<p>J1 (L-melphalanyl-L-<i>p</i>-fluorophenylalanyl ethyl ester) is a dipeptide derivative of the alkylating agent melphalan with increased cytotoxicity. In this thesis the preclinical pharmacology of J1 has been characterized.</p><p>Our results show that J1 rapidly enters the cells, where melphalan is released by hydrolysis. The maximum concentration (C_max) of melphalan was detected 15 min after exposure to J1 in human cancer cell lines. In comparison, melphalan exposure resulted in a 10-fold lower C_max that was shifted to later time points. J1 induced more DNA damage and apoptosis than melphalan. The cytotoxic activity and release of melphalan from J1 were inhibited by preincubating cells with the aminopeptidase inhibitor bestatin. In accordance with these results, we showed that J1 is a substrate for aminopeptidase N (APN), which may result in increased tumor selectivity.</p><p>J1 effectively inhibited cell growth in a set of neuroblastoma cell lines.<i> </i>Athymic mice carrying neuroblastoma xenografts were treated either with equimolar doses of melphalan or J1. J1 inhibited the tumor growth more effectively than melphalan and the untreated control, and was associated with higher caspase-3 activation, fewer proliferating tumor cells and decreased mean vascular density.</p><p>J1 and melphalan showed similar activity profiles when tested in 176 primary tumor cell cultures from patients, but J1 exhibited 50- to 100-fold higher potency. The difference was greater in some diagnoses (e.g. breast cancer, NHL and AML), and was exceptionally large in some breast cancer samples with aggressive phenotypes. A combination screening of J1 and standard chemotherapeutics yielded mostly additive interactions, except for etoposide which induced synergy in all tested cell lines.</p><p>In conclusion, the melphalan prodrug J1 is effectively transported into the cells, where aminopeptidases (for example APN) catalyze the formation of melphalan. J1 shows promising preclinical potential in the diagnoses neuroblastoma and breast cancer.</p>

<p>Cancer is a common disease and due to problems with resistance against cancer drugs and the limited benefit from chemotherapy in many diagnoses, there is a need to develop new cancer drugs. In this thesis new methods to screen for cancer drugs and to evaluate their mechanism of action are discussed. </p><p>In Paper I, it was found that by studying the gene expression of a cell line panel and combining the data with sensitivity data of a number of cytotoxic drugs, it was possible to cluster compounds according to mechanism of action as well as identifying genes associated with chemosensitivity.</p><p>In Paper II, studies of compounds with selective activity in drug-resistant cell lines revealed the glucocorticoids as a group of interesting compounds. The glucocorticoid receptor was overexpressed in 8226/Dox40 and the difference in sensitivity was abolished when the cells were treated with a glucocorticoid receptor antagonist.</p><p>In Paper III, an image-based screening method for new proteasome inhibitors was successfully developed and the compounds disulfiram, PDTC and NSC 95397 were identified as inhibitors of the proteasome.</p><p>In Paper IV, disulfiram and PDTC were shown to induce cytotoxic activity, to inhibit the activation of the transcription factor NFkappaB and to inhibit the degradation of proteins normally degraded by the proteasome.</p><p>In Paper V, NSC 95397 was shown to be cytotoxic to all cells in the resistance-based cell line panel as well as to patient samples from a variety of cancer diagnoses. Connectivity Map was successfully used as a tool to propose a new mechanism of action of NSC 95397. The gene expression induced by NSC 95397-treatment was similar to that induced by several proteasome inhibitors not present in the Connectivity Map.</p>

