**3. Developing models**

#### **3.1. Pain**

The treatment of chronic pain is the largest indication for medical cannabis [62–67]. This is not surprising given that endocannabinoids are known to act as retrograde transmitters blocking the transmission of pain signals at both GABAergic and glutamatergic synapses [68]. Unfortunately the etiology of pain is vast and thus there is not an all-encompassing treatment for pain. Often, an analgesic is only effective for a subset of patients or can only partially reduce pain, but cannot eliminate it [69, 70]. This often leads to multiple drugs being used in combination, which opens the door to various drug interactions that can lead to a number of potential adverse effects and an increased side effect profile.

It is now widely accepted that zebrafish have similar somatosensory systems to higher vertebrates and they can detect painful stimuli (nociception) [71–83]. The models that have been developed vary and include thermal and chemical stimuli that is either bath applied or focally by injection. The models also make use of both acute and chronic nociceptive stimuli and have been developed using larvae and adults. This then provides a number of platforms with which to test potential analgesics that may have links to different disease etiologies.

Recently, a novel model of nociception has been developed and used to test and compare a number of known therapeutics with THC and CBD. The model made use of a short-term exposure to acetic acid which led to tissue damage on the surface of zebrafish larvae and a distinct, reproducible, activity pattern that appears to indicate a multifaceted nociceptive response [83]. The study revealed that THC and CBD had different effects on the behavioral response pattern that varied from those of the known analgesics. Interestingly, of the compounds tested CBD had the most unique effect increasing the rate at which the larval activity pattern returned to that of controls. This would seem to suggest that CBD shortened the recovery from the nociceptive stimulus. This is consistent with literature that has suggested CBD is a strong candidate for pain management [84–86]. Importantly, this activity was found at a concentration of CBD that had no effect on baseline activity for controls, suggesting that there would be a low potential for side effects. One of the major issues surrounding the therapeutics currently used for pain management lies in their side effect profile, which is often vast and can range from relatively minor (constipation) to severe (addiction). This is especially evident for opioids, which are the most commonly used therapeutic for chronic pain, but have one of the highest addictive potentials [87]. While more work is required to test the effect of other cannabinoids and extracts on the various zebrafish models of nociception, the initial indications are that zebrafish will be valuable for assessing the efficacy of potential therapeutics for pain management.

#### **3.2. Addiction**

[60]. A previous study from the same group evaluating spatial memory and found that THC exposure did not affect associative memory but did impair spatial cognition and memory retrieval [60]. In addition to THC, high levels of CBD also appear to reduce memory retention in a spatial memory test [61]. While the number of studies testing the role of cannabinoids on learning and memory using zebrafish is currently limited, it appears the model has great potential in assessing the role of the endocannabinoid system in multiple aspects of learning

The treatment of chronic pain is the largest indication for medical cannabis [62–67]. This is not surprising given that endocannabinoids are known to act as retrograde transmitters blocking the transmission of pain signals at both GABAergic and glutamatergic synapses [68]. Unfortunately the etiology of pain is vast and thus there is not an all-encompassing treatment for pain. Often, an analgesic is only effective for a subset of patients or can only partially reduce pain, but cannot eliminate it [69, 70]. This often leads to multiple drugs being used in combination, which opens the door to various drug interactions that can lead to a number of

It is now widely accepted that zebrafish have similar somatosensory systems to higher vertebrates and they can detect painful stimuli (nociception) [71–83]. The models that have been developed vary and include thermal and chemical stimuli that is either bath applied or focally by injection. The models also make use of both acute and chronic nociceptive stimuli and have been developed using larvae and adults. This then provides a number of platforms with which to test potential analgesics that may have links to different disease

