<p>Studies of intestinal drug permeability have traditionally been performed in the colon-derived Caco-2 cell model. However, the permeability of these cell monolayers resembles that of the colon rather than that of the small intestine, which is the major site of drug absorption following oral administration. One aim of this thesis was therefore to develop a new cell culture model that mimics the permeability of the small intestine. 2/4/A1 cells are conditionally immortalized with a temperature sensitive mutant of SV40T. These cells proliferate and form multilayers at 33°C. At cultivation temperatures of 37 – 39°C, they stop proliferating and form monolayers. 2/4/A1 cells cultivated on permeable supports expressed functional tight junctions. The barrier properties of the tight junctions such as transepithelial electrical resistance and permeability to hydrophilic markers resembled those of the human small intestine <i>in vivo</i>. These cells lacked functional expression of drug transport proteins and can therefore be used as a model to study passive drug permeability unbiased by active transport. The permeability to diverse sets of drugs in 2/4/A1 was comparable to that of the human <i>jejunum</i> for both incompletely and completely absorbed drugs, and the prediction of human intestinal permeability was better in 2/4/A1 than in Caco-2 for incompletely absorbed drugs. The small intestinal-like paracellular permeability of 2/4/A1 thus enables better predictions of drug permeability in the small intestine than does Caco-2. </p><p>The studies of the paracellular route and its importance for intestinal drug permeability was also in focus in the second part of this thesis, in which a new principle for tight junction modulation was developed, based on the primary structure of the extracellular tight junction protein occludin. Peptides corresponding to the N-terminus of the first extracellular loop increased the permeability of the tight junctions, but lacked apical effect. This problem was solved by conjugation of one peptide to a lipoamino acid, resulting in two diastereomers with different effects. The L-isomer had a sustained apical effect, while that of the D-isomer was transient. In conclusion, conjugated occludin peptides constitute a new class of tight junction modulators that can enhance the tight junction permeability.</p>

<p>The use of nano- and micro-particulate carriers as delivery systems for oral vaccines has been under investigation for several decades. Surprisingly little is known of their uptake in the human intestine, despite the fact that substantial improvement is required to achieve adequate immune responses in man after oral administration. </p><p>In this thesis, various aspects of particle transcytosis across the human intestinal epithelium were studied, in order to identify strategies for improved uptake of nano- and micro-particulate drug delivery systems. </p><p>The follicle associated epithelium (FAE) overlying Peyer´s patches contains M-cells, which have an increased capacity for uptake of particulate antigens. Therefore, a model of human FAE was developed to study mechanisms of particle uptake and transport.</p><p>Receptors that could be used for targeting to the FAE had previously not been identified in humans. By use of the model FAE, two new targets were identified on human intestinal FAE; CD9 and β1-integrin. Furthermore, studies of isolated human intestinal tissue showed that an integrin-adherent peptide motif, RGD, could be utilized to achieve selective and improved transport of nanoparticles into human Peyer´s patches.</p><p>Studies of factors influencing intestinal particle uptake and transcytosis revealed that two cytokines, TNF-α and LTα1/β2, but also one growth factor, TGF-β1, induced uptake of particles in Caco-2 cells and transcytosis of particles in the model FAE. Furthermore, it was shown that an enteric bacterium, Yersinia Pseudotuberculosis, could trigger uptake and transcytosis of particles across model absorptive epithelial cells.</p><p>In conclusion, this thesis provides a platform for further investigations of particle transcytosis across the human intestinal epithelium. The identification of two new proteins with increased expression in human FAE and a targeting sequence that improves particle uptake into Peyer’s patches, gives new hope for the development of subunit oral vaccines.</p>

Studies of intestinal drug permeability have traditionally been performed in the colon-derived Caco-2 cell model. However, the permeability of these cell monolayers resembles that of the colon rather than that of the small intestine, which is the major site of drug absorption following oral administration. One aim of this thesis was therefore to develop a new cell culture model that mimics the permeability of the small intestine. 2/4/A1 cells are conditionally immortalized with a temperature sensitive mutant of SV40T. These cells proliferate and form multilayers at 33°C. At cultivation temperatures of 37 – 39°C, they stop proliferating and form monolayers. 2/4/A1 cells cultivated on permeable supports expressed functional tight junctions. The barrier properties of the tight junctions such as transepithelial electrical resistance and permeability to hydrophilic markers resembled those of the human small intestine in vivo. These cells lacked functional expression of drug transport proteins and can therefore be used as a model to study passive drug permeability unbiased by active transport. The permeability to diverse sets of drugs in 2/4/A1 was comparable to that of the human jejunum for both incompletely and completely absorbed drugs, and the prediction of human intestinal permeability was better in 2/4/A1 than in Caco-2 for incompletely absorbed drugs. The small intestinal-like paracellular permeability of 2/4/A1 thus enables better predictions of drug permeability in the small intestine than does Caco-2. The studies of the paracellular route and its importance for intestinal drug permeability was also in focus in the second part of this thesis, in which a new principle for tight junction modulation was developed, based on the primary structure of the extracellular tight junction protein occludin. Peptides corresponding to the N-terminus of the first extracellular loop increased the permeability of the tight junctions, but lacked apical effect. This problem was solved by conjugation of one peptide to a lipoamino acid, resulting in two diastereomers with different effects. The L-isomer had a sustained apical effect, while that of the D-isomer was transient. In conclusion, conjugated occludin peptides constitute a new class of tight junction modulators that can enhance the tight junction permeability.

