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ICR State Fiscal Year 2019

The Institute of Cannabis Research (ICR) proposed a new research vision to get insights into fundamental cannabis biology and cannabinoids chemistry to address the intrinsic questions of why Cannabis plants produce the secondary metabolites, how they exploit the molecules for their survival, and what evolutionary benefits they provide.

The ICR research team has launched a genomic consortium for comparative genomics of hemp varieties to better understand genomic structures and genetic regulations underlying cannabinoids biosynthesis and agronomically important traits. ICR also leads a variety of research projects to explore a new agricultural/ therapeutic use of cannabidiol (CBD) using an invertebrate model organism.

An ongoing investigation into the effects of medicinal cannabis on seizures in adults with medically refractory epilepsy

Principal Investigator: Dr. Barbara Brett-Green, Psychology

The primary objective for this project is to continue an ongoing study into the effects of medicinal cannabis on seizures in adults with medically refractory epilepsy. The Principal Investigator has established a partnership with Realm of Caring, a nonprofit organization, to provide support services for patients, and iC42, a bioanalytics company, to process participants’ biological samples (blood and urine) for antiepileptic medication and cannabinoid levels. Additional funding awarded by the ICR substantially improved the quality of the original study; however, it also lengthened the original study’s time frame.

Challenging aspects of conducting this research include the six-month length of the study and the expected length of the participant enrollment period. The Principal Investigator and research team are actively engaged in recruiting participants for this study. Recruitment is expected to continue for between six to 12 months until 34 participants are enrolled.

Data analysis and the production of articles for peer-review are expected to continue beyond 2019. Since the ICR initially awarded funding for this study, the Principal Investigator has been engaged in numerous research-related activities, including presenting a progress report at the first annual ICR conference, producing a technical report for the Pueblo County Commissioners, submitting multiple abstracts for presentations at upcoming regional and national conferences, accepting invitations to present at national cannabis conferences and other conferences, and preparing a review article for publication in the upcoming ICR journal.

The primary objective is to keep the Principal Investigator’s lab fully operational so that data collection for this study can be completed. Specific aims are to continue to support the costs of participant stipends, personnel, re-assigned time for the Principal Investigator, travel to present research results at scholarly conferences, and publishing. The Principal Investigator also plans to apply for funding external to the ICR.

Effects of dietary hempseed (Cannabis sativa L.) on growth patterns, body composition, bone mineral density, and gut microbiota diversity in female C57BL/6J mice

Principal Investigator: Dr. Annette Gabaldon, Biology 

The objective of this study is to understand the effects of dietary hempseed (Cannabis sativa L.) ingestion on growth patterns, body composition, bone mineral density, and gut microbiota diversity in female C57BL/6J mice. Hempseed is a nutrient-dense food which contains high amounts of protein, carbohydrate, fiber, lipids, vitamins, minerals, and phytochemicals, but its use as a dietary aide is controversial in the U.S. due to the presence of cannabinoids present in small amounts. The oil from cold-pressed hempseed is mostly polyunsaturated and 

rich in Omega-3 and Omega-6 fatty acids. The pressed cake is rich in proteins and carbohydrate. Hempseed oil and seeds are beginning to appear in the U.S. market for human consumption, but its use in agricultural animal feed has not gained approval from the FDA. Most of the research on hempseed nutritional properties and health benefits to animals has come from regions where it is a major gran, such as China, Canada, Australia, Italy, and France. Here, we will investigate the influence that dietary hempseed has on growth parameters and intestinal microbiota diversity in young female mice. Forty mice will be randomly divided into four groups: control diet (CON, no hempseed), 5% hempseed in deity (HS5), 10% hempseed in diet (HS10), and 20% hempseed in diet (HS20).

The individual groups of mice will be fed their respective diets daily from ages five to 30 weeks and growth parameters will be evaluated at monthly intervals. Body composition analysis will be performed using dual energy x-ray absorptiometry (DEXA) scanning, which reports lean mass, fat mass, bone area, bone mineral content, and bone mineral density. Somatometric measurements will include body mass, length, and surface area. At bi-weekly intervals, the mice will be placed singly into metabolic cages for measurement of food intake and collection of feces for microbiota diversity testing by colony forming unit (CFU) assay. Endpoint measurements at 30 weeks of age will include blood plasma total antioxidant capacity, visceral organ component analysis, and DEXA scan on selected individual skeletal bones which will then be tested for mechanical strength properties.

This is a new area of ICR research, but it complements well two ongoing ICR pilot projects. Study 1 is investigating the influence of hempseed supplementation on probiotic growth and synthesis of secondary metabolites in cultured Lactobacillus strains. Study 2 is evaluating new bone formation by cultured human osteoblast (HOB) cells in the presence of estrogen and endocannabinoids (2-AG and anandamide). These studies will help us to gain a better understanding of the functional significance of industrial hempseed and cannabinoids to human nutrition and health.

