ABSTRACT: UTILIZING NOVEL DOSE EQUIVALENCE METHODOLOGIES TO EXAMINE COCAINE'S EFFECTS ON THE VASCULATURE Neil S. Lamarre Doctor of Philosophy Temple University School of Medicine, 2013 Doctoral Advisory Committee Chair: Ronald J. Tallarida, Ph.D. Cocaine abuse and addiction is a serious health problem, resulting in thousands of emergency room visits and deaths each year in the United States. It is particularly toxic to the cardiovascular system, including deleterious effects on the peripheral vasculature. These effects are not well understood, but evidence suggests chronic cocaine use may lead to endothelial dysfunction, thereby increasing relative risk of a number of other cardiovascular diseases including stroke, aneurysm, myocardial infarction, hypertension, etc. Data from our lab, and others, suggest that the presence of a functional endothelium has a dramatic effect on the contractility of the rat aorta that is agonist-specific. Attenuation of this endothelium-dependent vasodilatory component of agonist action is a primary feature of endothelial dysfunction. We have utilized dose equivalence theory to calculate the dose response relationship for the endothelium-dependent vasodilatory component of an agonist causing overt vasoconstriction. This component cannot be measured directly, but our novel methodology allows us to quantitate agonist-specific impairment of vasodilation, and describe it using the familiar parameters of the dose response curve. Another strength of this method, relative to currently used in vitro methods, is that it also avoids the confounding variable of a second agonist used to produce the initial vasoconstriction. To validate the methodology, a pilot study was performed examining the endothelial dysfunction in STZ-induced diabetic rats, as a positive control for endothelial dysfunction. Interestingly, this treatment showed impairment in the endothelium-dependent vasodilatory component of action of norepinephrine, but not of angiotensin-II. Thus, our initial hypothesis was confirmed - that disruption of the vasodilatory components of various agonists are independent, and that agonist-specific information may prove useful. Next, we employed our new methodology utilizing the rat aorta as our vascular model to test the hypothesis that chronic cocaine administration causes endothelial dysfunction. We first examined the endothelium-dependent vasodilation component of a number of physiologically important vasoconstrictors, and attempted to determine which vasodilatory mediators contributed to the effect. We found the endothelium to have a profound effect on the dose response curve to three important endogenous agonists. These data suggest that under conditions of endothelial dysfunction exaggerated vasoconstriction could occur, even within normal plasma concentration ranges of these vasoconstrictors, resulting in elevated blood pressure and further damage to the endothelium over time. No endothelial dysfunction was observed with this treatment paradigm, using our methodology or the standard approach. This may be a result of insufficient duration of cocaine treatment, or a result of our selection of the rat aorta as a model. We wanted to further investigate which vasodilatory mechanisms were involved in this vasodilatory component of action. We inhibiting various endothelium-derived mediators of this vasodilatory component of action (such as nitric oxide or prostacyclin), which revealed differential activation of these mediators by the agonists examined. For example, inhibition of nitric oxide synthesis abolished the endothelium-dependent vasodilatory component of endothelin-1, but only partially attenuated that of angiotensin-II. Thus, the agonist-specific pattern of impairment may also prove useful in examining the underlying mechanisms of impaired vasodilation. Endothelial dysfunction is one reported consequence of long term cocaine abuse; however, there are conflicting reports on the acute vascular effects of cocaine, with some reports concluding that cocaine is a vasoconstrictor, and some reporting its action as a vasodilator. There are in vitro reports of cocaine causing release of vasoconstrictors from the endothelium, which supports the longstanding notion of cocaine as a vasoconstrictor. However, one recent report demonstrates a dose-dependent vasodilatory effect of cocaine in