This dissertation thesis consists of a series of chapters that are interwoven by solving interesting biological problems, employing various computational methodologies. These techniques provide meaningful physical insights to promote the scientific fields of interest. Focus of chapter 1 concerns, the importance of computational tools like docking studies in advancing structure based drug design processes. This chapter also addresses the prime concerns like scoring functions, sampling algorithms and flexible docking studies that hamper the docking successes. Information about the different kinds of flexible dockings in terms of accuracy, time limitations and success studies are presented. Later the importance of Induced fit docking studies was explained in comparison to traditional MD simulations to predict the absolute binding modes. Chapter 2 and 3 focuses on understanding, how sickle cell disease progresses through the production of sickled hemoglobin and its effects on sickle cell patients. And how, hydroxyurea, the only FDA approved treatment of sickle cell disease acts to subside sickle cell effects. It is believed the primary mechanism of action is associated with the pharmacological elevation of nitric oxide in the blood, however, the exact details of this mechanism is still unclear. HU interacts with oxy and deoxyHb resulting in slow NO production rates. However, this did not correlate with the observed increase of NO concentrations in patients undergoing HU therapy. The discrepancy can be attributed to the interaction of HU competing with other heme based enzymes such as catalase and peroxidases. In these two chapters, we investigate the atomic level details of this process using a combination of flexible-ligand / flexible-receptor virtual screening (i.e. induced fit docking, IFD) coupled with energetic analysis that decomposes interaction energies at the atomic level. Using these tools we were able to elucidate the previously unknown substrate binding modes of a series of hydroxyurea analogs to human hemoglobin, catalase and the concomitant structural changes of the enzymes. Our results are consistent with kinetic and EPR measurements of hydroxyurea-hemoglobin reactions and a full mechanism is proposed that offers new insights into possibly improving substrate binding and/or reactivity. Finally in chapter 4, we have developed a 3D bioactive structure of O6-alkylguanine-DNA alkyltransferase (AGT), a DNA repair protein using Monte Carlo conformational search process. It is known that AGT prevents DNA damage, mutations and apoptosis arising from alkylated guanines. Various Benzyl guanine analouges of O6- methylguanine were tested for activity as potential inhibitors. The nature and position of the substitutions methyl and aminomethyl profoundly affected their activity. Molecular modeling of their interactions with alkyltransferase provided a molecular explanation for these results. The square of the correlation coefficient (R2 ) obtained between E-model scores (obtained from GLIDE XP/QPLD docking calculations) vs log(ED)values via a linear regression analysis was 0.96. The models indicate that the ortho-substitution causes a steric clash interfering with binding, whereas the meta-aminomethyl substitution allows an interaction of the amino group to generate an additional hydrogen bond with the protein. Using this model for virtually screening studies resulted in identification of seven lead compounds with novel scaffolds from National Cancer Institute Diversity Set2.

One of the largest obstacles in drug development is to overcome solubility and bioavailability problems. Preformulation strategies such as nanoparticle formation are often employed but sometimes create new issues and are limited in their effectiveness and applications. Since the majority of drugs are marketed and sold as solid forms, drug delivery systems are not always desirable. This is where solid-state chemistry becomes important. Traditional solid-state chemistry approaches are often successful but are sometimes too restrictive and cannot be applied to certain compounds. Cocrystals have emerged as an alternative solid-state technique that can be applied to a broad range of compounds. However, the technology is still very new and its effectiveness in certain conditions had previously not been evaluated. The studies detailed herein investigated the ability of two different technology platforms for overcoming drug design challenges for two promising bioflavonoids: EGCg and quercetin. Studies have shown that EGCg might be useful for the treatment of Alzheimer's disease and other neurodegenerative diseases. Quercetin is being investigated for numerous bioactivities and is currently being marketed as an energy dietary supplement. Both of these bioflavonoids exhibit poor bioavailability and water solubilities that are at opposite ends of the spectrum. In the chapters to follow, nanoparticle technology was applied to EGCg and evaluated in cell models of AΒ production, a hallmark of Alzheimer's disease. Bioavailability improvements were also evaluated in rats. Additionally, new forms of both flavonoids were created using cocrystallization. These new cocrystals were characterized using powder and single crystal x-ray diffraction, differential scanning calorimetry, and thermogravimetric analysis. Solubility and bioavailability changes were also evaluated. These data have strong implications in drug development since they elucidated the strengths and weaknesses of two major technologies in compounds with different design challenges.

