Targeting drug-resistant prostate cancer with dual PI3K/mTOR inhibition

The phosphatidylinositol-3-kinase (PI3K)/Akt/mTOR pathway is one of the most frequently activated signaling pathways in prostate cancer cells, and loss of the tumor suppressor PTEN and amplification of PIK3CA are the two most commonly detected mechanisms for the activation of these pathways. Aberrant activation of PI3K/Akt/mTOR has been implicated not only in the survival and metastasis of prostate cancer cells but also in the development of drug resistance. As such, selective inactivation of this pathway may provide opportunities to attack prostate cancer from all fronts. However, while preclinical studies examining specific inhibitors of PI3K or mTOR have yielded promising results, the evidence from clinical trials is less convincing. Emerging evidence from the analyses of some solid tumors suggests that a class of dual PI3K/mTOR inhibitors, which bind to and inactivate both PI3K and mTOR, may achieve better anti-cancer outcomes. In this review, we will summarize the mechanisms of action of these inhibitors, their effectiveness when used alone or in combination with other chemotherapeutic compounds, and their potential to serve as the next generation therapies for prostate cancer patients, particularly those who are resistant to the frontline chemotherapeutic drugs.

Monitoring illicit drug use through wastewater analysis in south east Queensland, Australia

Introduction and Aims: Wastewater analysis has become a useful technique for monitoring illicit drug use in communities. Findings have been reported from different countries in Europe and North America. We applied this technique to gauge the illicit drug consumption in an urban catchment from South East Queensland, Australia. Design and Methods: The sampling campaigns were conducted in 2009 (21st November – 2nd December) and 2010 (19th – 25th November). We collected daily composite wastewater samples from the inlet of the sewage treatment plant using continuous flow-proportional sampling. Ten illicit drug residues (parent compounds and key metabolites) in the samples were measured using liquid chromatography coupled to tandem mass spectrometer. Results: Seven compounds were quantified in all the samples. Our data indicated higher drug consumption on weekends. Cannabis was the highest used drug in both sampling periods. Compared to the first sampling campaign which indicated that cocaine and methamphetamine use exceeded ecstasy usage, the second sampling campaign suggested the use of methamphetamine exceeded that of ecstasy which in turn exceeded cocaine use. Discussion and Conclusions: The observed weekly trend of drug use in our study is in agreement with findings in other studies. The variation between two sampling periods in the prevalence of drug use may relate to the availability and prices of the drugs on markets. The cocaine use we estimated in 2009 was much greater than estimations obtained through the national household survey [1], implying under- reporting of cocaine use in surveys. Future work is underway to tackle methodological challenges for more accurate estimation.

Using quantitative wastewater analysis to measure daily usage of conventional and emerging illicit drugs at an annual music festival

Introduction and Aims Wastewater analysis provides a non-intrusive way of measuring drug use within a population. We used this approach to determine daily use of conventional illicit drugs [cannabis, cocaine, methamphetamine and 3,4-methylenedioxymethamphetamine (MDMA)] and emerging illicit psychostimulants (benzylpiperazine, mephedrone and methylone) in two consecutive years (2010 and 2011) at an annual music festival. Design and Methods Daily composite wastewater samples, representative of the festival, were collected from the on-site wastewater treatment plant and analysed for drug metabolites. Data over 2 years were compared using Wilcoxon matched-pair test. Data from 2010 festival were compared with data collected at the same time from a nearby urban community using equivalent methods. Results Conventional illicit drugs were detected in all samples whereas emerging illicit psychostimulants were found only on specific days. The estimated per capita consumption of MDMA, cocaine and cannabis was similar between the two festival years. Statistically significant (P < 0.05; Z = −2.0–2.2) decreases were observed in use of methamphetamine and one emerging illicit psychostimulant (benzyl piperazine). Only consumption of MDMA was elevated at the festival compared with the nearby urban community. Discussion and Conclusions Rates of substance use at this festival remained relatively consistent over two monitoring years. Compared with the urban community, drug use among festival goers was only elevated for MDMA, confirming its popularity in music settings. Our study demonstrated that wastewater analysis can objectively capture changes in substance use at a music setting without raising major ethical issues. It would potentially allow effective assessments of drug prevention strategies in such settings in the future.

