The ABCG5 ABCG8 (G5G8) sterol transporter promotes cholesterol secretion into bile and opposes dietary sterol absorption in the small intestine. An emerging body of literature suggests that G5G8 links sterol flux to various risk factors for metabolic syndrome (MetS) and nonalcoholic fatty liver disease (NAFLD). Therapeutic approaches that accelerate G5G8 activity may augment reverse cholesterol transport (RCT) and provide beneficial effects in the prevention and treatment of cardiovascular and liver disease. Mice lacking leptin (ob/ob) or its receptor (db/db) are obese, insulin resistant in part due to the reduced levels of hepatic G5G8 and biliary cholesterol. The underlying mechanisms responsible for the reduced G5G8 protein expression in these mice may provide a clue to the drug development for this target. My studies show that neither acute leptin replacement nor liver-specific deletion of leptin receptor alters G5G8 abundance or biliary cholesterol. Similarly, hepatic vagotomy has no effect on G5G8 expression. Conversely, expression of the ER chaperone, GRP78, rescues G5G8 in db/db mice. Previous studies suggest an interdependent relationship between liver and intestine for cholesterol elimination. A combination therapy that increases G5G8-mediated biliary cholesterol secretion and simultaneously reduces intestinal absorption is likely to act additively in cholesterol elimination. My studies show that treatment with ursodiol (Urso) increases hepatic G5G8 protein and both biliary and fecal sterols in a dose-dependent manner. Ezetimibe (EZ), a potent inhibitor of intestinal cholesterol absorption, produces an additive and dose-dependent increase in fecal sterol excretion in the presence of Urso. However, the stimulatory effects of both Urso and Urso-EZ are not G5G8-dependent. Beyond increasing G5G8 protein expression and biliary cholesterol secretion, my studies also show that Urso stimulates ileal FGF15 expression in mice. Our data of the stimulated ileal FGF15 expression in LIRKO and reduced hepatic G5G8 protein levels in Atsb KO mice both indicate the previous unrecognized role of FGF15/19 in the regulation of G5G8 and its activity. Indeed, this is subsequently confirmed by our results from the direct test of recombinant human FGF19 on G5G8. Thus, FGF15/19 may provide an alternative strategy in drug development to target G5G8 activity and accelerate cholesterol elimination.

Methamphetamine interacts with vesicular monoamine transporter-2 (VMAT2) to inhibit dopamine (DA) uptake and promotes DA release from presynaptic vesicles, increasing cytosolic DA available for methamphetamine-induced reverse transport by DA transporters. By inhibiting VMAT2, lobelane, a defunctionalized, saturated lobeline analog, decreases methamphetamine-evoked DA release and methamphetamine self-administration in rats. In this dissertation structure-activity relationships around the lobelane structure were investigated on racemic lobelane analogs with varying methylene linker lengths at central piperidine ring. Affinity for dihydrotetrabenazine (DTBZ) sites on VMAT2 and for inhibition of VMAT2 function was determined to be 0.88-63 and 0.024-4.6 µM, respectively, and positively correlated. The most potent and selective analog, (±)-cis-2-benzyl-6-(3-phenylpropyl)piperidine [(±)-GZ-730B], for VMAT2 uptake was identified as the lead. The ability of (±)-GZ-730B to inhibit methamphetamine-evoked [3H]DA release from striatal synaptic vesicles and endogenous DA release from striatal slices was determined. The lead analog-induced inhibition of methamphetamine-evoked vesicular [3H]DA release did not translate to inhibition of methamphetamine-evoked DA release in the more intact striatal slices. Moreover, poor water solubility of these lobelane analogs prohibited further in vivo work. Subsequent work focused on analogs with the C-3 and C-4 carbons in the piperidine ring eliminated to afford racemic acyclic lobelane analogs. Generally, acyclic analogs exhibited greater water solubility and less lipophilicity compared to lobelane. Acyclic analogs exhibited affinities (Ki = 0.096-17 μM) for [3H]DTBZ sites that correlated positively with affinity (Ki = 3.3-300 nM) for inhibition of [3H]DA uptake. Pure enantiomers of potent racemic analogs were synthesized, and found to potently, selectively, and competitively inhibit [3H]DA uptake at VMAT2 and to release vesicular [3H]DA in a biphasic manner. Lead enantiomer (R)-N-(1-phenylpropan-2-yl)-3-phenylpropan-1-amine [(R)-GZ-924] inhibited methamphetamine-evoked [3H]DA release from striatal synaptic vesicles, but not from the more intact striatal slices. Surprisingly, (R)-GZ-924 inhibited nicotine-evoked [3H]DA overflow from striatal slices, revealing nonspecific effects. Importantly, (R)-GZ-924 inhibited methamphetamine self-administration in rats. However, the analog also inhibited food-maintained responding, revealing a lack of specificity. The lead analog will not be pursued further as a pharmacotherapy due to the lack of specificity. Further evaluation of the pharmacophore is needed to discover analogs which specifically inhibit the neurochemical and behavioral effect of methamphetamine.