Cocaine is a widely abused and addictive drug, resulting in serious medical and social problems in modern society. Currently, there is no FDA-approved medication specific for cocaine abuse treatment. The disastrous medical and social consequences of cocaine abuse have made the development of an anti-cocaine medication a high priority. However, despite decades of efforts, traditional pharmacodynamic approach has failed to yield a truly useful small-molecule drug due to the difficulties inherent in blocking a blocker like cocaine without affecting the normal functions of the transporters or receptors. An alternative approach, i.e. pharmacokinetic approach, is to interfere with the delivery of cocaine to its receptors/transporters and/or accelerate its metabolism in the body. It would be an ideal anti-cocaine medication to accelerate cocaine metabolism producing biologically inactive metabolites. Two natural enzymes may catalyze hydrolysis of cocaine: human butyrylcholinesterase (BChE) and bacterial cocaine esterase (CocE). However, the wild-type enzymes are not suitable as anti-cocaine therapeutics, due to the low catalytic activity, thermoinstability, or short biological half-life. In this investigation, we performed integrated computational-experimental studies to rationally design and discover mutants of these enzymes in order to improve the catalytic activity, thermostability, and/or biological half-life. To rationally design desirable mutants of the enzymes, we have successfully developed computational models, including those for BChE gating, glycosylated BChE structure, BChE-substrate complex structures, BChE dimer/tetramer structures, CocE monomer/dimer structures, and CocE-substrate complex structures. Development of the computational models enabled us to rationally design new amino-acid mutations that may improve the catalytic activity, thermostability, and/or prolonged biological half-life. The computational design was followed by wet experimental tests, including both in vitro and in vivo experiments, leading to discovery of new enzyme forms with not only a high catalytic efficiency against cocaine, but also an improved thermostability and/or prolonged biological half-life. The identified new mutants of BChE and CocE are expected to be valuable candidates for development of a more efficient enzyme therapy for cocaine abuse. The encouraging outcomes of the present study also suggest that the structure-and-mechanism-based design and integrated computational-experimental approach is promising for rational drug design and discovery.

Cocaine is a widely abused drug without an FDA-approved medication. It has been recognized as an ideal anti-cocaine medication to accelerate cocaine metabolism producing biologically inactive metabolites via a route similar to the primary cocaine-metabolizing pathway, i.e. butyrylcholinesterase (BChE)-catalyzed hydrolysis. However, the native BChE has a low catalytic activity against cocaine. We recently designed and discovered a set of BChE mutants with a high catalytic activity specifically for cocaine. An ideal, therapeutically valuable mutant of human BChE should have not only a significantly improved catalytic activity against cocaine, but also certain selectivity for cocaine over neurotransmitter acetylcholine (ACh) such that one would not expect systemic administration of the BChE mutant to interrupt cholinergic transmission. Through integrated computational-experimental studies, several BChE mutants were identified to have not only a considerably improved catalytic efficiency against cocaine, but also the desirable selectivity for cocaine over ACh. Representative BChE mutants have been confirmed to be potent in actual protection of mice from acute toxicity (convulsion and lethality) of a lethal dose of cocaine (180 mg/kg, LD100). Pretreatment with the BChE mutant (i.e. 1 min prior to cocaine administration) dose-dependently protected mice against cocaine-induced convulsions and lethality. The in vivo data reveal the primary factor, i.e. the relative catalytic efficiency, determining the efficacy in practical protection of mice from the acute cocaine toxicity and future direction for further improving the efficacy of the enzyme in the cocaine overdose treatment. For further characterization in animal models, we successfully developed high-efficiency stable cell lines efficiently expressing the BChE mutants by using a lentivirus-based repeated-transduction method. The large-scale protein production enabled us to further characterize the in vivo profiles of the BChE mutant concerning the biological half-life and potency in accelerating cocaine clearance. In particular, it has been demonstrated that the BChE mutant can rapidly metabolize cocaine and completely eliminate cocaine-induced hyperactivity in rodents, implying that the BChE mutant may be developed as a promising therapeutic agent for cocaine abuse treatment.

