Cbd oil for neovascularization

Cannabis and the Cornea

Address correspondence and reprint requests to Albert Y. Wu, MD, PhD, FACS, Stanford Institute for Stem Cell Biology and Regenerative Medicine, Stanford University School of Medicine, 265 Campus Drive West, Room G3155, Stanford, CA 94303. [email protected], Phone number: (650) 497-0758

Abstract

Purpose:

While cannabis has potential to reduce corneal pain, cannabinoids might induce side effects. This review article examines the effects of cannabinoids on the cornea. As more states and countries consider bills that would legalize medical and recreational adult use of cannabis, health care providers will need to recognize ocular effects of cannabis consumption.

Methods:

Studies included in this review examined the connection between cannabis and the cornea, more specifically anti-nociceptive and anti-inflammatory actions of cannabinoids. NCBI Databases from 1781 up to July 2019 were consulted.

Results:

Five studies examined corneal dysfunctions caused cannabis consumption (opacification, decreased endothelial cell density). 12 studies observed reduction in corneal pain and inflammation (less lymphocytes, decreased corneal neovascularization, increased cell proliferation and migration).

Conclusion:

More than half of the studies examined the therapeutic effects of cannabinoids on the cornea. As the field is still young, more studies should be conducted to develop safe cannabinoid treatments for corneal diseases.

INTRODUCTION

Due to the therapeutic and psychotropic properties of cannabinoids, cannabis is the most consumed and illegally traded drug worldwide. In 2016, more than 192 million people have used cannabis, either topically, orally, sublingually or more commonly through inhalation. After recreational cannabis’ legalization in Uruguay (2013) and in Canada (2018), more states and countries consider bills that would decriminalize and legalize adult use of cannabis. In the context of widespread use of recreational cannabis and cannabinoid-based treatments, health-care providers, including eye care professionals (ophthalmologists, optometrists), general internists, and emergency physicians, will need to recognize ocular effects of cannabis consumption in patients.

Corneal visual impairment is the fourth cause of blindness worldwide. Surgery is the only available treatment. As it requires graft donors, this treatment can be inaccessible. In order to prevent corneal visual impairment, it is important to treat corneal pain and inflammation effectively and safely.

A paucity of articles correlates cannabinoid pathways with reduction of corneal inflammation and pain. The objectives of this study are to summarize the current literature about cannabinoids and corneal hyperalgesia and assess if certain cannabinoids could be isolated and used as a reliable treatment to reduce the nociceptive and inflammatory effects associated to corneal diseases.

METHODS

Study Eligibility Criteria

Studies that were eligible examined the protective and toxic effects of cannabinoids on the function of the human or animal cornea.

Studies indirectly addressing the connection between cannabinoids and the cornea were excluded. Studies on glaucoma and inflammation, cannabinoids and intraocular pressure, transcorneal permeability of cannabinoids, corneal kindling model experiments using cannabinoids, as well as experiments using cannabinoids to treat pterygium were not included.

Search Methods and Terms Used

The literature search identified relevant articles in the NCBI Literature Databases (PubMed/PubMed Central) from 1781 (publication date of the oldest article available on PubMed) up to December 2019.

Article searches contained the following MeSH terms and keywords: cannabinoid receptors, cannabinoids, cannabis, cornea, corneal endothelial cell loss, corneal injuries, dry eye syndromes, marijuana abuse, marijuana smoking, marijuana use, medical marijuana.

Additional studies were found by consulting the reference lists of selected articles.

Results

Seventeen articles were selected. These studies examined the following cannabinoid effects on the cornea: corneal cellular properties (density, migration via chemotaxis, quantity), corneal opacification, corneal neovascularisation, corneal inflammation, and corneal pain. Only three review articles were included. The majority of the articles (10 out of 17) were experimental studies performed on animal models. The main limitation of these studies is the low number of experiment repetitions performed ( Table 1 ).

Table 1.