ABCD2 (D2) has been proposed as a peroxisomal long-chain acyl-CoA transporter that is essential for very long chain fatty acid metabolism. In the livers of mice, D2 is highly induced by fenofibrate, a PPARα ligand that has been widely used as a lipid lowering agent in the treatment of hypertriglyceridemia. To determine if D2 is a modifier of fibrate responses, wild-type and D2 deficient mice were treated with fenofibrate for 14 days. The absence of D2 altered expression of gene clusters associated with lipid metabolism, including PPARα signaling. Using 3T3-L1 adipocytes, which express high levels of D2, we confirmed that knock-down of D2 modified genomic responses to fibrate treatment. We next evaluated the impact of D2 on effects of fibrates in a mouse model of dietinduced obesity. Fenofibrate treatment opposed the development of obesity, hypertriglyceridemia, and insulin resistance. However, these effects were unaffected by D2 genotype. We concluded that D2 can modulate genomic responses to fibrates, but that these effects are not sufficiently robust to alter the effects of fibrates on diet-induced obesity phenotypes. Although proposed as a peroxisomal transporter, the intracellular localization of D2, especially in adipose tissue, has not been validated with direct experimental evidence. Sequential centrifugation of mouse adipose homogenates generated a fraction enriched with D2, but lacked well-known peroxisome markers including catalase, PEX19, and ABCD3 (D3). Electron microscopic imaging of this fraction confirmed the presence of D2 protein on an organelle with evidence of a dense matrix and a diameter of ~200 nm, the typical structure and size of a microperoxisome. D2 and PEX19 antibodies recognized distinct structures in mouse adipose. Immunoisolation of the D2-containing compartment from adipose tissue confirmed the scarcity of PEX19. Proteomic profiling of the D2 compartment revealed the presence of proteins associated peroxisome, endoplasmic reticulum (ER), and mitochondria. We conclude that D2 is localized to a distinct subclass of peroxisomes that lack many peroxisome proteins and may physically associate with mitochondria and the ER.

Supersaturating Drug Delivery Systems (SDDS) could enhance oral bioavailability of poorly water soluble drugs (PWSD). Precipitation inhibitors (PIs) in SDDS could maintain supersaturation by inhibiting nucleation, crystal growth, or both. The mechanisms by which these effects are realized are generally unknown. The goal of this dissertation was to explore the mechanisms underpinning the effects of model PIs including hydroxypropyl β-cyclodextrins (HP-β-CD), hydroxypropyl methylcellulose (HPMC), and polyvinylpyrrolidone (PVP) on the crystal growth of indomethacin, a model PWSD. At high degrees of supersaturation (S), the crystal growth kinetics of indomethacin was bulk diffusion-controlled, which was attributed to a high energy form deposited on the seed crystals. At lower S, indomethacin growth kinetics was surface integration-controlled. The effect of HP-β-CD at high S was successfully modeled using the reactive diffusion layer theory. The superior effects of PVP and HPMC as compared to HP-β-CD at high S were attributed to a change in the rate limiting step from bulk diffusion to surface integration largely due to prevention of the high energy form formation. The effects of PIs at low S were attributed to significant retardation of the surface integration rate, a phenomenon that may reflect the adsorption of PIs onto the growing surface. PVP was selected to further understand the relationship between adsorption and crystal growth inhibition. The Langmuir adsorption isotherm model fit the adsorption isotherms of PVP and N-vinylpyrrolidone well. The affinity and extent of adsorption of PVP were significantly higher than those of N-vinylpyrrolidone, which was attributed to cooperative interactions between PVP and indomethacin. The extent of PVP adsorption on a weight-basis was greater for higher molecular weight PVP but less on a molar-basis indicating an increased percentage of loops and tails for higher molecular weight PVPs. PVP significantly inhibited indomethacin crystal growth at high S as compared to N-vinylpyrrolidone, which was attributed to a change in the growth mechanism resulting in a change in the rate limiting step from bulk diffusion to surface integration. Higher molecular weight PVPs were better inhibitors than lower molecular weight PVPs, which was attributed to a greater crystal growth barrier provided by a thicker adsorption layer.

