Skin cancer is the most widely diagnosed form of cancer and it is split in to non-melanoma skin cancer (NMSC) and cutaneous malignant melanoma (CMM). Cutaneous melanoma has a high propensity for malignancy and it has the highest mortality rate of all skin cancers (de Gruijl, 1999:2004). The first line of treatment for most skin cancers is surgical excision but instances do arise in which surgery is not feasible due to the health of the patient or the location of the lesion. Therefore, viable alternatives are necessary in cases where surgery is not possible (Telfer et al., 2008:36). The skin is readily available for delivery of cytotoxic drugs to treat carcinomas and melanomas so the topical delivery of 5-fluorouracil was investigated in this study. 5-Fluorouracil is a pyrimidine anti-metabolite which interferes with deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) synthesis by inhibiting the nucleotide synthetic enzyme thymidylate synthase (TS) and by becoming misincorporated into RNA and DNA. Thymidylate is essential for replication as well as repair of DNA, in the event of TS inhibition thymidylate is not formed and “thymineless deaths” of cells occur (Chu & Sartorelli, 2009:935; Longley et al., 2003:330). This active pharmaceutical ingredient (API) causes death of atypical and rapidly dividing cells (Tsuji & Karasek, 1986:474). The intravenous and topical routes are approved for 5-fluorouracil and in the case of skin cancer the obvious choice would be topical application (Chu & Sartorelli, 2009:935). Topical application of 5-fluorouracil results in the occurrence of terrible side effects such as severe inflammation, stomatitis, photosensitivity and dermatitis. A reduction in side effects would reduce the stigma associated with topical 5-fluorouracil and in turn increase patient compliance. Topical drug delivery entails the delivery of an API onto or into the various layers of the skin (Flynn & Weiner, 1993:33) in order to treat conditions on or within the skin. Topical application of APIs is non-invasive, painless and simple plus the target site is readily accessible for topical therapy, thus the API is delivered directly to the site of action (Naik et al., 2000:318). In the case of skin cancer, 5-fluorouracil should be able to reach the epidermis because NMSC originates from the keratinocytes (Marks & Hanson, 2010:305) and CMM from melanocytes (de Gruijl, 1999:2004) which are both found in the epidermis. The barrier function of the skin limits the penetration of molecules into the skin and the rate-limiting step is usually penetration into the stratum corneum (Foldvari, 2000:418). The aim of this study was to investigate the diffusion of 5-fluorouracil from formulations into and through the skin. Two physico-chemical properties of 5-fluorouracil that influence skin permeation were determined (aqueous solubility and n-octanol-buffer partition coefficient (log D)). The Pheroid™ drug delivery system was used to enhance the delivery of 5-fluorouracil (Grobler et al., 2008:284). Pheroid™ is a novel technology that is used in the delivery of APIs in pharmaceutical products. It enhances the efficacy of delivered compounds while allowing for the reduction of unwanted adverse effects (Grobler et al., 2008:284). Franz cell skin diffusion studies and tape-stripping were conducted with Pheroid™ and non-Pheroid™ formulations to allow for comparison and determination of the effect of Pheroid™. The in vitro efficacy of 5-fluorouracil in inducing apoptosis of human melanoma cells was investigated using a flow cytometric apoptosis assay. Different concentrations of 5-fluorouracil in formulation were utilised in the experiments so as to observe the cytotoxic effect of 5-fluorouracil. The effect of the drug delivery vehicle on the efficacy of 5-fluorouracil was investigated by utilising API solutions in addition to Pheroid™ and non-Pheroid™ formulations in the experiments. Relatively high concentrations of 5-fluorouracil diffused into and through the skin with Pheroid™ formulations resulting in a greatly enhanced in vitro skin permeation of 5-fluorouracil. The tape-stripping revealed that the Pheroid™ lotions resulted in higher concentrations of 5-fluorouracil in the epidermis and dermis after 12 h as compared to the lotions. There was no deducible trend with respect to the distribution of 5-fluorouracil between the epidermis and dermis. Subsequent to the apoptosis assay it was found that 5-fluorouracil was able to induce apoptosis in A375 cells after a 24 h incubation period. The Pheroid™ treatment of cells resulted in a greater response (mean fluorescence intensity) as compared to treatments with the other drug delivery vehicles at three of the four concentrations. This showed that the drug delivery vehicle played a role in the in vitro efficacy of 5-fluorouracil. Further research must be done in order to combine these results. Optimum and highly effective topical formulations with low doses of 5-fluorouracil must be formulated for the purpose of treating cutaneous cancers with a reduced incidence of side effects. / Thesis (MSc (Pharmaceutics))--North-West University, Potchefstroom Campus, 2013.