Cocaine is one of the most reinforcing drugs of abuse and has caused serious medical and social problems. There is no FDA-approved medication specific for cocaine. It is of a high priority to develop an effective therapeutic treatment for cocaine abuse. Human butyrylcholinesterase (BChE) has been recognized as a promising candidate of enzyme therapy to metabolize cocaine into biologically inactive metabolites and prevent it from reaching central nervous system (CNS). However, the catalytic activity of wide-type human BChE against cocaine is not sufficiently high for treatment of cocaine abuse. Dr. Zhan’s lab has successfully designed and discovered a series of high-activity mutants of human BChE specific for cocaine metabolism. This dissertation is mainly focused to address the possible concerns in further development of promising human BChE mutants for cocaine detoxification, including whether the administration of this exogenous enzyme will affect the cholinergic system, whether it can efficiently hydrolyze cocaine’s toxic metabolites, and whether the commonly used therapeutic agents will significantly affect the catalytic activity of the BChE mutants against cocaine when they are co-administered. According to the results obtained, all of the examined BChE mutants have a considerably improved catalytic efficiency against (-)-cocaine, without significantly improving the catalytic efficiency against any of the other examined substrates, including neurotransmitter acetylcholine. Two representative mutants (including E12-7) also have a considerably improved catalytic activity against cocaethylene (formed from combined use of cocaine and alcohol) compared to wild-type BChE, and E12-7 can rapidly metabolize cocaethylene, in addition to cocaine, in rats. Further evaluation of possible drug-drug interactions between E12-7 and some other commonly used therapeutic agents revealed that all of the examined agents, except some tricyclic antidepressants, do not significantly inhibit E12-7. In addition, an effort to discover new mutants with further improved activity against cocaine led to the discovery of a new BChE mutant, denoted as E20-7, according to both the in vitro and in vivo assays. The encouraging outcomes of the present investigation suggest that it is possible to develop a more effective enzyme therapy for cocaine abuse treatment using one of the most promising BChE mutants, such as E12-7 or E20-7.

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.

One of the objectives of the U.S. Food and Drug Administration is to protect the public health through post-marketing drug safety surveillance, also known as Pharmacovigilance. An inexpensive and efficient method to inspect post-marketing drug safety is to use data mining algorithms on electronic health records to discover associations between drugs and adverse events. The purpose of this study is two-fold. First, we review the methods and algorithms proposed in the literature for identifying association drug interactions to an adverse event and discuss their advantages and drawbacks. Second, we attempt to adapt some novel methods that have been used in comparable problems such as the genome-wide association studies and the market-basket problems. Most of the common methods in the drug-adverse event problem have univariate structure and thus are vulnerable to give false positive when certain drugs are usually co-prescribed. Therefore, we will study applicability of multivariate methods in the literature such as Logistic Regression and Regression-adjusted Gamma-Poisson Shrinkage Model for the association studies. We also adopted Random Forest and Monte Carlo Logic Regression from the genome-wide association study to our problem because of their ability to detect inherent interactions. We have built a computer program for the Regression-adjusted Gamma Poisson Shrinkage model, which was proposed by DuMouchel in 2013 but has not been made available in any public software package. A comparison study between popular methods and the proposed new methods is presented in this study.

The topic of this thesis is a global pharmaceutical industry focussing on its geographical aspects. It is a progressive branch of industry with a specific structure and it is characterised by specific trends of development and localization. The first section of the thesis shows the basic characteristics of the pharmaceutical industry. The second section of the thesis deals primarily with a global pharmaceutical industry, important producers of pharmaceutics and pharmaceutical companies and also geographical organization and deployment of this branch of industry. This section brings also the issue of research in the pharmaceutical industry, the question of supranational activities of pharmaceutical industry or functioning of international pharmaceutical associations. The third last section deals with Czech pharmaceutical industry and its situation in the Czech Republic. The thesis focuses primarily on the domestic producers of pharmaceutics it describes their history and current state shows some geographical patterns of domestic pharmaceutical industry and distribution. Tables, graphs and maps make the integral part of this thesis which function primarily as an illustration and specification of acquired information.