This original table breaks down all the publications selected for this review article. It indicates their type (i.e. systematic review, random control trial, case report, case-control study, cohort study), their most important findings (i.e. corneal opacification, corneal neovascularization in humans, corneal endothelial cell density reduction) and their overall effects on corneal health (protective effects: +; toxic effects: -).

Title of Publications Type of Study Findings Effects
The ocular manifestations of the cannabinols 6 Review Article • Increase size and number of corneal nerves NA
Evidence for a GPR18 Role in Chemotaxis, Proliferation, and the Course of Wound Closure in the Cornea 7 Experimental study in culture bovine corneal cells and murine corneal explants • Induction of chemotaxis and proliferation after corneal GPR18 activation (wound healing process) +
The Cannabinoids Delta(8)THC, CBD, and HU-308 Act via Distinct Receptors to Reduce Corneal Pain and Inflammation 8 Experimental study on murine animals • Reduction of corneal pain and inflammation +
Topical cannabinoid agonist, WIN55,212-2, reduces cornea-evoked trigeminal brainstem activity in the rat 10 Experimental study on murine animals • Modulation of CB1R and corneal-responsive neurons +
The cannabinoid WIN 55,212-2 inhibits transient receptor potential vanilloid 1 (TRPV1) and evokes peripheral antihyperalgesia via calcineurin 11 Experimental study in murine animals • Co-Activation of CB1R and TRPV1 by Delta-8-THC +
Cannabinoid-induced chemotaxis in bovine corneal epithelial cells 12 Experimental study on bovine corneal epithelial cells • Induction of chemotaxis after CB1 activation in bovine cells +
Epidermal growth factor receptor transactivation by the cannabinoid receptor (CB1) and transient receptor potential vanilloid 1 (TRPV1) induces differential responses in corneal epithelial cells 13 Experimental study on human corneal epithelial cells • Proliferation and migration of corneal cells after CB1 and TRPV1 activation +
Cannabinoid receptor 1 suppresses transient receptor potential vanilloid 1-induced inflammatory responses to corneal injury 14 Experimental study on human and murine epithelial cells • Reduction of corneal opacification and inflammation +
Cannabinoid CB2R receptors are upregulated with corneal injury and regulate the course of corneal wound healing 15 Experimental study in an in vitro murine model • Increased activity of CB2R after corneal injury
• Chemorepulsion
+
Gene therapy for corneal dystrophies and disease, where are we? 16 Review Article • Reduction of corneal neovascularisation +
Genetic and pharmacologic inactivation of cannabinoid CB1 receptor inhibits angiogenesis 17 Experimental Study
Turning Down the Thermostat: Modulating the Endocannabinoid System in Ocular Inflammation and Pain 5 Systematic Review • Corneal cell proliferation and migration via chemotaxis +/−
Ocular hypotension, ocular toxicity, and neurotoxicity in response to marihuana extract and cannabidiol 20 Experimental study on cats • Corneal opacification (in cats and dogs)
Intraocular pressure, ocular toxicity and neurotoxicity in response to 11-hydroxy-delta 9-tetrahydrocannabinol and 1-nantradol 19 Experimental study on animal models (cats, rats, monkeys)
Intraocular pressure, ocular toxicity and neurotoxicity after administration of delta 9-tetrahydrocannabinol or cannabichromene 18 Experimental study on cats
Comparative Toxicities of Tetrahydropyridobenzopyrans 21 Experimental study on animal models (dogs, rhesus monkeys, rats)
Corneal endothelial changes in long-term cannabinoid users 22 Case-control study • Reduction in corneal cell density

DISCUSSION

Cannabis and Cannabinoids

Cannabis

Extracted from hybrid cannabis plants, cannabis is a medical and recreational drug used for its psychotropic and physical effects. These properties are inherent to cannabinoids, which are more than 100 chemical compounds that are found in the cannabis plant.

Cannabinoids

Cannabinoids adhere to cannabinoid and non-cannabinoid receptors in order to generate different reactions. There are many varieties of cannabinoids, including tetrahydrocannabinol (THC), cannabidiol (CBD), cannabichromene (CBC). tetrahydrocannabinolic acid (THCA) and cannabinol (CBN).

