How exactly does CBD research work?

25. 04. 2024

Over the past few decades, a number of studies have been published on the health benefits of hemp products and CBD. However, not all of them can be given equal importance. There is a big difference between studies looking at the effects of cannabinoids cell culture studies and large-scale, double-blind clinical trials involving living subjects. We have now entered a new era of legalisation of cannabis and, as a result, there is a growing effort to fill the gaps in our knowledge of this plant. And it is equally important to find out as much as possible about the health effects of cannabinoids.

The importance of CBD research

Since cannabis prohibition has been repealed or relaxed in most of the Western world, considerable confusion has arisen about how best to regulate CBD and other hemp-based products. Pharmaceutical companies are racing to create and patent cannabinoid-based prescription drugs such as Epidiolex or Sativex. Producers of herbal extracts are struggling to keep up with the growing demands of the market, but at the same time are faced with unclear regulatory rules and regulations.

The prescription for the emerging difficulties is the gradual convergence of these two conflict areas, and with this some important questions arise:

What do we actually know about the short- and long-term safety of CBD use?
For what ailments can CBD be used?
Are there health problems that could be aggravated by CBD use?
How does CBD interact with prescription and non-prescription drugs?

Analysis of CBD research types

With the rapid development of the CBD industry, there are endless questions that scientists will seek answers to. Their conclusions will depend on thorough scientific research. Through various levels of research, the properties of CBD can be examined under the same rigorous standards of safety and efficacy as any other drug or plant extract.

CBD research falls into three main categories at five levels:

Level 1: Basic research - low-level research mapping the structural composition and chemical properties of cannabidiol
Level 2: In vitro studies (using cell cultures) - low-level research that begins to investigate the toxicity of a substance in living cells and tissues
Level 3: In Vivo testing (using real live organisms) - higher level research involving live animals
Level 4: Clinical trials - conducted in live human subjects in 4 different phases (I, II, III and IV)
Level 5: Meta-analysis - a review of published preclinical and clinical research, providing the highest possible level of evidence by combining data from similar studies.

So how does the scientific process work and what are the methods and stages of research that will ultimately tell us everything we need to know about the benefits and risks of CBD use? Let's take a closer look at the different levels of scientific research on CBD. Below, we offer a glimpse into the process responsible for the fantastic reports of CBD's effects on health ailments ranging from acne to cancer that appear almost daily.

Level 1: Basic research - is CBD safe to use?

CBD is generally considered safe and non-toxic. This is great news. But is CBD completely free of side effects? That's what basic research studies are looking into. So they don't examine the specific benefits of the compound, but they do assess how much of the compound is needed to produce toxic effects on the body, and they also examine the chemical composition of CBD and hemp extracts. A common method of assessing toxicity is to administer increasing doses of a compound (such as CBD) to rats until 50% of the sample population dies. This gives us the LD50 (lethal dose for 50% of the population). The rats are carefully recorded as they die, giving us an idea of the toxic side effects of the compound.

During the first stages of basic research, the structural, molecular and chemical properties of the substance, in this case CBD, are determined. The results of this early research tell us that CBD is soluble in fats but not in water and that its molecular formula is C21H30O2. CBD looks something like this: (here is a picture of the chemical formula of CBD)

Examples of studies on the structural properties of CBD

Study topic	Authors	Level of study
Structure of cannabidiol	Mechoulam et al., 1963	1
Separation of cannabinoids	Lerner., 1963	1
Identification of CBD subgroups	Harvey., 1976	1

A) Structure of cannabidiol

A 1963 study determined the chemical structure of CBD, based on three possible structures suggested by an earlier report, which confirmed important details such as the positions of certain molecular groups and the positions of double bonds between carbon atoms.

Advantages and disadvantages of this type of study:

Benefits	Disadvantages
Advances in knowledge of the molecular structure of CBD	No

B) Separation of cannabinoids

In 1963, a group of scientists proposed a new method for the rapid separation of the main c annabinoids in cannabis. The findings of this research are still used today to isolate various cannabinoids during research and development of cannabis products.

Advantages and disadvantages of this type of study:

Benefits	Disadvantages
It has made laboratory procedures for studying CBD more efficient	No

C) Identification of CBD subgroups

A 1976 study identified subgroups of CBD, its homologues. These are CBD molecules, but with an extra molecular group created during the extraction procedure. This information helps to determine more accurately the amount of CBD present in a sample of cannabis. This research has contributed greatly to the extraction process in the production of products with added CBD.

Advantages and disadvantages of this type of study:

Benefits	Disadvantages
Improved accuracy of CBD extraction procedures	No

Level 2: In vitro studies (live cell testing)

Once the structural and chemical properties of CBD are determined and catalogued, controlled amounts of the compound are introduced into cell and tissue cultures. Tissue cultures are created from live cells that are grown in test tubes and Petri dish (in vitro, literally translated as 'in glass'). The aim of this research is to see how CBD will interact with live cells in a highly controlled environment.

