Browsing by Subject "Pharmacokinetics"
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Item Active efflux transport and CNS distribution of the novel antifolate pemetrexed.(2010-03) Li, LiPemetrexed (PMX, Alimta®) is a novel multi-targeted antifolate approved for the treatment of malignant pleural mesothelioma and non-small cell lung cancer (NSCLC). Given the high incidence of brain metastasis in NSCLC patients and wide use of the classic antifolate, methotrexate (MTX), in primary CNS lymphoma, the clinical use of PMX may eventually include the treatment of CNS tumors. However, previous studies in our laboratory indicated that, like MTX, PMX has difficulty in penetrating across the blood-brain barrier (BBB). Factors limiting the CNS distribution of PMX remain unidentified. One important determinant of CNS distribution is efflux transport by BBB transporters. The overall objective of this study was to characterize the brain-to-blood efflux transport of PMX and to examine role of BBB efflux transporters such as BCRP, MRP2 and other organic anion transporters in brain distribution of PMX. The interaction of PMX with BCRP was examined in vitro and in vivo. In vitro results revealed that PMX is a substrate for BCRP-mediated transport. In vivo examination indicated that deletion of Bcrpl has little influence on brain penetration of PMX. Using the brain efflux index method, the mechanism responsible for the brain efflux of PMX and MTX was investigated. The results revealed that brain elimination half-life of PMX and MTX were 48 and 32 minutes, respectively and both PMX and MTX undergo saturable efflux transport across the BBB. MRP2 does not play a role in the brain distribution of either antifolate. However, BCRP makes a significant contribution to brain elimination of MTX, but not PMX. In addition, it was observed that brain-to-blood transport of PMX and MTX was markedly inhibited by probenecid and benzylpenicillin, suggesting the involvement of organic anion transporters, possibly OAT3. Given the knowledge that solid tumors and some physiological barriers have an acidic extracellular environment, the effect of pH on transport activity of BCRP for PMX was examined. In addition, the molecular basis of the observed pH-dependency in BCRP transport activity was explored based on the recent homology model of BCRP. This study has important implications in the handling of PMX and other chemotherapy drug molecules in the acidic environment of tumors and in the distribution and elimination of the drug molecules. Studies presented in this dissertation provide useful information about the specific mechanisms involved in limiting the brain penetration of PMX and MTX. This new knowledge will help in formulating strategies to improve CNS delivery of these antifolates and maylead to more successful treatment of primary and secondary CNS tumors.Item A heroin/morphine vaccine: mechanism of action and extending its use to other abused opioids(2013-07) Raleigh, Michael DennisHeroin is more widely used than any other illicit opioid and mortality rates among heroin users are an average of 13 times higher than the general population. Intravenous heroin use is associated with crime, social disruption, and transmission of blood-born pathogens such as human immunodeficiency virus and hepatitis C. Effective pharmacotherapies are available to treat heroin abuse but have been largely unsuccessful because they require frequent dosing, have a high abuse potential, or have low compliance. Vaccines against heroin and its metabolites (e.g. morphine) are being considered as a complementary treatment for heroin abuse because they are long-acting, selective, have no abuse potential, and may benefit those unwilling to take the current pharmacotherapies. Vaccination with morphine-conjugate vaccines can elicit a strong immune response that reduces the behavioral effects of heroin in animals, presumably by morphine-specific antibodies binding opioids in blood and reducing their distribution to brain. This thesis explores the use of M-KLH, a morphine hapten conjugated to keyhole limpet hemocyanin (KLH) using a tetraglycine linker and mixed with either Freund's or alum adjuvant for increasing the immune response. Morphine vaccines present many challenges that make translation to clinical use difficult. Heroin is sequentially metabolized to its active intermediates 6-monoacetylmorphine (6-MAM), morphine, and then to morphine-6-glucuronide (in vivo and ex vivo). Heroin enters brain and is rapidly converted to 6-MAM, which is presumed to mediate most of heroin's early effects. With regard to the mechanism of action of morphine vaccines, it is unclear whether the antibodies they generate must bind heroin, its downstream metabolites, or both to prevent opioid distribution from plasma to brain