Genetic control of bone density and turnover: Role of the collagen 1α1, estrogen receptor, and vitamin D receptor genes

Genetic factors are known to influence both the peak bone mass and probably the rate of change in bone density. A range of regulatory and structural genes has been proposed to be involved including collagen 1α1 (COL1A1), the estrogen receptor (ER), and the vitamin D receptor (VDR), but the actual genes involved are uncertain. We therefore studied the role of the COL1A1 and VDR loci in control of bone density by linkage in 45 dizygotic twin pairs and 29 nuclear families comprising 120 individuals. The influences on bone density of polymorphisms of COL1A1, VDR, and ER were studied by association both cross-sectionally and longitudinally in 193 elderly postmenopausal women (average age, 69 years) over a mean follow-up time of 6.3 years. Weak linkage of the COL1A1 locus with bone density was observed in both twins and families (p = 0.02 in both data sets), confirming previous observations of linkage of this locus with bone density. Association between the MscI polymorphism of COL1A1 and rate of lumbar spine bone loss was observed with significant gene-environment interaction related to dietary calcium intake (p = 0.0006). In the lowest tertile of dietary calcium intake, carriers of "s" alleles lost more bone than "SS" homozygotes (p = 0.01), whereas the opposite was observed in the highest dietary calcium intake (p = 0.003). Association also was observed between rate of bone loss at both the femoral neck and the lumbar spine and the TaqI VDR polymorphism (p = 0.03). This association was strongest in those in the lowest tertile of calcium intake, also suggesting the presence of gene-environment interaction involving dietary calcium and VDR, influencing bone turnover. No significant association was observed between the PvuII ER polymorphism alone or in combination with VDR or COL1A1 genotypes, with either bone density or its rate of change. These data support the involvement of COL1A1 in determination of bone density and the interaction of both COL1A1 and VDR with calcium intake in regulation of change of bone density over time.

Interactions of natural products with β-lactoglobulins, members of the lipocalin family

Natural products constitute an important source of new drugs. The bioavailability of the drugs depends on their absorption, distribution, metabolism and elimination. To achieve good bioavailability, the drug must be soluble in water, stable in the gastrointestinal tract and palatable. Binding proteins may improve the solubility of drug compounds, masking unwanted properties, such as bad taste, bitterness or toxicity, transporting or protecting these compounds during processing and storage. The focus of this thesis was to study the interactions, including ligand binding and the effect of pH and temperature, of bovine and reindeer β-lactoglobulin (βLG) with such compounds as retinoids, phenolic compounds as well as with compounds from plant extracts, and to investigate the transport properties of the βLG-ligand complex. To examine the binding interactions of different ligands to βLG, new methods were developed. The fluorescence binding method for the evaluation of ligand binding to βLG was miniaturized from a quartz cell to a 96-well plate. A method of ultrafiltration sampling combined with high-performance liquid chromatography was developed to assess the binding of compounds from extracts. The interactions of phenolic compounds or retinoids and βLG were investigated using the 96-well plate method. The majority of flavones, flavonols, flavanones and isoflavones and all of the retinoids included were shown to bind to bovine and reindeer βLG. Phenolic compounds, contrary to retinol, were not released at acidic pH. Those results suggest that βLG may have more binding sites, probably also on the surface of βLG. An extract from Camellia sinensis (L.) O. Kunze (black tea), Urtica dioica L. (nettle) and Piper nigrum (black pepper) were used to evaluate whether βLG could bind compounds from plant extracts. Piperine from P. nigrum was found to bind tightly and rutin from U. dioica weakly to βLG. No components from C. sinensis bound to βLG in our experiment. The uptake and membrane permeation of bovine and reindeer βLG, free and bound with retinol, palmitic acid and cholesterol, were investigated using Caco-2 cell monolayers. Both bovine and reindeer βLG were able to cross the Caco-2 cell membrane. Free and βLG-bound retinol and palmitic acid were transported equally, whereas cholesterol could not cross the Caco-2 cell monolayer free or bound to βLG. Our results showed that βLG can bind different natural product compounds, but cannot enhance transport of retinol, palmitic acid or cholesterol through Caco-2 cells. Despite this, βLG, as a water-soluble binding protein, may improve the solubility of natural compounds, possibly protecting them from early degradation and transporting some of them through the stomach. Furthermore, it may decrease their bad or bitter taste during oral administration of drugs or in food preparations. βLG can also enhance or decrease the health benefits of herbal teas and food preparations by binding compounds from extracts.