Glycosaminoglycans (GAGs) are complex linear chain carbohydrate molecules found on virtually all animal cell surfaces. Owing to their negatively charged nature, GAGs interact with a number of different proteins. Thus, although they have great potential as therapeutic agents, their apparent promiscuous interactions increase their side effect risk. GAG mimetics, including GAG oligosaccharides and non-saccharide GAG mimetics (NSGMs) are viable approaches to address this. This work discusses sulfated benzofuran thrombin inhibitors with submaximal protease inhibition, sulfated diflavonoid inhibitors of plasmin and GAG oligosaccharides with selectivity for human neutrophil elastase (HNE). Anticoagulants are very important for the treatment of thrombotic diseases. The adverse effects associated with current clinically used anticoagulants warrant the continuous search for new agents. Thrombin, being the central player in the coagulation cascade, remains a very important target for anticoagulant therapy, however drugs inhibiting its activity carry the risk of prolonged bleeding. Based on a previously identified sulfated benzofuran thrombin inhibitor, we have developed analogs with submaximal inhibition of the protease. These agents inhibit thrombin with efficacies approaching 50%, for both chromogenic and macromolecular substrates, ensuring a basal level of thrombin activity even at saturating inhibitor concentrations. The most potent of these compounds had a potency of 1.8 µM, 2-3 fold better than the lead. Additionally, these compounds utilize an allosteric mechanism for thrombin inhibition. Further, studies have revealed structural features responsible for submaximal thrombin inhibition. Fibrinolysis is an important part of hemostasis and plasmin is the most important fibrinolytic enzyme. Anti-plasmin agents are thus important for conditions such as hemophilia; however, there are no clinically used direct plasmin inhibitors. By structural modifications of a previously identified sulfated diflavonoid plasmin inhibitor, we have achieved a compound with 12-fold better potency (IC50 = 6.3 ± 0.4 µM), and a selectivity index of at least 22 over closely related serine proteases. We have shown that this compound inhibits plasmin mediated clot lysis, and further demonstrated that its activity is reversible using protamine sulfate, indicating its potential as a lead for the development of clinical anti-plasmin agents. HNE, a serine protease associated with inflammatory diseases is known to be inhibited by GAGs. However, the interactions at the molecular level have remained elusive. Using biochemical methods, and by studying the inhibitory potency of different GAGs and GAG oligosaccharides, we have shown that an octasaccharide may be the ideal GAG length for the achievement of potent HNE inhibition. Under our assay conditions, the inhibition of HNE by an octasaccharide species was only 5-fold less than that of unfractionated heparin, whereas the hexasaccharide species was 30-fold less active. The data also suggests that the inhibition of HNE by GAGs is via an allosteric mechanism and using molecular modeling, we have identified putative GAG binding sites on HNE and further identified GAG species with potential selectivity for anti-HNE activity

INTRODUCTION: Diabetes mellitus is one of the most prevalent and costly chronic diseases in the United States (US). The healthcare and drug cost of diabetes has risen steadily and the increase in patients’ out-of-pocket drug expenditures are associated with a reduction in treatment adherence. The objectives of this study were to assess trends in insulin products prices in the period January 1983-July 2019, and to compare the price, acquisition costs and reimbursement amount of insulins available in the US. DATA AND METHODS: Data of insulin products marketed in the US during the period January 1983-July 2019 