Locomotor activation by theacrine, a purine alkaloid structurally similar to caffeine : involvement of adenosine and dopamine receptors

Purine compounds, such as caffeine, have many health-promoting properties and have proven to be beneficial in treating a number of different conditions. Theacrine, a purine alkaloid structurally similar to caffeine and abundantly present in Camellia kucha, has recently become of interest as a potential therapeutic compound. In the present study, theacrine was tested using a rodent behavioral model to investigate the effects of the drug on locomotor activity. Long Evans rats were injected with theacrine (24 or 48 mg/kg, i.p.) and activity levels were measured. Results showed that the highest dose of theacrine (48 mg/kg, i.p.) significantly increased locomotor activity compared to control animals and activity remained elevated throughout the duration of the session. To test for the involvement of adenosine receptors underlying theacrine's motor-activating properties, rats were administered a cocktail of the adenosine A₁ agonist, N⁶-cyclopentyladenosine (CPA; 0.1 mg/kg, i.p.) and A(2A) receptor agonist 2-p-(2-carboxyethyl)phenethylamino-5'-N-ethylcarboxamidoadenosine (CGS-21680; 0.2 mg/kg, i.p.). Pre-treatment with theacrine significantly attenuated the motor depression induced by the adenosine receptor agonists, indicating that theacrine is likely acting as an adenosine receptor antagonist. Next, we examined the role of DA D₁ and D₂ receptor antagonism on theacrine-induced hyperlocomotion. Both antagonists, D₁R SCH23390 (0.1 or 0.05 mg/kg, i.p.) and D₂R eticlopride (0.1 mg/kg, i.p.), significantly reduced theacrine-stimulated activity indicating that this behavioral response, at least in part, is mediated by DA receptors. In order to investigate the brain region where theacrine may be acting, the drug (10 or 20 μg) was infused bilaterally into nucleus accumbens (NAc). Theacrine enhanced activity levels in a dose-dependent manner, implicating a role of the NAc in modulating theacrine's effects on locomotion. In addition, theacrine did not induce locomotor sensitization or tolerance after chronic exposure. Taken together, these findings demonstrate that theacrine significantly enhances activity; an effect which is mediated by both the adenosinergic and dopaminergic systems.

An influenza virus inspired polymer system for the timed release of siRNA

Small interfering RNA silences specific genes by interfering with mRNA translation, and acts to modulate or inhibit specific biological pathways; a therapy that holds great promise in the cure of many diseases. However, the naked small interfering RNA is susceptible to degradation by plasma and tissue nucleases and due to its negative charge unable to cross the cell membrane. Here we report a new polymer carrier designed to mimic the influenza virus escape mechanism from the endosome, followed by a timed release of the small interfering RNA in the cytosol through a self-catalyzed polymer degradation process. Our polymer changes to a negatively charged and non-toxic polymer after the release of small interfering RNA, presenting potential for multiple repeat doses and long-term treatment of diseases.

Mathematical modelling of controlled drug release from polymer micro-spheres : incorporating the effects of swelling, diffusion and dissolution via moving boundary problems