rat aorta that is independent of the endothelium. This complexity is perhaps due, in part, to cocaine's affinity for a number of molecular targets, acting in combination. In examining the acute action of cocaine in our preparation, we observed an "inverted-U" shaped dose response, also referred to as a hormetic dose response curve. We then applied dose equivalence methodology in order to derive the "unknown" second component contributing the vasodilatory action of cocaine at higher doses. This methodology lets us calculate this unknown component, and describe it with the familiar parameters of a dose response curve, which could potentially aid in the identification of the unknown component. The preliminary studies with acute cocaine utilized a sub-maximal dose of phenylephrine in order to observe tension changes in either direction. This prompted us to further characterize the interaction of cocaine with other alpha adrenoceptor agonists. Importantly, because cocaine alone had no effect at doses up to 100 µM, but potentiated the vasoconstriction of alpha agonists, the interaction is therefore synergistic. This constitutes evidence of a previously undescribed mechanism contributing to cocaine's vasoconstricting effect. In vivo, reuptake inhibition is a major mechanism for cocaine-induced vasoconstriction, but is excluded in this experiment by virtue of low levels of sympathetic innervation in the rat aorta, and the use of methoxamine, an alpha agonist not subject to the reuptake mechanisms. This interaction may contribute to cocaine-induced vasoconstriction in the coronary arteries, especially in circumstances of endothelial dysfunction. In summary, the work presented in this dissertation applies new methodologies utilizing dose equivalence theory to the study of cocaine's effects on peripheral vasculature, and presents novel findings of synergy with respect to cocaine's enhancement on the action of alpha adrenoceptor-mediated vasoconstriction. / Pharmacology

O interesse em efeitos sinérgicos ou antagônicos através de experiências com associações cresceu imensamente nas últimas décadas. Com o objetivo de estudar a associação de dois herbicidas inibidores da ALS (sulfometuron-methyl e chlorimuron-ethyl) em Digitaria insularis, empregou-se dos métodos estatísticos fornecidos por meio das curvas dose-resposta e isobologramas. O conceito de curvas dose-resposta tem sido amplamente utilizado para avaliar os resultados das experiências com herbicidas. No entanto, a abordagem estatística em torno das curvas deve ser cuidadosa; cosiderar as curvas dose-resposta semelhantes quando elas não são pode influenciar muito na comparação entre as moléculas e afetar o cálculo da potência relativa. A potência relativa ajuda a entender qual é a relação de potência entre as moléculas, esse conhecimento é fundamental para os estudos com os herbicidas em associação. Sabendo-se disso foram conduzidos experimentos do tipo dose-resposta em dois tipos de substratos utilizando Digitaria insularis como planta daninha indicadora. Os experimentos foram conduzidos em casa de vegetação do Centro de Pesquisa e Desenvolvimento da DuPont em Paulínia (SP). Para cada molécula foram aplicadas, em pré-emergência, oito doses com quatro repetições cada e quatro plantas testemunha nos diferentes substratos. O substrato A é somente substrato comercial (marca Tropstrato HT®) puro; E o substrato B é uma mistura de solo arenoso, peneirado e livre de contaminação mais substrato comercial na proporção 3:1 v/v. Neste caso ficou evidente a maior capacidade do sulfometuronmethyl em reduzir a biomassa da espécie, as curvas dose-resposta foram descritas pelo modelo log-logístico com os valores de ED50 e as declividades de cada curva variando livremente, pois as hipóteses testadas nos diferentes substratos de curvas paralelas foram rejeitadas. O valor calculado da potência relativa para o nível de resposta de 50% entre os herbicidas CLASSIC® (chlorimuron-ethyl) e CURAVIAL® (sulfometuron-methyl) nas condições desse estudo foi de 8,596 e 44,047 nos Substrato A e B respectivamente. A partir dessa informação foram utilizadas as mesmas condições do experimento anterior para o experimento com as isoboles dos herbicidas em associação, cinco curvas dose-resposta, foram propostas, duas curvas para os produtos isolados e três curvas para as associações em diferentes