was derived from the FDA databases, the RedBook online, Medicaid.gov, the Department of Veterans Affairs, and the Centers for Medicare & Medicaid Services. Prices were adjusted using the consumer price index (CPI). The compounded average group rate (CAGR) was calculated for each insulin product. Data was analyzed by summary descriptive statistics. RESULTS: Human insulins had a CPI-adjusted AWP CAGR ranging 4.89%-8.89% from the first AWP effective date to July 2019 and insulin analogues had a CPI-adjusted AWP CAGR ranging 9.5%- 9.75%. The 2 follow-on (biosimilar) insulins; long-acting insulin glargine and rapid-acting insulin lispro experienced a negative adjusted CAGR (-1.20%, -33.70%, respectively). Insulin acquisition cost and reimbursement amounts showed a large variation when compared with the average wholesale (AWP) prices. The wholesale acquisition cost (WAC) was typically set at 83.33% of the AWP. Community pharmacies acquired insulins and analogues at a median of 80.27% of the AWP. Significant reductions in AWP were observed for Medicare Part D (78.80% of the AWP), and Federal Supply Schedule (FSS) /Big4 (25.89%). CONCLUSION: Manufacturer prices of insulins and analogues increased significantly during the period of 1983- July 2019. There are significant differences in the manufacturer prices, pharmacy acquisition costs and reimbursement rates of insulins and analogues.

Cannabidiol (CBD), a non-psychotropic phytocannabinoid with structural similarity to Δ 9 -tetrahydrocannabinol (THC), is currently being investigated as a therapeutic for its immunosuppressive effects. One disease for which CBD is extensively researched is multiple sclerosis (MS), a demyelinating, autoimmune disorder, and its murine model counterpart, experimental autoimmune encephalomyelitis (EAE). The focus of this dissertation aimed to analyze the transcriptomic brain pathways in EAE and its comparison to MS in addition to CBD’s immunosuppressive mechanisms in the innate and adaptive immune systems. Evidence presented here showed that transcriptomic signaling pathways in the EAE brain of mice with clinical symptoms were similar to the transcriptome of active lesions from MS patients. The transcriptomic analysis also presented two differentially expressed genes that were increased in CBD-treated, asymptomatic EAE mouse brains: oxytocin and vasopressin. Expression of these genes was also increased in naïve, CBD-treated mouse brains, which may indicate potential as efficacy biomarkers. Subsequently, as disease progression requires input from the innate and adaptive immune systems, the mechanisms of CBD were analyzed under naïve and stimulatory conditions in macrophages and splenocytes. In macrophages, CBD exerted an anti-inflammatory effect by dampening the M1 polarization phenotype, decreasing pro-inflammatory cytokines and chemokines, reducing TNF-α through intracellular TACE retention, and diminishing the translocation of RelA to the nucleus. Notably, similar impact of CBD on TACE was evident in naïve macrophages, suggesting that CBD exerted an effect under naïve conditions. In splenocytes, CBD exhibited a long-term effect on the percentage of various immune populations during naïve and splenic T cell activation (with anti-CD3/anti-CD28) conditions but only provided temporary relief and short-term from TNF-α and IFN-γ cytokine secretion. CBD also increased early mRNA expression of Tnfa in CBD in stimulated splenocytes. In naïve splenocytes, CBD impacted key immune mediators discovered from a transcriptomic re-analysis of human neuroblastoma cells, including decreased early expression of Noxo1 but increased expression of Ctsb. In summary, this dissertation presented evidence that CBD impacts the immune system from the transcriptional level in the brain, the innate and adaptive immune systems at the cellular level, and the overall EAE disease phenotype.

