbilical vein endothelial cells (HUVECs). In Chapter 3, the development and biochemical characterization of a long-term cultured human enteroid monolayer model was explored. Chapter 4 provides further characterization of the long-term cultured enteroid monolayer model, utilizing RNA-seq deconvolution methods to determine the cell type proportions present in our differentiated enteroid cultures. In Chapter 5, the utility of the enteroid monolayer model to recapitulate and uncover the mechanisms precipitating a select natural product-drug interaction between goldenseal and metformin is explored. This work presented in this dissertation suggests that human enteroid monolayers are a promising model to predict orally administered drug disposition for use in preclinical drug development. Additionally, human enteroid monolayers display promise as a screening tool to predict drug-drug and natural product-drug interactions in vitro to better inform prescribing clinicians and improve the health and safety of patients.
Study of the Impact of Isotretinoin Administration on CYP3A4 Activity Using 6β-Hydroxylation of Cortisol and Cortisone as Endogenous Biomarkers
(2024-10-16) LaFrance, Jeffrey Matthew; Isoherranen, Nina
Isotretinoin is an FDA approved treatment for severe cystic acne and neuroblastoma and has been deployed in several non-FDA approved treatments of various cancers.1-5 Cortisol (COL) and Cortisone (CON) are metabolized to their 6β-hydroxy metabolites primarily by CYP3A4.6 Cortisol and 6β-hydroxycortisol (6β-OHCOL) are converted by 11β-HSD type 2 to cortisone and 6β-hydroxycortisone (6β-OHCON) respectively. Inversely, cortisone and 6β- hydroxycortisone are converted to cortisol and 6β-hydroxycortisol by 11β-HSD type 1.6,7 Previous in vitro work has suggested that CYP3A4 in the liver is the main enzyme responsible for the 6β-hydroxylation of cortisol and cortisone making cortisol metabolism an attractive choice as an endogenous biomarker of CYP3A4 activity.6 This research investigated the effect of isotretinoin on CYP3A4 activity in severe acne patients using the 6β-hydroxylations of cortisol and cortisone as endogenous biomarkers. The plasma concentrations and renal clearance of cortisol, cortisone, 6β-hydroxycortisol, and 6β-hydroxycortisone are reported, along with the urinary and plasma metabolic ratios, and the apparent formation clearances of 6β- hydroxycortisol, and 6β-hydroxycortisone. The results show that there was an increase in the plasma concentrations of cortisol and 6β-hydroxycortisol suggesting that isotretinoin is impacting cortisol production. Furthermore, a decrease in 6β-hydroxycortisol renal clearance was found which supports isotretinoin impacting the interconversion of cortisol and cortisone in the kidney. Surprisingly, we saw a decrease in the formation clearance of 6β-hydroxycortisone which could imply CYP3A4 inhibition. Lastly, there was no change in plasma or urinary metabolic ratios which supports CYP3A4 not being induced by isotretinoin.
Impact of Aging and Sex on the Expression and Function of Xenobiotic Transporters at the Blood-Cerebrospinal Fluid Barrier
(2024-10-16) Shan, Tianzheng; Wang, Joanne
The blood-cerebrospinal fluid barrier (BCSFB), formed by the choroid plexuses (CP) in the brain ventricles, serves as a critical clearance organ in the brain by mediating bulk-flow CSF clearance and through the active removal of drugs and metabolites by transporters. The choroid plexuses epithelial (CPE) cells are understood to go through morphological and functional changes during aging, leading to a reduction in CSF secretion rate. Emerging evidence also indicate that drug transporter expression can exhibit sex differences in drug disposition organs such as the liver, potentially contributing to differences in drug response. However, currently little is known regarding the impact of aging and sex on transporter expression and function at the BCSFB, which is the focus of this master thesis. The mRNA expression of Slc transporters (Slc22a6, Slc22a8, Slc15a2, Slc29a4, Slco1a1, Slco1a4, Slco1a5, Slco1a6, Slco1c1, Slco3a1) and Abc transporters (Abcb1a, Abcb1b, Abcg2, Abcc1, Abcc4) were analyzed by RT-qPCR in young adult young adult (3 months) and old (20 months) mouse CP tissues.Â Aquaporin 1 (Aqp1) was used as a marker for CP epithelial cells, and Gapdh and GusB were used as the housekeeping genes. The results showed that while the majority of analyzed genes showed no difference in expression between the two age groups, Abcg2 (Bcrp) mRNA levels increased in aged mice by 70% and 100% in male and female CP tissues, respectively. In contrast, the expression of Slco3a1 (Oatp3a1) was decreased in aged mice by 30% in males and 40% in females. In young adult mice, no significant sex differences were observed for all analyzed transporter genes. However, in old mice, Slc22a8 (Oat3) CP expression in females was 40% lower compared to the male cohort. Using a quantitative real-time imaging method recently developed in our laboratory, the activity of Oatp1a – Mrp1/4 pathway was further investigated and the results showed that the activity of this main pathway for organic anions across the BCSFB is not influenced by aging or sex, consistent with minimal impact of aging on Oatp1a and Mrp1/4 mRNA expression. As BCSFB transporters play a crucial role in removing drugs, toxins, and metabolic wastes from the brain, knowledge gained from this master thesis contributes to understandings of the effect of aging and sex on major BCSFB transporter expression and activity, which may further influence the CSF drug concentrations and the pharmacokinetics of CNS-targeted drugs in geriatric patients and between genders.
