The overall objectives of this research project were i) to develop and evaluate methods of characterizing nasal spray products using realistic nasal airway models as more clinically relevant in vitro tools and ii) to develop and evaluate a novel high-efficiency antibiotic nanoparticle dry powder formulation and delivery device. Two physically realistic nasal airway models were used to assess the effects of patient-use experimental conditions, nasal airway geometry and formulation / device properties on the delivery efficiency of nasal spray products. There was a large variability in drug delivery to the middle passages ranging from 17 – 57 % and 47 – 77 % with respect to patient use conditions for the two nasal airway geometries. The patient use variables of nasal spray position, head angle and nasal inhalation timing with respect to spray actuation were found to be significant in determining nasal valve penetration and middle passage deposition of Nasonex®. The developed test methods were able to reproducibly generate similar nasal deposition profiles for nasal spray products with similar plume and droplet characteristics. Differences in spray plume geometry (smaller plume diameter resulted in higher middle passage drug delivery) were observed to have more influence on regional nasal drug deposition than changes to droplet size for mometasone furoate formulations in the realistic airway models. Ciprofloxacin nanoparticles with a mean (SD) volume diameter of 120 (10) nm suitable for penetration through mucus and biofilm layers were prepared using sonocrystallization technique. These ciprofloxacin nanoparticles were then spray dried in a PVP K30 matrix to form nanocomposite particles with a mean (SD) volume diameter of 5.6 (0.1) µm. High efficiency targeted delivery of the nanocomposite nasal powder formulation was achieved using a modified low flow VCU DPI in combination with a novel breathing maneuver; delivering 73 % of the delivered dose to the middle passages. A modified version of the nasal airway model accommodating Transwell® inserts and a Calu-3 monolayer was developed to allow realistic deposition and evaluation of the nasal powder. The nanocomposite formulation was observed to demonstrate improved dissolution and transepithelial transport (flux = 725 ng/h/cm2) compared to unprocessed ciprofloxacin powder (flux = 321 ng/h/cm2).

α7 Neuronal nicotinic acetylcholine receptors are one of two major classes of receptors responsible for cholinergic neurotransmission in the central nervous system. The existence of α7 neuronal nAChRs in different regions of the nervous system suggests their involvement in certain essential physiological functions as well as in disorders such as Alzheimer’s disease (AD), drug dependence, and depression. This project was aimed toward the discovery and development of small–molecule arylguanidines that modulate α7 nAChR function with improved subtype-selectivity through an allosteric approach. Identifying the required structural features of these small molecules allowed optimization of their negative allosteric modulator (NAM) actions at α7 neuronal nAChRs. MD-354 (3-chlorophenylguanidine) was the first small–molecule NAM at α7 nAChRs; however, it also binds at 5-HT3 receptors. The N-methyl analog of MD-354 appeared to be more selective toward α7 nAChRs than 5-HT3 receptors. Comparative studies using two series of novel compounds based on MD-354 and its N-methyl analog explored the aryl 3-position and investigated whether or not the MD-354 series and the N-methyl series bind in the same manner. Biological potencies of the MD-354 series and the N-methyl series of compounds, obtained from electrophysiological assays with Xenopus laevis oocytes expressing human α7 nAChRs in two-electrode voltage-clamp assays, showed that N-(3-iodophenyl)-N- methylguanidine (28) is the most potent analog at α7 nAChRs. Our comparative study and Hansch analyses indicated different binding modes of the two series. In addition, we investigated: i) the length/size of the aliphatic side chain at the anilinic nitrogen, ii) the effect of alkylating the guanidine nitrogen atoms, and iii) the necessity of the presence of these nitrogen atoms for the inhibitory effects of arylguanidines at α7 nAChRs. In efforts to explain the varied functional activity of these arylguanidines, homology models of the extracellular domain and the transmembrane domain of human α7 nAChRs were developed, allosteric sites identified, and docking studies and hydropathic analysis conducted. The 3D quantitative structure-activity relationships for our compounds were also analyzed using CoMFA. A pharmacophore for arylguanidines as α7 nAChR NAMs was identified. Together, these data should be useful for the subsequent design of novel arylguanidine analogs for their potential treatment of neurological disorders.

