Monoamine oxidase (MAO) is an enzyme located on the outer mitochondrial membrane and is considered to be a target for the treatment of diseases such as Parkinson’s disease and depression. MAO may be classified into two isoforms, MAO-A and MAO-B. Since MAO-A and MAO-B catalyzes the metabolism of serotonin and dopamine, respectively, MAO-A inhibitors are used in the therapy of depression while MAO-B inhibitors are useful in the treatment of Parkinson’s disease. The older nonselective and irreversible MAO inhibitors, however, are not frequently used because they may ellicit potentially dangerous side effects such as the “cheese reaction”. The cheese reaction occurs when irreversible MAO-A inhibitors block the metabolism of tyramine in the gastrointestinal tract. Excessive amounts of tyramine subsequently enter the systemic circulation and cause a hypertensive reaction. This problem may be overcome by the development of selective MAO-B inhibitors and reversible MAO-A inhibitors. Selective MAO-B inhibitors do not cause the cheese reaction, because tyramine is metabolized, in the intestines, by MAO-A. Tyramine also has the ability to displace reversible MAO-A inhibitors and can subsequently be normally metabolized, thus not causing the cheese reaction. Several reseach groups are therefore involved in the discovery of reversible MAO-A and MAO-B inhibitors. As mentioned above, such drugs may be used in the treatment of depression and Parkinson’s disease. One approach is the de novo design of novel molecules with affinities for MAO-A and MAO-B active sites. In a second approach, existing drugs may be reappropriated as MAO inhibitors. With this approach, approved drugs are screened for the possibility that they, in addition to their action at the indicated target, also act as inhibitors of MAO-A and/or MAO-B. Such drugs may then be applied as MAO inhibitors in the treatment of depression and Parkinson’s disease. From a toxicological point of view, it is also of importance to identify MAO-A inhibitory activities among existing drugs as this will alert to the occurance of potential side effects such as the cheese reaction. In this study the second approach will be followed. This study will screen a virtual library of approved drugs for inhibitory activity towards MAO-A and MAO-B. Molecular modeling may be used to screen virtual libraries of drugs as potential inhibitors of the MAO enzymes. This may conveniently be achieved by employing structure-based or ligand-based pharmacophore models. In this study a virtual library of approved drugs was screened for secondary inhibitory activities towards the MAO isoforms with the use of structure-based pharmacophore models. There are several advantages to this approach. Molecular modeling aims at reducing the overall cost associated with the discovery and development of a new drug by identifying the most promising candidates to focus the experimental efforts on. It aids in understanding how a ligand binds to the active site of an enzyme. It is relatively easier to re-register a drug for a second pharmacological activity. This approach may also lead to drugs with a multi-target mode of action. The structure-based pharmacophores were constructed using the known crystallographic structures of MAO-A and MAO-B with the inhibitors, harmine and safinamide, complexed in the active sites, respectively. Employing the MAO-A and MAO-B structure-based pharmacophore model in the virtual screening of a library of approved drugs, 45 compounds were found to map to the MAO-A and MAO-B pharmacophore models. Among the hits, 29 compounds were selected for in vitro evaluation as MAO-A and MAO-B inhibitors. The IC50 values for these compounds were determined. After in vitro evaluation, 13 compounds showed inhibitory activity towards MAO. Of the 13 compounds 3 showed interesting inhibitory activities. These compounds included caffeine (IC50 = 0.761 μM for MAO-A and 5.08 μM for MAO-B), esomeprazole (IC50 = 23.2 μM for MAO-A and 48.3 μM for MAO-B) and leflunomide (IC50 = 19.1μM for MAO-A and 13.7 μM for MAO-B). The MAO inhibitory properties of caffeine and esomeprazole were further investigated. The reversibility of MAO inhibition by caffeine and esomeprazole were determined by dialysis and dilution studies. Sets of Lineweaver-Burk plots were constructed to determine the modes of binding of these inhibitors to the MAO enzymes. Both caffeine and esomeprazole were found to be reversible and competitive inhibitors of MAO. Dialysis of mixtures of caffeine