I would like to thank the Sally McDonnell Barksdale Honors College for this opportunity to conduct my own research. I would also like to thank the National Institutes of Health (NIH) and the National Institute of General Medical Sciences Grant #P20GM104932 for funding this research. The most promising development was the 2018 Food and Drug Administration approval of the first marijuana-derived drug, Epidiolex (cannabidiol or CBD).
INTRODUCTION
- Cannabinoids as Therapeutics…
- Cannabis and Epilepsy
- Current Indications
- Epilepsy
- Epilepsy Genes Investigated in this Study
- Dravet Syndrome
- Zebrafish as an Epilepsy Model
- scn1a Zebrafish
- Legality Issues
- Study Goals
In his published case reports, he referred to the success of using 'Indian hemp' in the treatment of. In the Phase 3 clinical trials, Epidiolex significantly reduced seizure frequency compared to placebo in highly treatment-resistant patients (Thiele et al., 2018). Provide context and relevance through the discussion of the complicated legal status of marijuana in the United States.
MATERIALS AND METHODS
- Zebrafish Husbandry
- Acute Exposure
- Subchronic Exposures
- Trial 1
- Trial 2
- Trial 3
- Gas Chromatography Confirmation
- Behavioral Screening
- RNA Extraction, Reverse Transcription, PCR Amplification
- Statistics
Beginning at 8 dpf, larvae were fed Gemma Micro feed daily before the water change but after behavioral screening. Beginning at 8 dpf, larvae were fed Gemma microfeed daily before the water change but after behavioral screening. Larvae were transferred using transfer pipettes to a 96-well plate, 1 larva per well. well, for behavioral analysis every other day starting at 6 dpf.
RESULTS
Acute Exposure
Subchronic Exposures
On days 9-10 post-fertilization, larvae exposed to 0.18 mg/L CBD began to develop malformations, such as body axis curvatures, enlarged eyes, lethargy, and death. Because some of the fish died midway through the trial, we did not perform statistical analysis on these data to determine whether any of the days showed a significant difference in the duration of high activity. The behavior of zebrafish larvae was analyzed using the Viewpoint Zebrabox (45 min recording with 100% light) to record the duration of high activity.
Behavioral analysis was recorded daily from 4 dpf to 10 dpf, after their continuous exposure to CBD (0.18 mg/L) to determine whether CBD exhibits antiepileptic properties. Further statistical analysis was not performed to determine significance in the duration of high activity because some of the fish died mid-exposure. Given that larvae showed deformities in trial 1 after exposure to 0.18 mg/L CBD and homozygous larvae were difficult to identify at 3 dpf, in Trial 2 scn1a-/- and scn1a+/- larvae were exposed to control or 0.075 mg/L from 5-10 dpf in a 24-well plate.
Behavioral analysis was recorded daily from 6 to 10 dpf, after their continuous exposure to CBD (0.075 mg/L), to determine whether CBD has antiepileptic properties. Both control and 0.18 mg/L CBD treatments had very little activity in homozygous fish at 6 and 8 dpf. Behavioral analysis was recorded at 6 and 8 dpf, after continuous exposure to CBD (0.18 mg/L) to determine whether CBD has antiepileptic properties.
Further statistical analysis was not performed to determine significance in duration of major activity due to the premature death of 100% of the fish at 9 dpf.
Gene Expression
Gas chromatography mass spectrometry was used to confirm the cannabinoid water concentrations for each trial. Calculations for mean and standard deviation were calculated by constructing a standard calibration curve for and 10 mg/L for CBD. Gene expression was determined by qPCR after exposure to control and 0.18 mg/L CBD from 5-6 dpf.
DISCUSSION
Acute Exposure
Subchronic Exposures
The zebrafish were transferred from their respective vials to well plates for behavioral analysis, and then back to the vials for feeding/dosing. There was a decrease in the duration of major activity from 6 to 8 dpf for the homozygous group. In the heterozygous mutants, there was virtually no change in the duration of major activity between 6 and 8 dpf.
These findings may be particularly useful in describing the increase in activity from 4 to 5 dpf in trial 1. They also observed that the total movement of non-inflated swimbladder control larvae was lower compared to larvae of control with inflated swim bladders. In this study, the hyperactive behavior of scn1a mutants was not due to the lack of inflated swim bladder, but due to abnormal brain activity (Zhang et al., 2015).
All the homozygous fish in our study all had non-inflated swim bladders, while all the heterozygous fish had inflated swim bladders. The well plates used in our study were 24 well plates, an even smaller test arena than in the Colwill and Creton study, which in turn could have affected the development of the zebrafish, and consequently their behavior. An additional factor in the subchronic exposures was that the zebrafish were fed Gemma Micro Food starting at 8 dpf.
The fish may have been overfed, causing the nitrate levels in the water and the fish itself to rise.
Gene Expression
Federal restriction of cannabis use/sale first occurred with the passage of the Marihuana Tax Act in 1937. The criteria for a substance to be designated as Schedule I are no currently accepted medical use in the United States, high potential for abuse, and lack of accepted safety when using the drug or other substance under medical supervision (Mead, 2017). The most recent legislative advancement in marijuana was with the passage of the Hemp Farming Act of 2018.