Controlled drug delivery is a key topic in modern pharmacotherapy, where controlled drug delivery devices are required to prolong the period of release, maintain a constant release rate, or release the drug with a predetermined release profile. In the pharmaceutical industry, the development process of a controlled drug delivery device may be facilitated enormously by the mathematical modelling of drug release mechanisms, directly decreasing the number of necessary experiments. Such mathematical modelling is difficult because several mechanisms are involved during the drug release process. The main drug release mechanisms of a controlled release device are based on the device’s physiochemical properties, and include diffusion, swelling and erosion. In this thesis, four controlled drug delivery models are investigated. These four models selectively involve the solvent penetration into the polymeric device, the swelling of the polymer, the polymer erosion and the drug diffusion out of the device but all share two common key features. The first is that the solvent penetration into the polymer causes the transition of the polymer from a glassy state into a rubbery state. The interface between the two states of the polymer is modelled as a moving boundary and the speed of this interface is governed by a kinetic law. The second feature is that drug diffusion only happens in the rubbery region of the polymer, with a nonlinear diffusion coefficient which is dependent on the concentration of solvent. These models are analysed by using both formal asymptotics and numerical computation, where front-fixing methods and the method of lines with finite difference approximations are used to solve these models numerically. This numerical scheme is conservative, accurate and easily implemented to the moving boundary problems and is thoroughly explained in Section 3.2. From the small time asymptotic analysis in Sections 5.3.1, 6.3.1 and 7.2.1, these models exhibit the non-Fickian behaviour referred to as Case II diffusion, and an initial constant rate of drug release which is appealing to the pharmaceutical industry because this indicates zeroorder release. The numerical results of the models qualitatively confirms the experimental behaviour identified in the literature. The knowledge obtained from investigating these models can help to develop more complex multi-layered drug delivery devices in order to achieve sophisticated drug release profiles. A multi-layer matrix tablet, which consists of a number of polymer layers designed to provide sustainable and constant drug release or bimodal drug release, is also discussed in this research. The moving boundary problem describing the solvent penetration into the polymer also arises in melting and freezing problems which have been modelled as the classical onephase Stefan problem. The classical one-phase Stefan problem has unrealistic singularities existed in the problem at the complete melting time. Hence we investigate the effect of including the kinetic undercooling to the melting problem and this problem is called the one-phase Stefan problem with kinetic undercooling. Interestingly we discover the unrealistic singularities existed in the classical one-phase Stefan problem at the complete melting time are regularised and also find out the small time behaviour of the one-phase Stefan problem with kinetic undercooling is different to the classical one-phase Stefan problem from the small time asymptotic analysis in Section 3.3. In the case of melting very small particles, it is known that surface tension effects are important. The effect of including the surface tension to the melting problem for nanoparticles (no kinetic undercooling) has been investigated in the past, however the one-phase Stefan problem with surface tension exhibits finite-time blow-up. Therefore we investigate the effect of including both the surface tension and kinetic undercooling to the melting problem for nanoparticles and find out the the solution continues to exist until complete melting. The investigation of including kinetic undercooling and surface tension to the melting problems reveals more insight into the regularisations of unphysical singularities in the classical one-phase Stefan problem. This investigation gives a better understanding of melting a particle, and contributes to the current body of knowledge related to melting and freezing due to heat conduction.

Polymer nanocarrier system for endosome escape and timed release of siRNA with complete gene silencing and cell death in cancer cells

An influenza virus-inspired polymer mimic nanocarrier was used to deliver siRNA for specific and near complete gene knockdown of an osteoscarcom cell line (U-2SO). The polymer was synthesized by single-electron transfer living radical polymerization (SET-LRP) at room temperature to avoid complexities of transfer to monomer or polymer. It was the only LRP method that allowed good block copolymer formation with a narrow molecular weight distribution. At nitrogen to phosphorus (N/P) ratios of equal to or greater than 20 (greater than a polymer concentration of 13.8 μg/mL) with polo-like kinase 1 (PLK1) siRNA gave specific and near complete (>98%) cell death. The polymer further degrades to a benign polymer that showed no toxicity even at polymer concentrations of 200 μg/mL (or N/P ratio of 300), suggesting that our polymer nanocarrier can be used as a very effective siRNA delivery system and in a multiple dose administration. This work demonstrates that with a well-designed delivery device, siRNA can specifically kill cells without the inclusion of an additional clinically used highly toxic cochemotherapeutic agent. Our work also showed that this excellent delivery is sensitive for the study of off-target knockdown of siRNA.