Characterisation and processing of amorphous binary mixtures with low glass transition temperature

The number of drug substances in formulation development in the pharmaceutical industry is increasing. Some of these are amorphous drugs and have glass transition below ambient temperature, and thus they are usually difficult to formulate and handle. One reason for this is the reduced viscosity, related to the stickiness of the drug, that makes them complicated to handle in unit operations. Thus, the aim in this thesis was to develop a new processing method for a sticky amorphous model material. Furthermore, model materials were characterised before and after formulation, using several characterisation methods, to understand more precisely the prerequisites for physical stability of amorphous state against crystallisation. The model materials used were monoclinic paracetamol and citric acid anhydrate. Amorphous materials were prepared by melt quenching or by ethanol evaporation methods. The melt blends were found to have slightly higher viscosity than the ethanol evaporated materials. However, melt produced materials crystallised more easily upon consecutive shearing than ethanol evaporated materials. The only material that did not crystallise during shearing was a 50/50 (w/w, %) blend regardless of the preparation method and it was physically stable at least two years in dry conditions. Shearing at varying temperatures was established to measure the physical stability of amorphous materials in processing and storage conditions. The actual physical stability of the blends was better than the pure amorphous materials at ambient temperature. Molecular mobility was not related to the physical stability of the amorphous blends, observed as crystallisation. Molecular mobility of the 50/50 blend derived from a spectral linewidth as a function of temperature using solid state NMR correlated better with the molecular mobility derived from a rheometer than that of differential scanning calorimetry data. Based on the results obtained, the effect of molecular interactions, thermodynamic driving force and miscibility of the blends are discussed as the key factors to stabilise the blends. The stickiness was found to be affected glass transition and viscosity. Ultrasound extrusion and cutting were successfully tested to increase the processability of sticky material. Furthermore, it was found to be possible to process the physically stable 50/50 blend in a supercooled liquid state instead of a glassy state. The method was not found to accelerate the crystallisation. This may open up new possibilities to process amorphous materials that are otherwise impossible to manufacture into solid dosage forms.

The use of a synthetic prostaglandin-E1 analog (misoprostol) as an adjunct to pancreatic-enzyme replacement in cystic-fibrosis

Eleven cystic fibrosis children (mean age, 9.6 years) were chosen at random to participate in a study to observe the effects of concurrently stimulating gastric/duodenal bicarbonate secretion and inhibiting gastric acid secretion, using a methylated prostaglandin E1 analogue in patients with pancreatic insufficiency and taking pancreatic enzymes. Percentage fat absorption in 3-day stool collections were calculated before and after commencing therapy with misoprostol, 400 μg/day in divided doses. We found a significant reduction in fat output (14.7 ± 11.7 versus 7.5 ± 3.5 g/day, p < 0.05) in the study group as a whole and a significant reduction in steatorrhoeic level as a percentage of fat intake in all of the patients with abnormal base-line collections (23.1% versus 9.2%, p < 0.002). We conclude that misoprostol should be considered in cystic fibrosis patients with steatorrhoea as a means of improving nutrient absorption.

The social supply of cannabis among young people in Australia

Cannabis is the most prolifically used illicit drug in Australia, however, there is a gap in our understanding concerning the social interactions and friendships formed around its supply and use. The authors recruited cannabis users aged between 18 and 30 years throughout Australia, to explore the impact of supply routes on young users and their perceived notions of drug dealing in order to provide valuable insight into the influence that reciprocal relationships have on young people’s access to cannabis. Findings reveal that the supply of cannabis revolves around pre-existing connections and relationships formed through associates known to be able to readily source cannabis. It was found that motivations for proffering cannabis in a shared environment were related more to developing social capital than to generating financial gain. Given this, often those involved in supply do not perceive that they are breaking the law or that they are ‘dealers’. This social supply market appears to be built on trust and social interactions and, as such, presents several challenges to law enforcement. It is suggested that there would be benefit in providing targeted education campaigns to combat social supply dealing among young adults.