CBD is considered the non-psychoactive component of marijuana and has become the center of legality confusion, especially after the FDA approval of Epidiolex (a CBD-based epilepsy drug). To add another layer of complexity, the passage of the Hemp Agriculture Act of 2018 removed hemp from the list of controlled substances. Most knowledge about the negative effects of medical cannabis comes from limited clinical trial data and anecdotal studies of recreational users.
The vague indications and relatively high availability of the product can lead to overuse and misuse by patients (Sagy et al., 2018). Inconsistency within the two regulatory agencies of the FDA and the Environmental Protection Agency (EPA) has led to further confusion and risks associated with the medical cannabis industry as a whole. One question concerns how recreational users in states that allow recreational use of the drug would proceed with a new federal classification of marijuana.
As it stands, to address some of the major issues surrounding the marijuana industry, the drug needs to be federally legalized.
DISCUSSION OF MARIJUANA LEGALITY IN THE US
History
In the 19th and early 20th centuries, cannabis was widely used throughout the United States as a medicine and could be easily purchased at pharmacies and general stores. 34;Extractum Cannabis." Cannabis was listed as a treatment for various conditions such as neuralgia, tetanus, cholera, opiate addiction and convulsive disorders (Bridgeman & . Abazia, 2017). Prescriptions of the drug fell sharply after the passing of the law because doctors generally concluded , that it was easier not to prescribe marijuana than to struggle with the extra work imposed by this law (Pacula, 2002).
Following the 1937 law, cannabis was removed from the United States Pharmacopedia in 1942, causing the drug to lose its remaining therapeutic legitimacy. The level of control exercised by the Drug Enforcement Agency (DEA) is determined by a substance's classification into one of the five. In 2001, the Rohrabacher-Farr Amendment prohibited the Department of Justice from using federal funds to replace state laws in those states that have legalized the use of medical marijuana (U.S. Congress, 2019).
As of April 2019, 33 states and the District of Columbia have currently passed laws broadly legalizing marijuana in some form. This law removed hemp, a less potent cultivar of marijuana, from the list of controlled substances. The Hemp Bill 2018 defines hemp as all parts of the Cannabis sativa plant that do not exceed 0.3% THC by dry weight, including.
Prior to the passage of this bill, cultivated hemp was legal only federally under several state-sanctioned pilot programs.
Central Issues…
- Convoluted Law
- Adverse Effects of Cannabis Use
- Research Restrictions and Gaps
- FDA/EPA Regulation Inconsistency
In order to resolve the conflict, it is imperative to emphasize the importance of the role of science in this political debate. That being said, marijuana and its cannabinoid derivatives are classified as Schedule I drugs that currently have no accepted medical use, a high potential for abuse, and a lack of accepted safety for the use of the drug or other substance under medical supervision. This guidance led to the approval of the 2013 Cole Memorandum, which repealed the prosecution of marijuana in states where its use was legal.
Understanding the consequences of chronic cannabis use in terms of their sustainability and causality is inadequate and inconsistent. The FDA oversees approved cannabis medicines such as Epidiolex and Marinol, but does not regulate most medical marijuana products sold online or in dispensaries. The FDA's role in the drug approval and review process is designed to ensure that new drugs, including those derived from plants, are properly evaluated for safety, efficacy, and.
In another study conducted by the FDA in 2016, results showed that most online marijuana products contained little to no CBD, and other products contained much higher levels of THC than listed on the label (Mead, 2017). However, these claims are unfounded because federal regulation of the marijuana industry would allow for federal control. Removing marijuana from the CSA presents the greatest benefits to the industry as a whole.
Indiana University School of Medicine (2007). https://www.fda.gov/food/dietarysupplements/. Historical perspective on the medical use of. Cannabis for epilepsy: Ancient times to the 1980s. 1964) Isolation, Structure and Partial Synthesis of an Active Constituent of Hashish Journal of the American Chemical Society.
Potential Reform Options
- Federal Exemptions following State Compliance
- Rescheduling of Marijuana
- Removal of Marijuana from the CSA
Future of Marijuana Policy
The first and most vital step in the federal legalization process must be less restrictive research opportunities for marijuana. Once these questions are answered, the industry would be in a better position to defend and verify the therapeutic value of marijuana. Subsequently, the extensive and robust research will allow for the creation of an effective marijuana policy by scientists and legislators to ensure safety and stability.
It is unlikely that any short-term solutions will become the universal formula for the future of marijuana legalization in the United States, as it is abundantly clear that we do not have all the answers we need. What policies should be pursued to accelerate the research needed to fully exploit the therapeutic potential of marijuana. What will be the effects of chronic users of marijuana and how can they be remedied.
How could a comprehensive list of all possible drug interactions of marijuana and other substances be determined? Quantifying locomotor activity in larval zebrafish: considerations for the design of high-throughput behavioral studies. Altered mRNA expression for brain-derived neurotrophic factor and type II calcium/calmodulin-dependent protein kinase in the hippocampus of patients with intractable temporal lobe epilepsy.
Cannabidiol induces rapid and sustained antidepressant-like effects through increased BDNF signaling and synaptogenesis in the prefrontal cortex.