and reduce heroin's behavioral effects. However, because analytical assays to measure heroin and metabolite concentrations in tissues have used a wide range of conditions and varying degrees of stability have been reported, studying the effect of vaccination on heroin distribution is not straightforward. In addition, heroin and metabolite distribution after i.v. heroin administration, the most common route of abuse by humans, has not been well characterized in non-vaccinated rodents. Finally, blockade of heroin by vaccination may not prevent the abuse of structurally distinct opioids. The overall goal of this thesis was to better understand the mechanism of action of morphine vaccines and to extend their use to other abused opioids. The specific aims were to stabilize heroin in blood and brain tissues for subsequent pharmacokinetic studies, study distribution of heroin and its metabolites in non-vaccinated and vaccinated rats, explore the effects of vaccination on heroin-induced behaviors, and determine if vaccine efficacy is retained when combined with a vaccine targeting oxycodone, another commonly abused opioid. These aims were explored using clinically relevant drug doses. Heroin and metabolite degradation was significantly reduced by 1) the addition of ice-cold sodium fluoride (a general esterase inhibitor) and formate buffer (pH 3.0) in heroin-spiked tissues, 2) rapid removal of red blood cells via centrifugation, and 3) drying opioids after extraction from tissues prior to measuring their levels. Using these conditions heroin and its metabolites were stabilized in tissues for subsequent distribution studies. In non-vaccinated rats 6-MAM was the predominant metabolite in brain as early as one minute after administration of 0.26 mg/kg i.v. heroin, which is consistent with previous studies that suggest that 6-MAM mediates heroin's early behavioral effects. Vaccination with a morphine-conjugate vaccine (M-KLH) led to a reduction of 6-MAM and morphine, but not heroin, distribution to brain after heroin administration, suggesting that morphine vaccines reduce accumulation of 6-MAM in brain. The mechanism by which this occurs is likely through antibody binding of 6-MAM in plasma to prevent its distribution to brain and is consistent with very high plasma 6-MAM concentrations in vaccinated rats after i.v. heroin or 6-MAM administration. Vaccination with M-KLH led to a reduction of heroin-induced anti-nociception and locomotor activity and remained effective for up to 16 days after repeated dosing suggesting that heroin vaccines may have long-lasting efficacy. These results are consistent with findings from the distribution studies and support the hypothesis that morphine vaccines function by retaining 6-MAM in plasma and prevent its accumulation in brain. To determine whether opioid vaccines could be combined without reducing individual vaccine efficacy and prevent heroin addicts from abusing structurally distinct opioids, rats were vaccinated with M-KLH, an oxycodone-conjugate vaccine (Oxy-KLH), or the bivalent vaccine (both M-KLH and Oxy-KLH). Total morphine- and oxycodone-specific antibody titers were significantly increased in rats that received the bivalent vaccine compared to rats that received individual vaccines. Concurrent i.v. administration of 6-MAM and oxycodone in M-KLH vaccinated rats led to increased 6-MAM retention in plasma and reduced 6-MAM distribution in brain. A similar effect on oxycodone distribution was seen in Oxy-KLH vaccinated rats. There was a trend towards greater efficacy in altering both 6-MAM and oxycodone distribution in the bivalent group compared to individual vaccine groups. These data suggest that combining opioid vaccines will retain, and possibly enhance, individual vaccine efficacy and might be a viable option to prevent addicts from abusing structurally distinct opioids. These findings contribute to the understanding of how morphine vaccines elicit their effects on heroin-induced behaviors and suggest that morphine vaccines, alone or in combination with other pharmacotherapies, may benefit those seeking treatment for heroin addiction.Item Improving Delivery of Therapeutics to Targeted Tissues in Lysosomal Diseases(2021-12) Kim, SarahLysosomal diseases are a group of over 70 diseases with a combined incidence of approximately 1 in 7,700 live births. Most lysosomal diseases are caused by mutations in enzymes normally present in the lysosome. Lysosomal diseases are multi-systemic and progressive diseases. Currently, only 12 lysosomal diseases have treatments. A challenge in drug development is the lack of biomarkers that reflect disease progression or show response to therapy. Furthermore, current therapies have difficulty reaching certain tissues that are major contributors to morbidity and mortality, such as the central nervous system (CNS) and cardiac valves. The overall objective of this dissertation is to improve the delivery of therapeutics to targeted tissues in lysosomal diseases. To accomplish this objective, three main studies were performed. 