Social supply of cannabis in Australia

While cannabis is the most prolifically used illicit drug in Australia, there is a gap in our understanding concerning the social interactions and friendships formed around its supply and use. The authors recruited cannabis users aged between 18 and 30 years throughout Australia, to explore the impact of supply routes on young users and their perceived notions of drug dealing in order to provide valuable insight into the influence that reciprocal relationships have on young people’s access to cannabis. Findings reveal that the supply of cannabis revolves around pre-existing connections and relationships formed through associates known to be able to readily source cannabis. It was found that motivations for proffering cannabis in a shared environment were related more to developing social capital than to generating financial gain. Given this, often those involved in supply do not perceive that they are breaking the law or that they are ‘dealers’. This social supply market appears to be built on trust and social interactions and, as such, presents several challenges to law enforcement. It is suggested that there would be benefit in providing targeted education campaigns to combat social supply dealing among young adults.

Expression of cytochrome P450 enzymes and metabolism of timolol in human ocular tissue

Glaucoma is a group of progressive optic neuropathies causing irreversible blindness if not diagnosed and treated in the early state of progression. Disease is often, but not always, associated with increased intraocular pressure (IOP), which is also the most important risk factor for glaucoma. Ophthlamic timolol preparations have been used for decades to lower increased intraocular pressure (IOP). Timolol is locally well tolerated but may cause e.g. cardiovascular and pulmonary adverse effects due to systemic absorption. It has been reported that approximately 80% of a topically administered eye drop is systemically absorbed. However, only limited information is available on timolol metabolism in the liver or especially in the human eye. The aim of this work was to investigate metabolism of timolol in human liver and human ocular tissues. The expression of drug metabolizing cytochrome P450 (CYP) enzymes in the human ciliary epithelial cells was studied. The metabolism of timolol and the interaction potential of timolol with other commercially available medicines were investigated in vitro using different liver preparations. The absorption of timolol to the aqueous humor from two commercially available products: 0.1% eye gel and 0.5% eye drops and the presence of timolol metabolites in the aqueous humor were investigated in a clinical trial. Timolol was confirmed to be metabolized mainly by CYP2D6 as previously suggested. Potent CYP2D6 inhibitors especially fluoxetine, paroxetine and quinidine inhibited the metabolism of timolol. The inhibition may be of clinical significance in patients using ophthalmic timolol products. CYP1A1 and CYP1B1 mRNAs were expressed in the human ciliary epithelial cells. CYP1B1 was also expressed at protein level and the expression was strongly induced by a known potent CYP1B1 inducer 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). The CYP1B1 induction is suggested to be mediated by aryl hydrocarbon receptor (AHR). Low levels of CYP2D6 mRNA splice variants were expressed in the human ciliary epithelial cells and very low levels of timolol metabolites were detected in the human aqueous humor. It seems that negligible amount of CYP2D6 protein is expressed in the human ocular tissues. Timolol 0.1% eye gel leads to aqueous humor concentration high enough to achieve therapeutic effect. Inter-individual variation in concentrations is low and intraocular as well as systemic safety can be increased when using this product with lower timolol concentration instead of timolol 0.5% eye drops.