Recently, a novel model of nociception has been developed and used to test and compare a number of known therapeutics with THC and CBD. The model made use of a short-term exposure to acetic acid which led to tissue damage on the surface of zebrafish larvae and a distinct, reproducible, activity pattern that appears to indicate a multifaceted nociceptive response [83]. The study revealed that THC and CBD had different effects on the behavioral response pattern that varied from those of the known analgesics. Interestingly, of the compounds tested CBD had the most unique effect increasing the rate at which the larval activity pattern returned to that of controls. This would seem to suggest that CBD shortened the recovery from the nociceptive stimulus. This is consistent with literature that has suggested CBD is a strong candidate for pain management [84–86]. Importantly, this activity was found at a concentration of CBD that had no effect on baseline activity for controls, suggesting that there would be a low potential for side effects. One of the major issues surrounding the therapeutics currently used for pain management lies in their side effect profile, which is often vast and can range from relatively minor (constipation) to severe (addiction). This is especially evident for opioids, which are the most commonly used therapeutic for chronic pain, but have one of the highest addictive potentials [87]. While more work is required to test the effect of other cannabinoids and extracts

and how this can be influenced by various cannabinoids.

potential adverse effects and an increased side effect profile.

**3. Developing models**

18 Recent Advances in Cannabinoid Research

**3.1. Pain**

etiologies.

Recent data suggests that approximately 9% of individuals that use cannabis show symptoms associated with addiction, including tolerance and withdrawal [88]. Comparatively the rate of dependence for tobacco is 67.5% and for alcohol is 22.7% [89]. Zebrafish represent an underutilized model with which to study the addictive properties of cannabinoids. While it has been demonstrated that zebrafish can be used to study the pathology of addiction to numerous drugs of abuse, including, alcohol, cocaine, morphine, nicotine, amphetamine, diazepam and salvinorin A [90–95], thus far their use to study the addictive properties of cannabinoids has been minimal. Changes in both larval and adult zebrafish behavior can be linked to numerous phenotypes associated with addiction that include conditioned place preference for drugs of abuse, relapse, changes in social behavior, along with symptoms indicating the development of tolerance and withdrawal [90, 93, 96–99]. It has also been found that the genetic pathways linked to addiction are highly conserved in zebrafish [100]. Currently, with respect to cannabinoids, only one study has shown that zebrafish larvae develop tolerance to the effects of cannabinoids after chronic exposure [101]. As the levels of THC in cannabis plant strains is varied and the refinement and extraction processes allow for other cannabinoids to be used at higher levels both medicinally and recreationally, there is a need to develop models with which to test the addictive properties of both pure cannabinoids on their own, in combination and as part of a complex mixture or extract. Zebrafish have the potential to be such a model.

#### **3.3. Stress and anxiety**

One of the known difficulties in using cannabinoids as therapeutics lies in their effect on stress and anxiety. A sought after symptom of cannabis use is the euphoric feeling that often leads to it being considered an anxiolytic. However, it has been broadly shown that as the levels of cannabinoids (specifically THC) are elevated there can be an increase in anxiety-related effects [102]. This is important not only from a side effect perspective, but also becomes an issue when cannabinoids are used to treat anxiety related disorders such as PTSD.

Zebrafish provide numerous models with which to assess stress responses in both larvae and adults. Measurements such as scototaxis (light-dark preference), thigmotaxis (wall hugging), shoaling and the amount of time spent in the bottom of a tank are used as standard measures of stress. Induction of stress can occur by chemical means such as neuro-hyperactive compounds or exposure to the alarm substance. Stress can also be induced physically by touch or following the placement of a fish in a novel setting (novel tank response). Various visible stimuli can also lead to stress responses such as changes in back ground light/dark levels or the appearance of an image of prey. All of these models seem to activate both unique and overlapping neural pathways and thus could provide insight into the mechanism of action of any potential anxiolytic effect [103–105]. An example of the use of zebrafish stress models for testing cannabinoids was outlined in a recent paper that evaluated the acute effects of both THC and CBD on larval behavior [106]. Zebrafish larvae show a preference for light and a transition from a light to a dark setting results in an increase in activity in the form of darting type movements which are thought to be a stress response. It was found that while THC reduced the baseline activity in the light, the response to a light-dark transition was still evident. Exposure to CBD had a much different response with almost no effect on the baseline activity in the light accompanied by a concentration-dependant reduction in the light-dark transition until it was eliminated. This may suggest that CBD is showing anxiolytic effects at the levels tested [107].