Each cannabinoid can be further divided into components. For example, delta-9-THC and delta-8-THC are different psychoactive components of cannabis. THC is the major cannabinoid type causing cannabis’ mind-altering properties. CBD is a main cannabis component, which causes the drug’s neurologic and bioactive activity. Unlike THC’s psychoactivity, CBD triggers reactions that can be beneficial in the treatment of psychiatric diseases, epilepsy, and neurodegenerations.

Phytocannabinoids, Synthetic Cannabinoids and Endocannabinoids

The most common legalized cannabinoids are natural cannabinoids, which are extracted from hybrid cannabis plants. These phytocannabinoids differ from synthetic cannabinoids (which are produced in the laboratory) and endocannabinoids (which are physiologically important). ( Figure 1 ) In North America, synthetic cannabinoids pose a serious health threat, as they are new psychoactive substances that are unregulated.

Cannabinoids bind to cannabinoid receptors, which are predominantly found in corneal epithelial cells and the basal ganglia. Non-cannabinoid receptors modulate the cerebral cannabinoid-signaling pathway involved in the regulation of ocular pain and inflammation.

Endocannabinoids and Corneal Receptors

Cannabinoid Receptors and Location in the Cornea 1,2

Cannabinoid receptor 1 (CB1) and cannabinoid receptor 2 (CB2) are the most prominent cannabinoid receptors that are part of the endocannabinoid network. These G-protein receptors (GPCRs) constitute the largest membrane protein family and modulate important cell signaling molecules. Cannabinoid receptors are involved in various neural pathways associated with cannabis and can be found abundantly in the basal ganglia.

In the eye, CB1 receptors are predominantly located in the corneal epithelium and endothelium. While CB2 receptors are not present in the cornea, they are involved in the turnover of the aqueous humor. ( Figure 1 )

Corneal Endocannabinoids 3

Endocannabinoids that have been detected in the human cornea include: palmitoyethanolamide (PEA), 2-arachidonoylglycerol (2-AG) and anandamide (N-arachidonoylethanolamine, AEA). ( Figure 1 ) PEA promotes the activity of AEA. 2-AG is a physiologically important CB1R agonist. While AEA also activate cannabinoid receptors, AEA specifically mediates the transient receptor potential cation channel subfamily V member 1 (TRPV1).

Non-Cannabinoid Receptors 4

TRPV1 works concurrently with cannabinoid receptors in the modulation of corneal hyperalgesia and inflammation. Other non-cannabinoid receptors that are cannabinoid molecular targets include: transient receptor potential cation channel subfamily M member 8 (TRPM8), transient receptor potential ankyrin 1 (TRPA1), peroxisome proliferator-activated receptors (PPARs), and the G-protein-coupled receptor serotonin 1A receptor (5-HT1A). ( Figure 1 )

Cannabinoids’ actions are linked to various systems, including the gabaergic, serotonergic, cholinergic and dopaminergic pathways.

By modulating these receptors and systems, cannabis can produce anti-inflammatory and antinociceptive effects. However, the exact mechanisms responsible for the modulation of cannabinoids in corneal pain are still unknown. Studies are currently examining the correlation between corneal cell proliferation and migration via chemotaxis in the modulation of endocannabinoids and corneal receptors. 5

In Vivo Evidence-Based Effects of Cannabinoids on the Cornea

Corneal Neuropathic Pain and Short-Term Cannabis Usage 1

A study conducted from 1968 to 1973 examined a cohort of 350 cannabinol consumers and found that large and numerous corneal nerves were significant in a large number of patients. Short-term cannabis use might therefore be related to corneal neuropathic pain. 6

Corneal Endothelial Cells and Long-Term Cannabis Usage 17

Similarly to the process of aging, intraocular surgeries, glaucoma, trauma, alcohol, and tobacco, cannabis might cause a decrease in the endothelial cell count.