Effects of oral calcium sensitizers on experimental heart failure

Suun kautta annosteltava kalsiumherkistäjä parantaa sydämen vajaatoimintaan liittyvää pumppausvajetta kokeellisissa sydämen vajaatoimintamalleissa Huolimatta viime vuosikymmenien lääketieteellisestä kehityksestä krooninen sydämen vajaatoiminta on silti edelleen vakava, elämänlaatua voimakkaasti rajoittava sairaus. Kalsiumherkistäjät ovat uusi, sydämen pumppausvoimaa lisäävä lääkeryhmä. Levosimendaani, kotimaista alkuperää oleva kalsiumherkistäjä, on kliinisessä käytössä akuutin vajaatoiminnan hoitoon suonensisäisesti ja lyhytaikaisesti annosteltavana valmisteena. Levosimendaanilla on aktiivinen metaboliitti, OR-1896, jonka oletetaan olevan vuorokauden mittaisen levosimendaani-infuusion jälkeen havaittujen useita päiviä kestävien hyödyllisisten vaikutuksisten takana. Levosimendaanin kroonisen, suun kautta tapahtuvan annostelun vaikutuksista tieto on vähäisempää, mutta sillä näyttää olevan positiivisia vaikutuksia potilaiden raportoimana. FM Marjut Louhelainen on selvittänyt väitöskirjassaan suun kautta annosteltavan levosimendaanin ja sen pitkäkestoisen aktiivisen metaboliitin vaikutuksia kroonisen vajaatoiminnan hoidossa käyttämällä sekä hypertensiivisen sydäntaudin että 2 tyypin diabeteksen komplisoimaan sydäninfarktin kokeellisia malleja. Tutkimuksessa selvitettiin lisäksi vajaatoimintaan johtavia molekyylitason tapahtumia sydänlihaksessa. Tutkimuksessa osoitettiin, että krooninen suun kautta annosteltu hoito sekä kalsiumherkistäjä levosimendaanilla että sen aktiivisella metaboliitilla estää hypertensiiviseen sydämen vajaatoiminnan aikaasaamaa sydämen uudelleenmuovaantumista ja siihen liittyvää kuolleisuutta. Nämä vaikutukset välittyivät vähentyneen sydänlihassoluhypertrofian, solukuolleisuuden ja neurohumaraalisen aktivaation kautta. Levosimendaanin ja OR-1896:n osoitettiin myös parantavan sydämen pumppausfunktiota tyyppi 2 diabeteksen komplisoimassa sydäninfarktissa. Ei-diabeettiseen tilanteeseen verrattuna diabetekseen liittyvä infarktin jälkeinen vajaatoiminnan kehitys oli yhteydessä lisääntyneeseen tulehdukseen, fibroosiin, solukuolemaan, neurohumoraaliseen aktivaatioon ja ennenaikaiseen kudoksen vanhenemiseen. Sekä levosimendaani, että OR-1869 vähensivät tulehduksen, fibroosin ja solukuoleman merkkejä ja vaimensi neurohumoraalista aktivaatiota. OR-1896 myös vähensi solujen vanhenemiseen liittyvien merkkiaineiden ilmentymistä. Väitöskirjassa todettiin, että suun kautta annosteltuna sekä levosimendaani, että sen aktiivinen metaboliitti OR-1896, omaavat terapeuttista potentiaalia sekä hypertensiivisen sydäntaudin hoitoon että sydäninfarktin jälkeisen vajaatoiminnan estoon. FM Marjut Louhelaisen farmakologian alaan kuuluva väitöskirja Effects of oral calcium sensitizers on experimental heart failure tarkastetaan Helsingin yliopiston Lääketieteellisessä tiedekunnassa perjantaina 29.01.2010 klo 12 (Biomedicum Helsinki, luentosali 2, Haartmaninkatu 8, Helsinki). Vastaväittäjänä toimii professori Raimo Tuominen, Helsingin yliopiston Farmasian tiedekunnasta ja kustoksena professori Eero Mervaala Helsingin yliopiston Lääketieteellisestä tiedekunnasta.

Development of a cognitive framework for pharmaceutical care

Pharmaceutical Care is defined as “the responsible provision of drug therapy for the purpose of achieving definite outcomes that improve a patient’s quality of life”. One of the fundamental concepts in understanding needs for pharmaceutical care are Drug-Related Problems (DRPs). As the complexity of medication treatment increases, identification of drug-related problems (DRPs) by healthcare professionals remains vital to patient safety and Quality Use of Medicines(QUM). DRPs have been used by many researchers to evaluate the QUM in different settings. DRPs present, however, a list of potential problems not a strategic framework for assessing a medication regimen.