Association between migraine and a functional polymorphism at the dopamine β-hydroxylase locus

Migraine is a common neurological disorder with a significant genetic component. Although a number of linkage and association studies have been undertaken, the number and identity of all migraine susceptibility genes has yet to be defined. The existence of dopaminergic hypersensitivity in migraine has been recognised on a pharmacological basis and some studies have reported genetic association between migraine and dopamine-related gene variants. Our laboratory has previously reported association of migraine with a promoter STR marker in the dopamine beta hydroxylase (DBH) gene. In the present study, we analysed two additional DBH markers in two independent migraine case–control cohorts. These two markers are putative functional SNPs, one within the promoter (−1021C→T) and another SNP (+1603C→T) in exon 11 of the DBH gene. The results showed a significant association for allelic and genotypic frequency distribution between the DBH marker in the promoter and migraine in the first (P = 0.004 and P = 0.012, respectively) and the second (P = 0.013 and P = 0.031, respectively) tested cohorts. There was no association observed between either genotype and/or allelic frequencies for the DBH marker located in exon 11 and migraine (P ≥ 0.05). The promoter DBH marker, reported associated with migraine in this study, has been shown to affect up to 52% of plasma DBH activity. Varying DBH activity levels have been postulated to be involved in migraine process with an increase of dopamine, resulting from a lower DBH activity shown positively correlated with migraine severity. It is plausible that the functional promoter variant of DBH may play a role in the migraine disorder.

Association study of a functional variant in intron 8 of the dopamine transporter gene and migraine susceptibility

Migraine is a common, genetically influenced neurovascular disorder. The dopamine transporter gene is a candidate for migraine association studies. This study tested a functionally linked variable number tandem repeat (VNTR) in intron 8 of the dopamine transporter gene (DATInt8) in 550 migraine cases (401 with aura, 149 without aura) and 550 non-migraine controls. Chi-squared analysis of the DATInt8 revealed that the allele and genotype frequency distributions for migraine cases (including subtype analysis) and controls were not different (P > 0.1). These findings offer no evidence for an association of the DATInt8 with migraine with and without aura and therefore do not implicate the dopamine transporter gene as a modifier of migraine risk.

Association between a 19 bp deletion polymorphism at the dopamine beta-hydroxylase (DBH) locus and migraine with aura

Migraine is a debilitating neurological disorder, affecting 12% of Caucasian populations. It is well known that migraine has a strong genetic component, although the type and number of genes involved is unclear. Our previous work has investigated dopamine related migraine candidate genes and has reported a significant allelic association with migraine of a microsatellite localised to the promoter region of the dopamine beta-hydroxylase (DBH) gene. The present study performed an association analysis in a larger population of case-controls (275 unrelated Caucasian migraineurs versus 275 controls) examining two different genetic DBH polymorphisms (a functional insertion/deletion promoter and a coding SNP A444G polymorphism). Although no significant association was found for the SNP polymorphism, the results showed a significant association between the insertion/deletion variant and disease (chi(2)=8.92, P=0.011), in particular in migraine with aura (chi(2)=11.53, P=0.003) compared to the control group. Furthermore, the analysis of this polymorphism stratified by gender, revealed that male individuals with the homozygote deletion genotype had three times the risk of developing migraine, compared to females. The DBH insertion/deletion polymorphism is in linkage disequilibrium with the previously reported migraine associated DBH microsatellite and this insertion/deletion polymorphism is functional, which may explain a potential role in susceptibility to migraine.

Dopamine receptor genes and migraine with and without aura : an association study

OBJECTIVE: To investigate the role of the dopamine receptor genes, DRD1, DRD3, and DRD5 in the pathogenesis of migraine. BACKGROUND: Migraine is a chronic debilitating disorder affecting approximately 12% of the white population. The disease shows strong familial aggregation and presumably has a genetic basis, but at present, the type and number of genes involved is unclear. The study of candidate genes can prove useful in the identification of genes involved in complex diseases such as migraine, especially if the contribution of the gene to phenotypic expression is minor. Genes coding for proteins involved in dopamine metabolism have been implicated in a number of neurologic conditions and may play a contributory role in migraine. Hence, genes that code for enzymes and receptors modulating dopaminergic activity are good candidates for investigation of the molecular genetic basis of migraine. METHODS: We tested 275 migraineurs and 275 age- and sex-matched individuals free of migraine. Genotypic results were determined by restriction endonuclease digestion of polymerase chain reaction products to detect DRD1 and DRD3 alleles and by Genescan analysis after polymerase chain reaction using fluorescently labelled oligonucleotide primers for the DRD5 marker. RESULTS: Results of chi-square statistical analyses indicated that the allele distribution for migraine cases compared to controls was not significantly different for any of the three tested gene markers (chi2 = 0.1, P =.74 for DRD1; chi2 = 1.8, P =.18 for DRD3; and chi2 = 20.3, P =.08 for DRD5). CONCLUSIONS: These findings offer no evidence for allelic association between the tested dopamine receptor gene polymorphisms and the more prevalent forms of migraine and, therefore, do not support a role for these genes in the pathogenesis of the disorder.