Proteasome inhibitors bortezomib and carfilzomib are FDA-approved anticancer agents that have contributed to significant improvements in treatment outcomes. However, the eventual onset of acquired resistance continues to limit their clinical utility, yet a clear consensus regarding the underlying mechanisms has not been reached. Bortezomib and carfilzomib are known to target both the constitutive proteasome and the immunoproteasome, two conventional proteasome subtypes comprising distinctive sets of catalytic subunits. While it has become increasingly evident that additional, ‘intermediate’ proteasome subtypes, which harbor non-standard mixtures of constitutive proteasome and immunoproteasome catalytic subunits, represent a considerable proportion of the proteasome population in many cell types, less is known regarding their contribution to cellular responses to proteasome inhibitors. Importantly, previous studies in murine models have shown that individual proteasome subtypes differ in sensitivity to specific proteasome inhibitors. Furthermore, research efforts in our laboratory and others have revealed that proteasome catalytic subunit expression levels and activity profiles are altered when human cancer cells acquire resistance to proteasome inhibitors. We therefore hypothesized that changes in the relative abundances of individual proteasome subtypes contribute to the acquired resistance of cancer cells to bortezomib and carfilzomib. A major obstacle in testing our hypothesis was a lack of chemical probes suitable for use in identifying distinct proteasome subtypes. We addressed this by developing a series of bifunctional proteasome probes capable of crosslinking specific pairs of catalytic subunits colocalized within individual proteasome complexes and compatible with immunoblotting-based detection of the crosslinked subunit pairs. We confirmed the utility of these probes in discerning the identities of individual proteasome subtypes in a multiple myeloma cell line that abundantly expresses catalytic subunits of both the constitutive proteasome and immunoproteasome. Our findings indicate that constitutive proteasomes, immunoproteasomes, and intermediate proteasomes co-exist within these cells and support conclusions drawn from previous studies in other cell types. We also established non-small cell lung cancer cell line models of acquired bortezomib and carfilzomib resistance in which to test our hypothesis. Using immunoblotting and proteasome activity assays, we discovered that changes in the expression levels and activities of individual catalytic proteasome subunits were associated with the emergence of acquired resistance to bortezomib or carfilzomib. These changes were inhibitor-dependent and persisted after the selective pressure of the inhibitor was removed. Finally, results obtained using our bifunctional proteasome probes suggest that the altered abundance of an intermediate proteasome subtype is associated with acquired proteasome inhibitor resistance. Collectively, our results provide evidence linking changes proteasome composition with acquired proteasome inhibitor resistance and support the hypothesis that such changes are involved in resistance mechanisms to these inhibitors.

There has been a desire to accurately interpret the inhaled pharmacokinetic (PK) profiles of drugs in humans to aid successful inhaled drug and product developments. However, challenges are layered, as 1) the drug dose delivered to the lung (DTL) from inhalers is a portion of the formulated dose but rarely determined; 2) lung delivery and regional deposition differ, depending on drug, formulation and inhaler; 3) drugs are not only absorbed from the lung but may also be from the gastrointestinal (GI) tract; and 4) in addition to absorption into the systemic circulation, multiple non-absorptive processes also eliminate drugs from the lung, such as mucociliary clearance, metabolism, phagocytosis and tissue binding. Hence, this thesis project aims to develop new lung disposition model-based analyses to derive the meaningful kinetic descriptors for lung disposition from inhaled PK profiles in humans. Two approaches, curve fitting- and moment-based approaches, were developed. Both approaches modeled the kinetics of lung disposition rate-controlled by absorption (ka) and non-absorptive loss (knal), assuming no contribution of GI absorption. An exhaustive literature review found necessary data sets for three drugs, tobramycin, calcitonin and ciprofloxacin. In the curve fitting-based approach, each inhaled PK profile was fitted to the lung disposition model, while the DTL was obtained from corresponding -scintigraphic lung deposition and the kinetic parameters of systemic disposition were fixed by separate intravenous PK profile model analysis. In the moment analysis-based approach, the mean lung residence times (MLRT) and the DTL-based bioavailability (FL) were estimated and used to determine the ka and knal values in the lung disposition model, given FL = MLRTka = ka/(ka+knal). The ka and knal values were successfully derived for all the three drugs delivered by dry powder inhalers (DPIs) and/or nebulizers (NEB) through both approaches. Their “goodness-of-fit” was reasonably satisfactory. The