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2-AG – Not Such Eternal Bliss?

Ananda at the first Buddhist council

Ananda at the First Buddhist Council - Image: Wikipedia - Sacca

The cannabinoid receptors, CB1 and CB2, were first identified as the receptors for the active components of Cannabis sativa. The endogenous ligands for the cannabinoid receptors, anandamide (from ananda, a Sanskrit word meaning ‘bliss’) and 2-arachidonoylglycerol (2-AG), exert most of their analgesic affects through binding to the main cannabinoid receptor in the brain, CB1. Attempts to prepare CB1 agonists that mimic the analgesic effects of the main component of cannabis, Δ9-tetrahydrocannabinol (THC) but have reduced potential for memory impairment, locomotor dysfunction, and possibly addiction have met with limited success and a recent alternative approach has been to devise ways of preventing the breakdown of anandamide and 2-AG. Signalling by the endocannabinoids is terminated by enzymatic hydrolysis which, for anandamide, is mediated by fatty acid amide hydrolase (FAAH) and, for 2-arachidonoylglycerol, by monoacylglycerol lipase (MAGL).

Inhibitors of both FAAH and MAGL produce analgesic effects but scientists at the Scripps Research Institute, Virginia Commonwealth University and the Medical College of Wisconsin have now shown that the effects of blocking MAGL are short-lived compared with the effects of blocking FAAH. Although a single injection of the MAGL inhibitor, JZL184, reduced pain in the mouse, after six consecutive daily injections, the effect disappeared. The chronically treated animals also became less sensitive to THC or the synthetic CB1 agonist, WIN55,212-2, and showed characteristic drug withdrawal symptoms when treated with the CB1 blocker, rimonabant.

Further tests showed that CB1 receptors were down-regulated in some brain areas of mice treated for 6 days with JZL184. Genetic disruption of MAGL also resulted in elevated levels of 2-AG and desensitised the CB1 signalling system, suggesting that chronic inhibition of MAGL may not provide effective analgesia. In contrast, chronic treatment with a FAAH inhibitor to boost anandamide levels did not lead to desensitisation of the CB1 system.

Although the team do not yet understand why chronic administration of FAAH inhibitors and MAGL inhibitors should produce such strikingly different results, the study, which is published in Nature Neuroscience, suggests that complete blockade of MAGL may not provide effective analgesia over the longer term.


Mice Show Pain, Just As We Do

mouse and man

Image: Flickr - bjormeansbear

We are used to the idea that facial expressions can tell us a lot about what our fellow humans are experiencing and researchers in Canada and the Netherlands have now found that the same is true for mice. In human volunteers, grimacing has been shown to match self-reported pain intensity and facial expression is also used to evaluate pain in patients who are unable to communicate orally.

Existing pain remedies do not work well for all patients and, in the search for more effective pain relief, mice and other rodents are used to develop new treatments. The present study has shown that mice, like humans, express pain through facial expressions and should provide a more accurate way to ensure that laboratory animals do not suffer unnecessarily and could also lead to better drugs for humans. The team have developed a ‘Mouse Grimace Scale’ by analysing high-resolution video images of mice before and during moderately painful stimuli – pain that is suggested to be comparable to a headache or that associated with an inflamed and swollen finger. The researchers, who are leaders in developing standards for facial expression to assess pain in human infants and others unable to communicate verbally, have proposed that five facial features in the mice should be scored. Several of these criteria – which are orbital tightening (eye closing), nose and cheek bulges, and ear and whisker positions – are similar to those used to assess pain in humans.

The study, which is the first to propose such a standardised and accurate scoring system linking facial expression to pain in animals, is published in the journal Nature Methods. Based on facial expression, trained researchers were able to correctly and reliably assess pain levels in real time. Previously, assessment of pain was limited to measuring withdrawal responses to pressure and heat and the ‘Mouse Grimace Scale’ may provide a more accurate way of predicting whether new treatments will eventually work in humans as well as reducing unnecessary pain in laboratory animals.