with MAO-A and MAO-B resulted in the recovery of enzyme activity to levels of 97% and 96%, respectively. Dialysis of mixtures of esomeprazole with MAO-A and MAO-B resulted in the recovery of enzyme activity to levels of 93% and 88%, respectively. Similarly, dilution of mixtures containing esomeprazole and MAO-A/MAO-B resulted in the recovery of enzyme activity to levels of 94% and 87%, respectively.For the inhibition of MAO-A and MAO-B by caffeine and esomeprazole, the Lineweaver-Burk plots were indicative of a competitive mode of inhibition. In an attempt to gain further insignt, caffeine, esomeprazole and leflunomide were docked into models of the active sites of MAO-A and MAO-B. An analysis of the interactions between the enzyme models and the ligands were carried out and the results are discussed in the dissertation The results of the present study show that screening of a virtual database of molecules with a pharmacophore model may be useful in identifying existing drugs with potential MAO inhibitory activities. The search for new reversible MAO inhibitors for the treatment of diseases, including Parkinson’s disease and depression, may be facilitated by employing a virtual screening approach. Such an approach also may be more costeffective than de novo inhibitor design. In addition, the virtual screening approach may alert to potential side effects of existing drugs that may arise as a consequence of a secondary inhibition of MAO. / MSc (Pharmaceutical Chemistry), North-West University, Potchefstroom Campus, 2014

Monoamine oxidase (MAO) is an enzyme located on the outer mitochondrial membrane and is considered to be a target for the treatment of diseases such as Parkinson’s disease and depression. MAO may be classified into two isoforms, MAO-A and MAO-B. Since MAO-A and MAO-B catalyzes the metabolism of serotonin and dopamine, respectively, MAO-A inhibitors are used in the therapy of depression while MAO-B inhibitors are useful in the treatment of Parkinson’s disease. The older nonselective and irreversible MAO inhibitors, however, are not frequently used because they may ellicit potentially dangerous side effects such as the “cheese reaction”. The cheese reaction occurs when irreversible MAO-A inhibitors block the metabolism of tyramine in the gastrointestinal tract. Excessive amounts of tyramine subsequently enter the systemic circulation and cause a hypertensive reaction. This problem may be overcome by the development of selective MAO-B inhibitors and reversible MAO-A inhibitors. Selective MAO-B inhibitors do not cause the cheese reaction, because tyramine is metabolized, in the intestines, by MAO-A. Tyramine also has the ability to displace reversible MAO-A inhibitors and can subsequently be normally metabolized, thus not causing the cheese reaction. Several reseach groups are therefore involved in the discovery of reversible MAO-A and MAO-B inhibitors. As mentioned above, such drugs may be used in the treatment of depression and Parkinson’s disease. One approach is the de novo design of novel molecules with affinities for MAO-A and MAO-B active sites. In a second approach, existing drugs may be reappropriated as MAO inhibitors. With this approach, approved drugs are screened for the possibility that they, in addition to their action at the indicated target, also act as inhibitors of MAO-A and/or MAO-B. Such drugs may then be applied as MAO inhibitors in the treatment of depression and Parkinson’s disease. From a toxicological point of view, it is also of importance to identify MAO-A inhibitory activities among existing drugs as this will alert to the occurance of potential side effects such as the cheese reaction. In this study the second approach will be followed. This study will screen a virtual library of approved drugs for inhibitory activity towards MAO-A and MAO-B. Molecular modeling may be used to screen virtual libraries of drugs as potential inhibitors of the MAO enzymes. This may conveniently be achieved by employing structure-based or ligand-based pharmacophore models. In this study a virtual library of approved drugs was screened for secondary inhibitory activities towards the MAO isoforms with the use of structure-based pharmacophore models. There are several advantages to this approach. Molecular modeling aims at reducing the overall cost associated with the discovery and development of a new drug by identifying the most promising candidates to focus the experimental efforts on. It aids in understanding how a ligand binds