The neuropeptide Y (NPY) family of structurally related peptides includes NPY, peptide YY (PYY) and pancreatic polypeptide (PP). They bind to G-protein coupled receptors named Y receptors, and include in mammals Y1, Y2, Y4, Y5, Y6 and in non-mammalian vertebrates also Y7, Yb and Yc. Subtypes Y1 and Y5 stimulate appetite, while Y2 and Y4 have the opposite effect in mammals. The studies described here concern human Y1 and Y4, chicken Y6 and Y7, and zebrafish Y2. Site-directed mutagenesis of human Y1 identified sites important for binding of NPY and PYY as well as Y1 antagonists. The results clarify contradictory findings previously reported by others and identify new sites of interaction. A three-dimensional structural model of the Y1 receptor based upon the high-resolution structure of bovine rhodopsin was generated that increases our understanding of ligand-receptor interactions and hopefully will facilitate the design of novel subtype-selective agonists and antagonists. Two naturally occurring variants of human Y4 with substitutions R240C and V276M have been found in a sample of obese children. Functional studies in vitro showed that the cellular response to PP was greatly decreased for R240C and may provide a causative link to juvenile obesity. The genes for chicken Y6 and Y7 were found to be located ~1 megabase apart on chromosome 13 syntenic to the human Y6 pseudogene. Y6 mRNA has widespread expression whereas Y7 mRNA was found only in adrenal gland. Truncated fragments of PYY had lower affinity to chicken Y7 and zebrafish Y2 than to Y2 from mammals and chicken. The results suggest that Y2 in mammals acquired the ability to bind truncated PYY, i. e. PYY3-36, fairly recently, which has implications for its role in appetite inhibition. These differences between receptor subtypes in sequences and pharmacological properties will be useful to further elucidate the structure and activation of Y receptors by site-directed mutagenesis.

J1 (L-melphalanyl-L-p-fluorophenylalanyl ethyl ester) is a dipeptide derivative of the alkylating agent melphalan with increased cytotoxicity. In this thesis the preclinical pharmacology of J1 has been characterized. Our results show that J1 rapidly enters the cells, where melphalan is released by hydrolysis. The maximum concentration (Cmax) of melphalan was detected 15 min after exposure to J1 in human cancer cell lines. In comparison, melphalan exposure resulted in a 10-fold lower Cmax that was shifted to later time points. J1 induced more DNA damage and apoptosis than melphalan. The cytotoxic activity and release of melphalan from J1 were inhibited by preincubating cells with the aminopeptidase inhibitor bestatin. In accordance with these results, we showed that J1 is a substrate for aminopeptidase N (APN), which may result in increased tumor selectivity. J1 effectively inhibited cell growth in a set of neuroblastoma cell lines. Athymic mice carrying neuroblastoma xenografts were treated either with equimolar doses of melphalan or J1. J1 inhibited the tumor growth more effectively than melphalan and the untreated control, and was associated with higher caspase-3 activation, fewer proliferating tumor cells and decreased mean vascular density. J1 and melphalan showed similar activity profiles when tested in 176 primary tumor cell cultures from patients, but J1 exhibited 50- to 100-fold higher potency. The difference was greater in some diagnoses (e.g. breast cancer, NHL and AML), and was exceptionally large in some breast cancer samples with aggressive phenotypes. A combination screening of J1 and standard chemotherapeutics yielded mostly additive interactions, except for etoposide which induced synergy in all tested cell lines. In conclusion, the melphalan prodrug J1 is effectively transported into the cells, where aminopeptidases (for example APN) catalyze the formation of melphalan. J1 shows promising preclinical potential in the diagnoses neuroblastoma and breast cancer.