Detecting cannabinoid-induced alterations to cellular metabolism using a Seahorse XF Bioanalyzer and monitoring its impact on viral replication

Principal Investigators: Dr. Annette Gabaldon, Biology, and Dr. Brian Vanden Heuvel, Biology

Cannabinoid receptors are ubiquitous and are found on cells all throughout the body. Endogenous, exogenous, or synthetic cannabinoids can bind to the receptors and trigger a cellular response. It has been shown that activation of the CB1 receptor in liver cells increases metabolic pathways including fatty acid synthesis and gluconeogenesis.

Current published literature has shown basic trends of metabolic changes during cannabinoid receptor activation, but a detailed analysis of which cellular pathways are involved and real-time data showing those changes is lacking. Last year the ICR funded the purchase and installation of a Seahorse Bioanalyzer to measure metabolic alterations in living cells. Originally led by Dr. Jordan Steel, the work resulted in protocols and optimization of the Seahorse for measuring mitochondrial manipulations in our human liver cell line.

The initial data from the Seahorse is very promising and indicates that cannabinoids are inducing specific metabolic changes in cells expressing the CB1 receptor. A better and clearer understanding of how cannabinoids alter cellular metabolism will have a huge impact on cannabis research and provide critical data on how medicinal or recreational marijuana use impacts cellular physiology. This proposal is seeking funding for the next three years to support the collection of this important data.

Thus far, ICR funding provided Dr. Steel with the essential resources to collect preliminary data, which resulted in oral and poster presentations at the 2017 ICR conference and also the Conference on Pharmacology and Drug Abuse in Boston, Massachusetts in September 2017. Dr. Steel also submitted a manuscript to the upcoming ICR journal to publish findings from the pilot study looking at the impact of cannabinoid-induced metabolic changes on viral replication. Dr. Annette Gabaldon and Dr. Brian Vanden Heuvel will continue the project for 2018-2019.

Rheological characteristics of hemp-based filament composites for 3D printing

Principal Investigator: Dr. Neb Jaksic, Engineering 

This research investigates flow characteristics of various hemp-based composites used in 3D printing. It is an expansion to the research already in progress. While the current research is based on the goal of optimizing the hemp-plastic ratio in filament composites, this proposed research emphasizes understanding of underlying principles and it should further help in material optimization. This application seeks funding mainly for an instrument necessary for this research. We are also seeking limited funding for faculty and students compensation. In particular, the funding is requested for one rheometer, two faculty members (one from Engineering and one from Chemistry), two undergraduate students, and one graduate student.

3D printing is a prototyping and manufacturing process where molten plastic filament is extruded from a nozzle creating a layer of a 3D object. A number of these layers are fused together to create 3D objects. The process may create complicated (or personalized) objects and assemblies that cannot be created by any other manufacturing method. To minimize the cost of filament material and to increase the strength of the created objects, different reinforcing agents can be added to the filament composite. We are studying hemp as a reinforcing agent because hemp is stronger than plastic and is less expensive. However, if the hemp particles in the filament are too large they can clog the 3D printing nozzle and therefore stop the process. This results in 

wasted material and time (time to clear the nozzle and time to re-print the object). If the hemp particles are too small, then the created objects are not as strong as they could be.

In addition, we are considering recyclability of 3D printed objects. Namely, the process of creating new filament by directly recycling 3D printed parts does not yield useful filament – such filament clogs 3D printers’ nozzles. This research aims to characterize hemp-based material before and after 3D printing from the “viscosity” point of view. This research will provide the following analyses of polymers and hemp-based composites used in 3D printers: 1) Rheological analysis (viscosity) of molten (varied temperatures) filament before and after 3D printing using HempBioPlastic (HBP@), hemp-PLA, hemp-ABS, hemp-Nylon, and wood-PLA; and 2) Polymer analysis (structure and configuration) of HempBioPlastic, hemp-PLA, hemp-ABS, hemp-Nylon, and wood-PLA liquid solutions using Liquid Chromatography (LC) methods with appropriate detectors. We will verify our results by using instruments from engineering and chemistry department. We plan to submit abstracts for the ICR Conference (2019 and 2020), present our results at the ICR conference, and submit two manuscripts for the Journal of Cannabis Research (2019 and 2020).

Big data analytics in cannabis research

Principal Investigator: Dr. Yoanna Long, CIS, Hasan School of Business

The primary objective of this research is two-fold. First, as a continuation of the current ICR-funded research, we will establish a data center to host and analyze cannabis-related data. As a component of the data center, during the next stage we will introduce data analytics functionalities based on the Apache Hadoop framework in order to analyze large volumes of cannabis data.

Second, to evaluate the data analytics capabilities of the cannabis data center, we have collected and will continue to collect from online discussion forums  of people seeking advice and support for their marijuana-quitting attempts. We believe that online user generated data, as one of the main contributors of big data, is appropriate for use in the context of our research. Artificial intelligence and text mining algorithms will be applied to analyze online message content.