In vitro studies allow scientists to create precisely controlled environments in which all variables can be controlled, using a single cell or tissue type. At this stage of research, it is possible to design an experiment that investigates and answers a single question about CBD, which is not possible in a living human with many systems that interact and constantly interact with each other.

Examples of in vitro CBD studies

Study topic	Authors	Level of study
CBD and stroke	Hind et al., 2016	2
CBD and inflammatory bowel disease	Harvey et al., 2014	2
CBD and cancer cells	Ramer et al., 2010	2

A) CBD and stroke

There is evidence that CBD protects the blood-brain barrier, a selective filtering mechanism in the brain. Scientists are investigating whether CBD could be useful for patients recovering from a stroke, or whether it could prevent strokes from occurring in the first place.

A clinical study cannot be performed without sufficient basic research, so an in vitro experiment was performed using a tissue culture model composed of human blood-brain barrier cells. They first caused a "stroke" by depriving the experimental cells of oxygen, then measured the permeability of the barrier with and without CBD present.

Advantages and disadvantages of this in vitro study:

Benefits	Disadvantages
This study allowed scientists to look at key biomarkers in the brain associated with stroke, which is not possible directly in the brain cells of living human patients	The experimental model cannot accurately show the effects that might occur in the whole functioning human brain
The barrier model uses human cells

B) CBD and inflammatory bowel disease

This in vitro study used tissue culture from human colon cells to determine the potential anti-inflammatory benefits of CBD in patients suffering from inflammatory bowel disease (IBD). Researchers added inflammatory molecules to colon cells to induce inflammation and cause cell damage similar to that seen in IBD.

Then, three treatment lines were prepared in which one of the following treatment groups was used on the damaged cells:

Steroids - conventional treatment
Anandamide - a naturally occurring endocannabinoid produced by the body
CBD - the primary compound that scientists wanted to test

Results: cells improved with all three treatments. CBD and hydrokortison (corticosteroid) were similarly effective in attenuating the inflammatory response, and both agents were slightly more effective than anandamide.

Advantages and disadvantages of this in vitro model:

Benefits	Disadvantages
Very low cost model setup	Although this study used cultured human colon cells, it lacks a realistic view of the full immune response and other modulatory effects that would occur in an animal or human.
Using real human intestinal cells
The time frame of this study was significantly shorter than the clinical trials

C) CBD and cancer cells

Cannabis has been found to have anti-cancer effects, but the psychoactive effects of cannabis limit the usable dose. If CBD alone had anti-cancer effects, either alone or in combination with medical cannabis, it would be very beneficial. However, before cancer patients can be treated with CBD, preliminary studies need to be carried out, and so an in vitro experiment using cancer cell lines from two different types of highly invasive cancer: human cervical cancer and lung cancer, has been initiated to determine whether CBD could inhibit cells invading surrounding healthy cells. These cancers are known for their ability to aggressively infiltrate surrounding healthy tissue. The results confirmed reduced invasiveness of cancer cells (the ability to invade new tissue) within 24 hours, with the effects continuing for the 72-hour duration of the study.

In addition, four different concentrations of CBD were used, each 10 times higher than the previous one, and even at the lowest concentration level, CBD was able to significantly inhibit invasiveness.

Advantages and disadvantages of this study:

Benefits	Disadvantages
Scientists could control a single component of tumor cells independently while eliminating many factors that would be present in humans and could influence the results	Tumour cells in a petri dish behave differently to tumour cells in humans, so the data does not provide a complete picture of the effects of CBD on these cancers in live animals or humans
The results were known within hours, not days or even weeks

Level 3: In Vivo studies (live animal testing)

The next level of research is in vivo research, which takes place in live animals and allows scientists to obtain more accurate information about the efficacy, safety and toxicity of the test substance. Using real living creatures, scientists can test how a compound will affect the organism as a whole. In vivo testing allows the effects to be compared across different species. Mammals are usually chosen for in vivo medical research because their physiology is most similar to ours.

Research progresses gradually from rodents (mice, rats, rabbits, etc.) to larger species (cats, dogs, non-human primates). This may reveal how responses vary from one species to another, and it may also help researchers narrow down a safe initial dose when it comes time to test on humans. With this information, we can understand how CBD is likely to penetrate into and through the body and how much the body can safely handle. This information is being used as the basis for the next phases of research that look more closely at the actual effects that CBD has on the body.

