Chronic Obstructive Pulmonary Disease (COPD), which is characterized by limitation of pulmonary air flow, is now the third major cause of death worldwide. Barrero et al. have reported that the elevation of extracellular hyperacetylated histone H3.3 in the lungs of COPD patients is associated with cytotoxicity and disease progression. They found that extracellular hyperacetylated H3.3 was cytotoxic to lung structural cells and resistant to proteasomal degradation, and that mouse antibodies to either the C- or N- termini of H3.3 could partially reverse H3.3 toxicity in vitro. Thus, we hypothesize that human antibodies directed against H3.3 may be effective biotherapeutics useful to control progression of COPD in vivo. The discovery and development of human monoclonal antibodies (mAbs) is a fast growing field of biotherapeutics. In addition to full length mAbs, antibody fragments also have been used in antibody discovery research. We have used phage display technology in this project to discover human anti-H3.3 antibody Fab fragments. This technology utilizes genetically engineered phage particles containing genes encoding diverse Fab fragments displayed on the particles. The “Ylanthia” library from MorphoSys AG, a synthetic fully human Fab antibody phage display library with 1.3 x 1011 independent clones, was panned against purified recombinant human H3.3 immobilized on 96-well plates. Seven H3.3-binding Fab fragments with unique DNA sequences were isolated after four rounds of panning. Following their expression in E.coli and purification, Fab purities and electrophoretic mobilities were evaluated on SDS-PAGE. The concentration-dependent binding activities of all seven Fabs to human H3.3 were tested by ELISA. All seven Fabs were shown by ELISA to bind H3.3 but not histones 2A, 2B or 4. Since H3.3 is localized to the nucleus, western blotting was used to demonstrate that seven Fabs recognize purified, recombinant H3.3 and denatured natural histone(s) from nuclear extracts of human 293T cells. In order to characterize these molecules further, biological activity assays will be done to test their potential to reverse the toxic effects of H3.3 in cell culture. If these Fabs prove active in cell culture, they will be converted to IgGs and tested in animal models as potential biotherapeutics for COPD. / Pharmaceutical Sciences