The use of nano- and micro-particulate carriers as delivery systems for oral vaccines has been under investigation for several decades. Surprisingly little is known of their uptake in the human intestine, despite the fact that substantial improvement is required to achieve adequate immune responses in man after oral administration. In this thesis, various aspects of particle transcytosis across the human intestinal epithelium were studied, in order to identify strategies for improved uptake of nano- and micro-particulate drug delivery systems. The follicle associated epithelium (FAE) overlying Peyer´s patches contains M-cells, which have an increased capacity for uptake of particulate antigens. Therefore, a model of human FAE was developed to study mechanisms of particle uptake and transport. Receptors that could be used for targeting to the FAE had previously not been identified in humans. By use of the model FAE, two new targets were identified on human intestinal FAE; CD9 and β1-integrin. Furthermore, studies of isolated human intestinal tissue showed that an integrin-adherent peptide motif, RGD, could be utilized to achieve selective and improved transport of nanoparticles into human Peyer´s patches. Studies of factors influencing intestinal particle uptake and transcytosis revealed that two cytokines, TNF-α and LTα1/β2, but also one growth factor, TGF-β1, induced uptake of particles in Caco-2 cells and transcytosis of particles in the model FAE. Furthermore, it was shown that an enteric bacterium, Yersinia Pseudotuberculosis, could trigger uptake and transcytosis of particles across model absorptive epithelial cells. In conclusion, this thesis provides a platform for further investigations of particle transcytosis across the human intestinal epithelium. The identification of two new proteins with increased expression in human FAE and a targeting sequence that improves particle uptake into Peyer’s patches, gives new hope for the development of subunit oral vaccines.

This thesis explores the effectiveness of the Food and Drug Administration’s implementation of Breakthrough Therapy Designation (BTD), focusing on the low number of approval rates and repercussions of BTD for the development of new drugs for patients suffering serious life threatening illnesses. BTD, as an expedited review process, shows potential for improvement in its guidelines for necessary qualifications for BTD. Cutting costs, through a shortening in development time, and raising profits, through first mover status of new to market drugs, BTD is regarded by pharmaceutical executives as a tool to insure not only return on investment but also the rewards that accompanies a profitable blockbuster drug. Lessons learned from activism from 1980’s HIV/AIDS crisis show how advocates and “activist-experts” can rebalance and refocus more attention on the necessary beneficence for patients. A policy stipulation that insures all members: corporate, regulatory, and patient advocate, sit together at the decision making table will insure a more balanced discussion in regards to drug development.

Microneedle (MN) enhanced drug delivery is a safe, effective and efficient enhancement method for delivery of drug molecules across the skin. The “poke (press) and patch” approach employs solid stainless steel MN to permeablize the skin prior to application of a regular drug patch over the treated area. It has been previously shown that MN can be used to deliver naltrexone (NTX) at a rate that provides plasma concentrations in the lower end of the therapeutic range in humans. The drug delivery potential of this technique is, however, limited by the re-sealing of the micropores in a 48-72h timeframe. The goal of the current research was to optimize the formulation for a 7 day MN enhanced delivery system for NTX either by adding a second active pharmacological moiety or by optimizing formulation characteristics alone. Three different formulation strategies were explored: formulation pH optimization with NTX; a codrug approach with NTX and a nonspecific cyclooxygenase inhibitor, diclofenac (DIC); and a topical/transdermal approach with NTX and an enzyme inhibitor of the cholesterol synthesis pathway, fluvastatin (FLU). The results indicated that formulation pH cannot be used to extend micropore lifetime, although formulation optimization leads to enhanced transport and thus drug delivery across MN treated skin. The codrug approach was successful in extending the micropore lifetime and further screening of codrug structures and formulation optimization helped in selection of a codrug candidate suitable for evaluation in animal pharmacokinetic studies. Local treatment with FLU helped to keep the micropores open and enabled delivery of NTX for an extended period. The pores re-sealed on removal of treatment within a 30-45 minute timeframe, indicating that infection/irritation should not be a major issue, as in the case of other topical chemical enhancers. Thus, overall it can be concluded that different formulation strategies can be utilized to extend micropore lifetime and enhance delivery of drug molecules across the skin.

