The endocannabinoid system - current implications for drug development.

In this review, the state of the art for compounds affecting the endocannabinoid (eCB) system is described with a focus on the treatment of pain. Amongst directly acting CB receptor ligands, clinical experience with ∆9 -tetrahydracannabinol and medical cannabis in chronic non-cancer pain indicates that there are differences between the benefits perceived by patients and the at best modest effect seen in meta-analyses of randomized controlled trials. The reason for this difference is not known but may involve differences in the type of patients that are recruited, the study conditions that are chosen and the degree to which biases such as reporting bias are operative. Other directly acting CB receptor ligands such as biased agonists and allosteric receptor modulators have not yet reached the clinic. Amongst indirectly acting compounds targeting the enzymes responsible for the synthesis and catabolism of the eCBs anandamide and 2-arachidonoylglycerol, fatty acid amide hydrolase (FAAH) inhibitors have been investigated clinically but were per se not useful for the treatment of pain, although they may be useful for the treatment of post-traumatic stress disorder and cannabis use disorder. Dual-acting compounds targeting this enzyme and other targets such as cyclooxygenase-2 or transient potential vanilloid receptor 1 may be a way forward for the treatment of pain.

Sulfotransferase 1A3/4 copy number variation is associated with neurodegenerative disease.

The cytosolic aryl sulfotransferase genes SULT1A3 and SULT1A4 are located on chromosome 16p11.2 in a region of chromosomal instability. SULT1A3/4 are important enzymes in the metabolism of catecholamines linked to neurodegenerative diseases such as Parkinson's and Alzheimer's. In the present study, copy number variation of the SULT1A3/4 genes in healthy individuals, as well as a cohort of Parkinson's disease and Alzheimer's disease patients was examined. In all subjects, SULT1A3/4 copy number varied from 1 to 10. In Alzheimer's disease patients, there was a significantly lower copy number compared to controls, and a positive correlation between copy number and age of disease onset. By contrast, there were no differences in Parkinson's disease patients. However, when early-onset Parkinson's disease was evaluated separately, there appeared to be an association with gene copy number and risk. The current study shows that these neurodegenerative diseases may be related to SULT1A3/4 copy number.

Reduced TRPC6 mRNA levels in the blood cells of patients with Alzheimer's disease and mild cognitive impairment.

Transient receptor potential canonical 6 (TRPC6) inhibits ß-amyloid (Aß) production. Hyperforin, the TRPC6 agonist, reduces Aß levels and improves cognitive performance in Alzheimer's disease (AD) models. However, it's unknown whether TRPC6 expression is changed in AD patients. In this case-control study, we measured TRPC6 expression levels in the peripheral blood cells of four independent AD sets from five hospitals and one mild cognitive impairment (MCI) set from a local community (229 AD, 70 MCI, 40 Parkinson disease and 359 controls from China, total n=698) using quantitative real-time PCR assay. We found a specific reduction of TRPC6 mRNA levels in four AD sets and one MCI set. The median TRPC6 mRNA levels were lower in the following: (1) combined AD patients than in age-matched controls (0.78 vs 1.73, P<0.001); (2) mild-to-moderate AD patients than in age-matched controls (0.81 vs 1.73, P<0.001); and (3) MCI patients than in age-matched controls (0.76 vs 1.72, P<0.001). In the receiver-operating characteristic curve analysis, the area under curve was 0.85 for combined AD, 0.84 for mild-to-moderate AD and 0.79 for MCI. In a subgroup of AD patients with brain Aß examination, TRPC6 was associated with standardized uptake value ratio of Pittsburgh Compound B (Spearman's r=-0.49, P=0.04) and cerebrospinal fluid Aß42 (Spearman's r=0.43, P=0.04). The TRPC6 reduction in AD patients was further confirmed in blood RNA samples from The Australian Imaging, Biomarkers and Lifestyle Flagship Study of Aging, in post-mortem brain tissues from The Netherlands Brain Bank and in induced pluripotent stem cells-derived neurons from Chinese donors. We conclude that TRPC6 mRNA levels in the blood cells are specifically reduced in AD and MCI patients, and TRPC6 might be a biomarker for the early diagnosis of AD.

Delta(9) -tetrahydrocannabinol and cannabidiol as potential curative agents for cancer: A critical examination of the preclinical literature.