Hypoxia is a characteristic pathophysiological feature of many solid tumors, which contributes significantly to resistance to chemotherapy and radiotherapy. It also induces numerous intracellular signaling pathways, which in turn trigger the upregulation of various key proteins promoting tumor cell survival, progression and metastasis. In this context, novel therapeutic approaches are urgently needed to facilitate the early detection and improve the treatment of hypoxic tumors. Focusing on the hypoxic tumor microenvironment, one can recognize that the membrane bound carbonic anhydrase IX (CA IX) isozyme represents a potential biomarker and a compelling therapeutic target for better diagnosis and management of hypoxic tumors. CA IX is significantly overexpressed under hypoxic conditions as compared to normal tissues and it assists tumor cell to maintain neutral intracellular pH values. Building on this hypothesis, we are focusing our efforts in this thesis towards the development and the optimization of drug delivery nanosystems capable of selectively targeting CA IX that is overexpressed in the hypoxic tumor niche, which in turn will enhance the early detection of hypoxic tumors as well as improve the accumulation of chemotherapeutic drugs in hypoxic cancer cells. This strategy is expected to overcome the chemoresistance associated with tumor hypoxia and minimize the systemic side effects associated with chemotherapeutic drugs administration. In chapter 2, we focused our efforts towards the development of the in vitro biological models for testing our nanoparticles. This process was achieved through screening a series of cancer cell lines for the expression of our target epitope under hypoxic conditions. We induced hypoxia either chemically, using cobalt chloride, or physicochemically, using a hypoxia chamber purged with hypoxia gas mixture containing 1% O2. Screening for CA IX overexpression under hypoxic conditions was done both in 2D monolayer cells and 3D tumor spheroids, which become naturally hypoxic due to their 3D growth. Western blot analysis was used to confirm the expression of our target protein and we have identified three cell lines with a high level of expression of CA IX under hypoxic conditions, namely HT-29 colorectal cancer, SKOV-3 ovarian cancer and MDA-MB-231 breast cancer cell lines. In chapter 3, we optimized a theranostic liposomal delivery system through the use of a combination of zwitterionic amphiphilies of different packing parameters to encapsulate a potent fluorescent carbonic anhydrase inhibitor (CAI), as a novel approach to facilitate the detection of colorectal cancer. Our main focus was to increase the aqueous concentration of poorly water-soluble CAI, to correlate its delivery efficiency with the lipid type and composition of the liposomal nanosystem, as well as to enhance the tissue permeability, allowing easy detection of small tumor polyps. Our optimized DMPC/DOPE liposomal formulation demonstrated an optimum size, high encapsulation efficiency of CAI, and a phase transition temperature below 37 ᴼC that allows efficient delivery of CAI and good tissue penetrability towards the hypoxic tumor cells overexpressing CA IX. In chapter 4, we optimized a CAI-targeted long circulating liposomal delivery system encapsulating doxorubicin. Our main focus was to enhance the accumulation of doxorubicin in hypoxic tumors through targeting CA IX protein overexpressed under hypoxic conditions. This strategy proved to enhance the internalization of the drug carrier into hypoxic cancer cells thus overcoming chemoresistance associated with hypoxia and also minimize the systemic side effects associated with the intravenous administration of non-targeted Doxil®-like formulations. In chapter 5, we optimized a pH sensitive gold nanoplatform functionalized with CAI based moieties to enhance the selective delivery of doxorubicin to hypoxic tumors in a controlled release manner. Our main focus was to combine the advantage of targeting CA IX overexpressed under hypoxic conditions with the intracellular triggered release of doxorubicin in the lysosomes inside the cell in order to enhance the delivery of doxorubicin inside the cancer cells and to overcome the chemoresistance associated with hypoxia. / Pharmaceutical Sciences

Bakgrund: Antibiotika är en läkemedelsgrupp som används vid behandling av inflammatoriska sjukdomar. Antibiotika kan utsöndras i mjölken och därmed kan konsumenter utsättas för antibiotika i onödan. Syfte: Syftet med det här arbetet är att undersöka utsöndring av tetracykliner, makrolider och fluorokinloner från plasma till mjölk hos olika mjölkproducerande djur. En delfrågeställning är att undersöka om djurslag påverkar utsöndring av dessa läkemedel.  Metod: En systematisk litteratursökning genomfördes genom att söka vetenskapliga artiklar i databaserna PubMed och Scopus. Resultat: Sju läkemedelssubstanser (azitromycin, tildipirosin, tilmicosin, tylosin, tulatromycin, pefloxacin och enrofloxacin) passerade och tre läkemedelsubstanser (difloxacin, orbifloxacin och levofloxacin) passerade inte över till mjölken. Resultaten på fem läkemedelsubstanser (oxitetracyklin, erytromycin, moxifloxacin, danofloxacin och marbofloxacin) motsades varandra. Getter hade ofta högre läkemedelskoncentration i mjölken än får och ko. Diskussion: Basiska, höga molekylstorlekar och log P-värde kan vara de faktorer som ledde till att makrolider hade höga M/P-ratio. Erytromycin, orbifloxacin var lipofila och därmed skulle ansamlas mer i fårmjölken än getmjölken, vilken hade högre fetthalt. Dock det gav motsatta resultat i detta litteraturarbete.