Analytical methods to determine the degree of drug association for complex injectable drug-combination particles containing multiple drug substances
(2024-10-16) Xu, Guangyuan; Ho, Rodney
The aim of this research is to develop and characterize complex injectable products composed of multiple drug substances targeted to human immunodeficiency virus (HIV). Some of these product candidates are composed of current HIV drug substances that exhibit desperate water solubilities. For example, lopinavir (LPV), ritonavir (RTV) and dolutegravir (DTG) are water insoluble while tenofovir (TFV) and lamivudine (3TC) are water soluble. Water insoluble LPV/RTV and water soluble TFV (or TDF) prodrugs are given orally to provide sustained viral suppression as a one-pill-a-day treatment in people living with HIV (PLWH). Recently DTG, 3TC and TFV, referred to as TLD one-pill-a day, has been widely used in the low-to-middle income countries with the goal to bring down HIV infections worldwide. Unfortunately, PLWH express pill fatigue and missing a daily dose may lead to virus rebound and disease progression. In addition, uncontrolled virus levels in PLWH may relate to a continued increase in the number of people with HIV. This laboratory is developing a complex injectable LPV/RTV/TFV in a drug-combination nanoparticle (DcNP) product candidate, referred to as targeted long-acting combination antiretroviral therapy 101 (TLC-ART 101) that is currently under clinical testing. A next generation product, TLC-ART 301 composed of DTG, 3TC and TFV (or TLD) is currently under preclinical development. As DcNP enabled TLC-ART 101 and TLC-ART 301 products containing 3 drug substances assembled to associate with DcNP particles, a key performance characteristic is the degree of drug substance association to the DcNP product in the suspension. The initial approach is based on dialyzing the unbound 3 drug substances under sink-condition, to enable diffusion of free drug through a semi-permeable membrane across a large volume of solvent. This degree or percentage of DcNP association for each drug is based on the fraction of drug retained within the dialysis chamber and to account for dialysate (due to large volume and analytical assay sensitivity). With the development of a combination of increasing analytical assay and volume reduction dialysate (thus increasing drug concentrations amenable for drug analysis), this thesis research was able to determine the percentage of drug association to DcNP based on a mass-balance approach. We found that comparing the association efficiency (AE) analysis for the 3 drug substances based with retentate only, or with a mass-balance approach was well-correlated. In addition, under the sink conditions, only one of the two lipid excipients, sodium; [(2R)-2,3-di(octadecanoyloxy)propyl] 2- (2-methoxyethoxycarbonylamino)ethyl phosphate (mPEG-2000 DSPE), but not 1,2-Distearoyl-sn-glycero-3-phosphocholine (DSPC) are found in dialysate; suggesting that some of the water soluble drug TFV may have remained bound to mPEG-2000 DSPE and TFV-mPEG2000-DSPE or that micelles may have also contributed to apparently over 90% of the TFV behaving as a DcNP bound drug; reported in primates after TLC-ART 101 dosing via IV as well as SC route. The method of AE estimate – both retentate and mass-balance approaches and correlation, for the 3 drug substances in the TLC-ART 101, is demonstrated to extend to the TLC-ART 301 product composed of DTG, 3TC and TFV. Collectively, this thesis research was able to verify the analytical methods to characterize the degree of drug association for both abbreviated retentate as well as mass-balance approach for a complex injectable drug-combination formulated in drug-combination nanoparticle product. Having the validated method may help accelerate development of the drug-combination injectable nanoparticles, intended to provide long-acting and longer lasting viral suppression in people living with HIV.