An Internet search with search words "cannabis cures cancer" produce a wealth of sites claiming that cannabis has this effect. These sites are freely accessible to the general public and thus contribute to public opinion. But do delta(9) -tetrahydrocannabinol (Δ(9) -THC) and cannabidiol (CBD) cure cancer? In the absence of clinical data other than a safety study and case reports, preclinical data should be evaluated in terms of its predictive value. Using a strict approach where only concentrations and/or models relevant to the clinical situation are considered, the current preclinical data do not yet provide robust evidence that systemically administered Δ(9) -THC will be useful for the curative treatment of cancer. There is more support for an intratumoral route of administration of higher doses of Δ(9) -THC. CBD produces effects in relevant concentrations and models, although more data are needed concerning its use in conjunction with other treatment strategies.

Prognostic serum miRNA biomarkers associated with Alzheimer's disease shows concordance with neuropsychological and neuroimaging assessment.

There is no consensus for a blood-based test for the early diagnosis of Alzheimer's disease (AD). Expression profiling of small non-coding RNA's, microRNA (miRNA), has revealed diagnostic potential in human diseases. Circulating miRNA are found in small vesicles known as exosomes within biological fluids such as human serum. The aim of this work was to determine a set of differential exosomal miRNA biomarkers between healthy and AD patients, which may aid in diagnosis. Using next-generation deep sequencing, we profiled exosomal miRNA from serum (N=49) collected from the Australian Imaging, Biomarkers and Lifestyle Flagship Study (AIBL). Sequencing results were validated using quantitative reverse transcription PCR (qRT-PCR; N=60), with predictions performed using the Random Forest method. Additional risk factors collected during the 4.5-year AIBL Study including clinical, medical and cognitive assessments, and amyloid neuroimaging with positron emission tomography were assessed. An AD-specific 16-miRNA signature was selected and adding established risk factors including age, sex and apolipoprotein ɛ4 (APOE ɛ4) allele status to the panel of deregulated miRNA resulted in a sensitivity and specificity of 87% and 77%, respectively, for predicting AD. Furthermore, amyloid neuroimaging information for those healthy control subjects incorrectly classified with AD-suggested progression in these participants towards AD. These data suggest that an exosomal miRNA signature may have potential to be developed as a suitable peripheral screening tool for AD.

An anemia of Alzheimer's disease.

Lower hemoglobin is associated with cognitive impairment and Alzheimer's disease (AD). Since brain iron homeostasis is perturbed in AD, we investigated whether this is peripherally reflected in the hematological and related blood chemistry values from the Australian Imaging Biomarker and Lifestyle (AIBL) study (a community-based, cross-sectional cohort comprising 768 healthy controls (HC), 133 participants with mild cognitive impairment (MCI) and 211 participants with AD). We found that individuals with AD had significantly lower hemoglobin, mean cell hemoglobin concentrations, packed cell volume and higher erythrocyte sedimentation rates (adjusted for age, gender, APOE-ɛ4 and site). In AD, plasma iron, transferrin, transferrin saturation and red cell folate levels exhibited a significant distortion of their customary relationship to hemoglobin levels. There was a strong association between anemia and AD (adjusted odds ratio (OR)=2.43, confidence interval (CI) (1.31, 4.54)). Moreover, AD emerged as a strong risk factor for anemia on step-down regression, even when controlling for all other available explanations for anemia (adjusted OR=3.41, 95% CI (1.68, 6.92)). These data indicated that AD is complicated by anemia, which may itself contribute to cognitive decline.

Changes in cannabinoid CB(1) receptor functionality in the female rat prefrontal cortex following a high fat diet.

AIMS: A high fat diet (HFD) has been found to affect neurotransmission in the prefrontal cortex, but the effects of this dietary regime upon the endocannabinoid system has not been studied in this brain region. In consequence, in the present study, we have investigated the effect of HFD for up to 20 weeks upon the endocannabinoid system in the prefrontal cortex of female rats. MAIN METHODS: CB1 receptor functionality was measured using CP55,940-stimulated [(35)S]GTPγS autoradiography. Fatty acid amide hydrolase and monoacylglycerol lipase activities were analysed in brain regions by assessing rates of [(3)H]anandamide and JZL184-sensitive [(3)H]2-oleoylglycerol hydrolysis, respectively. KEY FINDINGS: In the prefrontal cortex, a significantly greater stimulation of [(35)S]GTPγS binding by CP55,940 was seen following 4-12, but not 16-20 weeks of HFD. No significant changes were seen for the caudate-putamen, CA1-CA3 region of the hippocampus or the dentate gyrus. The increased response for the 12 week animals was not accompanied by a significant change in the receptor density, measured with [(3)H]CP55,940 autoradiography. No significant changes in the activity of the endocannabinoid hydrolytic enzymes fatty acid amide or monoacylglycerol lipase were seen in the prefrontal cortex, hippocampus, amygdala or hypothalamus following either 12 or 20 weeks of HFD. SIGNIFICANCE: It is concluded that HFD produces an increased CB1 receptor functionality in the prefrontal cortex of female rats. Given that the endocannabinoid system regulates neurotransmission in the prefrontal cortex, the present data would implicate this system in the disturbed prefrontal cortical activity in this region following a high fat diet.