to the active site of an enzyme. It is relatively easier to re-register a drug for a second pharmacological activity. This approach may also lead to drugs with a multi-target mode of action. The structure-based pharmacophores were constructed using the known crystallographic structures of MAO-A and MAO-B with the inhibitors, harmine and safinamide, complexed in the active sites, respectively. Employing the MAO-A and MAO-B structure-based pharmacophore model in the virtual screening of a library of approved drugs, 45 compounds were found to map to the MAO-A and MAO-B pharmacophore models. Among the hits, 29 compounds were selected for in vitro evaluation as MAO-A and MAO-B inhibitors. The IC50 values for these compounds were determined. After in vitro evaluation, 13 compounds showed inhibitory activity towards MAO. Of the 13 compounds 3 showed interesting inhibitory activities. These compounds included caffeine (IC50 = 0.761 μM for MAO-A and 5.08 μM for MAO-B), esomeprazole (IC50 = 23.2 μM for MAO-A and 48.3 μM for MAO-B) and leflunomide (IC50 = 19.1μM for MAO-A and 13.7 μM for MAO-B). The MAO inhibitory properties of caffeine and esomeprazole were further investigated. The reversibility of MAO inhibition by caffeine and esomeprazole were determined by dialysis and dilution studies. Sets of Lineweaver-Burk plots were constructed to determine the modes of binding of these inhibitors to the MAO enzymes. Both caffeine and esomeprazole were found to be reversible and competitive inhibitors of MAO. Dialysis of mixtures of caffeine with MAO-A and MAO-B resulted in the recovery of enzyme activity to levels of 97% and 96%, respectively. Dialysis of mixtures of esomeprazole with MAO-A and MAO-B resulted in the recovery of enzyme activity to levels of 93% and 88%, respectively. Similarly, dilution of mixtures containing esomeprazole and MAO-A/MAO-B resulted in the recovery of enzyme activity to levels of 94% and 87%, respectively.For the inhibition of MAO-A and MAO-B by caffeine and esomeprazole, the Lineweaver-Burk plots were indicative of a competitive mode of inhibition. In an attempt to gain further insignt, caffeine, esomeprazole and leflunomide were docked into models of the active sites of MAO-A and MAO-B. An analysis of the interactions between the enzyme models and the ligands were carried out and the results are discussed in the dissertation The results of the present study show that screening of a virtual database of molecules with a pharmacophore model may be useful in identifying existing drugs with potential MAO inhibitory activities. The search for new reversible MAO inhibitors for the treatment of diseases, including Parkinson’s disease and depression, may be facilitated by employing a virtual screening approach. Such an approach also may be more costeffective than de novo inhibitor design. In addition, the virtual screening approach may alert to potential side effects of existing drugs that may arise as a consequence of a secondary inhibition of MAO. / MSc (Pharmaceutical Chemistry), North-West University, Potchefstroom Campus, 2014

Monoamine oxidase (MAO) is responsible for dopamine catabolism in the brain and therefore is especially important in the treatment of Parkinson's disease (PD). MAO–B inhibition provides symptomatic relief by indirectly elevating dopamine levels in the PD brain. PD is caused by the loss of dopaminergic neurons in the substantia nigra and the formation of proteinaceous structures in the brain. The cause of idiopathic PD is unknown, but one theory states that reactive oxygen species (ROS), partly derived from the catalytic cycle of MAO, may be to blame for damaging dopaminergic neurons. Since MAO inhibitors may reduce the MAO–catalyzed production of ROS, these compounds may protect dopaminergic neurons against degeneration in PD. It is commonly accepted that by the time PD symptoms manifest, about 80% of striatal dopamine has been lost. MAO is present as two subtypes in the human brain, namely MAO–A and MAO–B. MAOs are found mainly attached to the mitochondrial membrane and is responsible for the oxidative deamination of various monoamines, including dopamine. MAO is a dimeric enzyme which operates in conjunction with a co–factor, flavin adenine dinucleotide (FAD), to which it is covalently bound. The flavin is in a bent conformation, which assists the catalytic activity of MAO. As mentioned above, the catalytic action of MAO also produces harmful substances such as hydrogen peroxide, ammonia, aldehydes and may also