anti-seizure medications or drug cocktails. In general this leads to an ever increasing side effect profile that is often debilitating in and of itself. The treatment of seizures is one of the oldest reported uses of cannabis and it has recently garnered attention along with the use of pure cannabinoids (CBD) for their ability to treat severe forms of refractory childhood epilepsy (i.e. Dravet syndrome [112]). However, to date there still remains some controversy regarding its efficacy, with some groups suggesting there is no concrete evidence that it is effective [113]. While it has been purported that cannabinoids, in particular CBD, can mitigate, to some degree, epileptic seizures, unfortunately, with the exception of the childhood epilepsy study [112], the numerous human studies that have evaluated the effect of cannabinoids on seizures have either been from small sample groups, had insufficient controls or were not blinded, which confounds any potential outcomes of the studies [114]. The study was able to show that there was a reduction in seizure frequency in patients with Dravet syndrome following the addition of CBD to their current prescription regime. The one question that does remain is whether the reduction in seizures was due to the direct effect of CBD or if the effect was due

Zebrafish as a High-Throughput In Vivo Model for Testing the Bioactivity of Cannabinoids

http://dx.doi.org/10.5772/intechopen.79321

21

The lack of high-quality human trials for testing anti-epileptics stem from the difficulty in properly designing and/or interpreting the results of human studies. This is partially due to the fact that most study participants are already on another anti-epileptic drug, which often varies between participants in either the drug target or the dosage. During the course of the clinical trials often the levels of either the cannabinoid or the existing therapeutic have to be modified for an individual in order to resolve issues relating to side effects. This makes the proper group-

There are currently a number of zebrafish models of epilepsy that have been generated to provide a platform for identifying new seizure medications and potentially to understand the etiology of the disease [115]. For instance, a number of small molecules that target different receptors or ion channels can be used to induce seizures or neural hyperactivity in larvae [103, 116, 117]. These platforms provide high throughput testing models with multiple etiologies. While CBD has been shown to be effective in the treatment of some forms of epilepsy, the mechanism of action is still largely unknown. The existing zebrafish seizure models provide multiple platforms with which to evaluate both the efficacy and potentially the mechanism of action for cannabinoids in the treatment of epilepsy. The further development of these models will be of great benefit for discerning the true therapeutic potential of various cannabinoids

Currently the main delivery method for cannabinoids both medicinally and recreationally is by the inhalation of smoke from marijuana cigarettes. This is generally because of the rapid onset of effects compared with other delivery methods, which is beneficial from the perspective of symptom relief and also allows for a level of self-regulated dose control that is not possible with other delivery methods such as edibles, which can often take up to 90 min to reach peak effect [111]. Unfortunately, some of the major caveats to an inhaled product are that dosing is often inconsistent and difficult to titrate and they have the potential to have similar health risks as are found for smoked tobacco [118]. It has been suggested that high

to the effect of CBD on the patient's current medication.

ing of different treatment regimens difficult.

for the treatment of epilepsy.

**3.6. Smoke toxicity**

It is currently felt that there is insufficient evidence to support the use of cannabis for the treatment more complex stress disorders such as PTSD [108]. Recently work has begun to establish zebrafish models of complex disorders such as PTSD [109, 110]. The development of these models will provide additional systems with which to test the efficacy of various cannabinoids and combinations thereof in the treatment of anxiety related disorders and may help provide insight into their etiology.

#### **3.4. Uptake and metabolism**

The adsorption and bioavailability of cannabinoids provides a challenge for their use as therapeutics. This is particularly true for orally ingested cannabinoids, which show low and, at times, unpredictable bioavailability [111]. The interaction between various cannabinoids along with their interaction with other therapeutics can affect their bioavailability. This is important since the effects of various cannabinoids can be bimodal (hyperactivity at low concentrations and sedation at higher concentrations). Having the ability to measure their uptake, bioaccumulation and excretion will provide insights into the exact levels found within the fish. Knowing the true concentration response profile based on the amount of compound found within zebrafish may also allow for comparisons to be made to the dose-response patterns found for mammals.