Modulation of in vitro platelet 5-HT release by species of Erythrina and Cymbopogon

Extracts of Australian plants were screened to detect constituents affecting adenosine di-phosphate (ADP) induced platelet aggregation and [14C]5-hydroxytryptamine (5-HT) release. Extracts of four tested plants including, Eremophila gilesii, Erythrina vespertilio, Cymbopogon ambiguus, and Santalum acuminatum, were found to cause significant inhibition of platelet 5-HT release. Inhibition levels ranged from 56-98%, and was not due to the non-specific effects of protein binding tannins. These extracts, and those we have previously identified as being active, were examined further to determine if they affect epinephrine (EPN), arachidonic acid (A.A) or collagen stimulated platelet aggregation and 5-HT release. Among those extracts investigated, we found that both the methanolic extract of E. vespertilio and the dichloromethane (DCM) extract of C. ambiguus were most potent and caused significant inhibition of platelet activation induced by EPN, A.A and to a lesser extent by collagen. Inhibition of ADP induced platelet 5-HT release by both of these extracts, was dose-dependent, with IC50 values for E. vespertilio and C. ambiguus estimated to be 20.4 microl (1.855 mg/ml) and 8.34 microl (0.758 mg/ml), respectively. Overall, C. ambiguus exhibited most activity and also caused dose-dependent inhibition of A.A induced platelet activation. These results indicate that inhibition may occur specifically at a site within the A.A pathway, and suggest the presence of a cyclo-oxygenase inhibitor. Both E. vespertilio and C. ambiguus are reported to be traditional headache treatments, with the present study providing evidence that they affect 5-HT release.

Inhibition of platelet aggregation and 5-HT release by extracts of Australian plants used traditionally as headache treatments

To identify potential migraine therapeutics, extracts of eighteen plants were screened to detect plant constituents affecting ADP induced platelet aggregation and [14C]5-hydroxytryptamine (5-HT) release. Extracts of the seven plants exhibiting significant inhibition of platelet function were reanalysed in the presence of polyvinyl pyrrolidone (PVP) to remove polyphenolic tannins that precipitate proteins. Two of these extracts no longer exhibited inhibition of platelet activity after removal of tannins. However, extracts of Crataegus monogyna, Ipomoea pes-caprae, Eremophila freelingii, Eremophila longifolia, and Asteromyrtus symphyocarpa still potently inhibited ADP induced human platelet [14C]5-HT release in vitro, with levels ranging from 62 to 95% inhibition. I. pes-caprae, and C. monogyna also caused significant inhibition of ADP induced platelet aggregation. All of these plants have been previously used as traditional headache treatments, except for C. monogyna which is used primarily for protective effects on the cardiovascular system. Further studies elucidating the compounds that are responsible for these anti-platelet effects are needed to determine their exact mechanism of action.

Evidence for allelic association of the dopamine β-hydroxylase gene (DBH) with susceptibility to typical migraine