1) Validation of Chitotriosidase as a CNS Biomarker for Gangliosidoses. Chitotriosidase was investigated as a probable surrogate endpoint for clinical trials with gene therapy. The first objective was to validate chitotriosidase levels for important clinical outcomes in patients with lysosomal diseases. The second objective was to assess chitotriosidase’s ability to detect effective gene therapy in murine models of lysosomal diseases. In patients with gangliosidoses, the most severe infantile phenotype had higher chitotriosidase levels in the cerebrospinal fluid (CSF) and a different pattern over time than the more attenuated juvenile and late-onset forms. Chitotriosidase levels were also significantly associated with neurocognitive impairment. In mice with mucopolysaccharidosis type I (MPS I), there were significant differences among the untreated, gene-therapy treated, and mice heterozygous for a mutation in the IDUA gene. These results support the use of CSF chitotriosidase levels to diagnose different disease phenotypes and to monitor disease progression in patients. As a potential biomarker of neurological improvement, CSF chitotriosidase can aid in the development of therapies that target the CNS. 2) Investigation of Iduronidase Enzymes Linked to Pepcan to Improve Delivery to Targeted Tissues. The objective of this study was to determine if pepcan-12 can increase the uptake of iduronidase into the brain of MPS I mice. Pepcan-12 is a ligand for the cannabinoid receptor type 1 (CB1), a highly expressed receptor in the CNS. The hypothesis was that a fusion iduronidase containing pepcan-12 would have higher activity levels than iduronidase in the brain. Sequences of one of two linkers, Linker S or Linker T, were inserted between pepcan-12 and IDUA to conjugate the ligand and iduronidase. MPS I mice were injected with plasmids encoding either the native iduronidase or one of four fusion iduronidase enzymes containing: pepcan-12 + Linker S, pepcan-12 + Linker T, Linker S, or Linker T. The fusion enzymes and iduronidase had similar activity levels in the brain. Unexpectedly, the fusion enzymes had higher activity levels than iduronidase in the heart and plasma, which appears to be caused by the linkers. Therefore, these fusion enzymes may improve cardiovascular outcomes in MPS I. In several MPS disorders, the cardiac valves continue to worsen despite enzyme replacement therapies (ERT) and hematopoietic cell transplants. The small size of these linkers facilitates their use as fusion enzymes encoded in gene therapy or administered directly as ERT. Therefore, these linkers may aid in therapeutic development for other lysosomal diseases. 3) Pharmacokinetic Analysis of Iduronidase and a Fusion Iduronidase Enzyme Encoded in Gene Therapy. In the previous study, an iduronidase enzyme containing Linker T, termed Linker T iduronidase, had higher activity levels than iduronidase in the plasma and heart. This study sought to investigate the mechanism of Linker T iduronidase, but a gap between the fields of lysosomal diseases/gene therapy and pharmacokinetics (PK)/ pharmacodynamics (PD) became apparent. In the field of lysosomal diseases, the activity level of an enzyme is an important measurement of efficacy, because an enzyme’s activity levels are more predictive of efficacy than its physical levels. However, pre-clinical studies in lysosomal diseases lack well-described methods to quantify changes in enzyme activity levels over time. In contrast, the pharmacokinetic field has rigorous and reproducible methods to quantify changes in a therapy over time in the body. However, traditional pharmacokinetic methods face challenges in gene therapy because of the need for uniform or convertible units. Furthermore, absorption, distribution, metabolism, and elimination processes are well-characterized for small molecule drugs but not yet adapted for biological therapies. To bridge the fields of lysosomal diseases/gene therapy and PK/PD, I aimed to develop an approach incorporating values with greater prediction of efficacy from the field of lysosomal diseases and the quantitative methods from the field of pharmacokinetics. The objective of this study was to perform a pharmacokinetic analysis of iduronidase and Linker T iduronidase administered as gene therapies. The hypothesis was that Linker T iduronidase would have a higher area under the curve (AUC) or half-life, estimated with enzyme activity levels, than iduronidase in the plasma. MPS I mice were injected with plasmids encoding either iduronidase or Linker T