increase the levels of hydroxyl radicals. In the healthy brain, these substances are metabolized rapidly, but the PD brain may exhibit reduced clearance of these species. Thus the inhibition of MAOs may be beneficial to the PD sufferer as it indirectly increases dopamine levels in the brain and may also slow the formation of harmful substances. MAO inhibitors, of the MAO–A type, were first used as anti–depressants. It was these drugs that first prompted researchers to explore MAO inhibitors as novel anti–parkinsonian drugs, as MAO–A inhibition slows the degradation of dopamine. Two types of inhibition modes exist, irreversible and reversible inhibition. Irreversible inhibitors do not allow for competition with the substrate and inactivate the enzyme permanently. Selegiline, a propargyl amine derivative, is an example of an irreversible MAO–B selective inhibitor. The major disadvantage of irreversible inhibitors is that after terminating treatment, recovery of the enzyme activity may require several weeks, since the turnover rate for the biosynthesis of MAO in the human brain may be as much as 40 days. Reversible inhibitors have better safety profiles since they allow for competition with the substrate. (E)–8–(3–Chlorostyryl)caffeine (CSC) is an example of a reversible inhibitor of MAO–B and is also an antagonist of the adenosine A2A receptor. Since antagonism of A2A receptors also produces an antiparkinsonian effect, dual acting compounds such as CSC, which block both the A2A receptors and MAO–B, may have an enhanced therapeutic potential in PD therapy. Current PD therapy available only treats the symptoms of PD and do not halt or slow the progression of the neurodegenerative processes. There therefore exists the need for the development of antiparkinsonian drugs with neuroprotective effects. Since both MAO–B inhibitors and A2A receptor antagonists are reported to possess protective effects in PD and PD animal models, dual acting drugs, that antagonize A2A receptors and inhibit MAO–B, may be candidates for neuroprotection. Using the structure of CSC as lead, we investigate in the current study, the possibility that aminocaffeines may also possess potent MAO–B inhibitory properties. The structures of the aminocaffeine derivatives that were investigated bear close structural resemblance to CSC as well as to a series of alkyloxycaffeine analogues that was recently found to be potent MAO inhibitors. This study therefore further explores the structural requirements of caffeine derivatives to act as MAO inhibitors by examining the possibility that aminocaffeine derivatives may be MAO inhibitors. Such compounds may act as lead compounds for the development of improved PD therapy. In this study, a series of 8–aminocaffeine derivatives were synthesized and evaluated as inhibitors of human MAO–A and B. For this purpose, 8–chlorocaffeine was reacted with the appropriate amine at high temperatures to produce the desired 8–aminocaffeine derivatives. The inhibitory activities of the compounds were determined towards recombinant human MAO–A and B and expressed as IC50 values. The results showed that human MAO–B was most potently inhibited by 8–[methyl(4–phenylbutyl)amino]caffeine with an IC50 value of 2.97 ?M. Human MAO–A was most potently inhibited by 8–[2–(3–chlorophenyl)–ethylamino]caffeine with an IC50 value of 5.78 ?M. It was found that methylation of the amine group at C8 of the caffeine ring increases inhibition but also selectivity towards MAO–B inhibition. For example, 8–[4–(phenylbutylamino)]caffeine inhibits MAO–B with an IC50 value of 7.56 ?M whereas 8–[methyl(4–phenylbutyl)amino]–caffeine has an increased inhibition potency of 2.97 ?M. The selectivity for MAO–B inhibition also increases over MAO–A when the C8 amine is methylated. It was found that the aminocaffeine derivatives bind reversibly to both enzyme isoforms and the mode of inhibition is competitive for MAO–B. From these results it can be concluded that although the 8–aminocaffeine derivatives are only moderately potent MAO–B inhibitors, they may act as lead compounds for the design of more potent reversible MAO inhibitors. Docking studies revealed that the 8–aminocaffeine and 8–[(methyl)amino]caffeine derivatives traverse both the entrance and substrate cavities of the MAO–B enzyme, with the caffeinyl moiety oriented towards the FAD co–factor while the amino–side chain protrudes into the entrance cavity. / Thesis (M.Sc. (Pharmaceutical Chemistry))--North-West University, Potchefstroom Campus, 2012.