This analysis results will help us gain insights into marijuana users’ online interaction behavior, which can lead to better designed interventions from healthcare professionals and better designed marijuana discussion forums conducive of support exchange. Additionally, the collected data will continue to be a valuable source for various academic research as well as course teaching materials for the Data Analytics emphasis of the Computer Information Systems program. The established cannabis data center will also be a valuable data repository and analytics platform for researchers of cannabis.

K12 cannabis research project

Principal Investigator: Tim Peters

This study builds upon the work of the 2017-2018 K12 Pilot Study. The proposed 2018-2019 project will continue the focus on K12 student cannabis use, programs to prevent cannabis use, and interventions to address student violations of cannabis laws. Data will be collected from schools to determine trends in cannabis use among middle and high school students in southern Colorado. Cannabis use among LGBT students will be explored and prevention programming based on trauma informed schools will be developed and implemented locally. Restorative Justice programs for students with cannabis related infractions will be implemented and studied in Pueblo City Schools District 60 and Pueblo County School District 70. 

The 2017 K12 Pilot Study analyzed high school student use of cannabis based on data from the 2013 and 2015 Healthy Kids Colorado Survey which collected self-reported data from over 15,000 students across the state. This current proposal will synthesize data on cannabis infractions in middle and high schools through collating the years of data gathered by school district officials and law enforcement agencies in southern Colorado.

The K12 Pilot Study also focused on studying cannabis prevention education in regional middle and high schools and the effectiveness of specific marijuana education curriculum. The current proposal targets student populations who have high rates of cannabis use, specifically LGBT youth. Increased illicit drug use among LGBT youth is often reported to be connected to the unique stress factors of being a sexually identified minority. This study aims to explore the relationship between cannabis use and trauma faced by LGBT students struggling with minority sexual identification and to develop trainings for high school staff on trauma informed schools. Focus groups will be conducted with LGBT students, teachers and staff to explore cannabis use and identify needs within the school. Trauma Informed Schools training will then be developed and provided for teachers, staff, CSU-Pueblo students, and alumni.

Studies of intervention practices continue with the development of Restorative Justice Practices in schools in Pueblo County. With the support of community stakeholders regarding Restorative Justice Practices, this proposal expands Restorative Justice Practices in schools 

within Pueblo County with the long-term goal of developing a Center for Restorative Justice on the CSU-Pueblo campus. During this grant period, partnerships with D60 and D70 middle and high schools will be strengthened, a cadre of educators and other community members will be trained in Restorative Justice Practices, a qualitative study of Restorative Justice Practices in Pueblo will be conducted, and a prospectus for a Center of Restorative Justice will be written.

Modulation of NMDA-Receptor Dependent Neurological Function by Dexanabinol

Principal Investigator: Dr. Jeff Smith, Biology

The broad objectives of this proposed research are to characterize the inhibition of GluN2B subunit-containing NMDA receptors in mouse brain by the cannabinoid Dexanabinol (HU-211) and to demonstrate its relevance for modulating NMDA receptor-dependent learning and memory in mice at both the electrophysiological and behavioral levels.

The specific aims are to: 1) Characterize inhibitory effects of HU-211 on the function of NMDA receptors in mouse brain slices using whole-cell patch clamp electrophysiology; 2) To evaluate the extent to which GluN2B-containing NMDA receptors are selectively targeted by HU-211 in mouse brain slices using whole-cell patch clamp electrophysiology; 3) to determine whether long-term potentiation is affected by HU-211 in mouse brain slices using field potential recording; and 4) To determine whether NMDA receptor dependent learning and memory in live mice is affected by administration of HU-211 during automated fear learning and memory experiments.

The proposed work is significant because it provides missing knowledge needed to advance the effective implementation of HU-211 as a selective therapeutic for brain disorders involving impaired learning and memory including depression, post-traumatic stress disorder (PTSD), stroke, brain injury, and dementia such as Huntington’s and Alzheimer’s diseases. The work is also important because it will support the education of two graduate and two undergraduate students in the neuroscience lab at CSU-Pueblo. The proposed work is also important because it will promote the establishment of a new and meaningful collaboration between the Principal Investigator and a notable neuroscientist at the University of Montana.

Applications of industrial hemp

Principal Investigator: Dr. Brian Vanden Heuvel, Biology

This project enables an inter-departmental, multi-faculty collaborative workflow that builds on 18 months of previous achievements by the principal investigator and co-principal investigators and established infrastructure through previous ICR funding. We believe that it is time to extend our work to applied projects.

Specifically, this proposal asks: 1) Can industrial hemp be used as a remediation tool for metals and metalloids from soil and municipal sewage sludge?; 2) Can cannabinoid extraction methods developed in previous ICR projects be scaled up to industrial volumes?; 3) Can waste products from industrial hemp production (stems/leaves) be a reliable source of important biopolymers?; and 4) Can the recently sequenced genomes for industrial hemp be a framework for continued research into what genes are turned on and off during different growth stages, affecting important traits like disease resistance, drought intolerance, yield, specific concentrations of CBCs, THC, or other secondary chemicals?


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