<p dir="ltr">Biologic drugs are a key defense against many health issues. In many cases, biologic drugs are not stable in the solution state and must be lyophilized. Lyophilization in the presence of excipients increases the stability of the drug by interactions with the excipients through hydrogen bonding, which will lower the local mobility of the drug. Key threats to stability include: inhomogeneity of the drug substance and excipients, high mobility, and crystallization. Solid-state nuclear magnetic resonance spectroscopy was used to identify crystallization, assess homogeneity, and measure the local mobility of lyophilized protein and mRNA/LNP systems. </p><p dir="ltr">The impact of disaccharide type and concentration on protein stability was explored. Human serum albumin (HSA) was lyophilized with disaccharides (sucrose and/or trehalose) in different relative concentrations, and solid-state nuclear magnetic resonance spectroscopy (ssNMR) ¹H T₁ and ¹H T_1rho relaxation times were measured to determine the homogeneity of the lyophilized systems on 20-50 and 1-3 nm domains, and measure local mobility with ¹H T₁ relaxation times. HSA/sucrose systems had longer ¹H T₁ relaxation times and were slightly more stable than trehalose systems in almost all cases shown. HSA/sucrose/trehalose systems have ¹H T₁ relaxation times between the HSA/sucrose and HSA/trehalose systems and did not result in a more stable system compared to binary systems. Phase separation was evident in a sample containing relative concentrations of 10% HSA and 90% trehalose, suggesting trehalose crystallization during lyophilization. Under these stability conditions, a ¹H T₁ relaxation time below 1.5 s correlated with an unstable sample, regardless of disaccharide(s) used.</p><p dir="ltr">The effect of mannitol on protein stability was studied. Human serum albumin was lyophilized in binary systems with mannitol, and in ternary systems with sucrose or trehalose and mannitol. The monomer content of the HSA was monitored over 36 weeks of storage at 50 C. The amount of mannitol in the system dictated the ability of mannitol to crystallize, and the polymorph that mannitol crystallized into. In HSA/mannitol systems, mannitol crystallization caused inhomogeneity of the matrix, determined by ¹H T_1rho relaxation times. Adding a disaccharide to the matrix, however, increased the homogeneity of the matrix. Addition of mannitol to a HSA/disaccharide matrix resulted in less stability at similar HSA:disaccharide ratios.</p><p dir="ltr">The impact of storage temperature on protein stability was investigated. Human serum albumin was lyophilized with sucrose or trehalose in histidine, phosphate, or citrate buffer. ¹H T₁ relaxation times were measured by ssNMR and were used to assess the homogeneity and mobility of the samples after zero, six, and twelve months at different temperatures. The mobility of the samples decreased after 6 and 12 months storage at elevated temperatures, consistent with structural relaxation of the amorphous disaccharide matrix. Formulations with sucrose had lower mobility and greater stability than formulations with trehalose.</p><p dir="ltr">The effect of an RF-assisted lyophilization method on homogeneity, mobility, stability, and moisture content was explored. This method, utilizing 18 GHz microwave frequency to accelerate the lyophilization cycle, resulted in equivalent or better stability for attenuated live virus or protein formulations, respectively. ssNMR showed comparable amounts of homogeneity in the formulations, however mobility of the samples produced by RF-assisted lyophilization was slightly higher.</p><p dir="ltr">A lyophilized mRNA/LNP formulation was prepared. Disaccharide type, disaccharide concentration, and freezing rate were found to alter critical quality attributes of the system. When mRNA/LNP formulations were stored at 4 C, solution formulations outperformed lyophilized formulations for at least 6 months. When mRNA/LNP formulations were stored at room temperature, solution formulations were superior for the first three months, however lyophilized formulations outperformed solution formulations after 6 months, with less growth in particle size and less loss of encapsulation efficiency. ssNMR was used to assess the interactions between the formulation components.</p>

Objective. To develop and assess a toxicology module to teach pharmacy students about farming- and mining-related occupational exposures in the context of an existing toxicology elective course. Design. A teaching unit that included lectures and case studies was developed to address the unique occupational exposures of patients working in agricultural and mining environments. Upon completion of this 4-hour (2 class periods) module, students were expected to recognize the clinical signs and symptoms associated with these occupational exposures and propose acceptable therapeutic plans. Assessment. After completing the module, students scored significantly higher on a patient case involving suicide resulting from pesticide consumption. Seventy-three percent of the students scored higher than 90% on a 33-item multiple-choice examination. Eighty-two percent of students were able to correctly read a product label to determine the type of pesticide involved in an occupational exposure. Conclusion. Pharmacy students who completed a module on occupation exposure demonstrated competence in distinguishing occupational exposures from each other and from exposure to prescription and nonprescription drugs. This module can be used to educate future pharmacists about occupational health issues, some of which may be more prevalent in a rural setting.

