April 2010: A Mechanism by Which Cannabinoids Act as an Antidepressant is Illustrated. (Charles University; Prague, Czech Republic)

Note: Starting in May, there will consistently be two new summaries a week.

First some background: Mood disorders effect approximately 5-13% of the United States population, with major depressive disorder (unipolar depression) reflecting 4-9%.* Although the etiology of depression is not well understood, it is associated with decreased hippocampal volume. Within the hippocampus, a region named the subgranular zone is one of only two areas in the brain where new brain cells can be produced. This area of the hippocampus contains stem cells that form new neurons and differentiate in response to brain-derived neurotrophic factor (BDNF). Antidepressants work by increasing levels of serotonin, dopamine, and norepinephrine in the brain. Increased levels of serotonin and norepinephrine cause an increase in BDNF levels, thus causing an increase in hippocampal volume. Current pharmacological mechanisms for treating depression utilize reuptake inhibitors, which increase levels of these chemicals by inhibiting their reuptake into brain cells. However, older antidepressants, less favored now because of their side effects, are targeted to inhibit the enzyme monoamine oxidase (e.g. isocarboxazid, phelezine). Monoamines refer to a class of molecules which include the aforementioned neurotransmitters serotonin, dopamine, and norepinephrine as well as several others such as histamine. The enzyme monoamine oxidase catalyzes the degradation of these monoamines, thus a monoamine oxidase inhibitor (MAOI) would cause higher levels of these chemicals in the brain. There are two types of monoamine oxidase enzymes found in the human body, MAO-A and MAO-B. MAO-A is found mainly in brain cells that utilize norepinephrine and is able to degrade norepinephrine, serotonin, and dopamine most effectively; while MAO-B is found mainly in brain cells that utilize serotonin and is able to degrade β-phenylethanolamine and dopamine most effectively.

The new information: This experiment tested the effect of three cannabinoids on the activity of both monoamine oxidase enzymes. The three cannabinoids used were ∆9-tetrahydrocannabinol (THC), anandamide (a cannabinoid that occurs naturally in our body), and the synthetic cannabinoid WIN (WIN 55,212-2). The concentrations needed to inhibit 50% of the enzyme activity were then compared with the MAOI medication iproniazid. It was found that the MAO-A enzyme was blocked at lowest concentrations by WIN, followed closely by THC, with a large gap in concentration between THC and anandamide. The MAO-B enzyme was blocked at approximately equal concentrations of THC and WIN, with a large gap in concentrations between them and anandamide. Additionally, the concentrations at which all three blocked the MAOs were much greater than the concentration of iproniazid needed for the same result. The concentrations were measured in micromoles per liter, meaning that they were measured based on number of molecules and not their size or weight.

What this means: The results of this experiment illustrate in detail the dependence of the antidepressant effect of cannabis on concentration of cannabinoids. Because the effect of THC on monoamine oxidase is not as powerful as MAOI medications, there will not be the dangerous drug and food interactions that are notorious side effects of MAOIs. However, marijuana would nonetheless increase the amount of serotonin and norepinephrine in the brain, thus leading to an expansion in the size of the hippocampus. This neurogenerative effect is part of what leads to the antidepressant properties of marijuana. Additionally, damage to the hippocampus is also seen in Alzheimer’s disease, decreases in long-term memory, post-traumatic stress disorder, schizophrenia, and epilepsy caused by hippocampal sclerosis. Thus cannabis may hypothetically be helpful in the treatment of these conditions and more through its action as an inhibitor of the enzyme monoamine oxidase.

*Nestler, E.J., Hyman, S.E., and Malenka, R.C. Molecular Neuropharmacology: A Foundation for Clinical Neuroscience. New York: McGraw-Hill, 2009.

Fišar, Z. “Inhibition of Monoamine Oxidase Activity by Cannabinoids.” Naunyn-Schmiedeberg’s Archives of Pharmacology. (2010): preprint.