Previous work has shown that testing the uptake, metabolism and secretion of cannabinoids is possible using zebrafish larvae [106]. A number of important findings came from this study. First it was found that common pharmacokinetic cannabinoid metabolites are produced by the zebrafish larvae including the phase 1 and phase 2 metabolites hydroxylated THC (11-hyrdoxy-THC, 8-hydroxy-THC), 11-nor-9-carboxy THC, THC-glucuronide, hydroxyl-CBD and CBD-glucuronide. Both the cannabinoids and their metabolites were found to accumulate in the larvae with the metabolites eventually excreted into the bath. It was also shown that there appeared to be bioaccumulation of the cannabinoids in the larvae and a non-linear increase in the amount found in the larvae compared with the bath levels. The same study also revealed that when THC and CBD were co-administered the levels of metabolites that were produced was altered compared to when they were administered alone. This suggests that the complex chemical composition of various cannabis plant strains will also affect the normal metabolism of the individual cannabinoids. It then appears that it will be important to evaluate the uptake kinetics and metabolism of various cannabis derived compounds both alone and in combination.

#### **3.5. Seizures**

Approximately 1% of the world's population is purported to have epilepsy with 30% of those affected having multi-drug resistant epilepsy. This often leads to the requirement for strong anti-seizure medications or drug cocktails. In general this leads to an ever increasing side effect profile that is often debilitating in and of itself. The treatment of seizures is one of the oldest reported uses of cannabis and it has recently garnered attention along with the use of pure cannabinoids (CBD) for their ability to treat severe forms of refractory childhood epilepsy (i.e. Dravet syndrome [112]). However, to date there still remains some controversy regarding its efficacy, with some groups suggesting there is no concrete evidence that it is effective [113]. While it has been purported that cannabinoids, in particular CBD, can mitigate, to some degree, epileptic seizures, unfortunately, with the exception of the childhood epilepsy study [112], the numerous human studies that have evaluated the effect of cannabinoids on seizures have either been from small sample groups, had insufficient controls or were not blinded, which confounds any potential outcomes of the studies [114]. The study was able to show that there was a reduction in seizure frequency in patients with Dravet syndrome following the addition of CBD to their current prescription regime. The one question that does remain is whether the reduction in seizures was due to the direct effect of CBD or if the effect was due to the effect of CBD on the patient's current medication.

The lack of high-quality human trials for testing anti-epileptics stem from the difficulty in properly designing and/or interpreting the results of human studies. This is partially due to the fact that most study participants are already on another anti-epileptic drug, which often varies between participants in either the drug target or the dosage. During the course of the clinical trials often the levels of either the cannabinoid or the existing therapeutic have to be modified for an individual in order to resolve issues relating to side effects. This makes the proper grouping of different treatment regimens difficult.

There are currently a number of zebrafish models of epilepsy that have been generated to provide a platform for identifying new seizure medications and potentially to understand the etiology of the disease [115]. For instance, a number of small molecules that target different receptors or ion channels can be used to induce seizures or neural hyperactivity in larvae [103, 116, 117]. These platforms provide high throughput testing models with multiple etiologies. While CBD has been shown to be effective in the treatment of some forms of epilepsy, the mechanism of action is still largely unknown. The existing zebrafish seizure models provide multiple platforms with which to evaluate both the efficacy and potentially the mechanism of action for cannabinoids in the treatment of epilepsy. The further development of these models will be of great benefit for discerning the true therapeutic potential of various cannabinoids for the treatment of epilepsy.