Development and Characterization of an Orthotopic Pancreatic Ductal Adenocarcinoma (PDAC) Tumor Model for Evaluation of Drug-Combination Nanomedicine and Targeted Therapeutics
(2024-09-09) Chen, Xinyue; Ho, Rodney
Pancreatic ductal adenocarcinoma (PDAC) remains one of the deadliest cancers, with a dismal 5-year 10% pancreatic cancer survival rate. The lack of clinically relevant preclinical models that recapitulate the complex tumor microenvironment and metastatic patterns observed in pancreatic cancer patients has hindered the development of effective therapeutic strategies. To address this limitation, this thesis describes the research, development and characterization of a novel PDAC tumor model syngeneic C57BL/6 mice that suitable for evaluation of therapeutic interventions. Mice produced tumor consistently in the pancreas after intraperitoneal (IP) inoculation with Pan02 cell of PDAC origin. With luciferase expressed Pan02-luc cells, time-course studies of this orthotopic pancreatic tumor model, we found that Pan02 cells first localize in the pancreas, invade and grow in the pancreas before metastatically spread to other tissue and organ. The critical role of the inoculation route in determining tumor formation and metastatic patterns was established. Only inoculation of Pan02-luc cells through IP, but not IV route consistently leading to tumor development in the pancreas, followed by metastatic spread to the liver, gastrointestinal tract, and peritoneal wall. Time-course histological analysis revealed dynamic remodeling of the tumor stroma over time, mimicking the desmoplastic reaction observed in human PDAC. To elucidate the homing mechanisms driving Pan02 cell localization to the pancreas focused on evaluating the role of the epidermal growth factor receptor (EGFR) pathway, with flow cytometry experiments confirming EGFR expression on Pan02 cells. The introduction of the luciferase reporter gene did not significantly impact the sensitivity of Pan02 cells to gemcitabine but led to a striking 10-fold decrease in sensitivity to paclitaxel, highlighting the potential influence of genetic modifications on drug response mechanisms. Pharmacokinetic studies revealed that a novel gemcitabine-and-paclitaxel drug-combination-nanoparticle formulation (GT-in-DcNP) significantly improved the exposure and bioavailability of soluble gemcitabine and paclitaxel. Following IP administration in C57BL/6 mice, the study results suggest that the IP route, combined with drug combination nanoparticle formulation referred to as DcNP, may enhance the therapeutic index of gemcitabine-and-paclitaxel combination in the treatment for pancreatic cancer. Future research directions include transcriptomic or proteomic profiling to identify additional therapeutic targets, evaluation of combination therapies targeting multiple signaling pathways, longitudinal studies investigating the tumor microenvironment, mechanistic studies on paclitaxel sensitivity alterations, and preclinical evaluation of the GT-in-DcNP formulation as a novel product for the treatment of pancreatic cancer. This thesis research provides a novel pancreatic tumor model that is simple, robust that mimic many of characteristic of human pancreatic cancer of PDAC origin through a simple IP inoculation of Pan02 PDAC cell in syngeneic mouse. The subsequent investigation of two current chemotherapeutic drugs, gemcitabine and paclitaxel combination presented in DcNP nanoparticles suggest that DcNP nanoparticles may enhance exposure and effectiveness in the treatment of pancreatic cancer based on the newly established Pan02 orthotopic tumor model. Together, the model and novel GT-in-DcNP may offer a clinically relevant platform for advancing the development of novel targeted therapeutic strategies and improving the prognosis of pancreatic cancer.