June 2010: Cannabinoids inhibit a group of cancer-causing enzymes. (Hokuriku University; Kanazawa, Japan)

Note: June refers to the publication date

First some background: The human body contains an expansive number of enzymes, proteins which increase the rate of chemical reactions in our bodies. These enzymes typically facilitate the various molecular metabolic processes that are occurring at any given second within our cells, but some of their products and/or byproducts can be harmful, even carcinogenic (cause cancer). Perhaps the largest group of enzymes in our bodies is the cytochrome P450 (CYP) family, which catalyze the monooxidation (addition of one oxygen) of various organic molecules. One of the main functions of this enzyme family is the metabolism of drugs in the liver. However, some subfamilies, such as the CYP1 subfamily (enzymes CYP1A1, CYP1A2, CYP1B1), also induce the formation of carcinogenic compounds from polycyclic aromatic hydrocarbons. Polycyclic aromatic hydrocarbons are common constituents of smoke, especially cigarette smoke, and are known as procarcinogens. The label procarcinogen indicates that the molecule in and of itself will not cause cancer, but can be induced to cause cancer when altered by a metabolic process.

The new information: This experiment tested the effects of three cannabinoids found in marijuana on the catalytic effects of CYP1 enzymes. The three cannabinoids used were delta(9)-tetrahydrocannabinol (THC), cannabidiol, and cannabinol; it was found that all three cannabinoids inhibited all three CYP1 enzymes to some degree, with THC being the least potent inhibitor, cannabidiol inhibiting CYP1A1 most effectively, and cannabinol inhibiting CYP1A2 and CYP1B1 most effectively. Additionally, it was shown that all three cannabinoids were competitive inhibitors, meaning that at higher concentrations/potencies of other substrates for the CYP1 enzymes, the cannabinoids were displaced.

What this means: By illustrating that three of the major cannabinoids found in marijuana can cause potent inhibition of all three enzymes in the CYP1 subfamily, marijuana may prevent certain forms of cancer. Polycyclic aromatic hydrocarbons are common components in environmental pollution, and are usually inhaled, resulting in lung cancer. By inhibiting the enzyme that converts the procarcinogen into the cancer-causing compound, cannabis may be prophylactically used to prevent one of the main causes of lung cancer. Additionally, because CYP1 enzymes are also involved in drug metabolism, cannabis could be use to augment various pharmaceuticals for maximal effectiveness. In order for a drug to be excreted from the body, it generally first passes through at least two phases of metabolism, with cytochrome P450 enzymes representing one of the major components of the first phase. Thus if a drug is known to be metabolized by one of the CYP1 enzymes and cannabis is co-administered, it would take longer for the drug to be broken down in and removed from our bodies. Therefore, cannabis could extend the half-life of various medications, possibly reducing the cost to patients.

Yamaori, S., et al. “Characterization of Major Phytocannabinoids, Cannabidiol and Cannabinol, as Isoform-selective and Potent Inhibitors of Human CYP1 Enzymes.” Biochemical Pharmacology. 79.11(2010): 1691-8.

March 2010: Cannabinoids inhibit and may prevent neuropathic pain in diabetes. (University of Calgary; Alberta, Canada)

