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)

February 2010: Cannabinoids reduce the spread of damage following spinal cord injuries. (Hospital Nacional de Paraplejicos; Toledo, Spain)

First some background: The spinal cord is a bundle of nerve axons that descend from the brain down the back, to around the area of the waist. It is responsible for delivering and relaying messages traveling to and from the brain. The spinal cord is surrounded by bones known as vertebrae, which function to protect the spinal cord from damage or injury. However, it is still possible for damage to occur as a result of severe trauma, which tends to affect bodily functions below the area of injury. However, the initial trauma is not usually the major cause of cell death in the spinal cord. Necrosis occurs after a nerve cell axon is compressed, leading to swelling and eventually bursting. Additionally, a different process occurs known as apoptosis, or programmed cell death, in which neurons surrounding the initial area of damage receive a signal to essentially kill themselves. In spinal cord injuries this normally occurs in two waves: one wave eight hours after the initial injury that affects a specific cell type known as glial cells. The second wave occurs about seven days later in a different cell type known as oligodendrocytes, which can occur at areas distant from the epicenter of injury. This exacerbates initial damage and leads to increased loss of bodily functions.

The new information: It was found that by activating cannabinoid CB1 and CB2 receptors, neuronal axons were preserved at the immediate region of injury. Axons are long extensions of brain cells that form the actual spinal cord. These axons, also known as white matter, are coated with a fatty insulating material known as the myelin sheath, which is formed in the periphery by oligodendrocytes. It was shown that by activating these cannabinoid receptors, there was preservation of white matter and a decreased level of oligodendrocyte death at the epicenter. Additionally, the cannabinoid also inhibited myelin damage and oligodendrocyte loss at areas distant from the injury epicenter due to delayed apoptosis.

What this means: Currently, there are only two possible treatments for spinal cord injury that may help to halt the progression of neuronal damage: anti-inflammatory medication, and cold saline. Both of these work by decreasing the amount of signals that can be received by the cell processes in the spinal cord. However, anti-inflammatory medications may lead to an increased risk of infection, and administration of cold saline lacks empirical evidence to prove its effectiveness. This experiment showed that cannabis can possibly be used immediately following acute spinal cord injuries to decrease the amount of damage, and thus decrease the loss of function in patients.

Arevalo-Martin, A., et al. “The endocannabinoid 2-arachidonoylglycerol reduces lesion expansion and white matter damage after spinal cord injury.” Neurobiology of Disease. (2010): preprint.

January 2010: Cannabinoids inhibit a form of immunodeficiency due to HIV (Virginia Commonwealth University; Richmond, Virginia)

First some background: Immunodeficiency can be aptly described as the inability for the body's defense system to mount an effective response against invading pathogens, and is usually the result of a decreased number of white blood cells or a loss in ability to recognize the pathogen as foreign. HIV (Human Immunodeficiency Virus) leads to immunodeficiency via two main mechanisms: the direct killing of, or an increased rate of apoptosis (programmed cell death) in white blood cells. One of the cells that are targeted by HIV is the macrophage. Macrophages are involved in the initial response to an infection; foreign pathogens (i.e. bacteria) bind to surface receptors, causing the macrophage to envelop the bacterium and digest it. The macrophage then presents proteins of the digested pathogen to other cells, while also secreting chemical factors that attract other white blood cells. When macrophages are infected with HIV, they stop producing their own proteins and begin to produce and secrete viral toxic factors uncontrollably. One of these toxic factors is the protein Tat (transactivator), which serves as an attractant for monocytes, the precursor to macrophages. Once monocytes leave the blood stream and enter local tissues, they can develop into macrophages. By attracting other macrophages, HIV starts a vicious cycle leading to higher and higher levels of Tat in the human body. Additionally, Tat acts as a toxin by inducing apoptosis in T cells, one of the white blood cells responsible for mediating adaptive immunity. Adaptive immunity refers to the ability of the body to rapidly fight a pathogen upon re-infection. The death of the T cells leads to a loss in this adaptive immunity, which is a factor in the infection hypersensitization seen in HIV patients, especially those in which its progression has lead to the development of AIDS (Acquired Immune Deficiency Syndrome).