Including inherited genetic diseases, like lipoprotein lipase deficiency, and acquired diseases, such as cancer and HIV, gene therapy has the potential to treat or cure afflicted people by driving an affected cell to produce a therapeutic protein. Using primarily viral vectors, gene therapies are involved in a number of ongoing clinical trials and have already been approved by multiple international regulatory drug administrations for several diseases. However, viral vectors suffer from serious disadvantages including poor transduction of many cell types, immunogenicity, direct tissue toxicity and lack of targetability. Non-viral polymeric gene delivery vectors (polyplexes) provide an alternative solution but are limited by poor transfection efficiency and cytotoxicity. Microfluidic (MF) nano-precipitation is an emerging field in which researchers seek to tune the physicochemical properties of nanoparticles by controlling the flow regime during synthesis. Using this approach, several groups have demonstrated the successful production of enhanced polymeric gene delivery vectors. It has been shown that polyplexes created in the diffusive flow environment have a higher transfection efficiency and lower cytotoxicity. Other groups have demonstrated that charge-stabilizing polyplexes by sequentially adding polymers of alternating charges improves transfection efficiency and serum stability, also addressing major challenges to the clinical implementation of non-viral gene delivery vectors. To advance non-viral gene delivery towards clinical relevance, we have developed a microfluidic platform (MS) that produces conventional polyplexes with increased transfection efficiency and decreased toxicity and then extended this platform for the production of ternary polyplexes. This work involves first designing microfluidic devices using computational fluid dynamics (CFD), fabricating the devices, and validating the devices using fluorescence flow characterization and absorbance measurements of the resulting products. With an integrated separation mechanism, excess polyethylenimine (PEI) is removed from the outer regions of the stream leaving purified polyplexes that can go on to be used directly in transfections or be charge stabilized by addition of polyanions such as polyglutamic acid (PGA) for the creation of ternary polyplexes. Following the design portion of the research, the device was used to produce binary particle characterization was carried out and particle sizes, polydispersity and zeta potential of both conventional and MS polyplexes was compared. MS-produced polyplexes exhibited up to a 75% reduction in particle size compared to BM-produced polyplexes, while exhibiting little difference in zeta potential and polydispersity. A variety of standard biological assays were carried out to test the effects of the vectors on a variety of cell lines – and in this case the MS polyplexes proved to be both less toxic and have higher transfection efficiency in most cell lines. HeLa cells demonstrated the highest increase in transgene expression with a 150-fold increase when comparing to conventional bulk mixed polyplexes at the optimum formulation. A similar set of experiments were carried out with ternary polyplexes produced by the separation device. In this case it was shown that there were statistically significant increases in transfection efficiency for the MS-produced ternary polyplexes compared to BM-produced poyplexes, with a 23-fold increase in transfection activity at the optimum PEI/DNA ratio in MDAMB-231 cells. These MS-produced ternary polyplexes exhibited higher cell viability in many instances, a result that may be explained but the reduction in both free polymer and ghost particles.

The extensive and sometimes incorrect and noncompliant use of various types of antimicrobial agents has accelerated the development of antimicrobial resistance (AMR). In fact, AMR has become one of the greatest global threat to human health in this era. The broad-spectrum antibiotics aminoglycosides (AGs) display excellent potency against most Gram-negative bacteria, mycobacteria, and some Gram-positive bacteria, such as Staphylococcus aureus. The AG antibiotics amikacin, gentamicin, kanamycin, and tobramycin are still commonly prescribed in the U.S.A. for the treatment of serious infections. Unfortunately, bacteria evolve to acquire resistance to AGs via four different mechanisms: i) changing in membrane permeability to resist drugs from entering, ii) upregulating efflux pumps for active removal of intracellular AGs, iii) modifying the antimicrobial target(s) to prevent drugs binding to their targets, and iv) acquiring resistance enzymes to chemically inactivate the compounds. Amongst all, the acquisition of resistance enzymes, AG-modifying enzymes (AMEs), is the most common resistance mechanism identified. Depending on the chemistry each enzyme catalyzes, AMEs can be further divided into AG N-acetyltransferases (AACs), AG O-phosphotransferases (APHs), and AG O-nucleotidyltransferases. To overcome AME-related resistance, we need to better understand these resistance enzymes and further seek ways to either escape or inhibit their actions. In this dissertation, I summarized my efforts to characterize the AAC(6') domain and its mutant enzymes from a bifunctional AME, AAC(6')-Ie/APH(2")-Ia as well as another common AME, APH(3')-IIa. I also explained my attempt to inhibit the action of various AAC enzymes using metal salts. In an effort to explore the current resistance epidemic, I evaluated the resistance against carbapenem and AG antibiotics and the correlation between the resistance profiles and the AME genes in a collection of 122 Pseudomonas aeruginosa clinical isolates obtained from the University of Kentucky Hospital System. Besides tackling the resistance mechanisms in bacteria, I have also attempted to explore a new antifungal option by repurposing an existing antipsychotic drug, bromperidol, and a panel of its derivatives into a combination therapy with the azole antifungals against a variety of pathogenic yeasts and filamentous fungi.