Glucose and oxygen supply to the brain is critical for its proper function and when it is restricted as during a stroke, neurons and glial cells quickly become necrotic leading to structural damage as well as functional impairment and even death. To date there are few effective therapies that inhibit the neurodegenerative process and improves the outcome for the affected individual. One possible target is the cannabinoid system. Cannabinoid receptor agonists reduce ischemic volume, endogenous cannabinoid levels are elevated during neurodegenerative insults and mice devoid of the central cannabinoid receptor are more seriously affected by experimental stroke than wild type mice. The cannabinoids are also ascribed anti-inflammatory properties and post ischemic inflammation has been proposed to contribute to the evolution of the ischemic damage. In this thesis mechanisms that can contribute to cannabinoid neuroprotection have been studied. In papers I and II the chick was used as a model species, since preparation of embryonic primary neuronal cultures from chick is relatively simple and time efficient compared to rodent primary cultures. Both adult and embryonic chick brain membranes contain functional CB1 receptors and in the cultures they are coupled to inhibition of cAMP production. In embryonic primary cultures, neurons were not protected from glutamate toxicity by preincubation with CB receptor agonists suggesting that postsynaptic cannabinoid mediated neuroprotection is not effective in this system. The effect of cannabinoid agonists on neutrophil chemotaxis and transmigration was investigated in paper III. The CB1/CB2 agonist WIN 55,212-2 inhibited TNF-α-induced transmigration across ECV304 cell monolayers. The effect of WIN 55,212-2 on this process which was mediated by a reduction of IL-8 release from the ECV304 cells rather than a direct effect upon the migratory response to IL-8 was not possible to abolish with CB1 or CB2 agonists suggesting a mechanism distinct from the cannabinoid receptors is operative. In paper IV the photothrombotic ring stroke model was evaluated to determine if it is suitable in intervention studies targeting the cannabinoid system. Three major endpoints were of interest, ischemic volume, neutrophil infiltration and CB1 receptor function. Consistent with previous studies the ischemic volume peaked at 48 hours after irradiation. Neutrophil infiltration was quantified using a myeloperoxidase activity assay. The assay revealed an increase in myeloperoxidase activity 48 hours after irradiation, albeit at a modest level. The function of the CB1 receptor was assessed by radioligand binding and there was no change in either total binding or functional G-protein coupling following photothrombosis. Taken together these results indicate that it is feasible to undertake cannabinoid intervention studies in this model.

Cancer is a common disease and due to problems with resistance against cancer drugs and the limited benefit from chemotherapy in many diagnoses, there is a need to develop new cancer drugs. In this thesis new methods to screen for cancer drugs and to evaluate their mechanism of action are discussed. In Paper I, it was found that by studying the gene expression of a cell line panel and combining the data with sensitivity data of a number of cytotoxic drugs, it was possible to cluster compounds according to mechanism of action as well as identifying genes associated with chemosensitivity. In Paper II, studies of compounds with selective activity in drug-resistant cell lines revealed the glucocorticoids as a group of interesting compounds. The glucocorticoid receptor was overexpressed in 8226/Dox40 and the difference in sensitivity was abolished when the cells were treated with a glucocorticoid receptor antagonist. In Paper III, an image-based screening method for new proteasome inhibitors was successfully developed and the compounds disulfiram, PDTC and NSC 95397 were identified as inhibitors of the proteasome. In Paper IV, disulfiram and PDTC were shown to induce cytotoxic activity, to inhibit the activation of the transcription factor NFkappaB and to inhibit the degradation of proteins normally degraded by the proteasome. In Paper V, NSC 95397 was shown to be cytotoxic to all cells in the resistance-based cell line panel as well as to patient samples from a variety of cancer diagnoses. Connectivity Map was successfully used as a tool to propose a new mechanism of action of NSC 95397. The gene expression induced by NSC 95397-treatment was similar to that induced by several proteasome inhibitors not present in the Connectivity Map.

The aryl hydrocarbon receptor (Ah-receptor) is a member of the bHLH-PAS protein family. The Ah-receptor is a ligand dependent transcription factor, which activates a wide range of genes, most notably the xenobiotica metabolising genes, CYP1A1 and CYP1A2. The biological function of the Ah-receptor is still unknown and an endogenous ligand has yet not been identified. A possible Ah-receptor ligand is 6-formylindolo[3,2-b]carbazole (FICZ). FICZ has a high affinity for the Ah-receptor and is rapidly metabolised by CYP1A1, CYP1A2 and aldehydeoxidase (AOX). To try to trap FICZ or other possible endogenous Ah-receptor ligands, the metabolising enzymes CYP1A1, CYP1A2 and AOX were blocked. This was achieved through chemical blockage of CYP1A1 and CYP1A2 by ellepticin and through silencing with siRNA directed against CYP1A1 and CYP1A2. Successful blockage would be seen as an increase in Ah-receptor dependent XRE-luciferase activity. Chemical blockage of AOX with tungstate did not affect FICZ-dependent XRE-luciferase activation which could indicate that HepG2 cells lack AOX. The chemical blockage of CYP1A1 and CYP1A2 with ellepticin modified the XRE-luciferase response, but did not completely block Ah-receptor activation. In addition it is possible that ellepticin is a ligand for the Ah-receptor. The blockage of CYP1A1 by siRNA was successful; a silencing of CYP1A1 mRNA by at least 50 percent was detected. However due to lack of time it was not tested if the blockage of CYP1A1 and CYP1A2 was sufficient to trap Ah-receptor ligands.