proporções [(25% : 75%); (50% : 50%); (75% : 25%)] chlorimuron-ethyl / sulfometuron-methyl, totalizando 144 parcelas. Vários modelos isoboles foram testados, o modelo de Concentração Aditiva foi significativamente descartado, mas foi levado em consideração para testar teoricamente o grau de sinergia ou antagonismo que modelos mais complexos podiam fornecer. O modelo de isobole proposto por Hewlett e Plackett (1959) teve ajuste significativo e parâmetro de interação menor que um (λ=0,33). O modelo que mais se ajustou aos dados foi o proposto por Vølund e os resultados também mostraram que nessas condições a associação de sulfometuron-methyl com chlorimuron-ethyl foi antagônica; as isoboles são potentes ferramentas para capturar e descrever associações de herbicidas. Entretanto a literatura mostra que associar duas moléculas e testar em um sistema biológico complexo sofrendo variações das diferentes condições ambientais possíveis pode gerar respostas variáveis, portanto esses resultados devem ser repetidos em outras condições para validar as conclusões ou então serem executados em condições controladas de laboratório e usar organismos vivos de menor complexidade como bioindicador. / The interest in synergistic or antagonistic effects through experiences with associations has immensely grown in recent decades. In order to study the association of two ALS inhibitor herbicides (sulfometuron-methyl and chlorimuronethyl) in Digitaria insularis, the statistical methods provided by the dose-response curves and isobolograms were used. The concept of dose-response curves has been widely used to evaluate the results of herbicide experiments. However, the statistical approach around curves must be careful; considering the dose-response curves when they are not can greatly influence the comparison between molecules and affect the calculation of relative potency. Relative potency helps to understand what the power ratio is between molecules, this knowledge is fundamental for studies with herbicides in association. Knowing this, dose-response experiments were conducted on two types of substrates using Digitaria insularis as an indicator weed. The experiments were conducted in a greenhouse of the DuPont Research and Development Center in Paulínia (SP). For each molecule were applied, in pre-emergence, eight doses with four replicates each and four control plants on different substrates. Substrate A is pure commercial substrate (brand Tropstrato HT®); and substrate B is a mixture of sandy soil, sifted and contamination-free plus commercial substrate in the ratio 3:1 v/v. In this case it was evident the greater capacity of the sulfometuron-methyl to reduce the biomass of the species, the dose-response curves were described by the log-logistic model with the values of ED50 and the slopes of each curve varying freely, since the hypotheses tested in the different substrates of parallel curves were rejected. The calculated value of relative potency for the 50% response level between CLASSIC® (chlorimuron-ethyl) and CURAVIAL® (sulfometuron-methyl) herbicides under the conditions of this study was 8.596 and 44.047 on Substrate A and B respectively. From this information the same conditions of the previous experiment for the isoboles of the herbicides in association were used, five doseresponse curves were proposed, two curves for the isolated products and three curves for the associations in different ratios [25% : 75%); (50% : 50%); (75% : 25%)] chlorimuron-ethyl / sulfometuron-methyl, totaling 144 plots. Isoboles models were tested, the Concentration Additive model was significantly discarded, but was taken into account to theoretically test the degree of synergy or antagonism that more complex models could provide. The isobole model proposed by Hewlett and Plackett (1959) had a significant adjustment and interaction parameter smaller than 1 (λ = 0.33). The model that best fit the data was the one proposed by Vølund and the results also showed that under these conditions the association of sulfometuronmethyl with chlorimuron-ethyl was antagonistic; isoboles are potent tools for capturing and describing herbicide associations. So these results must be repeated in other conditions to validate the conclusions or to be executed in controlled laboratory conditions and to use living organisms of lesser complexity as a bioindicator.