An estimated 13.9% of Americans currently meet criteria for an alcohol (ethanol; EtOH) use disorder (AUD). While there are 4 medications approved by the Food and Drug Administration (FDA) to treat AUD, these treatments have demonstrated poor clinical efficacy. Our ongoing research program encompasses a multi-tiered screening of a natural product library and validation process to provide novel information about the mechanisms underlying EtOH-induced changes in neurobiology and to identify novel chemical scaffolds to be exploited in the development of pharmacological treatments for AUD in a rodent organotypic hippocampal slice culture model. Initial screens of several natural product compounds identified 3 compounds which attenuate 48 h EtOH-induced cytotoxicity in vitro. As analogs of natural products can be developed to have enhanced therapeutic potential over parental structures, Study 1 sought to extend on prior findings via the screening of several natural product analogs for their ability to attenuate EtOH-induced cytoxicity. Nine natural produce analogs demonstrated potent cytoprotective effects against EtOH-induced toxicity at 48 h. Several reports suggest EtOH-induced neurotoxicity may be secondary to the induction of persistent neuroimmune activation, and isoflavonoids have been shown to have effects on neuroimmune signaling. Thus, Study 2 compared the effects of compound 9b, an isoflavonoid analog identified in Study 1, to daidzein (DZ), a prototypical isoflavonoid, in the same 48 h model, with the addition of a neuroimmune component. Specifically, culture media was collected to assess for the release of the neuroimmune mediators HMGB1, TNF-α, IL-6, and IL-10 via ELISA. Compound 9b and DZ protected against EtOH-induced cytotoxicity at 48 h. EtOH exposure significantly increased secretion of HMGB1 and IL-6 into culture media at 48h. Compound 9b and DZ attenuated these increases at all concentrations tested. These results suggest potential neuroimmune modulating properties of isoflavonoids which may contribute to their neuroprotective effects against EtOH in vitro. These findings highlight the potential applications DZ and the novel isoflavonoid analog 9b for use in the treatment of AUD.

Introduction: Myocardial infarction (MI) remains the leading cause of morbidity and mortality worldwide. Induced by cardiomyocyte death, MI initiates a prolonged and uncontrolled inflammatory response which impairs the healing process. Immune cells, such as macrophages, play a central role in organizing the early post-MI inflammatory response and the subsequent repair phase. Two activation states of macrophages have been identified with distinct and complementary functions (inflammatory vs. reparatory). This bimodal pattern of macrophage activation is an attractive therapeutic target to favorably resolve post-MI inflammation and enhance recovery. It has been demonstrated that azithromycin (AZM), a commonly used antibiotic with immunomodulatory effects, polarizes macrophages towards the reparatory phenotype. AZM has an excellent safety profile and has been approved for human use. We hypothesize that AZM reduces inflammation and improves heart function in MI. Methods and results: In our initial studies, we demonstrated that oral free AZM (160 mg/kg daily for 7 days), initiated 3 days prior to MI, enhances post-MI cardiac recovery as a result of shifting macrophages to the reparatory state. We observed a significant reduction in mortality with AZM therapy. AZM-treated mice showed a significant decrease in pro-inflammatory and an increase in reparative macrophages, decreasing the pro-inflammatory/reparative macrophage ratio. Macrophage changes were associated with a significant decline in pro- and an increase in anti-inflammatory cytokines. Additionally, AZM treatment was correlated with a distinct decrease in neutrophil count due to apoptosis, a known signal for shifting macrophages towards the reparative phenotype. Finally, AZM treatment improved cardiac recovery, scar size, and angiogenesis. We designed this proof of concept study using pre-MI AZM therapy to achieve steady state levels prior to injury. Therefore, in our follow-up studies we targeted inflammatory macrophages using a non-Pegylated liposomal formulation of AZM (Lazm) which has been shown in multiple studies to promote drug efficacy and minimize off-target effects. To test the hypothesis that Lazm is more effective and safer than free AZM, low doses of free/liposomal AZM (10 or 40 mg/kg, administered intravenously) were initiated immediately after MI. We observed that Lazm induces early resolution of the post-MI inflammatory response as evidenced by switching of the activation state of monocytes/macrophages towards the reparatory phenotype. Neutrophils were substantially decreased, particularly pro-inflammatory neutrophils. Cytokine profiles were also shifted to the anti-inflammatory status with Lazm therapy. Taken together, AZM treatment resulted in a significant shift in macrophage activation towards the reparatory state. The shift in