#### **3.6. Smoke toxicity**

thought to be a stress response. It was found that while THC reduced the baseline activity in the light, the response to a light-dark transition was still evident. Exposure to CBD had a much different response with almost no effect on the baseline activity in the light accompanied by a concentration-dependant reduction in the light-dark transition until it was eliminated. This

It is currently felt that there is insufficient evidence to support the use of cannabis for the treatment more complex stress disorders such as PTSD [108]. Recently work has begun to establish zebrafish models of complex disorders such as PTSD [109, 110]. The development of these models will provide additional systems with which to test the efficacy of various cannabinoids and combinations thereof in the treatment of anxiety related disorders and may

The adsorption and bioavailability of cannabinoids provides a challenge for their use as therapeutics. This is particularly true for orally ingested cannabinoids, which show low and, at times, unpredictable bioavailability [111]. The interaction between various cannabinoids along with their interaction with other therapeutics can affect their bioavailability. This is important since the effects of various cannabinoids can be bimodal (hyperactivity at low concentrations and sedation at higher concentrations). Having the ability to measure their uptake, bioaccumulation and excretion will provide insights into the exact levels found within the fish. Knowing the true concentration response profile based on the amount of compound found within zebrafish may also allow for comparisons to be made to the dose-response patterns

Previous work has shown that testing the uptake, metabolism and secretion of cannabinoids is possible using zebrafish larvae [106]. A number of important findings came from this study. First it was found that common pharmacokinetic cannabinoid metabolites are produced by the zebrafish larvae including the phase 1 and phase 2 metabolites hydroxylated THC (11-hyrdoxy-THC, 8-hydroxy-THC), 11-nor-9-carboxy THC, THC-glucuronide, hydroxyl-CBD and CBD-glucuronide. Both the cannabinoids and their metabolites were found to accumulate in the larvae with the metabolites eventually excreted into the bath. It was also shown that there appeared to be bioaccumulation of the cannabinoids in the larvae and a non-linear increase in the amount found in the larvae compared with the bath levels. The same study also revealed that when THC and CBD were co-administered the levels of metabolites that were produced was altered compared to when they were administered alone. This suggests that the complex chemical composition of various cannabis plant strains will also affect the normal metabolism of the individual cannabinoids. It then appears that it will be important to evaluate the uptake kinetics and metabolism of various cannabis derived compounds both

Approximately 1% of the world's population is purported to have epilepsy with 30% of those affected having multi-drug resistant epilepsy. This often leads to the requirement for strong

may suggest that CBD is showing anxiolytic effects at the levels tested [107].

help provide insight into their etiology.

**3.4. Uptake and metabolism**

20 Recent Advances in Cannabinoid Research

found for mammals.

alone and in combination.

**3.5. Seizures**

Currently the main delivery method for cannabinoids both medicinally and recreationally is by the inhalation of smoke from marijuana cigarettes. This is generally because of the rapid onset of effects compared with other delivery methods, which is beneficial from the perspective of symptom relief and also allows for a level of self-regulated dose control that is not possible with other delivery methods such as edibles, which can often take up to 90 min to reach peak effect [111]. Unfortunately, some of the major caveats to an inhaled product are that dosing is often inconsistent and difficult to titrate and they have the potential to have similar health risks as are found for smoked tobacco [118]. It has been suggested that high doses of THC containing products are associated with an increased risk of developing respiratory infections [119]. However, it has been difficult to establish a clear relationship between smoked cannabis and more severe lung disorders, such as cancer, since tobacco use is often co-morbid with cannabis. While in general the number of smoked cannabis cigarettes is lower on a per day basis, this risk cannot be overlooked. Additionally, the inhalation delivery methods for cannabis smoke are somewhat more diverse than for tobacco and include pipes, water pipes, burning on metal and vaporizers. All of these delivery methods produce smoke with its own set of chemical characteristics that depend on the temperature at which the smoke was created and any filtering that occurred before the smoke was inhaled. Processing of the plant material into oils or resins before combustion adds another level of complexity to the potential chemical diversity of the smoke that is inhaled.

of interacting with other therapeutics. It has been suggested that large-scale zebrafish behavioral testing models can be used to help discern the polypharmacological mechanisms of neuroactive compounds [126]. This provides an ideal platform with which to test cannabis derivatives.