Skin cancer is the most widely diagnosed form of cancer and it is split in to non-melanoma skin cancer (NMSC) and cutaneous malignant melanoma (CMM). Cutaneous melanoma has a high propensity for malignancy and it has the highest mortality rate of all skin cancers (de Gruijl, 1999:2004). The first line of treatment for most skin cancers is surgical excision but instances do arise in which surgery is not feasible due to the health of the patient or the location of the lesion. Therefore, viable alternatives are necessary in cases where surgery is not possible (Telfer et al., 2008:36). The skin is readily available for delivery of cytotoxic drugs to treat carcinomas and melanomas so the topical delivery of 5-fluorouracil was investigated in this study. 5-Fluorouracil is a pyrimidine anti-metabolite which interferes with deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) synthesis by inhibiting the nucleotide synthetic enzyme thymidylate synthase (TS) and by becoming misincorporated into RNA and DNA. Thymidylate is essential for replication as well as repair of DNA, in the event of TS inhibition thymidylate is not formed and “thymineless deaths” of cells occur (Chu & Sartorelli, 2009:935; Longley et al., 2003:330). This active pharmaceutical ingredient (API) causes death of atypical and rapidly dividing cells (Tsuji & Karasek, 1986:474). The intravenous and topical routes are approved for 5-fluorouracil and in the case of skin cancer the obvious choice would be topical application (Chu & Sartorelli, 2009:935). Topical application of 5-fluorouracil results in the occurrence of terrible side effects such as severe inflammation, stomatitis, photosensitivity and dermatitis. A reduction in side effects would reduce the stigma associated with topical 5-fluorouracil and in turn increase patient compliance. Topical drug delivery entails the delivery of an API onto or into the various layers of the skin (Flynn & Weiner, 1993:33) in order to treat conditions on or within the skin. Topical application of APIs is non-invasive, painless and simple plus the target site is readily accessible for topical therapy, thus the API is delivered directly to the site of action (Naik et al., 2000:318). In the case of skin cancer, 5-fluorouracil should be able to reach the epidermis because NMSC originates from the keratinocytes (Marks & Hanson, 2010:305) and CMM from melanocytes (de Gruijl, 1999:2004) which are both found in the epidermis. The barrier function of the skin limits the penetration of molecules into the skin and the rate-limiting step is usually penetration into the stratum corneum (Foldvari, 2000:418). The aim of this study was to investigate the diffusion of 5-fluorouracil from formulations into and through the skin. Two physico-chemical properties of 5-fluorouracil that influence skin permeation were determined (aqueous solubility and n-octanol-buffer partition coefficient (log D)). The Pheroid™ drug delivery system was used to enhance the delivery of 5-fluorouracil (Grobler et al., 2008:284). Pheroid™ is a novel technology that is used in the delivery of APIs in pharmaceutical products. It enhances the efficacy of delivered compounds while allowing for the reduction of unwanted adverse effects (Grobler et al., 2008:284). Franz cell skin diffusion studies and tape-stripping were conducted with Pheroid™ and non-Pheroid™ formulations to allow for comparison and determination of the effect of Pheroid™. The in vitro efficacy of 5-fluorouracil in inducing apoptosis of human melanoma cells was investigated using a flow cytometric apoptosis assay. Different concentrations of 5-fluorouracil in formulation were utilised in the experiments so as to observe the cytotoxic effect of 5-fluorouracil. The effect of the drug delivery vehicle on the efficacy of 5-fluorouracil was investigated by utilising API solutions in addition to Pheroid™ and non-Pheroid™ formulations in the experiments. Relatively high concentrations of 5-fluorouracil diffused into and through the skin with Pheroid™ formulations resulting in a greatly enhanced in vitro skin permeation of 5-fluorouracil. The tape-stripping revealed that the Pheroid™ lotions resulted in higher concentrations of 5-fluorouracil in the epidermis and dermis after 12 h as compared to the lotions. There was no deducible trend with respect to the distribution of 5-fluorouracil between the epidermis and dermis. Subsequent to the apoptosis assay it was found that 5-fluorouracil was able to induce apoptosis in A375 cells after a 24 h incubation period. The Pheroid™ treatment of cells resulted in a greater response (mean fluorescence intensity) as compared to treatments with the other drug delivery vehicles at three of the four concentrations. This showed that the drug delivery vehicle played a role in the in vitro efficacy of 5-fluorouracil. Further research must be done in order to combine these results. Optimum and highly effective topical formulations with low doses of 5-fluorouracil must be formulated for the purpose of treating cutaneous cancers with a reduced incidence of side effects. / Thesis (MSc (Pharmaceutics))--North-West University, Potchefstroom Campus, 2013.