The micturition switch and its forebrain influences.

Dr DeGroat and Wickens has reviewed the central neural mechanisms controlling the lower urinary tract with a major focus on the brain stem circuitry that mediates the switch-like characteristics of micturition, in particular the periaqueductal grey and the pontine micturition centre (de 2012). The review culminates in a computer model of how the brainstem switch operates in animals in which forebrain influences on micturition have been removed by decerebration. In this complementary paper, we review the mechanisms of forebrain involvement in the voluntary control of human micturition and the maintenance of continence with evidence based heavily on the results of functional brain imaging experiments.

Botulinum toxin-A for the treatment of overactive bladder: UK contributions.

BACKGROUND: Botulinum toxin-A (BoNT/A) is now established second-line management for refractory overactive bladder (OAB) and recognised in many incontinence guidelines and pathways. For those with neurogenic detrusor overactivity secondary to spinal cord injury or multiple sclerosis, the toxin is currently licensed in certain parts of the world, including the UK. It is an effective treatment in those in whom antimuscarinics and conservative measures have failed who have symptoms of OAB and or detrusor overactivity (DO). METHODS: Treatment can be given in an outpatient setting and can be administered under local anaesthesia. Its efficacy lasts for between six and 12 months. RESULTS: It has an acceptable safety profile with the biggest risk being urinary tract infection and difficulty emptying the bladder, necessitating clean intermittent self-catheterisation (CISC). Medium-term follow-up suggests repeated injections are also safe and efficacious. CONCLUSIONS: The mechanism of action of the toxin is more complicated than originally thought, and it seems likely that it affects motor and sensory nerves of the bladder. In the last 10 years much of the progress of this treatment from early experimental trials to mainstream clinical use, and a better understanding of how it works in the bladder, are as a result of research conducted in the UK. This review summarises the significant and substantial evidence for BoNT/A to treat refractory OAB from UK centres.

Monoacylglycerol lipase - a target for drug development?

The endocannabinoid (eCB) system is involved in processes as diverse as control of appetite, perception of pain and the limitation of cancer cell growth and invasion. The enzymes responsible for eCB breakdown are attractive pharmacological targets, and fatty acid amide hydrolase inhibitors, which potentiate the levels of the eCB anandamide, are now undergoing pharmaceutical development. 'Drugable' selective inhibitors of monoacylglycerol lipase, a key enzyme regulating the levels of the other main eCB, 2-arachidonoylglycerol, were however not identified until very recently. Their availability has resulted in a large expansion of our knowledge concerning the pharmacological consequences of monoacylglycerol lipase inhibition and hence the role(s) played by the enzyme in the body. In this review, the pharmacology of monoacylglycerol lipase will be discussed, together with an analysis of the therapeutic potential of monoacylglycerol lipase inhibitors as analgesics and anticancer agents.

Randomized controlled trial of Sativex to treat detrusor overactivity in multiple sclerosis.

BACKGROUND: Bladder dysfunction is a common feature of multiple sclerosis (MS). OBJECTIVE: In this study we aimed to assess the efficacy, tolerability and safety of Sativex(®) (nabiximols) as an add-on therapy in alleviating bladder symptoms in patients with MS. METHODS: We undertook a 10-week, double-blind, randomized, placebo-controlled, parallel-group trial in 135 randomized subjects with MS and overactive bladder (OAB). RESULTS: The primary endpoint was the reduction in daily number of urinary incontinence episodes from baseline to end of treatment (8 weeks). Other endpoints included incidence of nocturia and urgency, overall bladder condition (OBC), daytime frequency, Incontinence Quality of Life (I-QOL), Patient's Global Impression of Change (PGIC) and volume voided. The primary endpoint showed little difference between Sativex and placebo. Four out of seven secondary endpoints were significantly in favour of Sativex: number of episodes of nocturia (adjusted mean difference -0.28, p = 0.010), OBC (-1.16, p = 0.001), number of voids/day (-0.85, p = 0.001) and PGIC (p = 0.005). Of the other endpoints, number of daytime voids was statistically significantly in favour of Sativex (-0.57, p = 0.044). The improvement in I-QOL was in favour of Sativex but did not reach statistical significance. CONCLUSIONS: Although the primary endpoint did not reach statistical significance, we conclude that Sativex did have some impact on the symptoms of overactive bladder in patients with MS, providing evidence of some improvement in symptoms associated with bladder dysfunction in these subjects.