inflammatory state was accompanied by a decrease in apoptosis and infarct size in the injured heart, as well as enhanced angiogenesis and LV functional recovery in our long-term studies. In addition, Lazm was protective against off-target effects of AZM on the heart. Conclusion: This is the first evidence of a novel and clinically-relevant therapeutic strategy to modulate post-MI inflammation. We found that AZM reduces cardiac inflammation and improves adverse cardiac remodeling after infarction via promoting a shift of macrophage activation state. The overarching significance of this work is the modulation of sterile inflammation, which can be a viable therapeutic target in many conditions including stroke and heart attack. Additionally, this is the first study to demonstrate the immune modulation properties of liposomal AZM, which has wide potential therapeutic applications beyond the cardiovascular field. Importantly, liposomal formulation of AZM is protective from its cardiac off-target effects. Our findings strongly support clinical trials using AZM as a novel and clinically relevant therapeutic target to improve cardiac recovery and reduce heart failure post-MI in humans.

Nanoparticulate drug delivery systems (NDDS) such as nanocrystals, nanosuspensions, solid-lipid nanoparticles often formulated for the bioavailability enhancement of poorly soluble drug candidates are stabilized by a mixture of excipients including surfactants and polymers. Most literature studies have focused on the interaction of excipients with the NDDS surfaces while ignoring the interaction of excipients in solution and the extent to which the solution-state interactions influence the affinity and capacity of adsorption. Mechanisms by which excipients stabilize NDDS and how this information can be utilized by formulators a priori to make a rational selection of excipients is not known. The goals of this dissertation work were (a) to determine the energetics of interactions between HPMC and model surfactants and the extent to which these solution-state interactions modulate the adsorption of these excipients onto solid surfaces, (b) to determine and characterize the structures of various aggregate species formed by the interaction between hydroxypropyl methylcellulose (HPMC) and model surfactants (nonionic and ionic) in solution-state, and (c) to extend these quantitative relationships to interpret probable mechanisms of mixed adsorption of excipients onto the model NDDS surface. A unique approach utilizing fluorescence, solution calorimetry and adsorption isotherms was applied to tease apart the effect of solution state interactions of polymer and surfactant on the extent of simultaneous adsorption of the two excipients on a model surface. The onset of aggregation and changes in aggregate structures were quantified by a fluorescence probe approach with successive addition of surfactant. In the presence of HPMC, the structures of the aggregates formed were much smaller with an aggregation number (Nagg) of 34 as compared to micelles (Nagg ~ 68) formed in the absence of HPMC. The strength of polymer-surfactant interactions was determined to be a function of ionic strength and hydrophobicity of surfactant. The nature of these structures was characterized using their solubilization power for a hydrophobic probe molecule. This was determined to be approximately 35% higher in the polymer-surfactant aggregates as compared to micelles alone and was attributed to a significant increase in the number of aggregates formed and the increased hydrophobic microenvironment within these aggregates at a given concentration of surfactant. The energetics of the adsorption of SDS, HPMC, and SDS-HPMC aggregate onto nanosuspensions of silica, which is the model solid surface were quantified. A strong adsorption enthalpy of 1.25 kJ/mol was determined for SDS adsorption onto silica in the presence of HPMC as compared to the negligible adsorption enthalpy of 0.1 kJ/mol for SDS alone on the silica surface. The solution depletion and HPMC/ELSD methods showed a marked increase in the adsorption of SDS onto silica in the presence of HPMC. However, at high SDS concentrations, a significant decrease in the adsorbed amount of HPMC onto silica was determined. This was further corroborated by the adsorption enthalpy that showed that the silica-HPMC-SDS aggregation process became less endothermic upon addition of SDS. This suggested that the decrease in adsorption of HPMC onto silica at high SDS concentrations was due to competitive adsorption of SDS-HPMC aggregates wherein SDS is displaced/desorbed from silica in the presence of HPMC. At low SDS concentrations, an increase in adsorption of SDS was due to cooperative adsorption wherein SDS is preferentially adsorbed onto silica in the presence of HPMC. This adsorption behavior confirmed the hypothesis that the solution-state interactions between pharmaceutical excipients such as polymers and surfactants would significantly impact the affinity and capacity of adsorption of these excipients on NDDS surfaces.