Monoamine oxidase (MAO) is responsible for dopamine catabolism in the brain and therefore is especially important in the treatment of Parkinson's disease (PD). MAO–B inhibition provides symptomatic relief by indirectly elevating dopamine levels in the PD brain. PD is caused by the loss of dopaminergic neurons in the substantia nigra and the formation of proteinaceous structures in the brain. The cause of idiopathic PD is unknown, but one theory states that reactive oxygen species (ROS), partly derived from the catalytic cycle of MAO, may be to blame for damaging dopaminergic neurons. Since MAO inhibitors may reduce the MAO–catalyzed production of ROS, these compounds may protect dopaminergic neurons against degeneration in PD. It is commonly accepted that by the time PD symptoms manifest, about 80% of striatal dopamine has been lost. MAO is present as two subtypes in the human brain, namely MAO–A and MAO–B. MAOs are found mainly attached to the mitochondrial membrane and is responsible for the oxidative deamination of various monoamines, including dopamine. MAO is a dimeric enzyme which operates in conjunction with a co–factor, flavin adenine dinucleotide (FAD), to which it is covalently bound. The flavin is in a bent conformation, which assists the catalytic activity of MAO. As mentioned above, the catalytic action of MAO also produces harmful substances such as hydrogen peroxide, ammonia, aldehydes and may also increase the levels of hydroxyl radicals. In the healthy brain, these substances are metabolized rapidly, but the PD brain may exhibit reduced clearance of these species. Thus the inhibition of MAOs may be beneficial to the PD sufferer as it indirectly increases dopamine levels in the brain and may also slow the formation of harmful substances. MAO inhibitors, of the MAO–A type, were first used as anti–depressants. It was these drugs that first prompted researchers to explore MAO inhibitors as novel anti–parkinsonian drugs, as MAO–A inhibition slows the degradation of dopamine. Two types of inhibition modes exist, irreversible and reversible inhibition. Irreversible inhibitors do not allow for competition with the substrate and inactivate the enzyme permanently. Selegiline, a propargyl amine derivative, is an example of an irreversible MAO–B selective inhibitor. The major disadvantage of irreversible inhibitors is that after terminating treatment, recovery of the enzyme activity may require several weeks, since the turnover rate for the biosynthesis of MAO in the human brain may be as much as 40 days. Reversible inhibitors have better safety profiles since they allow for competition with the substrate. (E)–8–(3–Chlorostyryl)caffeine (CSC) is an example of a reversible inhibitor of MAO–B and is also an antagonist of the adenosine A2A receptor. Since antagonism of A2A receptors also produces an antiparkinsonian effect, dual acting compounds such as CSC, which block both the A2A receptors and MAO–B, may have an enhanced therapeutic potential in PD therapy. Current PD therapy available only treats the symptoms of PD and do not halt or slow the progression of the neurodegenerative processes. There therefore exists the need for the development of antiparkinsonian drugs with neuroprotective effects. Since both MAO–B inhibitors and A2A receptor antagonists are reported to possess protective effects in PD and PD animal models, dual acting drugs, that antagonize A2A receptors and inhibit MAO–B, may be candidates for neuroprotection. Using the structure of CSC as lead, we investigate in the current study, the possibility that aminocaffeines may also possess potent MAO–B inhibitory properties. The structures of the aminocaffeine derivatives that were investigated bear close structural resemblance to CSC as well as to a series of alkyloxycaffeine analogues that was recently found to be potent MAO inhibitors. This study therefore further explores the structural requirements of caffeine derivatives to act as MAO inhibitors by examining the possibility that aminocaffeine derivatives may be MAO inhibitors. Such compounds may act as lead compounds for the development of improved PD therapy. In this study, a series of 8–aminocaffeine derivatives were synthesized and evaluated as inhibitors of human MAO–A and B. For this purpose, 8–chlorocaffeine was reacted with the appropriate amine at high temperatures to produce the desired 8–aminocaffeine derivatives. The inhibitory activities of the compounds were determined towards recombinant human MAO–A and B and expressed as IC50 values. The results showed that human MAO–B was most potently inhibited by 8–[methyl(4–phenylbutyl)amino]caffeine with an IC50 value of 2.97 ?M. Human MAO–A was most potently inhibited by 8–[2–(3–chlorophenyl)–ethylamino]caffeine with an IC50 value of 5.78 ?M. It was found that methylation of the amine group at C8 of the caffeine ring increases inhibition but also selectivity towards MAO–B inhibition. For example, 8–[4–(phenylbutylamino)]caffeine inhibits MAO–B with an IC50 value of 7.56 ?M whereas 8–[methyl(4–phenylbutyl)amino]–caffeine has an increased inhibition potency of 2.97 ?M. The selectivity for MAO–B inhibition also increases over MAO–A when the C8 amine is methylated. It was found that the aminocaffeine derivatives bind reversibly to both enzyme isoforms and the mode of inhibition is competitive for MAO–B. From these results it can be concluded that although the 8–aminocaffeine derivatives are only moderately potent MAO–B inhibitors, they may act as lead compounds for the design of more potent reversible MAO inhibitors. Docking studies revealed that the 8–aminocaffeine and 8–[(methyl)amino]caffeine derivatives traverse both the entrance and substrate cavities of the MAO–B enzyme, with the caffeinyl moiety oriented towards the FAD co–factor while the amino–side chain protrudes into the entrance cavity. / Thesis (M.Sc. (Pharmaceutical Chemistry))--North-West University, Potchefstroom Campus, 2012.