iduronidase. At ten time points, ranging from 0.5 to 168 hours post-injection, the enzymes’ physical levels in the liver, activity levels in the liver, and activity levels in the plasma were measured. In the liver, both the physical and activity levels over time were similar between the native iduronidase and Linker T iduronidase. In contrast, enzyme activity levels over time in the plasma showed differences between the native iduronidase and Linker T iduronidase. The time curves of activity in the plasma showed biphasic profiles for both enzymes. Iduronidase had a sharper decline between 24 and 48 hours, and both enzymes had approximately parallel slopes between 96 and 168 hours. The Linker T iduronidase had a two-fold higher AUC of activity than the normal iduronidase in the plasma. The AUC of plasma activity and other PK parameters were contextualized in gene therapy, and experimental data were used to deduce the mechanism of Linker T. These results suggest that Linker T iduronidase may have a distinct property that protects the enzyme from degradation or inactivation in the plasma. The enzymes were estimated to have a half-life of activity in the plasma under noncompartmental analysis. Future studies with compartmental analysis would better characterize half-lives of activity in biphasic profiles. This study performs a novel approach of conducting a formal pharmacokinetics analysis on enzyme activity levels, a traditionally pharmacodynamic outcome. The resulting PK parameters can be interpreted and used to gain mechanistic insight on gene therapy, by integrating concepts from pharmacokinetics and gene therapy In summary, these findings improve the therapeutic delivery in lysosomal disease through the validation of a CNS biomarker for lysosomal diseases, creation of a fusion enzyme with improved activity in the heart and plasma, and a novel approach and interpretation of pharmacokinetics to gain mechanistic insight on gene therapy.Item Improving hematopoietic cell transplantation therapeutics:emphasis in pharmacokinetic-pharmacodynamic relationships and pharmacogenomics.(2009-12) Long-Boyle, Janel ReneeTreatment-related mortality and acute graft vs host disease remain prominent clinical problems in nonmyeloablative allogeneic hematopoietic cell transplantation (HCT). Hence, the need for improved preparative regimens and immunosuppressive strategies in HCT persists. The research presented in my dissertation will be focused on defining pharmacokinetic-pharmacodynamic relationships, and pharmacogenomics involving two antineoplastic agents, fludarabine and clofarabine, and the immunosuppressive agent, mycophenolate all of which are used in the setting of HCT. Fludarabine is a purine analog commonly used in both adult and pediatric nonmyeloablative allogeneic HCT. Although the pharmacokinetics of fludarabine have been extensively studied in a variety of malignant diseases, very little data is available in nonmyeloablative HCT and the relationship between fludarabine pharmacokinetic parameters and clinical outcomes such as treatment-related mortality have yet to be evaluated. Similarly, no PK data is available for clofarabine; a newer purine analog currently used pediatric patients undergoing HCT for non-hematologic malignancies. Finally, mycophenolic acid pharmacokinetics in HCT recipients displays wide inter- and intra-patient variability in plasma concentrations and low mycophenolate exposure is associated with lower rates of engraftment and greater risk of acute graft vs host disease. Patient characteristics such as weight or body surface area, or clinical markers for hepatic and renal function incompletely explain pharmacokinetic variability suggesting there may be genetic factors influencing mycophenolate metabolism or transport. The methodologies and techniques employed to evaluate each individual agent will differ, including pharmacokinetic and statistical analyses. However, all projects share the common goal of improving patient outcomes and reducing toxicity in this very complex patient population.Item Improving The Delivery Of Novel Molecularly-Targeted Therapies For The Treatment Of Primary And Metastatic Brain Tumors(2019-01) Gampa, GauthamTumors in the brain are challenging to diagnose and are associated with poor survival outcomes. Brain tumors are difficult to treat, in part, due to restricted drug delivery across the blood-brain barrier (BBB). Although the BBB is compromised in some regions of brain tumors, the degree of disruption is not uniform and certain tumor locations have a functionally intact BBB. A critical component of BBB that restricts entry of therapeutics into brain is active efflux. The objective of this work is to examine brain distribution of novel molecularly-targeted therapies, including evaluation of influence of