Migraine is a debilitating neurological disorder characterized by recurrent attacks of severe headache. The disorder is highly prevalent, affecting approximately 12% of Caucasian populations. It is well known that migraine has a strong genetic component, although the type and number of genes involved is not yet clear. However, the calcium channel gene, CACNA1A, on chromosome 19 contains mutations responsible for familial hemiplegic migraine, a rare and severe subtype of migraine. There is also evidence to suggest that serotonin- and dopamine-related genes may be involved in the pathogenesis of migraine. This study employed a linkage and association approach to investigate neurotransmitter-related migraine candidate genes. Polymorphisms within the dopamine beta-hydroxylase (DBH) gene, serotonin transporter gene (SERT), and dopamine receptor gene (DRD2) were tested in 177 unrelated Caucasian migraineurs and 182 control individuals. In addition, an independent sample of 82 families affected with migraine was examined. Unrelated case-control association analysis of a DBH intragenic dinucleotide polymorphism indicated altered allelic distribution between migraine and control groups (L2=16.53, P=0.019). Furthermore, the transmission/disequilibrium test, which was implemented on the family data, also indicated distortion of allele transmission for the same DBH marker (L2=4.44, P=0.035). Together, these results provide evidence for allelic association of the DBH gene with typical migraine susceptibility (Fisher's combined P value=0.006) and indicate that further research into the role of the DBH gene in the etiology of migraine is warranted.

Converse consumption : the converse blank canvas project

Research background For almost 80 years the Chuck Taylor (or Chuck T's) All Star basketball shoe has been an iconic item of fashion apparel. The Chuck T's were first designed in 1921 by Converse, an American shoe company and over the decades they became a popular item not purely for sports and athletic purposes but rather evolved into the shoe of choice for many subcultural groups as a fashion item. In some circles the Chuck Taylor is still seen as the "coolest" sneaker of all time - one which will never go out of fashion regardless of changing trends. With over 600 millions pairs sold all over the world since its release, the Converse shoe is representative of not only a fashion culture - but also of a consumption culture - that evolved as the driving force behind the massive growth of the Western economic system during the 20th Century. Artisan Gallery (Brisbane), in conjunction with the exhibition Reboot: Function, Fashion and the Sneaker, a history of the sneaker, selected 20 designers to customise and re-design the classic Converse Chuck Taylor All Stars shoe and in doing so highlighted the diversity of forms possible for creative outcomes. As Artisan Gallery Curator Kirsten Fitzpatrick states “We were expecting people to draw and paint on them. Instead, we had shoes... mounted as trophies.." referring to the presentation of "Converse Consumption". The exhibition ran from 21 June – 16 August 2012: Research question The Chuck T’s is one of many overwhelmingly commercially successful designs of the last century. Nowadays we are faced with the significant problems of overconsumption and the stress this causes on the natural ecosystem; and on people as a result. As an active member of the industrial design fraternity – a discipline that sits at the core of this problem - how can I use this opportunity to comment on the significant issue of consumption? An effective way to do this was to associate consumption of goods with consumption of sugar. There are significant similarities between our ceaseless desires to consume products and our fervent need to consume indulgent sweet foods. Artisan Statement Delicious, scrumptious, delectable... your pupils dilate, your blood pressure spikes, your liver goes into overdrive. Immediately, your brain cuts off the adenosine receptors, preventing drowsiness. Your body increases dopamine production, in-turn stimulating the pleasure receptors in your brain. Your body absorbs all the sweetness and turns it into fat – while all the nutrients that you actually require are starting to be destroyed, about to be expelled. And this is only after one bite! After some time though, your body comes crashing back to earth. You become irritable and begin to feel sluggish. Your eyelids seem heavy while your breathing pattern changes. Your body has consumed all the energy and destroyed all available nutrients. You literally begin to shut down. These are the physiological effects of sugar consumption. A perfect analogy for our modern day consumer driven world. Enjoy your dessert! Research contribution “Converse Consumption” contributes to the conversation regarding over-consumption by compelling people to reflect on their consumption behaviour through the reconceptualising of the deconstructed Chuck T’s in an attractive edible form. By doing so the viewer has to deal with the desire to consume the indulgent looking dessert with the contradictory fact that it is comprised of a pair of shoes. The fact that the shoes are Chuck T’s make the effect even more powerful due to their iconic status. These clashing motivations are what make “Converse Consumption” a bizarre yet memorable experience. Significance The exhibition was viewed by an excess of 1000 people and generated exceptional media coverage and public exposure/impact. As Artisan Gallery Curator Kirsten Fitzpatrick states “20 of Brisbane's best designers were given the opportunity to customise their own Converse Sneakers, with The Converse Blank Canvas Project.” And to be selected in this category demonstrates the calibre of importance for design prominence.