Syftet med studien är att belysa hur barn i åldrarna tre till fem år tänker kring genus. Barnen fick lyssna på sagan ”Prinsessan Papperspåse” (Robert N. Munsch, 1980) och intervjuades sedan med utgångspunkt från sagan. Resultatet visar att barnen tänker relativt lika kring genus oberoende av kön och ålder. Både flickorna och pojkarna hade tydliga föreställningar om vad som är ett acceptabelt uppträdande av det motsatta könet. Framför allt hade de klara tankar om hur en flicka ska se ut om hon ska gifta sig. Att vara smutsig är för barnen inte acceptabelt, vare sig man är flicka eller pojke. Utan undantagsvis lade flickorna fokus på relationen mellan prinsessan och prinsen medan pojkarna lade fokus mer på draken och prinsen.

Breast cancer is one of the deadliest forms of cancers in the world. About 15–30% of all breast cancers are HER2-positive, which involves overexpression of HER2-receptors on the surface of tumor cells. HER2-positive breast cancer is viewed as an aggressive form of cancer because the overexpression of the HER2-receptor causes dimerization with other receptors of the same family; together they bind to ligands and become activated. The activation causes fast, uncontrolled cell proliferation that often results in the formation of a tumor. Cancer can be divided into different stages, at stage 4 the cancer cells from the original tumor breaks away, follows the bloodstream, and forms metastases in other places of the body. When cancer evolves and becomes metastatic the prognosis drastically worsens, and the treatment options are limited. Patients often need several lines of cancer treatment. The first line of treatment is usually trastuzumab in combination with pertuzumab and a taxane, second line is usually trastuzumab emtansine. There is no conclusive third line treatment for HER2-positive metastatic breast cancer (MBC). Due to the development of new anti-HER2 treatments over the last two decades, less patients are dying from breast cancer, however most patients diagnosed with HER2-positive MBC are estimated to face an early death.  The objective of this study was to analyze the pharmaceutical advances that has improved the prognosis for patients diagnosed with HER2-positive MBC. The material, on which this study was based on, was obtained from the Pubmed database via the Linnaeus University Library. Five articles were chosen based on criteria relevant to the topic. The articles were published between 2001 and 2021; all of them were randomized controlled trials (RCT).  The subject of the articles was to compare the efficacy and safety of different forms of anti-HER2 treatments, using patient populations diagnosed with HER2-positive MBC. Mainly the patients' disease progression, treatment response and survival time was analyzed. Pharmaceutical safety was assessed by the rate of adverse events. A total of 2513 patients participated in the studies. Among all the different treatment options that were analyzed in the articles, one treatment combination yielded some of the best results. Pyrotinib in combination with capecitabine increased the disease progression-free time, had the highest proportion of patients who responded to treatment as well as the highest proportion of patients with size-reducing lesions for the longest time. However, the patient group receiving pyrotinib also had the highest incidence of serious adverse events and had the largest percentage of patients who chose to discontinue the study due to adverse events.  Analysis of the five articles concludes that the prognosis for patients diagnosed with HER2-positive MBC has been improved by pharmaceutical advances regarding tyrosine kinase inhibitors, pan-HER inhibitors, combination therapy with monoclonal antibodies (single, mixed, modified or conjugated with other drugs) and chemotherapy with different mechanisms of action. The results from the studies indicated that treatment with a single anti-HER2 drug had the lowest effectiveness, and that some drug combinations had better synergistic effects than others, reflecting on patient survival data. Despite the pharmaceutical advances of the past two decades, the prognosis for HER2-positive MBC can still be considered bleak due to its high death rate. Resistance to anti-HER2 drugs is an ongoing concern that requires more research and development of new treatments.