In vivo research finds the following:

How CBD moves through the body - how quickly and efficiently it is absorbed into the bloodstream
CBD breaks down - whether it's in the kidneys, liver or elsewhere in the body
How safe or toxic is CBD and its metabolites - sometimes a compound appears safe in in vitro research but shows toxic effects when administered to live animals. For example, there is currently no known LD50 value for CBD in humans. However, a 2011 study found that doses of 200-300 mg/kg were lethal in some rhesus macaque monkeys.
How the compound is excreted from the body - compounds can leave the body in urine, faeces or even through the skin
CBD's effects on certain ailments and diseases - using animals with health problems, we can test CBD to see if it improves or worsens the condition A potential limitation of in vivo research on CBD is that each species contains different numbers of receptors. Although all mammals have endocannabinoid system, the density and distribution of endocannabinoid receptors can vary greatly between species, and thus the effects of CBD will vary.

For example, rats and rhesus macaque monkeys have a higher density of cannabinoid receptors in some parts of their brains than humans and a lower density in other parts. This makes it difficult to extrapolate how CBD will affect humans compared to an experimental animal. During and after research, it is important to remember that drugs that prove safe and effective in animal testing may have different effects on humans.

Examples of in vivo studies

Study topic	Authors	Level of study
CBD in wound healing	Klein et al., 2018	3
CBD in the treatment of fear and anxiety	Norris et al., 2016	3
CBD in the treatment of epilepsy	Jones et al., 2010	3

A) CBD in wound healing

An in vivo study has been conducted on rats looking at the ability of CBD to accelerate wound healing. Animals that suffered small wounds were given CBD injections into their abdomens daily for 7 days. By the third day, the CBD-treated lesions were significantly less inflamed. However, the improvement did not continue during the 7-day experiment, and on the last day, the CBD-treated wounds had similar levels of inflammation to the control group.

Advantages and disadvantages of this in vivo model:

Benefits	Disadvantages
This test worked with the whole immune system of the animal, not with individual isolated cells.	Test subjects were given CBD by injection, which is not a common way for people to use CBD.
This study provided insight into the different stages of inflammation, not just one isolated process.	The study used synthetic CBD and not naturally extracted CBD.
	The CBD extract in the study did not contain any other cannabinoids or hemp terpenes.
	The study lasted only 7 days and thus did not cover all the time necessary for wound healing.

B) CBD in the treatment of fear and anxiety

CBD is known for its calming effects on the nervous system. To find out if CBD can help reduce fear and anxiety, the researchers began an in vivo experiment in which they induced fear in a group of rats, and then injected CBD directly into areas of the brain controlling emotional control.

They found that CBD helped reduce the fear response by activating receptors for serotonin, one of the brain's calming neurotransmitters. To see if CB1 receptors were also involved, they set up a branch of the experiment in which they blocked CB1 receptors. They achieved the same results, suggesting that the effect on serotonin was carried solely by CBD. In another part of the study, they examined the effect of CBD on dopamine, a brain neurotransmitter that enhances the fear response, and GABA, which has calming effects. They found that CBD reduced activity in dopamine-promoting areas while increasing GABA activity. In addition, they used a number of different doses so they were able to determine the most effective one.

Advantages and disadvantages of this in vivo model:

Benefits	Disadvantages
The use of animals has allowed scientists to observe behavioural changes in response to induced fear instead of looking for physiological changes.	Injecting CBD directly into the brain does not reflect how CBD is commonly taken.
CBD was injected directly into the brains of the rats, ensuring that high doses were achieved.
The researchers were able to set up a number of separate study arms and collect a significant amount of data easily and simply in a laboratory setting.

C) CBD in the treatment of epilepsy

One of the most studied effects of CBD is its anti-seizure effect. An in vivo study used rats in which an epilepsy-like condition was induced by blocking the activity of the brain-soothing neurotransmitter GABA. The rats were then given one of three different doses of CBD and seizure activity was recorded. When the results were evaluated, it showed that CBD did not prevent seizures, but at the highest dose level, the severity of seizures was dramatically reduced, with 53% of the most severe seizure types without CBD administration dropping to just 7% in CBD-treated animals.

Advantages and disadvantages of this in vivo model:

Benefits	Disadvantages
Animal model effectively simulated the type of disorder in humans	The method of administration, injection into the abdomen, does not reflect the way most patients will use CBD.

Level 4: Clinical research (human testing)

Once sufficient in vitro and in vivo research has been conducted to determine whether the use of CBD is reasonably effective and safe for a particular purpose, clinical trials, that is, experiments on human volunteers, can begin. Clinical research is considered the highest level of experimental research because it is conducted directly on human subjects. These studies can be used to confirm pharmacokinetics from in vivo studies and to test whether the benefits observed in laboratory animals can be replicated in humans.