[The primary disorder of sphincter relaxation or Fowler's syndrome]. / Le désordre primaire de la relaxation sphinctérienne ou syndrome de Fowler.

The primary disorder of sphincter relaxation or Fowler's syndrome constitutes the first cause of urinary retention in young women after ruling neurological, iatrogenic and local origins out. It includes painless urinary retention, polycystic ovaries in more than 50% of the cases, high maximum urethral closure pressure, increase in sphincter volume and striated urethral sphincter electromyography abnormalities. So far, the only treatment restoring micturation in this condition is sacral neuromodulation.

Inhibition of monoacylglycerol lipase by troglitazone, N-arachidonoyl dopamine and the irreversible inhibitor JZL184: comparison of two different assays.

BACKGROUND AND PURPOSE: Drugs used clinically usually have a primary mechanism of action, but additional effects on other biological targets can contribute to their effects. A potentially useful additional target is the endocannabinoid metabolizing enzyme monoacylglycerol lipase (MGL). We have screened a range of drugs for inhibition of MGL and compared the observed potencies using different MGL enzyme assays. EXPERIMENTAL APPROACH: MGL activity was screened using recombinant human MGL (cell lysates and purified enzyme) with 4-nitrophenyl acetate (NPA) as substrate. 2-Oleolyglycerol metabolism by rat cerebellar cytosolic MGL and by recombinant MGL was also investigated. KEY RESULTS: Among the 96 compounds screened in the NPA assay, troglitazone, CP55,940, N-arachidonoyl dopamine and AM404 inhibited NPA hydrolysis by the lysates with IC(50) values of 1.1, 4.9, 0.78 and 3.1µM, respectively. The potency for troglitazone is in the same range as its primary pharmacological activity, activation of peroxisome proliferator-activated receptor (PPAR) γ. Among PPARγ ligands, the potency order towards human MGL was troglitazone > ciglitazone > rosiglitazone > 15-deoxy-Δ(12,14) -prostaglandin J(2) ≈ CAY 10415 > CAY 10514. In contrast to the time-dependent inhibitor JZL184, the potency of troglitazone was dependent upon the enzyme assay system used. Thus, troglitazone inhibited rat cytosolic 2-oleoylglycerol hydrolysis less potently (IC(50) 41µM) than hydrolysis of NPA by the human MGL lysates. CONCLUSIONS AND IMPLICATIONS: 'Hits' in screening programmes for MGL inhibitors should be assessed in different MGL assays. Troglitazone may be a useful lead for the design of novel, dual action MGL inhibitors/PPARγ activators.

Biochanin A, a naturally occurring inhibitor of fatty acid amide hydrolase.

BACKGROUND AND PURPOSE: Inhibitors of fatty acid amide hydrolase (FAAH), the enzyme responsible for the metabolism of the endogenous cannabinoid (CB) receptor ligand anandamide (AEA), are effective in a number of animal models of pain. Here, we investigated a series of isoflavones with respect to their abilities to inhibit FAAH. EXPERIMENTAL APPROACH: In vitro assays of FAAH activity and affinity for CB receptors were used to characterize key compounds. In vivo assays used were biochemical responses to formalin in anaesthetized mice and the 'tetrad' test for central CB receptor activation. KEY RESULTS: Of the compounds tested, biochanin A was adjudged to be the most promising. Biochanin A inhibited the hydrolysis of 0.5 microM AEA by mouse, rat and human FAAH with IC(50) values of 1.8, 1.4 and 2.4 microM respectively. The compound did not interact to any major extent with CB(1) or CB(2) receptors, nor with FAAH-2. In anaesthetized mice, URB597 (30 microg i.pl.) and biochanin A (100 microg i.pl.) both inhibited the spinal phosphorylation of extracellular signal-regulated kinase produced by the intraplantar injection of formalin. The effects of both compounds were significantly reduced by the CB(1) receptor antagonist/inverse agonist AM251 (30 microg i.pl.). Biochanin A (15 mg.kg(-1) i.v.) did not increase brain AEA concentrations, but produced a modest potentiation of the effects of 10 mg.kg(-1) i.v. AEA in the tetrad test. CONCLUSIONS AND IMPLICATIONS: It is concluded that biochanin A, in addition to its other biochemical properties, inhibits FAAH both in vitro and peripherally in vivo.

Neural control of the lower urinary and gastrointestinal tracts: supraspinal CNS mechanisms.