siRNA therapy is an emerging technique that reduces protein expression in cells by degrading their mRNAs via the RNA interference pathway (RNAi). Diseases such as cancer often proliferate due to increased protein expression and siRNA therapy offers a new method of treatment for those diseases. Although siRNA therapy has shown success in vitro, it often fails in vivo due to instability in the blood stream. To overcome this limitation, delivery vehicles are necessary for successful transfection of siRNA into target cells and cationic polymers have been widely studied for this purpose. However, complexes between siRNA and delivery vehicles made from cationic polymers exhibit stability issues in the blood stream which results in toxicity and low transfection. This work hypothesizes that improvement of vehicle/siRNA complex stability will improve siRNA transfection efficiency. To test this, the contributions and outcomes of poly(ethylene glycol) [PEG] shell and hydrophobic core modification to a polyethylenimine (PEI) based tethered nanoassemblies (TNAs) were examined. Initially, hydrophobic modification of palmitate (PAL) to the core of the TNA yielded improved transfection efficiency due to an enhanced endosomal escape capability. However, this modification also reduced the TNA/siRNA complex stability. This indicated that the core hydrophobicity must be balanced in order increase stability while increasing transfection efficiency. Additionally, TNAs made from PEG and PEI did not cause transfection in our initial study. The PEG shell density was found to be too great and thereby reduced transfection efficiency. Reducing the PEG density by lowering PEG molecular weight, reducing attachment percentage, and removing small PEI impurities from the synthesis stock increased overall transfection efficiency and unimolecularity of the TNA complexes. This indicated that the shell composition of the TNA must be tuned in order to improve particle design. Further study of the hydrophobically modification to TNAs yielded unintended effects on the transfection efficiency evaluation assay. These particles exhibited an siRNA independent reduction in the reporter protein used to observe transfection, or a false positive effect, that was not previously observed. It was found that this false positive was influence mainly by the hydrophobic group rather than the cationic polymer backbone. Cellular stress was observed in cells dosed with the hydrophobically modified TNAs which lead to over ubiquitination and rapid degradation of the luciferase protein. This demonstrated that core components of TNAs could cause cellular stress and influence interaction outside of the TNA. Overall, this work demonstrates that hydrophobic core and PEG shell modification require balancing and consideration to improve properties of future cationic polymer based siRNA delivery vehicle design.

“Bath salts” and “plant food”, which were legally marketed synthetic cathinones, have a high potential for abuse. Several recent studies indicate that 3,4-methylenedioxypyrovalerone (MDPV) and 3,4-methylenedioxymethcathinone (methylone), two common drugs of this type, have similar pharmacology to controlled psychostimulants such as cocaine, methamphetamine, and methylphenidate. MDPV acts as a norepinephrine (NE) and dopamine (DA) reuptake inhibitor via blockade of their transporters (DAT and NET), whereas methylone is a substrate for the NE, DA, and serotonin (5-HT) transporters, increasing the non-vesicular release of these monoamines. Both drugs cause significant increases in the levels of these neurotransmitters in the cleft. Increases in DA are associated with euphoric effects and thus promote drug abuse and addiction, hence the high addiction potential of MDPV and methylone. Indeed, MDPV is 50 times more potent at the DAT and 10 times more potent at the NET than cocaine. Here, we examined the pharmacokinetics of MDPV and methylone in the brain and plasma, following intraperitoneal injection in mice. These types of injections have similar pharmacokinetics to insufflation (snorting), which is the manner in which MDPV and methylone are commonly abused. Briefly, adolescent male Swiss-Webster mice were injected intraperitoneally with either 10 mg/kg MDPV or 10 mg/kg methylone, and brains and plasma were collected at the following time points: 1, 10, 15, 30, 60, and 120 minutes. Samples were then flash-frozen and stored at -70°C until analysis. Samples were spiked with deuterium-labeled MDPV or methylone (internal standards), and the drugs were extracted from tissue using a previously published solid phase extraction method. Chromatographic separation of the compounds was achieved using a HILIC column with a gradient elution of acetonitrile and 5 mM ammonium formate at a flow rate of 0.2 mL/min. Mass spectrometric detection utilized a Shimadzu IT-TOF system with the electrospray source running in positive mode. Data acquisition utilized a direct MS-MS method using a precursor ion of 276.3 m/z for MDPV and methylone. The calibration curve ranged from 100 ng/ml to 0.1 ng/ml. These conditions allowed for a lower limit of detection (LLOD) of less than or equal to 1 ng/mL and a lower limit of quantification (LLOQ) of less than or equal to 5 ng/mL for MDPV and methylone. MDPV and methylone peak concentrations in plasma were seen immediately at 1 min, while brain concentrations peaked at 15 min; however, MDPV reached higher concentrations in the brain the methylone. This is consistent with MDPV’s higher lipophilicity (logP value). In conclusion, the pharmacokinetic profile of these drugs reflects a quick uptake and distribution of the drugs to the brain, followed by the quick distribution out of the brain, which likely contributes to the binge use of these drugs.