Caffeine is frequently used in cosmetics due to its well-characterised skin permeation properties and is widely incorporated in cosmetic-related products intended for skin (Samah & Heard, 2013:631). Despite its polar characteristics (Dias et al., 1999:41), caffeine is an important biologically and cosmetically active compound (Herman & Herman, 2012:13). This active pharmaceutical ingredient (API) has a broad range of advantages in the world of cosmetics, including the improvement of microcirculation in the capillaries (Lupi et al., 2007:107), showing anti-cellulite activity in the fatty tissue (Velasco et al., 2008:24), anti-oxidation activity in sunscreens & anti-ageing products (Koo et al., 2007:964) and the stimulation of hair growth (Fisher et al., 2007:27). Caffeine has also shown significant decreases in UV-induced skin tumour multiplicity (Lu et al., 2001:5003, 5008) and has been proven to prevent photo-damaged skin, which includes the formation of wrinkles and histological alterations (Mitani et al., 2007:86). It is therefore clear that the challenge for the dermal delivery of the hydrophilic caffeine is for it to be retained in the specific skin layers (dermal delivery) where it can exert its action, rather than to permeate through the skin and into the hydrophilic systemic circulation (transdermal delivery) (Wiechers et al., 2008:10). In this study the calculated skin delivery gap (SDG) values, and the transdermal and dermal delivery of caffeine from three different semi-solid topical formulations were compared. The SDG theory was developed to evaluate the effectiveness of dermal delivery of API from topical formulations and is known as the ratio between the concentration required to achieve minimum effect relative to the concentration obtained at the target site (JW Solutions, 2011). During this study the principle of the SDG was investigated by using the formulating strategy, Formulating for Efficacy (FFE™), which aims to optimise skin delivery of APIs from different formulations. The SDG was therefore implemented and in vitro transdermal studies were utilised to ultimately prove or disprove the hypothesis of SDG on the prediction of the topical delivery of caffeine. The human skin consists of two distinctive layers namely the epidermis (including the stratum corneum (SC) and viable dermis) and the dermis (Menon, 2002:S3). The main barrier to dermal and transdermal permeation is the outermost layer of the skin, the SC (Fang et al., 2007:343). The difference between the target site for dermal and transdermal delivery of APIs is crucial to be mentioned. Dermal delivery includes the delivery of an API to the skin surface, SC, viable epidermis or dermis, whereas transdermal delivery requires the API to permeate all the way through the various skin layers and into the systemic circulation (Wiechers, 2000:42). Since this study involves the optimisation of the topical delivery of caffeine, the physicochemical properties of this API as well as those of the skin should be considered. As mentioned before, caffeine is a rather polar molecule (Dias et al., 1999:41), whereas the SC (lipophilic) provides the rate-limiting barrier to the percutaneous absorption of polar (hydrophilic) molecules, such as caffeine (Barry, 1983:105). Caffeine was incorporated into three different formulations: a gel and two creams (differing only in the ratio of the primary and secondary emollient). The three topical formulations each had different polarities, where the Gel represented the hydrophilic formulation (more polar than the skin), whereas the first cream, Cream 1 (containing 5% DMI and 9% glycerine), served as the intermediate formulation (similar polarity as the SC), and the second cream, Cream 2 (10% DMI and 4% glycerine), was the formulation less polar (therefore more lipophilic) than the SC. Franz cell type transdermal diffusion studies were performed on the three semi-solid formulations (Gel, Cream 1 and Cream 2). The diffusion studies were conducted over a period of 12 h, followed by the tape stripping of the skin directly after each diffusion study. Caucasian female abdominal skin was obtained with consent from willing donors. Ethical approval for the acquisition and use of the donated skin was granted under reference number NWU-00114-11-A5. The formulations each contained 1% of caffeine as API. The skin used for the diffusion studies was prepared with the use of a Zimmer Dermatome®. The receptor phase of each Franz cell was withdrawn at predetermined time intervals and subsequently analysed with high performance liquid chromatography (HPLC) in order to determine the concentration of caffeine that permeated through the skin. Stratum corneum-epidermis (SCE) and epidermis-dermis (ED) samples were prepared and left overnight at a temperature of 4 °C, and they were analysed the following day with the use of HPLC in order to determine the concentration of caffeine that had accumulated in the particular skin layers. The SDG value for each caffeine formulation was calculated