Das maligne Melanom zählt zu den aggressivsten und behandlungsresistentesten aller Krebsarten. In den letzten 20 Jahren hat sich die Rate der Melanom-Erkrankungen innerhalb der weißen Bevölkerung verdreifacht. Mittlerweile liegen eine Reihe von Untersuchungen zu den molekularbiologischen Mechanismen der Entwicklung und Progression des malignen Melanoms vor. Aktuelle Forschungsvorhaben beschäftigen sich vor allem mit der Identifizierung Melanom-spezifischer Biomarker, die diagnostische und prognostische Informationen liefern sowie die Entwicklung einer zielgerichteten, kombinierten und individualisierten Therapie des metastasierenden Melanoms ermöglichen. In diesem Kontext soll die vorliegende Arbeit einen weiteren Beitrag zum Verständnis der Metastasierungskaskade und der daran beteiligten Proteine leisten. Aufgrund der Überexpression in einer Reihe von Tumoren und seiner geringen Molmasse von lediglich 11,5 kDa bietet sich das S100A4-Protein als Marker mit hoher prognostischer Signifikanz für verschiedene Tumorentitäten an. Jedoch ist die Beteiligung von S100A4 bei der Ausbildung des invasiven Tumorphänotyps noch nicht vollständig aufgeklärt. S100A4 besitzt zahlreiche intra- und extrazelluläre Bindungspartner, wobei die Metastasierung scheinbar ausschließlich durch das extrazelluläre Protein beeinflusst wird. S100A4 wechselwirkt extrazellulär beispielsweise mit dem Rezeptor für fortgeschrittene Glykierungsendprodukte (RAGE). Ziel dieser Arbeit war es, speziell die Bedeutung von S100A4 und seiner Interaktion mit RAGE für das prometastatische Verhalten von Melanomzellen in vitro und in vivo näher zu charakterisieren. Darüber hinaus sollte die Beteiligung von S100A4 bei der Gehirn-Metastasierung untersucht werden, wobei insbesondere die Regulierung der Endothelzell-Permeabilität und der transendothelialen Migration der Melanomzellen im Vordergrund stand. Im Rahmen dieser Arbeit wurde gezeigt, dass S100A4 und die Interaktion mit RAGE die prometastatischen Eigenschaften der A375-Melanomzellen förderte. Zudem verringerte extrazelluläres S100A4 die Zell-Integrität von Gehirn-Endothelzellen und erleichterte somit die Durchdringung der Blut-Hirn-Schranke. Diese Erkenntnis lässt sich möglicherweise auf andere Blut-Gewebe-Schranken übertragen. Die In-vivo-Orientierungsstudie zeigte, dass S100A4- und RAGE-überexprimierende Zellen zu einer verstärkten disseminierten Metastasierung führten, wobei sich zwei unterschiedliche Verteilungsmuster ergaben. Darüber hinaus führten beide Zelllinien vereinzelt zur Bildung von Gehirnmetastasen, wodurch sich die intrakardiale Injektion durchaus als Modell für weitere Therapiestudien mit dem Augenmerk der S100A4-RAGE-stimulierten Metastasierung eignet. Die genauere Kenntnis regulativer Mechanismen bei der Synthese und Sekretion von S100A4 sowie die pathophysiologische Differenzierung der S100A4-Interaktion mit RAGE eröffnen neue Wege, die S100A4-vermittelten Effekte therapeutisch zu beeinflussen. Daraus lassen sich möglicherweise neue zielgerichtete Radionuklid-basierte Therapieansätze für das metastasierende Melanom ableiten.