First some background: According to the World Health Organization, more than 220 million people worldwide are living with diabetes. Within the United States, the National Diabetes Fact Sheet cites 23.6 million people, or 7.6% of the population, as currently living with diabetes; and an additional 1.6 million as diagnosed each year. There are two common forms of diabetes: type I and type II. Type I diabetes usually affects an individual at birth, as they are unable to produce insulin in sufficient quantity. Type II diabetes typically occurs later in an individual's life and reflects a decreased ability of cells to utilize insulin. Insulin is a hormone secreted from Beta cells of the pancreas mainly in response to increased glucose levels in the blood. Insulin acts to allow glucose to be taken up by cells in muscle, liver, and fat, and subsequently being converted into stored forms of energy. With the accompanying lack of insulin or its function in diabetes, glucose, the simplest form of sugar, accumulates in the bloodstream, leading to a multitude of pathologies including neuropathic pain. Hyperglycemia (elevated blood glucose) causes small blood vessels to uptake higher levels of glucose, leading to thicker as well as weaker blood vessel membranes. With these thicker blood vessels comes a reciprocal decrease in blood flow, leading to decreased oxygen levels in many organs, including the brain. The decreased oxygen levels in the brain decreases the conduction velocity of neurons and may cause structural changes in brain cells. Additionally, Hyperglycemia causes the upregulation of oxygen radicals as well as the activation of microglia, both of which can damage nerve cells. Microglia are a type of brain cell that act as immune cells of the brain, they respond to infections of the brain and spinal cord. However, their activation in diabetes causes them to release cytotoxic chemicals in absence of infection, which can damage nerve cells. This damage and structural change in nerve cells is thought to be responsible for the phenomenon of diabetic peripheral neuropathy. It has been previously shown that inhibiting microglial activation leads to a dissipation of neuropathic pain in mouse models of diabetes.* It is also well known that both neurons and microglial cells express cannabinoid receptors.

The new information: This experiment involved inducing diabetes in mice in the presence and absence of cannabinoid agonists and observing the mice over a course of 8 months. There were six main experimental groups, one of which diabetes was induced without cannabinoid treatment, serving as a control. In a second group, diabetes was induced in conjunction with cannabidiol treatment. It was found that in this second group, neuropathic pain did not develop over the course of 8 months and the levels of activated microglia in the spinal cord were greatly reduced compared to the control. Additionally, when cannabidiol treatment was stopped, the mice continued to show reduced microglia as well as no signs of neuropathic pain. A third and fourth group involved the induction of diabetes and treatment with both CB1 and CB2 cannabinoid receptor agonists once symptoms of neuropathic pain started. The results indicated that both CB1 and CB2 agonists inhibited the symptoms of neuropathic pain, but the pain returned after treatment was stopped. The last two groups involved treatment with CB1 and CB2 cannabinoid receptor antagonists, which block the effect of cannabinoids, and no change was seen in the levels of pain compared to the control group.

What this means: This experiment provided more evidence that cannabinoids may be used in the treatment of neuropathic pain. However, the novel information obtained is much more surprising. When treated with cannabidiol at the onset of diabetes, the diabetic mice did not have any symptoms of neuropathic pain even when treatment was stopped. This suggests that treatment with cannabidiol at the onset of diabetes may produce permanent protective changes for nerve cells. Therefore, cannabis could hypothetically be used short-term at the onset of type II diabetes in adults for lifetime or long-term prevention of diabetic peripheral neuropathy.

*Tsuda, M., et al. “Activation of Dorsal Horn Microglia Contributes to Diabetes-induced Tactile Allodynia via Extracellular Signal-regulated Protein Kinase Signaling.” Glia. 56.4(2008): 378-86.

Toth, C., et al. “Cannabinoid-mediated Modulation of Neuropathic Pain and Microglial Accumulation in a Model of Murine Type I Diabetic Peripheral Neuropathic Pain.” Molecular Pain. 6.16(2010).

March 2010: A novel process by which cannabinoids alleviate pain has been determined molecularly (Medizinische Hochschule Hannover; Hannover, Germany)