Normal urinary function is contingent upon a complex hierarchy of CNS regulation. Lower urinary tract afferents synapse in the dorsal horn of the spinal cord and ascend to the midbrain periaqueductal gray (PAG), with a separate nociception path to the thalamus. A spino-thalamo-cortical sensory pathway is present in some primates, including humans. In the brainstem, the pontine micturition center (PMC) is a convergence point of multiple influences, representing a co-ordinating center for voiding. Many PMC neurones have characteristics necessary to categorize the center as a pre-motor micturition nucleus. In the lateral pontine brainstem, a separate region has some characteristics to suggest a "continence center." Cerebral control determines that voiding is permitted if necessary, socially acceptable and in a safe setting. The frontal cortex is crucial for decision making in an emotional and social context. The anterior cingulate gyrus and insula co-ordinate processes of autonomic arousal and visceral sensation. The influence of these centers on the PMC is primarily mediated via the PAG, which also integrates bladder sensory information, thereby moderating voiding and storage of urine, and the transition between the two phases. The parabrachial nucleus in the pons is also important in behavioral motivation of waste evacuation. Lower urinary tract afferents can be modulated at multiple levels by corticolimbic centers, determining the interoception of physiological condition and the consequent emotional motor responses. Alterations in cognitive modulation, descending modulation, and hypervigilance are important in functional (symptom-based) clinical disorders.

A UK consensus on the management of the bladder in multiple sclerosis.

Bladder symptoms in multiple sclerosis (MS) are common and distressing but also highly amenable to treatment. A meeting of stakeholders involved in patients' continence care, including neurologists, urologists, primary care, MS nurses and nursing groups was recently convened to formulate a UK consensus for management. National Institute for Health and Clinical Excellence (NICE) criteria were used for producing recommendations based on a review of the literature and expert opinion. It was agreed that in the majority of cases, successful management could be based on a simple algorithm which includes using reagent sticks to test for urine infection and measurement of the post micturition residual urine volume. This is in contrast with published guidelines from other countries which recommend cystometry. Throughout the course of their disease, patients should be offered appropriate management options for treatment of incontinence, the mainstay of which is antimuscarinic medications, in combination, if necessary, with clean intermittent self-catheterisation. The evidence for other measures, including physiotherapy, alternative strategies aimed at improving bladder emptying, other medications and detrusor injections of botulinum toxin A was reviewed. The management of urinary tract infections as well as the bladder problems as part of severe disability were discussed and recommendations agreed.

A UK consensus on the management of the bladder in multiple sclerosis.

Bladder symptoms in multiple sclerosis (MS) are common and distressing but also highly amenable to treatment. A meeting of stakeholders involved in patients' continence care, including neurologists, urologists, primary care, MS nurses and nursing groups was recently convened to formulate a UK consensus for management. National Institute for Health and Clinical Excellence (NICE) criteria were used for producing recommendations based on a review of the literature and expert opinion. It was agreed that in the majority of cases, successful management could be based on a simple algorithm which includes using reagent sticks to test for urine infection and measurement of the post micturition residual urine volume. This is in contrast with published guidelines from other countries which recommend cystometry. Throughout the course of their disease, patients should be offered appropriate management options for treatment of incontinence, the mainstay of which is antimuscarinic medications, in combination, if necessary, with clean intermittent self-catheterisation. The evidence for other measures, including physiotherapy, alternative strategies aimed at improving bladder emptying, other medications and detrusor injections of botulinum toxin A was reviewed. The management of urinary tract infections as well as the bladder problems as part of severe disability were discussed and recommendations agreed.

Rescue from failed growth factor and/or chemotherapy HSC mobilization with G-CSF and plerixafor (AMD3100): an institutional experience.

Auto-SCT has been shown to be a potentially curative treatment for a variety of hematological malignancies. Auto-SCT is dependent on the successful mobilization and collection of hematopoietic stem cells to ensure engraftment. The inability to mobilize sufficient number of hematopoietic stem cells using standard cytokine-assisted mobilization strategies excludes eligible patients from potentially curative auto-SCT. Plerixafor (AMD3100; Mozobil), a novel bicyclam antagonist of the SDF-1alpha/CXCR4 complex, has been reported previously to augment PBSC mobilization in patients undergoing their first planned stem cell mobilization and collection attempt. In our experience, 17 of 20 patients otherwise eligible for auto-SCT who failed previous mobilization attempts had successful mobilization of CD34(+) hematopoietic stem cells with one apheresis procedure, and an additional patient required two aphereses procedures, when treated with the combination of plerixafor and G-CSF on a compassionate use protocol available at our institution.