Substance use disorders are serious health concerns in the United States. Furthermore, the National Survey on Drug Use and Health reports a continuous increase in substance use disorders in the United States during the last 10 years. However, there are not many effective pharmacotherapeutics available for substance use disorders. The current dissertation is focused on research aimed at discovering pharmacotherapeutics for substance use disorders. First part of dissertation focused on discovering methamphetamine (METH) use disorder therapeutics targeting specific mechanism of METH action on dopaminergic neurons. The second part of dissertation focused on opioids and cocaine use disorder therapeutics targeting rewarding pathway commonly activated by opioids and cocaine. With respect to METH, it induces release of dopamine (DA) in neuronal terminals by interacting with the vesicular monoamine transporter-2 (VMAT2) and DA transporter (DAT). VMAT2 inhibitors have been found by our research group to decrease METH-evoked DA release, METH-induced hyperlocomotion, and METH self-administration in rats. However, these VMAT2 inhibitors lacked selectivity and tolerance developed to these pharmacologic effects after repeated administration, thereby limiting their potential as pharmacotherapeutics for METH use disorders. In the current study, analogs from a novel scaffold were found to selectively inhibit VMAT2 and were evaluated using neurochemical and behavioral pharmacological approaches. R- and S-3-(4-methoxyphenyl)-N-(1-phenylpropan-2-yl)propan-1-amine (GZ-11610 and GZ-11608, respectively) exhibited 94- to 3450-fold selectivity for VMAT2 over human-ether-a-go-go (hERG) channel, DAT, serotonin transporter, and nicotinic acetylcholine receptors. GZ-11608 competitively and concentration-dependently inhibited METH-evoked DA release via VMAT2. Also, GZ-11610 (56-300 mg/kg, oral) and GZ-11608 (300 mg/kg, oral; 10-30 mg/kg, s.c.) reduced METH-induced hyperlocomotor activity in METH-sensitized rats. Furthermore, GZ-11608 (1-30 mg/kg, s.c.) inhibited METH self-administration, cue- and METH-induced reinstatement in a dose-dependent manner, and 30 mg/kg (s.c.), 10 mg/kg (s.c.), and 17 mg/kg (s.c.) produced significant effect, respectively. Importantly, the GZ-11608-induced decrease in METH self-administration was not surmounted by increasing the amount of METH available. GZ-11608 did not substitute for METH and did not serve as a reinforcer in rats self-administering METH and drug naïve rats, respectively. Thus, these VMAT2 inhibitors incorporating a new scaffold are novel leads for new pharmacotherapeutics to treat METH use disorders. Substances with high abuse potential including opioids and cocaine elevate extracellular DA concentration in the nucleus accumbens, and this mechanism has long been considered to underly substance-induced reward. DA in the nucleus accumbens originates from DA neuron cell bodies located in the ventral tegmental area in the midbrain. Interestingly, M5 muscarinic acetylcholine receptors (mAChRs) are proteins that are highly expressed on ventral tegmental area DA neurons. Also, studies investigating M5 mAChRs knockout mice showed reduced responding for cocaine in cocaine self-administration and decreased time spent in cocaine-paired and morphine-paired place preference studies. Pharmacological inhibition of M5 mAChRs function via microinfusing mAChR antagonists exhibiting no selectivity among M1-M5 mAChRs subtypes into the ventral tegmental area where expression of M5 mAChRs are dominant, reduced morphine-induced hyperlocomotion and cocaine seeking behaviors in rats. These studies support therapeutic potential of M5 mAChRs selectivity antagonists in opioids and cocaine use disorders. Thus, in the current study, affinity of a series of pethidine and quinuclidinyl N-phenylcarbamate analogs for M5 mAChRs was evaluated using in vitro and ex vivo neuropharmacological assays. Among the pethidine analogs, compound 6a showed the highest binding affinity at M5 (Ki = 0.38 µM), but also high affinity at M1 and M3 mAChRs (0.67 and 0.37 µM, respectively). Among the quinuclidinyl N-phenylcarbamate analogs, compound 13c exhibited the highest affinity at M5 (Ki = 1.8 nM), but also high affinity at M1, M2, M3 and M4 mAChRs (Ki = 1.6, 13, 2.6, 2.2 nM, respectively). Also, 13c acted as an agonist of mAChRs on oxotremorine-induced DA release from rat striatal slices. In addition, compound 13b was found exhibiting the highest selectivity (17-fold) at M3 over M2 mAChRs, suggesting potential of 13b as a chronic obstructive pulmonary disease therapeutics. Taken together, these novel analogs serve as leads for further discovery of subtype-selective M5 mAChR antagonists that may have potential as therapeutics for substance use disorders, as well as for chronic obstructive pulmonary disease.