Towards Safer Narcotic Pain Relief

Combat medics Image: Flickr - U.S. Army

Combat medics

Image: Flickr - U.S. Army

Morphine provides very effective pain relief for acute traumatic injury but the hypoxia and hypotension associated with narcotic use can lead to cardio-respiratory collapse and, potentially, death. One approach to reducing the risk of respiratory failure, whilst maintaining analgesia, would be to use low-level narcotic antagonist feedback. Naloxone is an effective, short-acting opioid antagonist that, at low doses, can reverse morphine-induced respiratory depression with relatively little effect on analgesia. Scientists at the University of Michigan have now described a pro-drug approach that releases naloxone only when tissues become hypoxic. Indolequinone-containing pro-drugs show good naloxone releasing properties under hypoxic conditions and, in the present study, a linker was attached so that the pro-drug can be tethered to other macromolecules to enhance retention time in vivo. Pro-drug (2) was stable for 24 hours at 37oC in PBS buffer but was cleaved to give naloxone on addition of the reductase, DT-Diaphorase. Naloxone was also released from pro-drug (2) in fresh human plasma under conditions of low oxygen pressure, but not under normoxic conditions.
Naloxone prodrug, Compound 2

Naloxone prodrug, Compound 2


The primary aim of the study, which is published in the journal Bioorganic and Medicinal Chemistry Letters, was to provide safer pain relief for soldiers injured in combat who are typically treated with morphine on the battlefield, but such a system could also potentially be used to improve pain management for many patients with chronic illnesses. The team plan to test their pro-drug approach in animal studies and also to develop dendrimer nanoparticles to which the pro-drug can be tethered to enhance in vivo performance.


Cannabinoids – Are They a Pain?

painMany groups developing cannabinoid receptor agonists for the treatment of pain have sought to identify selective CB2 receptor agonists to avoid the psychotropic effects associated with CB1 receptor agonists and a recent report in the journal Science may provide another reason to aim for selectivity. Although there is compelling evidence that cannabis and cannabis-based medicines can help reduce chronic pain, the new research suggests that endocannabinoids acting via the CB1 receptor can amplify and prolong pain rather than alleviate it.

The spinal cord contains both excitatory and inhibitory neurons which together regulate the transmission of information, including pain signals, to the brain. In ex vivo experiments using mouse spinal cord tissue, researchers found that the mixed CB1/CB2 receptor agonist, WIN 55212-2, reduced the amplitude of inhibitory postsynaptic currents, an effect that was reversed by the CB1 receptor antagonist/inverse agonist, AM251. In anaesthetised rats, local spinal application of AM251 reversed the increase in action potential firing triggered by mechanical stimulation in an area surrounding a capsaicin injection site on the hind paw and, in mice, intrathecal administration of AM251 also reversed mechanical sensitisation. Studies in knockout animals indicated that CB1 receptors on inhibitory dorsal root neurons, rather than those on primary nociceptors, were responsible for capsaicin-induced secondary hyperalgesia. Mechanical sensitisation could also be evoked by intrathecal injection of the CB1/CB2 agonist, CP 55,940.

In human volunteers, 10-day treatment with the CB1 receptor antagonist/inverse agonist, rimonabant, had no effect on acute pain ratings induced by electrical stimulation of the forearm, but reduced the sizes of hyperalgesic and allodynic skin areas to around 50% of pre-treatment values. The study reveals an unexpected role for endocannabinoids acting on CB1 receptors on neurons in the dorsal horn in diminishing inhibitory control of pain signals.

Another recently published study concluded that activation of spinal CB1 receptors by administration of the CB1 agonist, arachidonyl-2-chloroethylamine reduced bone cancer-related pain and that presynaptic inhibition may contribute to this analgesic effect.

Localisation of CB1 receptors and precise pathways of pain signalling may explain the differing effects of CB1 agonists in pain models and human patients.


Itching is Not Just Toned-Down Pain

Most animals and people experience itching from time to time, but the unpleasant sensation is usually short-lived and can be relieved by scratching. mouse scratchingItching caused by insect bites or allergic reactions can be treated with anti-histamines but, for some people, chronic generalised itching associated with conditions including eczema, psoriasis, liver or kidney disease, HIV and certain cancers, as well as the use of some drugs, is resistant to anti-histamine treatment and can be a debilitating condition.

Many scientists have regarded itching as simply a toned down version of pain but researchers at Washington University School of Medicine have now shown that itch-specific neurons exist in mice. Two years ago, the team identified the first ‘itch gene’ in the spinal cord. They showed that mice lacking the gene for the gastrin-releasing peptide receptor (GRPR) scratched less than normal littermates when exposed to itch-inducing stimuli. The team have now shown that if they destroy GRPR-expressing neurons in the spinal cord using a GRPR ligand conjugated to a neurotoxin (bombesin-saporin), scratching was reduced by more than 80% and, in some cases, eliminated completely. The mice showed normal motor control and continued to respond normally to pain, suggesting that there is an itch-specific neuronal pathway in the spinal cord. This is the first behavioural evidence for itch-specific neurons and could eventually lead to novel treatments which alleviate chronic itching without affecting the pain response.