This diploma thesis dealt with the comparison of different methods of amination of polycaprolcatone in terms of their effectiveness for tissue engineering. A polycaprolactone membrane was prepared by an electrospinning method, which was subsequently modified by three different amination methods. Selected types of amination were plasma polymerization with cyclopropylamine monomer, hybrid modification using plasma and N-allylmethylamine monomer, and chemical amination using aminolysis with diaminohexane. Surface amines were subsequently characterized by electron scanning microscopy (SEM), X-ray photoelectron spectroscopy (XPS), attenuated total reflection infrared spectroscopy (ATR-FTIR) and contact angle measurement. A cell culture designated A375 (Human malignant melanoma cell lines A375® CRL-1619®) was cultured on the thus modified membranes, which was analyzed by optical microscopy, and a proliferation assay was performed by determining the relative amount of ATP. Based on the experimental results, we can confirm the success for all types of amination. In terms of efficiency for tissue engineering, the amination method by plasma polymerization with the monomer cyclopropylamine has the most satisfactory results.

Das maligne Melanom zählt zu den aggressivsten und behandlungsresistentesten aller Krebsarten. In den letzten 20 Jahren hat sich die Rate der Melanom-Erkrankungen innerhalb der weißen Bevölkerung verdreifacht. Mittlerweile liegen eine Reihe von Untersuchungen zu den molekularbiologischen Mechanismen der Entwicklung und Progression des malignen Melanoms vor. Aktuelle Forschungsvorhaben beschäftigen sich vor allem mit der Identifizierung Melanom-spezifischer Biomarker, die diagnostische und prognostische Informationen liefern sowie die Entwicklung einer zielgerichteten, kombinierten und individualisierten Therapie des metastasierenden Melanoms ermöglichen. In diesem Kontext soll die vorliegende Arbeit einen weiteren Beitrag zum Verständnis der Metastasierungskaskade und der daran beteiligten Proteine leisten. Aufgrund der Überexpression in einer Reihe von Tumoren und seiner geringen Molmasse von lediglich 11,5 kDa bietet sich das S100A4-Protein als Marker mit hoher prognostischer Signifikanz für verschiedene Tumorentitäten an. Jedoch ist die Beteiligung von S100A4 bei der Ausbildung des invasiven Tumorphänotyps noch nicht vollständig aufgeklärt. S100A4 besitzt zahlreiche intra- und extrazelluläre Bindungspartner, wobei die Metastasierung scheinbar ausschließlich durch das extrazelluläre Protein beeinflusst wird. S100A4 wechselwirkt extrazellulär beispielsweise mit dem Rezeptor für fortgeschrittene Glykierungsendprodukte (RAGE). Ziel dieser Arbeit war es, speziell die Bedeutung von S100A4 und seiner Interaktion mit RAGE für das prometastatische Verhalten von Melanomzellen in vitro und in vivo näher zu charakterisieren. Darüber hinaus sollte die Beteiligung von S100A4 bei der Gehirn-Metastasierung untersucht werden, wobei insbesondere die Regulierung der Endothelzell-Permeabilität und der transendothelialen Migration der Melanomzellen im Vordergrund stand. Im Rahmen dieser Arbeit wurde gezeigt, dass S100A4 und die Interaktion mit RAGE die prometastatischen Eigenschaften der A375-Melanomzellen förderte. Zudem verringerte extrazelluläres S100A4 die Zell-Integrität von Gehirn-Endothelzellen und erleichterte somit die Durchdringung der Blut-Hirn-Schranke. Diese Erkenntnis lässt sich möglicherweise auf andere Blut-Gewebe-Schranken übertragen. Die In-vivo-Orientierungsstudie zeigte, dass S100A4- und RAGE-überexprimierende Zellen zu einer verstärkten disseminierten Metastasierung führten, wobei sich zwei unterschiedliche Verteilungsmuster ergaben. Darüber hinaus führten beide Zelllinien vereinzelt zur Bildung von Gehirnmetastasen, wodurch sich die intrakardiale Injektion durchaus als Modell für weitere Therapiestudien mit dem Augenmerk der S100A4-RAGE-stimulierten Metastasierung eignet. Die genauere Kenntnis regulativer Mechanismen bei der Synthese und Sekretion von S100A4 sowie die pathophysiologische Differenzierung der S100A4-Interaktion mit RAGE eröffnen neue Wege, die S100A4-vermittelten Effekte therapeutisch zu beeinflussen. Daraus lassen sich möglicherweise neue zielgerichtete Radionuklid-basierte Therapieansätze für das metastasierende Melanom ableiten.