O interesse em efeitos sinérgicos ou antagônicos através de experiências com associações cresceu imensamente nas últimas décadas. Com o objetivo de estudar a associação de dois herbicidas inibidores da ALS (sulfometuron-methyl e chlorimuron-ethyl) em Digitaria insularis, empregou-se dos métodos estatísticos fornecidos por meio das curvas dose-resposta e isobologramas. O conceito de curvas dose-resposta tem sido amplamente utilizado para avaliar os resultados das experiências com herbicidas. No entanto, a abordagem estatística em torno das curvas deve ser cuidadosa; cosiderar as curvas dose-resposta semelhantes quando elas não são pode influenciar muito na comparação entre as moléculas e afetar o cálculo da potência relativa. A potência relativa ajuda a entender qual é a relação de potência entre as moléculas, esse conhecimento é fundamental para os estudos com os herbicidas em associação. Sabendo-se disso foram conduzidos experimentos do tipo dose-resposta em dois tipos de substratos utilizando Digitaria insularis como planta daninha indicadora. Os experimentos foram conduzidos em casa de vegetação do Centro de Pesquisa e Desenvolvimento da DuPont em Paulínia (SP). Para cada molécula foram aplicadas, em pré-emergência, oito doses com quatro repetições cada e quatro plantas testemunha nos diferentes substratos. O substrato A é somente substrato comercial (marca Tropstrato HT®) puro; E o substrato B é uma mistura de solo arenoso, peneirado e livre de contaminação mais substrato comercial na proporção 3:1 v/v. Neste caso ficou evidente a maior capacidade do sulfometuronmethyl em reduzir a biomassa da espécie, as curvas dose-resposta foram descritas pelo modelo log-logístico com os valores de ED50 e as declividades de cada curva variando livremente, pois as hipóteses testadas nos diferentes substratos de curvas paralelas foram rejeitadas. O valor calculado da potência relativa para o nível de resposta de 50% entre os herbicidas CLASSIC® (chlorimuron-ethyl) e CURAVIAL® (sulfometuron-methyl) nas condições desse estudo foi de 8,596 e 44,047 nos Substrato A e B respectivamente. A partir dessa informação foram utilizadas as mesmas condições do experimento anterior para o experimento com as isoboles dos herbicidas em associação, cinco curvas dose-resposta, foram propostas, duas curvas para os produtos isolados e três curvas para as associações em diferentes proporções [(25% : 75%); (50% : 50%); (75% : 25%)] chlorimuron-ethyl / sulfometuron-methyl, totalizando 144 parcelas. Vários modelos isoboles foram testados, o modelo de Concentração Aditiva foi significativamente descartado, mas foi levado em consideração para testar teoricamente o grau de sinergia ou antagonismo que modelos mais complexos podiam fornecer. O modelo de isobole proposto por Hewlett e Plackett (1959) teve ajuste significativo e parâmetro de interação menor que um (λ=0,33). O modelo que mais se ajustou aos dados foi o proposto por Vølund e os resultados também mostraram que nessas condições a associação de sulfometuron-methyl com chlorimuron-ethyl foi antagônica; as isoboles são potentes ferramentas para capturar e descrever associações de herbicidas. Entretanto a literatura mostra que associar duas moléculas e testar em um sistema biológico complexo sofrendo variações das diferentes condições ambientais possíveis pode gerar respostas variáveis, portanto esses resultados devem ser repetidos em outras condições para validar as conclusões ou então serem executados em condições controladas de laboratório e usar organismos vivos de menor complexidade como bioindicador. / The interest in synergistic or antagonistic effects through experiences with associations has immensely grown in recent decades. In order to study the association of two ALS inhibitor herbicides (sulfometuron-methyl and chlorimuronethyl) in Digitaria insularis, the statistical methods provided by the dose-response curves and isobolograms were used. The concept of dose-response curves has been widely used to evaluate the results of herbicide experiments. However, the statistical approach around curves must be careful; considering the dose-response curves when they are not can greatly influence the comparison between molecules and affect the calculation of relative potency. Relative potency helps to understand what the power ratio is between molecules, this knowledge is fundamental for studies with herbicides in association. Knowing this, dose-response experiments were conducted on two types of substrates using Digitaria insularis as an indicator weed. The experiments were conducted in a greenhouse of the DuPont Research and Development Center in Paulínia (SP). For each molecule were applied, in pre-emergence, eight doses with four replicates each and four control plants on different substrates. Substrate A is pure commercial substrate (brand Tropstrato HT®); and substrate B is a mixture of sandy soil, sifted and contamination-free plus commercial substrate in the ratio 3:1 v/v. In this case it was evident the greater capacity of the sulfometuron-methyl to reduce the biomass of the species, the dose-response curves were described by the log-logistic model with the values of ED50 and the slopes of each curve varying freely, since the hypotheses tested in the different substrates of parallel curves were rejected. The calculated value of relative potency for the 50% response level between CLASSIC® (chlorimuron-ethyl) and CURAVIAL® (sulfometuron-methyl) herbicides under the conditions of this study was 8.596 and 44.047 on Substrate A and B respectively. From this information the same conditions of the previous experiment for the isoboles of the herbicides in association were used, five doseresponse curves were proposed, two curves for the isolated products and three curves for the associations in different ratios [25% : 75%); (50% : 50%); (75% : 25%)] chlorimuron-ethyl / sulfometuron-methyl, totaling 144 plots. Isoboles models were tested, the Concentration Additive model was significantly discarded, but was taken into account to theoretically test the degree of synergy or antagonism that more complex models could provide. The isobole model proposed by Hewlett and Plackett (1959) had a significant adjustment and interaction parameter smaller than 1 (λ = 0.33). The model that best fit the data was the one proposed by Vølund and the results also showed that under these conditions the association of sulfometuronmethyl with chlorimuron-ethyl was antagonistic; isoboles are potent tools for capturing and describing herbicide associations. So these results must be repeated in other conditions to validate the conclusions or to be executed in controlled laboratory conditions and to use living organisms of lesser complexity as a bioindicator.