Dietary elecrolytes and cyclosporine toxicity. Experimental studies in spontaneuosly hypertensive rats

Cyclosporine-A (CsA) is widely used after organ transplantation to prevent rejection and in the treatment of autoimmune diseases. Hypertension and nephrotoxicity are common side-effects of CsA. Studies in patients on the prevention of the side-effects of CsA are difficult to conduct because the patients often receive a combination of different drugs thus making study of the side-effects of a single drug impossible. A challenge in experimental studies has been the lack of an animal model in which the side-effects concomitantly occur. Epidemiological data show an association between sodium (Na) intake and blood pressure. There is also evidence on low dietary intake of magnesium (Mg) and potassium (K) and high blood pressure. Our study was designed to develop an experimental model to study the side-effects of CsA in spontaneously hypertensive rats (SHR). On high dietary sodium, CsA caused hypertension, left ventricular hypertrophy (LVH), narrowing of the coronary arteries, small myocardial infarctions, and proteinuria, reduced creatinine clearance and histopathological renal injury in SHR. Loss of Mg into the urine caused by CsA resulted in Mg depletion in the tissues. Renal excretion of dopamine was reduced and the renin-angiotensin-aldosterone system was activated. We investigated the effects of dietary Mg and/or K and the calcium antagonist drug, isradipine, on the prevention of CsA toxicity. Dietary supplementation of Mg alone or in combination with K prevented from the deleterious pathophysiological and histopathological changes in the kidneys and the heart. K alone had little effect. Isradipine protected better than Mg from LVH, but the combination of isradipine and Mg was the most effective. Isradipine did not, however, protect against Mg loss. In our animal model, the combination of high dietary Na and treatment with CsA accelerated the development of the cardiovascular and renal changes clinically known as the side-effects of CsA. Dietary supplementation of Mg and K and reduction of Na intake and the calcium antagonist drug isradipine prevent from the deleterious effects of CsA.

Age-related changes in hepatic function: An update on implications for drug therapy

The accumulation of deficits with increasing age results in a decline in the functional capacity of multiple organs and systems. These changes can have a significant influence on the pharmacokinetics and pharmacodynamics of prescribed drugs. Although alterations in body composition and worsening renal clearance are important considerations, for most drugs the liver has the greatest effect on metabolism. Age-related change in hepatic function thereby causes much of the variability in older people’s responses to medication. In this review, we propose that a decline in the ability of the liver to inactivate toxins may contribute to a proinflammatory state in which frailty can develop. Since inflammation also downregulates drug metabolism, medication prescribed to frail older people in accordance with disease-specific guidelines may undergo reduced systemic clearance, leading to adverse drug reactions, further functional decline and increasing polypharmacy, exacerbating rather than ameliorating frailty status. We also describe how increasing chronological age and frailty status impact liver size, blood flow and protein binding and enzymes of drug metabolism. This is used to contextualise our discussion of appropriate prescribing practices. For example, while the general axiom of ‘start low, go slow’ should underpin the initiation of medication (titrating to a defined therapeutic goal), it is important to consider whether drug clearance is flow or capacity-limited. By summarising the effect of age-related changes in hepatic function on medications commonly used in older people, we aim to provide a guide that will have high clinical utility for practising geriatricians.

Biochemical effects of 3,5-diethoxycarbonyl-1, 4-dihydrocollidine in mouse liver

3,5-Diethoxycarbonyl-1,4-dihydrocollidine (DDC) is a porphyrinogenic agent and is a powerful inducer of δ-aminolaevulinate synthetase, the first and rate-limiting enzyme of the haem-biosynthetic pathway, in mouse liver. However, DDC strikingly inhibits mitochondrial as well as microsomal haem synthesis by depressing the activity of ferrochelatase in vivo. The drug on repeated administration to female mice has been found to elicit hypertrophic effects in the liver microsomes initially, but the effects observed at later stages denote either hyperplasia or increase in polyploidal cells. The microsomal protein concentration shows a striking decrease with repeated doses of the drug. The rate of microsomal protein synthesis in vivo as well as in vitro shows an increase with two injections of DDC but decreases considerably with repeated administration of the drug. The activities of NADPH-cytochrome creductase and ribonuclease are not affected in the liver microsomes of drug-treated animals when expressed per mg of microsomal protein. DDC has also been found to cause degradation of microsomal haem, which is primarily responsible for the decrease in cytochrome P-450 content. The drug also leads to a decrease in mitochondrial cytochrome c levels due to inhibition of haem synthesis and also due to degradation of mitochondrial haem at later stages. The biochemical effects of the drug are compared and discussed with those reported for allylisopropylacetamide and phenobarbital.