P-gp and Bcrp-mediated efflux at the BBB, assessment of spatial heterogeneity in drug distribution to brain tumors, and comparison of unbound (active) drug exposures with in vitro efficacy. Ispinesib is a KIF11 inhibitor that inhibits both tumor proliferation and invasion in glioblastoma (GBM). We demonstrate that ispinesib has a limited brain delivery due to efflux by P-gp and Bcrp, and ispinesib delivery is heterogeneous to areas within a tumor in a GBM model. Furthermore, predicted unbound-concentrations in brain were less than in vitro cytotoxic concentrations, suggesting that delivery may limit in vivo efficacy. Also, pharmacological inhibition of efflux transport (elacridar co-administration) improves brain delivery of ispinesib, and future studies will evaluate if enhanced delivery will improve efficacy. CCT196969, LY3009120 and MLN2480 are panRAF inhibitors with minimal paradoxical activation of MAPK pathway and may overcome resistance observed with BRAF inhibitor therapy in melanoma. MEK inhibition is used in combination with BRAF inhibitors to delay resistance. E6201 is a potent MEK inhibitor with a unique macrocyclic structure. While brain distribution of panRAF inhibitors is limited by efflux, E6201 has a favorable brain distribution profile and interacts minimally with P-gp and Bcrp. The delivery of E6201 is variable to regions of tumor in an intracranial melanoma model. However, predicted unbound-concentrations in brain achieve levels higher than in vitro cytotoxic concentrations for LY3009120 and E6201, suggesting possible efficacy in melanoma brain metastases. Future studies evaluating in vivo efficacy in preclinical models will reveal the utility of selected compounds in brain tumor treatment, and if improved delivery translates to superior efficacy.Item Metabolism and CNS Distribution of Selected Histone Deacetylase Inhibitors(2024-03) Zhang, WenqiuBrain tumors are the leading cause of cancer-related death in children and efficacious treatment remains a critical unmet need. The blood-brain barrier (BBB) is a major hurdle for effective delivery of treatments for tumors in the central nervous system (CNS). While the paracellular transport of large, hydrophilic molecules is largely limited by tight junctions, efflux transporter systems are a key element of the BBB that can limit the penetration of lipophilic drugs. Histone deacetylase inhibitors (HDACIs) have been widely explored for their application in oncology, including the field of neuro-oncology. Despite their high in vitro potency and CNS-penetration-favorable physicochemical properties, the in vivo efficacy of HDACIs has been poor for CNS tumor treatment. This lack of in vitro-in vivo correlation may be in part attributed to poor CNS distribution. In this thesis project, we investigated the CNS distribution of three potent HDACIs, panobinostat, vorinostat, and quisinostat, following systemic administration. We characterized the systemic pharmacokinetics and CNS distributional kinetics of these compounds in wild-type and transgenic mice lacking p-glycoprotein (P-gp) and/or breast cancer resistance protein (Bcrp), two major efflux transporters expressed at the BBB. The in vitro stability studies show that all three hydroxamic acid-based HDACIs are enzymatically metabolized in mouse plasma, highlighting the need for careful sample handling to have accurate measurements of in vivo drug concentrations. In vivo experiments in the different mouse genotypes show that the CNS distribution of panobinostat and quisinostat is moderately limited by P-gp, but not Bcrp. Although the CNS penetration of vorinostat was not restricted by P-gp and Bcrp, its small unbound CNS tissue-to-plasma partition coefficients suggest that other efflux transporters could be involved. In addition, our results show that a tolerable dosing regimen of panobinostat would not result in adequate CNS exposure of unbound panobinostat in patients. In summary, our data show that the lack of adequate exposure of the active moieties can be a major reason for the lack of efficacy of these HDACIs in the CNS when systemically delivered. This result indicates that alternative approaches to improve delivery (e.g., convection-enhanced delivery or focused ultrasound) should be considered.Item Passive Immunization to treat nicotine dependence in rats.