Clinical studies can be used to assess the following characteristics:

The pathways by which CBD passes through the body, from absorption to distribution to excretion
Clinical effects of CBD (i.e. its health benefits), absence or presence of side effects
Optimal dose required for effective use of CBD in humans
The quality of clinical trials can vary widely and there are some key elements to consider when assessing the quality of a particular study.

Control group taking placebo

A placebo control is an essential part of a clinical trial to ensure that the results of the study were not due to the participants' expectations. This control is done by giving one group a sham treatment (an inactive compound that looks like CBD but is not). This procedure is designed to eliminate the so-called placebo effect, in which symptoms may improve simply because the patient believes he or she has received the drug, even though there were no active compounds in the substance used. The placebo effect has been found to occur in about 40% of study participants.

Randomization

To ensure that there are no patterns in either the treatment group or the placebo control group that could affect the results, statistical randomization is used to allocate participants. This process ensures that participants are evenly distributed across each arm of the study. Randomisation prevents all participants with a particular characteristic, such as age, gender or genetic risk factor, from being assigned to the same group. It also prevents the experiment from being influenced by conscious or unconscious biases of the researcher, such as placing all participants most likely to give a good (or bad) response in the same group.

Blinded

Blinding is used to keep study participants and researchers from knowing which participants are receiving CBD and which are receiving placebo.

There are two categories of blindness:

Simply blinded study: the participant is not informed about the treatment they are receiving This prevents participants' preconceived ideas or expectations from influencing their responses. Simple blinding can help eliminate the placebo effect. Many studies also rely on participants' subjective reports of their symptoms and overall experience. Simple blinding eliminates these potential perceptions. If people think they are receiving the treatment, they may answer questions differently than if they knew they were taking a placebo. So it is best to keep this part concealed until the study is over.

A double-blind study: neither the participant nor the researcher knows which treatment each participant is receiving. This prevents the researcher from consciously or subconsciously treating participants differently and thus influencing the results of the study. The best clinical trials use double-blinding in their research design.

Blinding is also important for statistics

In addition, even the statistician, a member of the study team who is crunching the numbers to determine the results of the experiment, should not be aware of the groupings until all the numbers have been evaluated. At any point in a clinical trial, the trial can be stopped if the drug is judged to be ineffective or too toxic and cannot be safely used by humans for its intended purpose. For pharmaceutical companies trying to develop a CBD-based drug, there are four phases of clinical trials that a drug must go through before it can be marketed for human administration.

Different levels of clinical trials

A) Phase I

Phase I studies are the first studies in which the drug is given to a small group of people (up to 80). In this phase, the best route of administration, i.e. oral, inhalation, external administration, injection into the bloodstream, etc., as well as the ideal dosage levels and frequency are determined. If this study proves to be safe and shows improvement, the study may proceed to phase II.

B) Phase II

During the Phase II study, further safety knowledge is being gained and efficacy data are being collected. These studies involve larger numbers of participants, usually between 100 and 300.

C) Phase III

Phase III clinical trials compare a new drug with a drug that is commonly used as the standard of care for specific conditions. These studies use large numbers of participants (from 300 to 3,000 or more). Once the drug is approved (in our country, this is the responsibility of the State Institute for Drug Control - SÚKL), the manufacturer may decide to conduct a fourth phase of testing, or it may be required by regulatory authorities.

D) Phase IV

Phase IV clinical trials are looking at long-term side effects and examining the risk-benefit ratio for someone who may need to take the drug for a long time. These studies use the largest number of people, with participants potentially numbering in the thousands. They usually take place over several months or even years, during which time long-term health data is collected.

E) Epidemiological research

Once a CBD-based medicine is approved by the authorities, it can be included in epidemiological research. Epidemiology is the study of factors affecting the health of a population.

Level 5: Meta-analysis (comparison of data from multiple studies)

A meta-analysis is a type of systematic review (study of studies) in which data from multiple studies on a particular aspect of CBD are combined and evaluated together. A meta-analysis may include preclinical or clinical studies. The higher the level of the studies used in the meta-analysis, the stronger the evidence for a particular finding. For example, a meta-analysis that uses double-blind, placebo-controlled, randomised human studies will provide stronger evidence than a meta-analysis of animal studies.

Summary: Types of CBD Research

Cannabis research has been restricted for nearly 80 years due to prohibition. Scientists are now working hard to fill knowledge gaps, but at the same time the regulatory environment is not entirely clear and the information available to CBD users can be confusing.

Scientists are using different research models to answer some of the biggest questions surrounding CBD use:

How safe is CBD?
How can CBD help?
Can CBD make some diseases worse?
How does CBD interact with drugs?

Knowing the procedures and reasons behind scientific research can help you understand the latest news about CBD so you can become an informed consumer.

Author: Canatura

PHOTO: Shutterstock

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