First some background: Chronic pain is often a difficult condition to treat and sometimes even diagnose. Originating as a protective mechanism, pain notifies us when an external stimulus may cause us harm or when something internal start to go awry. However, in certain types of chronic pain and what is referred to as neuropathic pain, this once protective mechanism exhibits functional degeneracy, where its function in the human body is not established. What has been well established however, is the process by which we feel this pain. When peripheral cells are damaged, an inflammatory response ensues, leading to the release of chemicals such as bradykinin, histamine, prostanoids, and tachykinins. These chemicals as well as physical pressure and severe temperatures act on dendritic terminals of nociceptive neurons, mostly activating TRP (transient receptor potential) channels. These TRP channels are a family of stimulus-sensitive non-selective cation channels, thus permeable to sodium, calcium, magnesium, and other positively charged ions. Activation of TRP channels causes a signal to be sent along this nociceptive (pain) neuron, whose cell body resides in the dorsal root ganglion. These cell bodies then relay their signal to a different neuron in the spinal cord. This spinal cord neuron, located in the dorsal horn, also receives input from several other neurons, dictating the level of pain felt and are usually inhibitory. It is well documented that cannabinoids can act in a retrograde fashion at these synapses utilizing CB1 (cannabinoid receptor 1) in order to inhibit the signal coming from the primary afferent neuron (the one that originally sensed the pain). Additionally, it has been established that cannabinoids may act at TRP channels directly, desensitizing them to painful stimuli. However, in recent years, it has emerged that cannabinoids may also act on other parts of the pain pathway.

The new information: Although it has been previously noted that cannabinoids act on different parts of the pain pathway, including glycine receptors, the exact molecular mechanism has not been established. The modulatory inhibitory neurons utilize one of two neurotransmitters to decrease the painful signal coming from the primary afferent neuron: GABA (gamma-aminobutyric acid) and glycine. It is known that cannabinoids somehow act on glycine receptors in order to decrease the sensation of pain. This experiment involved mutating the glycine receptor in specific regions to determine how cannabinoids, specifically cannabidiol, interact with the receptor. By mutating an amino acid in the second transmembrane domain from serine (polar) to isoleucine (nonpolar), cannabidiol had no effect on the receptor. However, in absence of the mutation, cannabidiol caused both co-activation and direct activation of the glycine receptor. Co-activation is also referred to as positive allosteric modulation, where the cannabinoid by itself will not activate the receptor, but in presence of glycine (the receptor agonist), there is an increased intracellular response. Additionally, cannabidiol was shown to directly activate this receptor, causing inhibition of the noxious (painful) signal.

What this means: As mentioned in previous entries, THC (∆9-tetrahydrocannabinol) is not the only cannabinoid found in plants of the Cannabis genus. The remaining cannabinoids all have differing structures, properties, and functions. However, the current pharmaceutical market utilizes only THC containing medication, which cannot fully utilize the benefits of Marijuana. By showing the exact molecular mechanism by which cannabidiol interacts with glycine receptors, another means by which cannabis lead to analgesia has been established.

Foadi, N., et al. “Lack of Positive Allosteric Modulation of Mutated Alpha(1)S267I Glycine Receptors by Cannabinoids.” Naunyn-Schmiedeberg's Archives of Pharmacology. (2010): preprint.

March 2010: Cannabinoids have a role in reducing heart disease. (Shanghai Jiaotong University; Shanghai, China)

First some background: According to the World Health Organization (WHO), heart disease accounts for approximately 12 million deaths worldwide per year; and within the United States, about 2,600 people die per day from its complications. Although heart disease can manifest itself in several forms, the most common and most lethal is coronary artery disease, or atherosclerosis of the heart arteries. Atherosclerosis refers to the thickening of artery walls due to deposits of cholesterol shuttles such as LDL (low-density lipoprotein). Atherosclerosis develops when LDL molecules become oxidized by free oxygen radicals such as superoxide, a by-product of cellular reactions. Oxidized species such as the newly formed LDL cause damage upon contact with the endothelial cells lining arteries. When these cells are damaged, the body’s immune system tries to repair the damage and break down the oxidized LDL, but are unable to, and instead release more reactive oxygen species (ROS) and tumor necrosis factor alpha (TNF-α). This starts a vicious cycle leading to greater and greater levels of inflammation, causing the artery to harden, narrow, and eventually be completely blocked. It is known that subtypes of immune system cells such as macrophages and T cells contain cannabinoid receptor 2 (CB2).