The study was published online on August 6th in Science.


Ampakines Protect against µ-Opioid Respiratory Depression

Respiratory depression is a leading cause of death from overdose of some classes of abused drugs but can also arise during medically supervised procedures such as perisurgical anaesthesia. Fentanyl, a µ-opioid agonist which is approximately 100 times more potent than morphine, is widely used to treat acute, postoperative, and chronic pain. Activation of opiate receptors can, however, lead to significant respiratory depression in a subset (10-15%) of patients. This has commonly been reversed by administration of a µ-opioid antagonist such as naloxone, albeit with the undesirable effect of blocking pain relief.

Writing in the journal Anesthesiology, researchers at the University of Alberta have now described the use of an Ampakine compound, CX717, for treating fentanyl-induced respiratory depression without interfering with analgesia. GluR2 Crystal StructureAmpakines bind allosterically to α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-type glutamate receptors and are undergoing clinical trials as potential treatments for cognitive disorders and as enhancers of memory and attentiveness. AMPA receptors mediate fast synaptic transmission in the CNS and play a central role in maintaining respiratory rhythm. In the present study, pre-administration of CX717 to rats was found to markedly attenuate fentanyl-induced respiratory depression and post-administration of CX717 rescued the animals from a lethal dose of fentanyl. The effective doses of CX717 were within the range considered to be safe from clinical study data (ca 10mg/kg bid) and, importantly, did not reverse the analgesia, suggesting that co-administration of Ampakines with fentanyl could improve pain management in a variety of clinical settings.

Older people, those with a history of sleep apnea, obese individuals and those with a history of chronic obstructive pulmonary disease are particularly at risk for fentanyl-induced respiratory depression during surgery. In a small Phase IIa study in alfentanil-induced respiratory depression, a single dose of 1500mg of CX717 achieved statistical significance over placebo on the primary endpoint measure of spontaneous basal respiration without affecting analgesia.

CX546 structureAnother ampakine compound, CX546, has previously been shown to reverse respiratory distress in rats treated with fentanyl or the barbiturate, phenobarbitol, without compromising the analgesic effect.


New Insights into Morphine Hyperalgesia

poppiesMorphine was first extracted from the opium poppy in the early years of the nineteenth century and is still the drug of choice for the treatment of severe acute and chronic pain. Paradoxically, chronic morphine use can lead to reduced analgesia and even increased pain sensitivity. A study by researchers at the Leiden University Medical Center and the City University of New York, published in the June edition of the journal Anesthesiology now provides further insights into why this happens. Initially, engagement of opioid receptors was believed to be critical for opioid-induced hyperalgesia but, more recently, morphine has been shown to increase pain sensitivity in triple knockout mice lacking μ-, κ-, and δ-opioid receptors. Hyperalgesia has also been observed in normal mice during co-administration with the non-selective opioid receptor antagonist, naltrexone. In humans, morphine is metabolised primarily to morphine-3-glucuronide (M3G) and morphine-6-glucuronide (M6G). M3G has no analgesic effect and accumulation of M3G has been suggested to underlie morphine hyperalgesia in humans. M6G, on the other hand, has analgesic properties similar to those of morphine although some earlier studies had indicated that it can also increase pain sensitivity in both mice and humans.

papaver somniferumIn the present study, M6G was shown to increase pain sensitivity in both mice lacking opioid receptors and in mice treated with the opioid receptor blocker, naltrexone. The N-methyl-D-aspartate (NMDA) receptor antagonist, MK-801, was shown to block or reverse the increased pain sensitivity caused by acute injection or continuous infusion of M6G in naltrexone-treated mice. In human volunteers, a single intravenous injection of M6G was found to increase heat pain sensitivity for at least 6 hours and the hyperalgesia was not blocked by simultaneous continuous infusion of the opioid receptor blocker, naloxone.