and it was compared to the flux and tape stripping results obtained from the diffusion studies. To ultimately prove or disprove the SDG theory, the skin diffusion studies and tape stripping results were used to determine whether any difference occurred in the absorption or penetration of the API from the different formulations into the skin. The formulation with the intermediate polarity (Cream 1) produced the highest transdermal flux of caffeine due to the hydrophilic and lipophilic nature of caffeine and the formulation, respectively. Cream 1 is sufficiently lipophilic to transport caffeine into the SC and at the same time sufficiently hydrophilic (more polar than Cream 2) to cause a greater driving force of caffeine through to the more hydrophilic epidermis, dermis and systemic circulation. The results from the tape stripping yielded that Cream 2 (the more lipophilic formulation) produced the highest concentration of caffeine into the SCE due to the hydrophilic and lipophilic nature of caffeine and the formulation, respectively. The difference in polarity between the formulation and the API in Cream 2 was the greatest compared to the other formulations, which significantly increased the driving force of caffeine to partition into the SC (Wiechers et al., 2004:177). The hydrophilic gel showed the highest concentration of caffeine in the ED layer of the skin due to the hydrophilic compounds formulated in the Gel, which showed greater ability to partition into the aqueous dermis and viable epidermis (Imai et al., 2013:372). Cream 2 had the lowest calculated SDG value compared to that of the Gel and Cream 1. The smaller the delivery gap, the greater the delivery of the API should be into the skin (Wiechers, 2010). Considering this, it was expected that Cream 2 would deliver greater amounts of caffeine into the skin than the more hydrophilic formulations. Cream 2, which showed the lowest calculated SDG value delivered the highest amount of caffeine into the SCE during the diffusion studies. The calculated SDG values therefore are consistent with the concentration of caffeine in the SCE (the lowest SDG value produced the highest concentration of API in the SCE). However, no correlations were found between the calculated SDG values and ED delivery or the flux of caffeine. The final conclusion for this study is that the SDG theory proved to be effective and trustworthy regarding the delivery of caffeine into the SC. / MSc (Pharmaceutics), North-West University, Potchefstroom Campus, 2014

Caffeine is frequently used in cosmetics due to its well-characterised skin permeation properties and is widely incorporated in cosmetic-related products intended for skin (Samah & Heard, 2013:631). Despite its polar characteristics (Dias et al., 1999:41), caffeine is an important biologically and cosmetically active compound (Herman & Herman, 2012:13). This active pharmaceutical ingredient (API) has a broad range of advantages in the world of cosmetics, including the improvement of microcirculation in the capillaries (Lupi et al., 2007:107), showing anti-cellulite activity in the fatty tissue (Velasco et al., 2008:24), anti-oxidation activity in sunscreens & anti-ageing products (Koo et al., 2007:964) and the stimulation of hair growth (Fisher et al., 2007:27). Caffeine has also shown significant decreases in UV-induced skin tumour multiplicity (Lu et al., 2001:5003, 5008) and has been proven to prevent photo-damaged skin, which includes the formation of wrinkles and histological alterations (Mitani et al., 2007:86). It is therefore clear that the challenge for the dermal delivery of the hydrophilic caffeine is for it to be retained in the specific skin layers (dermal delivery) where it can exert its action, rather than to permeate through the skin and into the hydrophilic systemic circulation (transdermal delivery) (Wiechers et al., 2008:10). In this study the calculated skin delivery gap (SDG) values, and the transdermal and dermal delivery of caffeine from three different semi-solid topical formulations were compared. The SDG theory was developed to evaluate the effectiveness of dermal delivery of API from topical formulations and is known as the ratio between the concentration required to achieve minimum effect relative to the concentration obtained at the target site (JW Solutions, 2011). During this study the principle of the SDG was investigated by using the formulating strategy, Formulating for Efficacy (FFE™), which aims to optimise skin delivery of APIs from different formulations. The SDG was therefore implemented and in vitro transdermal studies were utilised to ultimately prove or disprove the hypothesis of SDG on the prediction of the topical delivery of caffeine. The human skin consists of two distinctive layers namely the epidermis (including the stratum corneum (SC) and viable dermis) and the dermis (Menon, 2002:S3). The main barrier to dermal and transdermal permeation is the outermost layer of the skin, the SC (Fang et al., 2007:343). The difference between the