(2009-01) Roiko, Samuel A.Cigarette smoking is the leading preventable cause of death in the United States, primarily due to nicotine addiction. Currently available medications are only partially effective in increasing smoking cessation, and additional therapies are needed. Immunotherapy is an alternative strategy for attenuating nicotine's addictive effects. Vaccination against nicotine alters nicotine pharmacokinetics and nicotine's behavioral effects in rats. In clinical trials, nicotine vaccines reduce smoking, but efficacy is limited by high variability and low mean serum nicotine-specific antibody (NicAb) levels. Passive immunization is an alternative method of immunization, and can potentially circumvent the limitations of vaccination by allowing control over the dose and timing of NicAb administration. The overall goal of this thesis was to examine the use of passive immunization to treat tobacco addiction. The specific aims were to characterize the efficacy of passive immunization on nicotine pharmacokinetics and nicotine-induced behavior, explore the effects of combining vaccination with passive immunization, and examine a potential adverse effect of passive immunization, the precipitation of withdrawal. Passive immunization with the nicotine-specific monoclonal antibody Nic311 reduced nicotine clearance and steady-state volume of distribution, and prolonged nicotine's elimination half-life, demonstrating the underlying mechanism of immunotherapy efficacy. Nic311 attenuated nicotine-induced locomotor sensitization, and combining Nic311 with vaccination resulted in greater reductions in brain nicotine levels and nicotine-induced locomotor sensitization compared to either immunotherapy alone, demonstrating the behavioral efficacy of Nic311 and suggesting a possible clinical role as a supplement for vaccination. Administration of Nic311 to nicotine-dependent rats substantially reduced brain nicotine levels but did not elicit a nicotine withdrawal syndrome. These findings demonstrate the benefits and safety of passive immunization with Nic311, and point to its potential in treating nicotine addiction.Item Personalizing Therapy In Transplantation: Focus On Pharmacokinetics, Pharmacodynamics And Pharmacogenomics Of Drugs Used In Hematopoeitic Stem Cell And Kidney Transplant(2016-05) Sanghavi, KinjalPatients treated with a standardized dosing strategy often demonstrate a substantial variability in drug response. Number of factors influences systemic exposure of the drug and its effect on the biological targets. The central objective of this thesis was to identify biomarkers and develop personalized dosing of drugs used in hematopoietic stem cell transplant (HSCT) and kidney transplant to improve outcomes. Fludarabine is a chemotherapeutic drug used in reduced intensity conditioning (RIC) HSCT. High fludarabine exposure is associated with greater treatment related mortality (TRM). Fludarabine dose reductions are commonly empirical for obese and/or those with renal dysfunction. We developed a dosing equation, accounting for creatinine clearance and body size. Using this model to make dose reductions will reduce the probability of fludarabine overexposure and reduce TRM. Cyclophosphamide (Cy) is another chemotherapeutic agent used in RIC HSCT, associated with high toxicity and TRM. Due to complex metabolic pathway it is unclear which metabolite is most important to predict Cy’s efficacy and toxicity. We evaluated the association between the active metabolite, phosphoramide mustard (PM), exposure and TRM. We found that higher PM AUC of was associated with greater TRM. We further identified creatinine clearance and gender to influence PM clearance and volume of distribution respectively. Tacrolimus is an immunosuppressant used in kidney transplant recipients. African Americans show very high variability in tacrolimus exposure and poor outcomes. We developed a tacrolimus dosing model, taking into account the clinical and genetic variants to individualize dose in African Americans that could help achieve the target concentrations quicker and improve outcomes. Mycophenolic acid (MPA) is another immunosuppressant used in kidney transplant recipients. Enterohepatic recycling and high variability in trough concentrations make it very difficult to use MPA concentrations for routine therapeutic monitoring. We conducted an RNA sequencing analysis to measure gene expression to identify novel biomarkers to predict MPA efficacy and toxicity. We identified transient changes in gene expression post MPA administration and that expression of 3 genes out of ~20000 were significantly associated with MPA trough concentrations. Additional studies are required to identify if transient changes in gene expression are associated with MPA related outcomes.Item Pharmacokinetics and Pharmacodynamics of Strategically Substituted Agmatines(2022-10) Clements, BenjaminChronic pain remains a major issue affecting patients, with current pharmacotherapy limited to drugs with low efficacy, negative side effects, and/or social stigma. Thus, there is a critical need to develop novel pharmacotherapies that are effective in reducing chronic pain while being safe for long-term use. Agmatine has been extensively shown to reduce chronic pain behaviors in animal