<p>Epilepsi är ingen speciell sjukdom utan ett symtom på en hjärnskada eller störd nervcellsfunktion i hjärnan. Epileptiska anfall beror på abnorm urladdning i hjärnans nervceller. Idag lever omkring 60 000 d.v.s. 0,5-1 % av Sveriges befolkning med epilepsi. Risken att drabbas är störst under det första levnadsåret och efter 65-årsålder då risken att drabbas av stroke är som störst. Behandling av epilepsi används i syfte att hindra uppkomst av anfall och göra det möjligt för den drabbade att leva ett relativt normalt liv. Antiepileptika dämpar aktiviteten i hjärnan och reducerar därmed risken för anfall. Under flera år har man försökt utveckla nya antiepileptika mot andra möjliga targets än de som finns idag, bl.a. GABA-transporthämmare. Det enda förekommande läkemedlet med GABA transporthämmande effekt är tiagabin men detta är inte registrerat som läkemedel i Sverige. Syftet med denna studie var att undersöka om GABA-transporthämmare skulle kunna användas som läkemedel mot epilepsi. Metoden som användes var en litteraturstudie där vetenskapliga artiklar hämtades från PubMed, ELIN, Cochrane och Google Scholar. Arbetet baseras på 4 experimentella originalartiklar och en metaanalys. Artiklarna beskriver antiepileptiska effekter och/eller relaterade egenskaper för olika substanser med hämmande effekter på olika GABA- transportörer. Dessa hämmare, ensamma eller i kombination, visades ge kramplösande effekt i olika djurmodeller av epilepsi. Hämmare av olika GABA-transportörer, till exempel tiagabin och EF1502, gav synergistisk effekt, medan hämmare av samma GABA-transportör, till exempel tiagabin och LU-32-176B, resulterade i additiv effekt. Hämning av olika GABA-transportörer i olika celltyper i och runt synapsklyftan verkar därför kunna ge synergistisk effekt. Ingen synergistisk effekt observerades för toxiska effekter. Det finns anledning att tro att ytterligare läkemedel med effekter på GABA-transportörer kan komma att finnas i framtiden för behandling av epilepsi.</p> / <p>Epilepsy is not a specific disease but a symptom of brain injury or impaired nerve cell function in the brain. Epileptic seizures are symptoms of abnormal activity in the brain neurons. Today, about 60 000 i.e. 0.5-1% of the Swedish population live with epilepsy. The risk of being affected is greatest during the first year of life and after the age of 65 years when the risk for stroke is greatest. The treatment of epilepsy is used in order to prevent the onset of seizures and to allow the patient to live a relatively normal life. Anticonvulsants dampen the activity in the brain and thus reduce the risk of seizures.</p><p>During many years, attempts have been made to develop new anticonvulsants against other potential targets than those that exist today, for example GABA-transporter inhibitors. The only presently used medicine with GABA-transporter inhibiting effect is tiagabine, but this is not licensed as a pharmaceutical drug in Sweden.</p><p>The aim of this study was to investigate whether GABA-transport inhibitors could be used as medication for epilepsy. The method that was used was a literature study in which scientific articles were chosen from PubMed, ELIN, Cochrane and Google Scholar. The work is based on 4 original research articles and one meta-analysis. The articles describe antiepileptic effects and/or related properties of various substances with inhibitory actions on different GABA-transporters. These inhibitors, alone or in combination, were shown to have anticonvulsant effects in several different animal models of epilepsy. Inhibitors of different GABA transporters, such as tiagabine and EF1502, resulted in synergistic effects, while inhibitors of the same GABA transporter, such as tiagabine and LU-32-176 B, resulted in additive effects. Inhibition of various GABA transporters in different cell types in and around synapses therefore seems to provide synergistic effects. No synergistic effect was observed for toxic effects. There is reason to believe that additional drugs with effects on GABA transporters may be used in the future for the treatment of epilepsy.</p>