Intestinal Dysbiosis Alters Seizure Burden and Antiseizure Medicine Activity Profile in the Theiler’s Virus Model of Acute Encephalitis
(2024-09-09) Erickson, Inga; Barker-Haliski, Melissa; Thummel, Kenneth
Background: Epilepsy is a neurological disorder characterized by recurring, unprovoked seizures and chronic cognitive comorbidities. Epilepsy can arise due to numerous factors, including genetics, brain injury, and central nervous system infection. Brain infection with Theiler’s murine encephalomyelitis virus (TMEV) in C57BL/6J mice is thus a relevant animal model of infection-induced acute seizures and epileptogenesis. Acute seizures in this model are driven by brain infiltration of peripheral immune system macrophages, which secrete pro- inflammatory cytokines that further accelerate brain inflammation1. Diet formulation can dramatically modify the presentation of acute seizures in TMEV-infected mice and influence the diversity and composition of the intestinal microbiome (Zierath et al, Epilepsia 2024)2 that critically shapes immune system reactivity. However, whether antibiotic-induced gut microbiome dysbiosis influences the phenotype of symptomatic seizures after TMEV infection is unclear. Further, no study has assessed whether antibiotic-induced intestinal dysbiosis influences antiseizure medicine activity (ASM) in the TMEV model. We thus sought to define the extent towhich antibiotic administration influenced acute seizure presentation, the anticonvulsant activity of the prototype ASM, carbamazepine (CBZ), and pharmacokinetics of this ASM in this mouse seizure model. We hypothesized that there would be an effect of intestinal dysbiosis on seizure burden and ASM efficacy in the TMEV model and that this change would be independent of pharmacokinetic differences. Methods: Male C57BL/6J mice (4-5 weeks-old) received a broad-spectrum antibiotic cocktail (ABX) containing ampicillin, metronidazole, neomycin sulfate, and vancomycin (n=55) or vehicle (n=60) by oral gavage (p.o.) once daily beginning at arrival (Day -2) to Day 7 post- TMEV infection (p.i.) to induce intestinal dysbiosis. Mice were then infected with either intracerebral TMEV or PBS on Day 0. Mice received carbamazepine (CBZ; 20 mg/kg) or vehicle (VEH, 0.5% MC) intraperitoneally (i.p.) twice daily Days 3-7 p.i. and were assessed for handling-induced seizures 30 min after CBZ treatment. Plasma was collected on Day 7 p.i. at 15 and 60 min post-CBZ treatment to quantify the extent to which ABX-induced gut dysbiosis influences ASM pharmacokinetics. Results: TMEV infection induced acute symptomatic seizures, regardless of pretreatment and CBZ history. There were 18/25 (72%) ABX-CBZ mice, 7/20 (35%) ABX-VEH mice, 7/20 (35%) SAL-CBZ mice, and 15/20 (75%) SAL-VEH mice that presented with seizures during the 7-day monitoring period. Average seizure burden was: 12.5 in ABX-CBZ, 4.7 in ABX-VEH, 5.7 in SAL-CBZ, and 16.1 in SAL-VEH mice. There was a significant pretreatment x ASM interaction (F (1, 81) = 16.0, p=0.0001), with post-hoc tests revealing marked differences in seizure burden in SAL- versus ABX-pretreated mice (p=0.004). Further, CBZ administration significantly increased the latency to Stage 5 seizure during days 3-7 p.i.; an effect that was not present in ABX-treated mice similarly administered CBZ. In TMEV-infected mice, spleens were 0.32% of body weight in ABX-CBZ mice, 0.34% in ABX-VEH mice, 0.36% in SAL-CBZ mice, and 0.38% in SAL-VEH mice. In sham-infected mice, spleens were 0.45% of body weight in ABX-CBZ mice, 0.65% in SAL-CBZ mice, and 0.36% in SAL-VEH mice. Plasma CBZ concentrations did not differ between SAL and ABX pretreatment groups (F (3, 37) = 0.3468), suggesting that ABX history did not influence CBZ pharmacokinetics. Conclusion: Antibiotic-induced gut dysbiosis markedly altered the presentation of symptomatic seizures and acute disease burden in the TMEV mouse model, reflecting a novel therapeutic target for seizure control: the gut microbiome. ABX-induced gut dysbiosis also significantly changed acute seizure control by CBZ, but did not significantly influence plasma concentrations of CBZ. The gut-brain axis is thus a relevant contributor to the clinical course of TMEV infection. This study altogether demonstrates that the gut-brain axis is an understudied therapeutic target in epilepsy that may benefit from greater investigation.