The new information: Administration of cannabinoids lead to an inhibition in the migration of monocytes due to Tat. By activating the CB2 cannabinoid receptor, it was shown that monocytes and macrophages did not respond to this attractive factor. The experiment proved this using three separate mechanisms. First, a cannabinoid receptor agonist was administered, which lead to the activation of the CB2 receptor on macrophages and inhibition of migration in response to Tat. Secondly, a cannabinoid receptor antagonist was administered, which blocks the CB2 receptor on macrophages and lead to migration. Lastly, the DNA of the macrophage was altered so that the CB2 receptor was not produced, and this lead to migration even in the presence of cannabinoid.

What this means: By halting one of the vicious cycles that lead to AIDS, cannabinoids can potentially stop the progression of HIV (AIDS is defined by a CD4+ (helper) T cell count below 200 cells per microliter). By decreasing the levels of HIV-induced release of Tat by macrophages, the level of T cell death due to Tat would decline. Thus, cannabis could potentially slow the progression of HIV and AIDS by disallowing widespread cellular infection. Currently, the standard treatment for HIV/AIDS is HAART (Highly Active Antiretroviral Therapy), which utilizes several of what are known as anti-retroviral drugs, which inhibit an enzyme responsible for converting the HIV genes into a format that can be read by human cells. While this form of treatment is effective in preventing cellular infection, it cannot target cells already infected with the virus. Therefore, macrophages already producing Tat will continue to produce it, attracting other macrophages for infection, and causing the continued death of white blood cells. By administering cannabis concurrently, it would add an additional level of protection by reducing the spread of HIV to attracted macrophages. Additionally, HAART is very expensive, with an approximate average cost of $1,500 per month. By utilizing cannabis in conjunction with more cost-effective anti-retroviral medications, the cost of treatment could be reduced to as little as $100 a month.

Raborn, E. and G. Cabral. “Cannabinoid Inhibition of Macrophage Migration to the Tat Protein of HIV-1 is Linked to the CB2 Cannabinoid Receptor.” The Journal of Pharmacology and Experimental Therapeutics. (2010): preprint.

February 2010: Cannabinoids sensitize cancer cells to lethal signals. (Universita di Palermo; Palermo, Italy)

First some background: According to the American Cancer Society, in 2009, approximately 22,620 cases of hepatic (liver) cancer were diagnosed, with an approximate 82% mortality rate. Although this is a relatively rare form of cancer, it can be caused by hepatitis or excessive alcohol consumption, and has one of the highest mortality rates. Hepatic cancer is one of the hardest cancers to diagnose, and according to the National Cancer Institute, approximately only 10-20% of liver tumors can be fully removed during surgery. If not removed, liver cancer it is usually deadly within three to six months. One of the molecular causes of cancer is known to be decreased apoptotic ability. Apoptosis refers to programmed cell death, in which a cell receives a specific signal, activating “suicidal” pathways that eventually terminate in the cell’s death. This process is used to control the proliferation of cells in our bodies, allowing us to keep a relatively constant numbers of each cell type. If a cell undergoes a mutation that leads to an inability to perform apoptosis, cell growth can no longer be controlled and a tumor is formed. If the cells within this tumor are capable of recruiting blood vessels and traveling to other parts of the body, they are referred to as malignant tumors, causing what is commonly known as cancer.

The new information: When cannabinoids were administered to human hepatocellular carcinoma (HHC) cells, it caused an up-regulation of a receptor known as DR5 (Death Receptor 5). This receptor allows binding of molecules known as TNFs (Tumor Necrosis Factors), and leads to the activation of an apoptotic pathway. When the DR5 receptors are up-regulated, there are more binding sites for TNFs, which leads to higher levels of cell death. Additionally, administration of the cannabinoid lead to a significant decrease in survival factors, which have the ability to halt the process of apoptosis. The cannabinoid in this study was co-administered with TRAIL (TNF-related apoptosis inducing ligand), leading to a significantly higher level of apoptosis than administration with TRAIL alone.

What this means: This illustrates a novel treatment for liver cancer; because it is one of the hardest carcinomas to remove surgically and its high mortality rate, liver cancer remains one of the deadliest forms. This study proved that administration of cannabinoids lead to an increased sensitivity of hepatic cancer cells to factors that lead to their death. Therefore, co-administration of cannabis with current cancer treatments can lead to an increase in their effectiveness.

Pellerito, O., et al. “The synthetic cannabinoid WIN sensitizes hepatocellular carcinoma cells to TRAIL-induced apoptosis by activating p8/CHOP/DR5 axis.” Molecular Pharmacology. (2010): preprint.