Organic cation transporters (OCTs) play a pivotal role in the absorption, tissue distribution, and excretion of a diverse array of substances, and currently the nature of the biochemical interactions between substrate and OCTs are unknown. Therefore, identifying which amino acid residues are critical for OCT-substrate interactions is of central importance to understanding and predicting interactions between drugs and OCTs. A three-dimensional (3-D) homology model of human OCT3 was generated using the crystal structure of a high affinity phosphate transporter from Piriformospora indica (PiPT) as template, and putative binding pocket for the prototypical hOCT3 ligand 1-methyl-4-phenylpyridinium (MPP+) was identified through docking studies. Five residues, Phe36, Val40, Trp358, Glu451 and Asp478, were identified as potentially mediating hOCT3-MPP+ interactions, and confirmed through in vitro studies. Additionally, 3-D homology modeling of the functional hOCT3 mutant Val40Leu, and all non-functional hOCT3 mutants, indicated changes in binding pocket architecture consistent with weakening of ligand-transporter interactions. Docking of structurally divergent hOCT3 substrates indicated binding interactions in the same general region as that identified for MPP+, albeit with mostly unique residues. Interspecies differences were explored by generating 3-D homology models for rat and murine Oct3. Results from docking studies using compounds exhibiting vastly different binding affinities (Km or IC50) towards the OCT3/Oct3 orthologs were consistent with varying strength in ligand-transporter binding pocket interactions. Finally, a series of novel compounds exhibiting anti-depressant-like activity was screened for OCT interaction in vitro, and demonstrated significant inhibitory effects on OCTs for many of the compounds.

The current environment for drug discovery and disease treatment relies heavily on genomic analysis, structural biology and chemical biology techniques. With the enormous advances in genomic analysis and structural biology, the use of and desire for targeted therapies has increased. However, as more genomic data for cancer disease state pathology becomes available we must ask increasingly difficult questions and even produce new technologies, such as activity-based probes, to answer these questions. In particular, targeted kinase inhibitors for the treatment of cancer has become a mainstay for drug development for both industry and academia, but it is evident that the genomic data is not always indicative of protein expression. Additionally, protein expression alone does not completely characterize functional activity. Therefore, in order to more accurately validate drug targets and predict drug efficacy, we must not only identify possible targets but also determine their activity in vivo. The goal of this work was to develop a probe for Protein Kinase A that would act by alkylating a conserved cysteine in the substrate-binding pocket of the enzyme. We hypothesized that by targeting the substrate-binding pocket we could effectively utilize the natural substrate selectivity filters as well as take into account multiple endogenous regulatory mechanisms. We produced probes utilizing portions of the pseudosubstrate inhibitor PKI that demonstrate the ability to label the catalytic subunit of Protein Kinase A in an activity-dependent manner, thus making it an important first step in a new class of activity-based probes for the kinome.

Numerous studies have demonstrated the impact of organic cation (OCTs; SLC22 family) and anion transporters (OATs; SLC22 family) on the efficacy and safety of clinically important therapeutics. To be specific, OCTs and OATs have been identified as determinants for uptake into and secretion from enterocytes, hepatocytes and renal proximal tubular cells, and are frequent sites of drug-drug interaction (DDI). In addition, OCTs expressed in brain are components of the low-affinity, high capacity clearance pathway (uptake-2) for biogenic monoamine neurotransmitters. As a result, OCTs may represent novel targets for mood disorders. The inhibitory effects of several therapeutic agents, designed drugs and novel compounds were assessed on the function of OCTs/Octs and OATs/Oats. Among these compounds, the anthraquinone rhein showed significant inhibition on hOATs. While the antituberculosis drug ethambutol, the herbal products matrine and oxymatrine, synthetic cathinones, and all quinazoline and guanidine compounds produced significant inhibition on hOCT activity with most IC50 values in the micro- and even nanomolar ranges. Considering the clinically relevant unbound concentrations in biofluids, significant DDI potentials were found for rhein, ethambutol, matrine, oxymatrine and several synthetic cathinones affecting enterocytes, hepatocytes and/or proximal tubules. As hOCT2 and hOCT3 may participate in modulating neurotransmitter homeostasis in the CNS, these findings also suggested that the CNS pharmacological effects of synthetic cathinones, quinazoline and guanidine compounds might be due to their inhibitory effects on OCTs; although their impact may be limited solely to clearance of these compounds. Based upon their in vitro OCT/Oct inhibition profiles, three lead quinazoline and guanidine compounds were chosen for in vivo studies. Potent antidepressant-like effects of one lead hOCT-interacting compound (KEO-099) were re-confirmed in the tail suspension test. While in vivo results of the two newly identified hOCT-interacting lead compounds were somewhat less clear. Finally, homology modeling and docking studies for hOCT3 identified key amino acid residues that might be involved in interaction between hOCT3 and small molecules. Subsequent experiments confirmed a competitive mode of interaction between MPP+ and lead compounds on hOCT3. Thus, preliminary analysis indicates our hOCT3 homology model can be used to support rational drug design and high-throughput screening of novel hOCT substrates/inhibitors.