ka values appeared to be primarily described by partition-based diffusion affected by the three hydrophilic drug’s molecular weight. In contrast, the knal values differed, yet appeared to become plausible, with a notion of additional non-absorptive confoundedness due to lung tissue binding (tobramycin) and metabolism (calcitonin), in addition to mucociliary clearance. The ka and knal values derived by the two approaches were comparable in majority of the cases. The success of these PK modeling analyses enabled further attempts to identify most influential attributes by simulation. The systemic PK and lung exposure profiles were predicted by simulation upon ±20 % changes in each of the DTL, ka and knal values to examine changes in the systemic PK metrics (Cmax, AUC and Tmax) and local lung exposure metrics (AUClung and LRT0.5). For all three drugs, the Cmax and AUC changes were identical to changes in the DTL without changing the Tmax. In contrast, impacts of the ka and knal changes differed between drugs, depending on the relative contribution of the rate constant to their sum (ka+knal). It appeared that the major contributor of the sum (ka+knal) was that rate-controlling the kinetics of lung disposition. In conclusion, this thesis project has successfully proposed two new approaches of curve fitting and moment-based analysis by accurately deriving the kinetic descriptors of lung disposition (ka and knal) for three drugs from the inhaled PK profiles in humans. Their applications were extended to predict likely changes in the systemic PK and local lung exposure metrics by simulation. While attempts should continue with more drugs, these approaches are believed to be useful in identifying critical attributes to determine the lung disposition kinetics and thus predicting the lung kinetic behavior and systemic PK profiles of new drug entities in humans.

The administration of pharmaceutical aerosols to infants on mechanical ventilation needs to be improved by increasing the efficiency of delivery devices and creating better ways of evaluating potential therapies. Aerosolized medicines such as surfactants have been administered to ventilated infants with mixed results, but studies have shown improvement in respiratory function with a much lower dose than with liquid instillation through an endotracheal tube (ETT). An aerosolized medicine must be transported through the ventilation tubing and deposit in the lungs to have the desired therapeutic response. This work has taken a systematic approach to (i) develop new devices for the efficient production of small sized charged pharmaceutical aerosols, (ii) adapt a lead device to an infant ventilation system, (iii) develop a novel breathing infant lung (BIL) in vitro model capable of capturing lung delivery efficiency in an infant without the need for human subjects testing, and (iv) evaluate the hypothesis that small sized charged pharmaceutical aerosols can improve drug delivery efficiency to the lungs of a ventilated infant. Three new devices were developed and screened for the efficient generation of small sized charged pharmaceutical aerosols, which were: wick electrospray, condensational vapor, and a modified vibrating mesh nebulizer in a streamlined low flow induction charger (LF-IC). Of these devices, only the LF-IC produced a small [mean(SD) = 1.6(0.1) micrometers] and charged (1/100 Rayleigh limit) aerosol at a pharmaceutically relevant production rate [mean(SD) = 183(9) micrograms per minute]. The LF-IC was selected as a lead device and adapted for use in an infant ventilation system, which produced an increase in in vitro lung filter deposition efficiency from 1.3% with the commercial system to 34% under cyclic ventilation conditions. The BIL model was first shown to produce a realistic pressure-volume response curve when exposed to mechanical ventilation. The optimized LF-IC was then implemented in the BIL model to demonstrate superior reduction in inspiratory resistance when surfactant was delivered as an aerosol compared to liquid instillation. For the delivery of an aerosolized medication, the lung deposition efficiency increased from a mean(SD) 0.4(0.1)% when using the conventional delivery system to 21.3(2.4)% using the LF-IC in the BIL model, a 59-fold increase. The charged aerosol produced by the LF-IC was shown to have more depositional loss in the LF-IC than an uncharged aerosol, but the charge decreased the exhaled fraction of aerosol by 17%, which needs additional study to achieve statistical significance. Completion of this work has produced a device that can achieve lung delivery efficiency that is 59-fold greater than aerosols from conventional vibrating mesh nebulizers in invasively ventilated infants using a combination of small particle size, synchronization with inspiration and appropriate charge. The BIL model produced in this work can be used to test clinically relevant methods of administering medications to infants and can be used to provide more accurate delivery estimates for development of new nebulizers and inhalers. The LF-IC developed in this work could be used for controlled and efficient delivery of aerosolized antibiotics, steroids, non-steroidal anti-inflammatories, surfactants, and vasodilators.