Oral Drug Pharmacokinetics and Treatment Effects in Populations Affected by Diarrheal Diseases
(2024-09-09) Zhang, Cindy; Arnold, Samuel L.M.
Diarrheal diseases are a major global health concern. Globally, over 4.5 billion episodes of diarrhea occur among all ages every year. Diarrhea disproportionally affects young children and those living in low- and middle-income countries. In 2019 alone, diarrhea was responsible for approximately 1.5 million deaths among people of all ages, including over 500,000 deaths in children under 5 years of age (Dadonaite et al., 2024). Moreover, around 90% of diarrheal mortalities occur in sub-Saharan Africa and South Asia, where undernutrition, poor sanitation, and limited access to medical care are common (GBD 2016 Diarrhoeal Disease Collaborators, 2018). The high prevalence of diarrheal diseases creates a challenge for oral drug administration, as physiological changes associated with diarrhea can impact key processes involved in oral drug absorption, thus leading to alterations in oral drug pharmacokinetics (PK). As a result, the safety and effectiveness of oral drugs may be altered when taken during episodes of diarrhea. The understanding of how diarrheal diseases affect the oral drug PK, whether this impact influences treatment outcomes, and if dosage adjustments are necessary is essential for making accurate dosing decisions and optimizing the likelihood of treatment success. To this end, we leveraged PK modeling approaches to characterize and simulate oral drug PK and make predictions about treatment efficacy for populations affected by diarrheal diseases.In Chapter 2, to estimate the magnitude of diarrhea-associated impact on oral clofazimine PK, we developed a population PK model using clofazimine PK data collected from a phase 2a clinical trial of HIV-infected adults with/without cryptosporidiosis and diarrhea. Among potential covariates of cryptosporidiosis-associated diarrhea severity, HIV infection burden, baseline demographics, and study assignment, maximum diarrhea grade over the study duration was significantly associated with clofazimine bioavailability. Our model quantified a 6- and 22-fold reduction in clofazimine bioavailability associated with mild and severe diarrhea, respectively. Moreover, the model also estimated clofazimine PK parameters apparent clofazimine clearance (3.71 L/h), intercompartmental clearance (18.2 L/h; inter-individual variability [IIV] 45.0%), central volume of distribution (473 L; IIV 3.46%), peripheral volume of distribution (3434 L), absorption rate constant (0.625 h-1; IIV 149%), and absorption lag time (1.83 h), all of which were aligned with literature values. To determine whether cryptosporidiosis-associated diarrhea reduced clofazimine levels below levels associated with efficacy against Cryptosporidium infection, a pharmacokinetic/pharmacodynamic (PK/PD) modeling approach was undertaken in Chapter 3. Exposure-response relationships were determined using data collected in a preclinical mouse model of Cryptosporidium infection. Emax and logistic models were constructed, supporting predictions of efficacious clofazimine concentrations. By comparing the observed clofazimine concentrations from an unsuccessful phase 2a clinical trial to our predicted efficacious target, it was shown that the observed concentrations were well below concentrations associated with anti-Cryptosporidium efficacy. Thus, the prescribed doses were inadequate, even without any impact of diarrhea. Since the effect of diarrhea on oral drug absorption is likely disease- and drug-specific, tebipenem for shigellosis treatment in Bangladeshi pediatric patients was studied in Chapter 4. By validating an existing tebipenem pediatric population PK model with observed tebipenem PK data collected from the pilot study of an ongoing clinical trial, we first demonstrated that the existing model adequately predicted tebipenem PK in a population with diarrhea. Then, we performed population PK simulations using the validated model, incorporating demographic characteristics of the target Bangladeshi pediatric population. The simulated tebipenem PK profiles were used to predict the probability of achieving maximum treatment effect in the target population and the main trial’s chance of success, comparing two different dosing regimens. The findings of Chapter 4 showed that the effect of shigellosis-associated diarrhea on tebipenem PK was minimal. Moreover, Chapter 4 demonstrated an effective modeling workflow that can be adapted to support dose evaluation and efficacy predictions for other drugs and disease areas.

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