PF-3845 – A Potent and Selective FAAH Inhibitor

crystal structure of FAAHThe discovery of the cannabis receptors CB1 and CB2 and the subsequent discovery of anandamide (N-arachidonoylethanolamine), the first endogenous agonist of both receptors, provided a rationale for the known analgesic properties of Cannabis sativa. Anandamide is rapidly hydrolyzed and inactivated by the serine hydrolase, fatty acid amide hydrolase (FAAH), leading to the hypothesis that inhibition of FAAH would be an effective analgesic strategy. Writing in the journal Chemistry & Biology, scientists at the Scripps Research Institute and Pfizer Inc. have now described the discovery of PF-3845, a highly efficacious and selective inhibitor of FAAH. Mechanistic and structural studies confirmed that PF-3845 acts as a covalent inhibitor and carbamylates the active site serine of FAAH. PF-3845 structureInitial experiments showed that PF-3845 is 10 to 20 times more potent than other FAAH inhibitors and has superior pharmacokinetic properties. In animal studies, PF-3845 raised brain anandamide levels for up to 24 hours and produced a significant cannabinoid receptor-dependent reduction in inflammatory pain. PF-750 structureEven if PF-3845 is not itself progressed to the clinic, the team believe that it will be a valuable pharmacological tool for further in vivo characterization of the endocannabinoid system.


Long Lasting Pain Relief

saxitoxinA new slow-release anaesthetic drug-delivery system could potentially revolutionize the treatment of pain during and after surgery, and may also have a large impact on chronic pain management. Researchers at Children’s Hospital Boston have developed a liposomal delivery system for the ultra-potent local anaesthetic, saxitoxin. Saxitoxin is one of a family of neurotoxic alkaloids produced by aquatic microorganisms which block voltage-gated sodium channels on nerve cells. In studies in rats, liposomal delivery of saxitoxin blocked sciatic nerve transmission without causing significant nerve or muscle damage. The team found that liposomes containing only saxitoxin produced nerve blocks that lasted for two days whilst liposomes containing saxitoxin together with dexamethasone – a steroid known to enhance the action of encapsulated anaesthetics – caused a nerve block that lasted for seven days without significant damage to surrounding nerves or muscle. Despite the extreme potency of saxitoxin, systemic toxicity occurred only with high loadings of dexamethasone which increased release of the anaesthetic. Previous attempts to develop slow-release anaesthetics have been limited by short duration of action, toxicity to the surrounding tissue and/or systemic toxicity. The team hope that eventually a single injection that could cause a nerve block lasting for weeks – or even months – could be used to manage chronic pain and are optimising a formulation that would be suitable for clinical trials.

The research is published in the April 13th online edition of PNAS.


Pain Target Linked to Cancer

Blockade of the transient receptor potential vanilloid channel, TRPV1, which is widely expressed in both central and peripheral nervous tissue, has been considered by many groups to be an attractive approach to pain relief. chilli pepperThe receptor is activated by a number of endogenous and exogenous stimuli including the endocannabinoid, anandamide; capsaicin, the ‘hot’ component of chilli peppers; low pH; and heat. The sensitivity of TRPV1 to heat has suggested a role in maintenance of body temperature, and clinical trials of at least one TRPV1 antagonist were stopped because of unacceptable levels of hyperthermia.

A new study published in the January 19th Online First edition of the journal Cancer Research now suggests a link between TRPV1 and the development of cancer. The authors show that TRPV1 interacts with epidermal growth factor receptor (EGFR), a receptor tyrosine kinase that is overexpressed in many human epithelial cancers. Interaction of TRPV1 with EGFR was found to recruit the ubiquitin ligase, Cbl, leading to ubiquitylation and lysosomal degradation of EGFR.

In a further set of experiments, the authors showed that mouse epidermal cells over-expressing TRPV1 were significantly less likely to undergo malignant transformation when stimulated with EGFR, either with or without a tumour promoter. TRPV1 was next shown to be expressed in the skin of wild type mice but not TRPV1 knock-out mice; the knock-out mice also had elevated levels of EGFR protein in the skin. When exposed to the tumour initiator, 7,12-dimethylbenz[a]anthracene (DMBA) and the promoter, 12-O-tetradecanoylphorbol-13-acetate (TPA), almost all TRPV1 knock-out mice developed larger and more numerous tumours than age- and sex-matched wild type animals. Pretreatment of all mice with the EGFR antagonist, AG1478, significantly suppressed tumour formation, but the effect was much greater in the TRPV1 knock-out animals.

The authors suggest that channels such as TRPV1 are able have a direct effect which is independent of their function as ion channels; the TRP family of proteins seems to show different levels of expression in cancer tissues, although whether these changes are cause or effect is not known.