Hydropathic molecular modeling is a computer-aided molecular design technique for obtaining, representing, and understanding the properties and interactions of biomacromolecular complexes in the biological environment. Hydropathic INTeraction (HINT) is a novel empirical force field to calculate the free energy of intermolecular interaction based on experimentally determined partition coefficients (log Po/w). It includes all the expected interactions between molecules such as hydrogen bonding, hydrophobic, electrostatic, acid-base, and Coulombic interactions, entropy, solvation and others. HINT tools were used to determine, evaluate, and analyze protein-ligand interactions in different research projects: 1) We used these tools to discover small molecule inhibitors of PsaA, a potential target for Streptococcus pneumoniae. We screened and scored potential molecules to obtain hits. After the growth conditions for both the wild type and PsaA mutant of S. pneumoniae were optimized, we then tested our hits. A few compounds passed through the three-stage assay protocol and confirmed the inhibition of PsaA with MICs between 125-250 μM. 2) The SAR of C-3 and C-5 pyrrole-based antitubulin agents at the colchicine-binding site with explicitly solvated models was performed. After docking with GOLD at the colchicine site, post-docking scoring and evaluation were performed with HINT. The total HINT score correlates with binding and activity; similarly, the significance of individual functional groups, protein residues and interactions amongst a collection of compounds can be quantitated. The possibility of water-mediated interactions in a way solvent accessible part of the pocket was considered by subjecting molecular models to MD simulations. Several water molecules were identified to be contributing to the binding and were confirmed by HINT scoring. Finally, using hydropathic molecular modeling tools helped us to understand, evaluate, analyze, and improve protein-ligand interactions in different biological systems.

Using a semi-PBPK modeling/quantitative meta-analysis approach, this project investigated what factors affect pulmonary and systemic exposures of Budesonide (BUD), Tobramycin (TOB), and Ciprofloxacin (CIP) after inhalation: Three structurally different pulmonary disposition models were developed for each drug, including pulmonary absorption (all three), excretion (TOB and CIP) and sequestration (TOB) in a peripheral and central lung compartment. Systemic disposition parameters were estimated using available human mean plasma (cp(t)) and sputum (cs(t)) concentration profiles after IV administration, and GI absorption parameters were estimated from these profiles after oral administration. Pulmonary disposition parameters were estimated from cp(t) and cs(t) profiles after inhalation using various devices along with their published pulmonary deposition characteristics. Appropriate covariate models accounted for effects of Cystic Fibrosis on the systemic disposition/GI absorption for TOB and CIP. Monte Carlo Simulations (MCS) were used to optimize parameters and validate the final models and parameter spaces against published data. Despite limited available data, especially cs(t) for BUD and CIP (after IV administration), the point estimates for the final model parameters were mechanistically plausible for all three drugs and consistent with their known differences in physicochemical and ADME properties. Model predictions adequately described the observed cp(t) and cs(t) profiles as well as exposure metrics across studies. As the most lipophilic drug, BUD showed the fastest pulmonary absorption rates and highest Fpul (83%). TOB, a very hydrophilic drug, exhibited (intracellular) pulmonary sequestration, resulting in slow pulmonary absorption and excretion and low Fpul (10%). CIP - as zwitterion - showed relatively slow pulmonary absorption and excretion, leading to low Fpul (8%); pulmonary excretion accounted for 27% of CIP overall elimination. Results of a formal parameter sensitivity analysis demonstrated that, for all three drugs, after inhalation, (1) their systemic exposures (cp(t)) depend primarily on CLtot along with Fpul/sequestration combined with Foral; (2) increasing pulmonary exposures (cs(t)) can be accomplished by slowing down pulmonary absorption rates (kca) and/or slowing down mucociliary clearance from the lungs into the GI tract (kcm) – affirming the overall hypothesis guiding the project.