The new information: In this experiment, macrophages were isolated from model mice and rats and exposed to oxidized LDL in the presence and absence of a cannabinoid agonist. The levels of reactive oxygen species (ROS) and TNF-α as well as intracellular signaling molecules were then measured. It was found that in the absence of the cannabinoid, the oxidized LDL strongly induced the generation of ROS and TNF-α. However, in the presence of the cannabinoid, the levels of ROS and TNF-α were greatly reduced, which was shown to occur via a mechanism of inhibiting intracellular signaling pathways within the macrophage. When the macrophage was exposed to both cannabinoid and a cannabinoid receptor blocker, the oxidized LDL once again strongly induced the generation of ROS and TNF-α, suggesting that the reduction was a direct product of the cannabinoid.

What this means: By illustrating that cannabinoids effectively reduce the inflammatory response of macrophages to oxidized LDL, this study shows that cannabinoids may be used as a prophylactic measure in preventing coronary artery disease. Additionally, cannabinoids may have therapeutic benefits in the treatment of atherosclerosis, as it would greatly decrease further inflammation and the appearance of plaques. Therefore use of cannabis in patients with coronary artery disease may reduce their risk of heart attack.

Hao, M.X., et al. “The Cannabinoid WIN55, 212-2 Protects Against Oxidized LDL-induced Inflammatory Response in Murine Macrophages.” Journal of Lipid Research. (2010): preprint.

April 2010: Cannabinoids inhibit highly invasive cancer metastasis. (University of Rostock; Rostock, Germany)

Note: April refers to publication date, which is April 1; the actual study was conducted in November 2009. Additionally, to all the avid readers, I apologize for the wait, it's been a busy couple of weeks.

First some background: Most of the research summaries featured within this blog involve the effects of synthetic cannabinoid receptor agonists rather than the actual substances found within plants of the Cannabis genus. Although the ∆9-Tetrahydrocannabinol (THC) found in Cannabis is a CB1 and CB2 cannabinoid receptor agonist, it is still rarely used experimentally. Rarer still, is experimentation with one of the several other cannabinoids found within Cannabis plants. There are four other known cannabinoids that have been derived from Cannabis: Cannabidiol (CBD), Cannabinol (CBN), Tetrahydrocannabivarin (THCV), and Cannabichromene (CBC). When a patient considers the alternatives to medical marijuana, there is only one that supposedly is comparable, Dronabinol, marketed by Abbott (formally Solvay) pharmaceuticals as Marinol. Dronabinol is essentially synthetically produced THC, and thus contains only one of the substances in Cannabis that has shown therapeutic potential. Additionally, not all cannabinoids found in Cannabis act on the primary cannabis receptors CB1 and CB2; therefore in order to achieve the full medical benefits of marijuana, the other substances must be consumed as well. One of the other cannabinoids found in Cannabis plants is cannabidiol. Although it has not been researched as extensively as THC, it has been shown to generally make up 40% of extracts from the Cannabis plant.¹ Cannabidiol’s exact physiological functions have not been fully understood, but it has been previously shown to interact with TRPV1 (transient receptor potential cation channel, subfamily V, member 1) receptors and have anti-cancer properties.

The new information: In this experiment, two different cancer cell lines were treated with cannabidiol, and both showed impaired invasion. The cell lines were of highly invasive human cervical cancer (HeLa, C33A) and human lung cancer (A549). The cannabidiol-driven impaired invasion was shown to be reversed by both an antagonist to CB1 and CB2 cannabinoid receptors as well as an antagonist to TRPV1 receptors. Although this did not represent particularly novel information, it was also found that the decrease in invasion occurred concurrently with an increase in TIMP-1 (tissue inhibitor of matrix metalloprotease-1). When the cell lines were genetically altered to be unable to produce TIMP-1, cannabidiol showed no effect in impairing cancer invasion. Additionally, the human lung cancer cell line was induced in thymic-aplastic nude mice, which lack a functioning immune system that could possibly defend against the cancer, where it was found that treatment with cannabidiol caused significant inhibition of lung metastasis.