Purpose: Ivermectin (IVM) is a lipophilic BCS-II compound (molecular weight=875 g/mole, LogP=3.22, intrinsic solubility=700 ug/L). IVM is used as antiparasitic drug in both humans and animals. IVM is known to have a half-life of 12-56 hours in humans. Strongyloidiasis is a chronic parasitic infection of humans caused by Strongyloides stercoralis, with an estimated 30-100 million people infected worldwide. Infection may be severe and even life-threatening in cases of immunodeficiency. Patients with disseminated strongyloidiasis are usually bedridden hospitalized patients that show symptoms such as paralytic ileus and reduced plasma albumin and cholesterol. Oral IVM is the only FDA-approved treatment but may not be effective in patients with disseminated disease. Veterinary subcutaneous formulations have been used in severe infections. We hypothesized that IVM PK in patients with disseminated strongyloidiasis can be predicted using PBPK model originally built and refined in healthy human and animal species. This hypothesis was tested and shown to be valid. Methods:A systematic method was used to build and refine different parts of the PBPK model. The process involved construction of models, parameterization of these models, evaluation of the effect of uncertainty in model parameters on model prediction via local and global sensitivity analyses and finally, refinement of model predictions. Two disposition models that differ in the rate limiting step in drug distribution were constructed and include perfusion-limited and permeability-limited distribution models. The ability of each model to predict IVM disposition was evaluated using plasma PK data in rat after intra-arterial dosing and in dog after intravenous bolus dosing. Then the disposition model was scaled to humans and an oral input model was constructed as a modification on the well-known ACAT model. The oral input model was coupled with the disposition model and used to predict IVM plasma concentration-time profile in healthy fasted human subject after oral dosing. Two subcutaneous (SQ) input models were constructed and used to evaluate the effect of IVM precipitation at the injection site. Plasma PK data in dog after SQ dosing was used to refine the constructed SQ input models. The refined disposition, oral input and SQ input physiologically-based models were used to predict IVM PK in patients with disseminated strongyloidiasis after a complex dosing regimen. The physiological parameters of the model were modified to account for the effect of the disease-induced pathophysiological changes on the body physiology and hence on the drug PK. Plasma PK data from hospitalized subjects with disseminated strongylidiasis was used in this part. Results and conclusions:The disposition model with assumption of permeability-limited distribution was more capable of describing IVM disposition in rat after intra-arterial dosing compared to when perfusion-limited distribution was assumed. The model predicted that hepatic clearance is the most impactful parameter on model-predicted plasma concentration of the drug. Also, IVM was shown to have low hepatic extraction ratio along with high binding in plasma and large volume of distribution, which collectively may explain the long half-life in the plasma of 63 hours in rat after intra-arterial dosing. The oral input model predicted that the oral input is limited by drug dissolution in the GI lumen and that a very small fraction of oral tablet dose (0.03) is available in the systemic circulation in healthy fasted human subjects. Both of the studied SQ input models predicted that majority of IVM absorption after SQ dosing is via the lymphatic route and that drug precipitation at the injection site can further slowdown the drug absorption after SQ administration. The PBPK model was able achieve the main goal of this research which is to predict IVM pharmacokinetics in patients with disseminated strongyloidiasis after a complex dosing regimen of multiple oral and SQ dosing. This was achieved by modifying the most impactful physiological parameters of the model affected by the disease state and that are related to drug binding in the plasma (fraction unbound), the GI motility (gastric emptying rate) and the lymphatic flow rate. Based on our analysis, we recommend measurement of plasma IVM concentrations early after initiation of therapy to exclude treatment failure due to reduced oral and/or SQ absorption. Also, we recommend measurement of plasma lipoprotein levels and their composition in these patients to differentiate between low total plasma concentrations due to low binding plasma as opposed to low drug input. Finally, interventional procedures that enhance lymphatic flow rate to site of SQ injection are recommended to enhance SQ absorption.