Quantification of ebullitive and diffusive methane release to atmosphere from a water storage

Accurately quantifying total freshwater storage methane release to atmosphere requires the spatial–temporal measurement of both diffusive and ebullitive emissions. Existing floating chamber techniques provide localised assessment of methane flux, however, significant errors can arise when weighting and extrapolation to the entire storage, particularly when ebullition is significant. An improved technique has been developed that compliments traditional chamber based experiments to quantify the storage-scale release of methane gas to atmosphere through ebullition using the measurements from an Optical Methane Detector (OMD) and a robotic boat. This provides a conservative estimate of the methane emission rate from ebullition along with the bubble volume distribution. It also georeferences the area of ebullition activity across entire storages at short temporal scales. An assessment on Little Nerang Dam in Queensland, Australia, demonstrated whole storage methane release significantly differed spatially and throughout the day. Total methane emission estimates showed a potential 32-fold variation in whole-of-dam rates depending on the measurement and extrapolation method and time of day used. The combined chamber and OMD technique showed that 1.8–7.0% of the surface area of Little Nerang Dam is accounting for up to 97% of total methane release to atmosphere throughout the day. Additionally, over 95% of detectable ebullition occurred in depths less than 12 m during the day and 6 m at night. This difference in spatial and temporal methane release rate distribution highlights the need to monitor significant regions of, if not the entire, water storage in order to provide an accurate estimate of ebullition rates and their contribution to annual methane emissions.

Electrochemical detection of dopamine and cytochrome c at a nanostructured gold electrode

A nanostructured gold surface consisting of closely packed outwardly growing spikes is investigated for the electrochemical detection of dopamine and cytochrome c. A significant electrocatalytic effect for the electrooxidation of both dopamine and ascorbic acid at the nanostructured electrode was found due to the presence of surface active sites which allowed the detection of dopamine in the presence of excess ascorbic acid to be achieved by differential pulse voltammetry. By simple modification with a layer of Nafion, the enhanced electrocatalytic properties of the nanostructured surface was maintained while increasing the selectivity of dopamine detection in the presence of interfering species such as excess ascorbic and uric acids. Also, upon modification of the nanostructured surface with a monolayer of cysteine, the electrochemical response of immobilised cytochrome c in two distinct conformations was observed. This opens up the possibility of using such a nanostructured surface for the characterisation of other biomolecules and in bio-electroanalytical applications.

Exenatide extended-release; clinical trials, patient preference, and economic considerations

Type 2 diabetes remains an escalating world-wide problem, despite a range of treatments. The revelation that insulin secretion is under the control of a gut hormone, glucagon-like peptide 1 (GLP-1) led to a new paradigm in the management of type 2 diabetes, medicines that directly stimulate, or that prolong the actions of the endogenous GLP-1, at its receptors. Exenatide is an agonist at the GLP-1 receptors, and was initially developed as a subcutaneous twice daily medication, ExBID. The clinical trials with ExBID established a role for exenatide in the treatment of type 2 diabetes. Subsequently, once weekly exenatide (ExQW) was shown to have advantages over ExBID, and there is now more emphasis on the development of ExQW. ExQW alone reduces glycosylated haemoglobin (HbA1c) and body weight, and is well tolerated. ExQW has been compared to sitagliptin, pioglitazone and metformin, and shown to have a greater ability to reduce HbA1c than these other medicines. The only preparation of insulin, which ExQW has been compared to, is insulin glargine, and the ExQW has some favourable properties in this comparison, notably causing weight loss, compared to the gain with insulin glargine. ExQW has been compared to another GLP-1 receptor agonist, liraglutide, and ExQW is non-inferior to liraglutide in reducing HbA1c. The small amount of evidence available, shows that subjects with type 2 diabetes, prefer ExQW to ExBID, and that adherence was high to these in the clinical trial setting. Healthcare and economic modelling suggests that ExQW will reduce diabetic complications and be cost-effective, compared to other medications, with long term use. Little is known about whether subjects with type 2 diabetes prefer ExQW to other medicines, and whether adherence is good to ExQW in practice, and these important topics require further study.