models with no cardiac, motor, or neurological adverse effects1. This reduction is due to antagonism of the N-methyl-D-aspartate (NMDA) receptor, specifically at the GluN2B subunit2-4. This specificity allows agmatine to modulate the biological changes underlying chronic pain without the negative side effects observed with complete inhibition of the NMDA receptor. However, agmatine is a polar small molecule and does not efficiently diffuse passively across biological membranes. It has been shown to cross the intestinal epithelium and blood-brain barrier (BBB)5,6, but these distributions are limited, requiring high doses to generating quantifiable disposition. Additionally, the barrier in drug appearance in the CNS necessitates high doses (30-300 mg/kg in animal models) to achieve pharmacological effects. Furthermore, although agmatine has a long half-life in the CNS, its short elimination half-life in plasma restricts efficacy as a systemic therapeutic7. Therefore, our team and collaborators have designed and synthesized a series of agmatine-based prodrugs, the Strategically Substituted Agmatines (SSAs), which are designed to have increased lipophilicity, prolonged plasma half-lives, and equivalent pharmacological activity to agmatine. The central hypothesis of this thesis research is that the SSAs reduce pain behaviors in preclinical models of chronic pain in a manner comparable to agmatine while exhibiting improved pharmacokinetic parameters in rat. From this hypothesis, my objective has been to answer several questions: How can these compounds be measured in plasma and the central nervous system (CNS)? Do strategic substitutions improve pharmacokinetic parameters over agmatine? Do the SSAs distribute more readily to the CNS? Are the SSAs metabolized to agmatine within the CNS? And do the SSAs show improved potency over agmatine following oral administration? To answer these questions, my goals have been to develop techniques to quantify agmatine and the SSAs in complex tissues, determine the pharmacokinetic parameters of agmatine and the SSAs after IV and oral administration, investigate the prodrug activity of the SSAs, and explore the efficacy on chronic pain of these compounds as oral therapeutics. I accomplished these goals by completing the following specific aims: Specific Aim 1: Develop and Validate Analytical Methods to Accurately Quantify Agmatine and the SSAs in Complex MatricesUsing HPLC-MS/MS, a series of tissue preparation methods and chromatographic techniques were developed and validated according to FDA guidance8 to determine the concentration of agmatine, SSA1, SSA2, SSA3, and SSA4 in rat plasma, and quantify agmatine and SSA3 in rat brain and spinal cord. Specific Aim 2: Determine the Plasma Pharmacokinetic Parameters of Agmatine and the SSAs in Rat Following Intravenous and Oral DeliveryI hypothesized that the lipophilic substitutions of the SSAs would increase plasma half-life and volume of distribution over agmatine, as well as improve the oral bioavailability over agmatine. Sprague-Dawley rats with surgically implanted catheters received IV or oral agmatine, SSA1, SSA2, SSA3, or SSA4. Serial blood samples were collected, and plasma was analyzed using HPLC-MS/MS. These plasma concentrations were used to generate individual pharmacokinetic profiles of each drug in individual rats. Specific Aim 3: Assess the Tissue Distribution Profiles of Agmatine and SSA3 in RatI hypothesized that SSA3 would show increased CNS distribution across all tissues compared to agmatine. Furthermore, I hypothesized that SSA3 would be metabolized to agmatine within the CNS. Following IV administration of agmatine or SSA3 via tail-vein in Sprague-Dawley rat, multiple tissues from within the CNS were collected and heat-treated, along with plasma. Agmatine and SSA3 content were assessed using HPLC-MS/MS in each tissue, from which I determined distribution and pharmacokinetic profiles of each compound within individual tissues, such as spinal cord, cortex, ventral tegmental area (VTA), and nucleus accumbens (NAcc). Agmatine content was also assessed in CNS tissues following IV SSA3 and SSA4 to estimate prodrug activity. Specific Aim 4: Evaluate the Efficacy of Agmatine and the SSAs on NMDA-Evoked Responses and Chronic Pain Following Systemic Administration Intrathecal NMDA evokes characteristic behaviors in mice, including biting/scratching and tail-flick hyperalgesia. I hypothesized that the SSAs, due to their increased lipophilicity, would have increased systemic activity at lower doses than agmatine in the NMDA-evoked behavioral model. Additionally, I hypothesized that increased activity at lower doses would be observed in reversal of tactile hypersensitivity in inflammatory pain. I assessed changes in NMDA-evoked behaviors following subcutaneous