target site for dermal and transdermal delivery of APIs is crucial to be mentioned. Dermal delivery includes the delivery of an API to the skin surface, SC, viable epidermis or dermis, whereas transdermal delivery requires the API to permeate all the way through the various skin layers and into the systemic circulation (Wiechers, 2000:42). Since this study involves the optimisation of the topical delivery of caffeine, the physicochemical properties of this API as well as those of the skin should be considered. As mentioned before, caffeine is a rather polar molecule (Dias et al., 1999:41), whereas the SC (lipophilic) provides the rate-limiting barrier to the percutaneous absorption of polar (hydrophilic) molecules, such as caffeine (Barry, 1983:105). Caffeine was incorporated into three different formulations: a gel and two creams (differing only in the ratio of the primary and secondary emollient). The three topical formulations each had different polarities, where the Gel represented the hydrophilic formulation (more polar than the skin), whereas the first cream, Cream 1 (containing 5% DMI and 9% glycerine), served as the intermediate formulation (similar polarity as the SC), and the second cream, Cream 2 (10% DMI and 4% glycerine), was the formulation less polar (therefore more lipophilic) than the SC. Franz cell type transdermal diffusion studies were performed on the three semi-solid formulations (Gel, Cream 1 and Cream 2). The diffusion studies were conducted over a period of 12 h, followed by the tape stripping of the skin directly after each diffusion study. Caucasian female abdominal skin was obtained with consent from willing donors. Ethical approval for the acquisition and use of the donated skin was granted under reference number NWU-00114-11-A5. The formulations each contained 1% of caffeine as API. The skin used for the diffusion studies was prepared with the use of a Zimmer Dermatome®. The receptor phase of each Franz cell was withdrawn at predetermined time intervals and subsequently analysed with high performance liquid chromatography (HPLC) in order to determine the concentration of caffeine that permeated through the skin. Stratum corneum-epidermis (SCE) and epidermis-dermis (ED) samples were prepared and left overnight at a temperature of 4 °C, and they were analysed the following day with the use of HPLC in order to determine the concentration of caffeine that had accumulated in the particular skin layers. The SDG value for each caffeine formulation was calculated and it was compared to the flux and tape stripping results obtained from the diffusion studies. To ultimately prove or disprove the SDG theory, the skin diffusion studies and tape stripping results were used to determine whether any difference occurred in the absorption or penetration of the API from the different formulations into the skin. The formulation with the intermediate polarity (Cream 1) produced the highest transdermal flux of caffeine due to the hydrophilic and lipophilic nature of caffeine and the formulation, respectively. Cream 1 is sufficiently lipophilic to transport caffeine into the SC and at the same time sufficiently hydrophilic (more polar than Cream 2) to cause a greater driving force of caffeine through to the more hydrophilic epidermis, dermis and systemic circulation. The results from the tape stripping yielded that Cream 2 (the more lipophilic formulation) produced the highest concentration of caffeine into the SCE due to the hydrophilic and lipophilic nature of caffeine and the formulation, respectively. The difference in polarity between the formulation and the API in Cream 2 was the greatest compared to the other formulations, which significantly increased the driving force of caffeine to partition into the SC (Wiechers et al., 2004:177). The hydrophilic gel showed the highest concentration of caffeine in the ED layer of the skin due to the hydrophilic compounds formulated in the Gel, which showed greater ability to partition into the aqueous dermis and viable epidermis (Imai et al., 2013:372). Cream 2 had the lowest calculated SDG value compared to that of the Gel and Cream 1. The smaller the delivery gap, the greater the delivery of the API should be into the skin (Wiechers, 2010). Considering this, it was expected that Cream 2 would deliver greater amounts of caffeine into the skin than the more hydrophilic formulations. Cream 2, which showed the lowest calculated SDG value delivered the highest amount of caffeine into the SCE during the diffusion studies. The calculated SDG values therefore are consistent with the concentration of caffeine in the SCE (the lowest SDG value produced the highest concentration of API in the SCE). However, no correlations were found between the calculated SDG values and ED delivery or the flux of caffeine. The final conclusion for this study is that the SDG theory proved to be effective and trustworthy regarding the delivery of caffeine into the SC. / MSc (Pharmaceutics), North-West University, Potchefstroom Campus, 2014