Zebrafish as a High-Throughput In Vivo Model for Testing the Bioactivity of Cannabinoids

http://dx.doi.org/10.5772/intechopen.79321

23

One of the unique characteristics of researching the effects of cannabis and cannabinoids using various models and for various disease indications is that often there is already clinical data on the effects in humans. While much of the data is often anecdotal in nature, it does allow for animal model testing to be used to back validate the findings of the clinical trials. By designing top-down translational research studies we can begin to elucidate the biological basis of the clinical findings and potentially provide information on the mechanism of action of therapeutic compounds. This is particularly true for cannabis uses where the cannabinoid mechanism of action is often difficult to discern. The use of animal models of disease may

As outlined in this chapter, zebrafish have an established endocannabinoid system that is highly analogous to that of humans. Additionally, as a model system both adults and larval zebrafish provide numerous models of disease that have been shown to be efficacious for testing the therapeutic potential of novel compounds. Importantly the response patterns in these various disease models following exposure to different cannabinoids reveal unique characteristics for each cannabinoid. Thus far, only a limited number of these models have been used to test the efficacy of cannabinoids. However, the framework is in place for an expansion of

[1] Steenbergen PJ, Richardson MK, Champagne DL. The use of the zebrafish model in stress research. Progress in Neuro-Psychopharmacology & Biological Psychiatry. 2011;

[2] Howe K et al. The zebrafish reference genome sequence and its relationship to the

[3] Grunwald DJ, Eisen JS. Headwaters of the zebrafish—Emergence of a new model verte-

help to elucidate these mechanisms and further define the etiology of the disease.

**4. Discussion**

the use of zebrafish in this field.

Address all correspondence to: lee.ellis@nrc-cnrc.gc.ca

human genome. Nature. 2013;**496**(7446):498-503

brate. Nature Reviews. Genetics. 2002;**3**(9):717-724

National Research Council of Canada, Halifax, Nova Scotia, Canada

**Author details**

Lee Ellis

**References**

**35**(6):1432-1451

Zebrafish larvae are an established model for testing vertebrate toxicity, teratogenicity and environmental risk assessment [120–122]. The use of these models has proven to be a valuable resource for testing the toxicity of various extracts and condensates obtained from tobacco cigarette smoke [6, 123, 124]. It was found that smoke from tobacco cigarettes was more toxic and produced different phenotypes than that of nicotine alone, suggesting, that the other toxic components found in tobacco smoke are having an effect. The use of the previously validated smoke testing models for testing cannabis smoke has the potential to provide information on developmental, cardiac, behavioral/neural and acute toxicity.

#### **3.7. Multi-drug interactions and polypharmacology**

One of the major complexities of working with cannabis is the fact that it is currently known to be comprised of 500+ constituents and more than 100 cannabinoid molecules [125]. The potential of interactions between many of these compounds is high and has been widely demonstrated for THC and CBD. It has been shown that the zebrafish can be used to assess the interaction of THC and CBD with respect to their effects on locomotor activity along with the uptake and metabolism of each compound [106]. Many of the aforementioned zebrafish models have the potential to be used to assess the potential interaction of THC and CBD and likely other cannabinoids. This is important as one of the next steps in the use of the zebrafish models for testing cannabinoids is to begin to test various extracts and isolates from cannabis for their bioactivity. Having an understanding of how the pure compounds interact in an *in vivo* system and how this relates to their activity in complex mixtures derived from plant material will be extremely valuable. In addition to the interaction of the various compounds found within cannabis, the use of cannabis or cannabinoids as therapeutics is also complicated by the fact that many patients are already taking a prescription drug for their particular indication. The zebrafish testing platforms appear to have the potential to characterize some of these interactions as well.

Understanding the pharmacokinetics and pharmacodynamics of cannabis is complicated by the fact that CBD (and potentially other cannabinoids) has numerous targets and mechanisms of action that contribute to its various biological effects. Similar to CBD a high percentage of neuroactive compounds have multiple targets and act on them within similar concentration ranges. This polypharmacology has both advantages and disadvantages. As many disease etiologies are not entirely known and may be multifactorial, there may be a substantial benefit of having activity on multiple targets. However, this may also increase the side effect potential and the potential of interacting with other therapeutics. It has been suggested that large-scale zebrafish behavioral testing models can be used to help discern the polypharmacological mechanisms of neuroactive compounds [126]. This provides an ideal platform with which to test cannabis derivatives.