The oral route of administration remains the preferred route of administrating drugs due to patient acceptance and compliance. Therapeutic proteins are currently mainly administered by means of the parenteral route because of its low intestinal epithelial permeation capability. The major challenges for oral delivery of proteins and peptides are pre-systemic enzymatic degradation and poor penetration of the intestinal mucosa. The latter can be overcome by including safe and effective absorption enhancers in dosage forms. Aloe vera, Aloe ferox and Aloe marlothii gel materials as well as N-trimethyl chitosan chloride (TMC) were shown to be capable of increasing peptide drug transport across in vitro models such as Caco-2 cell monolayers. The purpose of this study is to investigate binary combinations of chemical drug absorption enhancers and to determine if synergistic drug absorption enhancement effects exist. A. vera, A. ferox and A. marlothii leaf gel materials as well as with N-trimethyl chitosan chloride (TMC) were combined in different ratios and their effects on the transepithelial electrical resistance (TEER) as well as the transport of FITC-dextran across Caco-2 cell monolayers were measured. The isobole method was applied to determine the type of interaction that exists between the absorption enhancers combinations. The TEER results showed synergism existed for the combinations between A. vera and A. marlothii, A. marlothii and A. ferox as well as A. vera and TMC. Antagonism interactions also occurred and can probably be explained by chemical reactions between the chemical permeation enhancers such as complex formation. In terms of FITC-dextran transport, synergism was found for combinations between A. vera and A. marlothii, A. marlothii and A. ferox, A. vera and TMC, A. ferox and TMC and A. marlothii and TMC, whereas antagonism was observed for A. vera and A. ferox. The combinations where synergism was obtained have the potential to be used as effective drug absorption enhancers at lower concentrations compared to single components. / MSc (Pharmaceutics), North-West University, Potchefstroom Campus, 2015

The oral route of administration remains the preferred route of administrating drugs due to patient acceptance and compliance. Therapeutic proteins are currently mainly administered by means of the parenteral route because of its low intestinal epithelial permeation capability. The major challenges for oral delivery of proteins and peptides are pre-systemic enzymatic degradation and poor penetration of the intestinal mucosa. The latter can be overcome by including safe and effective absorption enhancers in dosage forms. Aloe vera, Aloe ferox and Aloe marlothii gel materials as well as N-trimethyl chitosan chloride (TMC) were shown to be capable of increasing peptide drug transport across in vitro models such as Caco-2 cell monolayers. The purpose of this study is to investigate binary combinations of chemical drug absorption enhancers and to determine if synergistic drug absorption enhancement effects exist. A. vera, A. ferox and A. marlothii leaf gel materials as well as with N-trimethyl chitosan chloride (TMC) were combined in different ratios and their effects on the transepithelial electrical resistance (TEER) as well as the transport of FITC-dextran across Caco-2 cell monolayers were measured. The isobole method was applied to determine the type of interaction that exists between the absorption enhancers combinations. The TEER results showed synergism existed for the combinations between A. vera and A. marlothii, A. marlothii and A. ferox as well as A. vera and TMC. Antagonism interactions also occurred and can probably be explained by chemical reactions between the chemical permeation enhancers such as complex formation. In terms of FITC-dextran transport, synergism was found for combinations between A. vera and A. marlothii, A. marlothii and A. ferox, A. vera and TMC, A. ferox and TMC and A. marlothii and TMC, whereas antagonism was observed for A. vera and A. ferox. The combinations where synergism was obtained have the potential to be used as effective drug absorption enhancers at lower concentrations compared to single components. / MSc (Pharmaceutics), North-West University, Potchefstroom Campus, 2015