What this means: The results of this study indicate that current pharmaceutical capabilities to utilize the substances found in the Cannabis plant are severely underdeveloped. In order to utilize the full therapeutic potential of marijuana, it must be ingested along with other substances naturally occurring in the plant. This particular experiment elucidated the molecular mechanism of cannabidiol-induced inhibition of cancer metastasis, which along with studies pertaining to anti-cancer effects of strictly cannabinoid receptor agonists, start to form a complete picture of the cancer-inhibiting capabilities of Cannabis.

¹Grlie, L. "A Comparative Study on Chemical and Biological Characteristics of Various Samples of Cannabis Resin." Bulletin on Narcotics. 14(1976): 37–46.

Ramer, R., et al. “Cannabidiol Inhibits Cancer Cell Invasion via Upregulation of Tissue Inhibitor of Matrix Metalloproteinases-1.” Biochemical Pharmacology. 79.7(2010): 955-66.

February 2010: Cannabinoids inhibit pain and bone loss induced by bone cancer (The University of Arizona; Tucson, Arizona)

First some background: Malignant bone cancer refers to a number of diverse tumor types, including osteosarcoma, chondrosarcoma, fibrosarcoma, cordoma, and Erwig’s sarcoma. Although the physiological mechanisms leading to tumor formation and malignancy may differ, the main symptoms of most forms of bone cancer are severe pain and bone loss. Thus, in standard treatment regiments for bone cancer, opiates are used in addition to chemotherapy and radiotherapy to abate the pain. However, use of opiates for analgesia has several downsides: physical addiction, high abuse potential, and rapid tolerance to name a few. Additionally, two side effects of chronic opiate use lead to an exacerbation of bone cancer symptoms. The first is pain hypersensitization. When the body is exposed to constant levels of any drug that acts as a receptor agonist, it induces a protective response to maintain its original state. Therefore when exposed to chronic opiate medications, the body reduces expression of opioid receptors, leading to decreased pain inhibition and thus increased sensitivity to pain. The second is hypogonadism. Opiates act on what is known as the hypothalamic-pituitary axis, causing decreased levels of hormone release. One of these hormones is GnRH (gonadotropin releasing hormone). GnRH causes release of two hormones from the anterior pituitary: LH (luteinizing hormone) and FSH (follicle stimulating hormone). These two hormones are responsible for regulating the amount of testosterone in both males and females. Although testosterone is widely known for being the main sex hormone in males, it is also present in lesser amounts in females with a common protective function of maintaining bone density. Thus chronic use of opiate medications will lead to an increased level of bone loss.

The new information: Cannabinoids have been shown to be a more valid alternative for treating bone cancer-mediated pain. The experiment was carried out by inducing bone cancer in mice and performing both behavioral and radiologic image interpretation of symptoms. After confirming the development of cancer, the mice were shown to have experienced both spontaneous and touch-evoked behavioral signs of pain. By administrating cannabinoids to the mice, both the spontaneous and stimulated pain was inhibited. Additionally, a sustained treatment regimen of cannabinoids led to significant reductions in bone loss, manifesting as a decreased likelihood of cancer-induced bone fractures.

What this means: By showing the benefits of utilizing cannabinoids as an alternative analgesic for bone cancer patients, cannabis may be a healthier alternative than opiates in treating pain associated with the cancer. Chronic use of opiates can cause more harm than good, as they often exacerbate the symptoms of bone cancer via patient hypersensitivity to pain and decreased bone mineral density. Cannabinoids on the other hand not only provide a non-physically addictive alternative, but also have been shown to attenuate the bone loss seen in cancer patients.

Lozano, A., et al. “A Cannabinoid 2 Receptor Agonist Attenuates Bone Cancer-induced Pain and Bone Loss.” Life Sciences. 2010: (preprint)