RATIONALE: The purpose of this study is to investigate the stored stability of dilutions of neuromuscular blocking agents (NMBAs), namely cisatracurium, rocuronium, and vecuronium, for skin prick/intradermal testing. METHODS: Concentrations of NMBAs were monitored by liquid chromatography-mass spectrometry (LC-MS/MS) for a period of 14 days. Dilutions of NMBAs were prepared in saline by factors of 10x, 100x, 1,000x, and 100,000x as sensitivity of the assay allowed. Diluted drug products were stored in a laboratory refrigerator until sampling. On sampling days, aliquots of each dilution were removed and compared to a freshly prepared set of reference dilutions. RESULTS: The results are measured as beyond use date (BUD) defined as recovery of drug versus the reference (90-110%). Based on the LC-MS/MS data, the BUD for cisatracurium diluted to 10x and 100x is 96 hours. Higher dilutions (1,000x to 100,000x) should be used immediately following preparation (within less than 24 hours). Vecuronium at 10x and 100x also has a BUD of 96 hours, and the 1,000x dilution is stable for 24 hours. The 10,000x dilution should be used immediately. Rocurium at 10x to 1,000x has a BUD of 48 hours, yet higher dilutions (10,000x and 100,000x) should be used immediately. CONCLUSIONS: With increasing dilution factors, the stability of these drugs in saline decreases, increasing deviation between samples and references. The most stable dilutions for each of the drugs tested were 10x and 100x. Stability of these drugs is likely compromised by hydrolysis of the ester bonds in the drug molecules.

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Objective. To assess the impact of a case-based toxicology elective course on student learning in related required courses and student performance on the Pharmacy Curriculum Outcomes Assessment (PCOA) examination. Design. A case-based clinical toxicology elective course that contained topics from 2 required courses, Pharmacology III and Pharmacotherapy II, was offered in the spring 2009 to second- and third-year pharmacy students. Assessment. Scores on the Toxicology subsection of the PCOA of students enrolled in the elective were higher than those of students not enrolled (91.3% ± 4.1 vs. 67.2% ± 5.7). Enrollment in the elective was related to increased examination scores among Pharmacotherapy II students (89.5% ± 2.0 vs. 83.9% ± 1.8). Students indicated on course survey instruments that they were satisfied with the new elective offering. Conclusions. A toxicology elective provided a clinically relevant, active-learning experience for pharmacy students that addressed a curricular need within the college and increased examination scores.

Methamphetamine is the most common amphetamine used and, along with 3,4-methylenedioxymethamphetamine (MDMA, Ecstasy), is considered part of a worldwide drug epidemic. Monitoring metham-phetamine levels in the body is important for purposes of drug screening for employment, criminal investigations, and therapeutic drug monitoring. While methamphetamine is suitable for detection using immunoassay techniques, these methods tend to have significant cross reactivity with other compounds. Over the last decade, more than eighty different quantitative, confirmatory analytical methods for measuring methamphetamine in biological samples have been published in the scientific literature. Analytical instrumentation used in these methods includes gas chromatography-mass spectrometry (GC-MS), liquid chromatography-mass spectrometry (LC-MS), capillary electrophoresis (CE), among others. These assays are capable of quantifying methamphetamine concentrations in a variety of biological matrices, including blood, plasma, urine, hair, and fingernails. Some of these techniques can achieve detection as low as 0.1 ng/mL (1 ppb) concentra-tions. The strengths and limitations of these methodologies will be discussed in the context of methamphetamine analysis. Additionally, methods that can simultaneously measure methamphetamine levels as well as metabolites and other drugs of abuse will be highlighted.