and oral administration of agmatine and the SSAs to determine the CNS effects of these compounds from non-central delivery. Furthermore, these compounds were tested after oral administration for reduction of tactile hypersensitivity following inflammatory injury via CFA administration.Item Trafficking of Amyloid beta protein at the Blood Brain Barrier: Novel Insights in Alzheimer's Disease Pathogenesis(2016-12) Sharda, NidhiAlzheimer’s disease (AD) is the most common form of dementia in elderly population. Unfortunately, the current treatment approaches for AD are symptomatic and do not interfere with the disease progression at any stage. The risk of AD increases with age; from 12 %, above 70 years of age, to 50 % beyond 80 years of age. Due to an improvement in average life span in many regions across the globe, AD is being recognized as a major socio-economic health problem that is expected to worsen in the near future. Also, with 99.6 % of current clinical trials failing in AD due to insufficient knowledge of the disease targets and lack of early diagnostic methods; detailed investigations into AD pathogenesis is critically important. Development of amyloid beta (Aβ) plaques in the brain is one of the primary pathological hallmarks of AD. Impaired clearance and not overproduction of toxic Aβ proteins leads to their accumulation and subsequent plaque formation in sporadic AD that accounts for more than 90 % of total AD cases. Blood brain barrier (BBB) is expected to be the primary clearance portal of Aβ from the brain. Being at the interface between brain and plasma, the BBB also maintains the dynamic equilibrium of brain and plasma Aβ pools. This equilibrium is believed to be perturbed in AD due to BBB dysfunction. However, the manifestations of BBB dysfunction and the precise mechanisms that may trigger it, are not clearly understood. Two major Aβ isoforms, Aβ40 and Aβ42, play a predominant role in AD pathogenesis. Aβ42 is believed to be pathological whereas Aβ40 is constitutively expressed and believed to have protective properties against neurological anomalies. Also, Aβ40 expression is 10- and 1.5- fold higher than Aβ42 in cerebral spinal fluid (CSF) and plasma, respectively. This ratio of Aβ42:Aβ40 increases in brain and decreases in plasma, respectively, during disease progression. However, the pathophysiological mechanisms driving this clinical observation is not known yet. In my thesis, I demonstrate distinct age dependent changes in the BBB permeability and plasma exposure of Aβ40 and Aβ42 at the BBB endothelium in wild type (WT) and AD-transgenic (APP,PS1) mice. Further, to investigate the pathophysiological mechanisms driving these observations, I conducted in-vitro studies in polarized hCMEC/D3 monolayers, a widely used BBB model, and demonstrated that Aβ40 and Aβ42 are internalized at the BBB endothelium via clathrin- and lipid raft-mediated endocytosis, respectively. This mechanistic difference offers an opportunity to the BBB to independently handle and modulate the clearances of Aβ40 and Aβ42. Also, in-vivo investigations coupled with quantitative modeling, have indicated that Aβ40 may accumulate more than Aβ42 at the BBB endothelium. This may decrease Aβ40 transcytosis at the BBB and increase the exposure of the BBB endothelium to the vasculotropic Aβ40. To investigate the mechanisms driving this observation, I tested the hypothesis published by our lab previously that the impaired transcytosis of Aβ40 at the BBB endothelium is due to the perturbed vesicular exocytosis. Also, synaptic transmission involving SNARE exocytosis machinery is known to be impaired in AD. Thus, I investigated the ability of Aβ40 and Aβ42 to perturb the SNARE exocytosis machinery at the BBB endothelium and in neurons, in comparison with tetanus neurotoxin, a well-established disruptor of the VAMP-2 mediated exocytosis. My findings reveal that Aβ40 and Aβ42 isoforms use VAMP-2 (vesicular SNARE) to exocytose at the BBB endothelium and neurons. Further, using fluorescence resonance energy transfer (FRET) and lifetime microscopy (FLIM), I have demonstrated that Aβ40 and Aβ42 interfere with the functioning of SNARE fusion between VAMP-2 and SNAP-25. Moreover, my findings suggest that Aβ40 may be more efficient than Aβ42 in perturbing this process, and consequently may interfere with its own exocytosis. In summary, my work provides evidence for the presence of distinct clearance mechanisms of Aβ40 and Aβ42 isoforms at the BBB endothelium. This novel assertion provides a framework to explain the disrupted clearance of Aβ, perturbed Aβ42:Aβ40 ratios and dysregulated transport of Aβ and other endogenous proteins at the BBB endothelium observed in AD patients. These findings could be applied to identify new drug targets to ameliorate BBB dysfunction in AD.