One study has noted the long-term effects of cannabis use in human eyes. Corneal endothelial cell density (number of cells per square millimeter) was significantly reduced in the cannabinoid group. It has been suggested that this reduction in the corneal endothelial density is the result of endothelial cell death.

On the other hand, the coefficient of variation in cell size and the hexagonal cell ratio in cannabinoid users did not significantly change compared to the control group. It is likely that cannabinoid toxicity counters cellular growth and migration, which differs from the findings in previous studies using animal models and acute administration of cannabinoids. The mitotic rate of endothelial cells was insufficient to restore the endothelium due to cannabinoid toxicity.

It is plausible that the cornea is particularly vulnerable to cannabinoid toxicity due to the profusion of CB1 receptors in the anterior segment of the eye. The literature supports that the activation of CB1 receptors promotes oxidative stress, mitogen-activated protein kinase (MAPK) pathways, and apoptosis. However, these studies on the protective effects of inactive CB1 receptors in endothelial cells are not specific to the human cornea.

There were no clinically significant observations (i.e. absence of corneal edema). The reduction in CD was not sufficient to significantly reduce the central corneal thickness (CCT). This result might be explained by the selection criteria for the cannabinoid group: this study included 28 patients who have only been using cannabis during the past year at a frequency of three times per week or more (and who received a formal psychiatric diagnostic of cannabinoid use disorder). In order to clarify the relationship between CCT and cannabis, it would be important to examine research participants who have been consuming cannabis regularly for more than a year.

Cannabinoids’ Protective Effects on the Cornea

Cannabinoids and Corneal Inflammation and Pain

Recent studies on mice have further validated that cannabis has an influence on corneal pain. Cannabinoids would reduce corneal pain and inflammation by activating receptors involved in cannabinoid pathways. The nociceptive actions of capsaicin on cauterized murine eyes were significantly reduce by topical applications of cannabinoids, including the synthetic derivative HU-308 at a concentration of 1.5%, CBD at 5% and Delta-8-THC at 1%. Corneal inflammation was also reduced at those same cannabinoid concentrations, as a reduction of neutrophil infiltration was quantified through immunohistochemistry. ( Table 2 ) It appears that delta-8-THC is a CB1R agonist because mice were treated with AM251 (a CB1R antagonist) did not benefit from delta-8-THC anti-sensitization properties. 7,8

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Table 2.

This original table illustrates the findings from the 2018 study by Thapa et al. 8

Animal Topical applications of cannabinoids Dose prescribed (% w/v) Significant Pain Score Reduction Significant Neutrophil Number Reduction
Mice Delta-8-THC (CB1R agonist) 0.2 No Not examined
0.4 No Not examined
0.5 Yes Not examined
1 Yes Yes
CBD (5-HT1A agonist) 3 No Not examined
5 Yes Yes
HU-308 (CB2R agonist) 1 No Not examined
1.5 Yes Yes

Delta-8-THC is thought to be involved in the activation of CB1R and TPRV1 because the co-localization of these two has been well established. Studies have demonstrated that human corneal epithelial turnover was favored by the activation of CB1 and TRPV1. 9 Both receptors induce differential responses through epidermal growth factor receptor-mediated MAPK pathways. While TRPV1 activation by capsaicin leads to more IL-6 and IL-8 release through a EGFR-independent pathway, active CB1 blocks the IL-8 release induced by TRPV1. 10,11 (Figure 2)

As CB1 and CB2 have opposite effects, their balance is important in the regulation of efficient cell migration via chemotaxis when needed. Without the actions of these cannabinoid receptors, corneal wound healing is considerably impaired.

Other important receptors responsible for cannabinoids anti-inflammatory effects include the 5-HT1A receptor activated by CBD and GPR18, which was conducive to wound healing and triggered cell proliferation in vitro. 7

A study done in bovine corneal epithelial cells (bCEC) observed that the directed migration of bCEC is enhanced by 2-AG and halted by a CB1R antagonist (SR141716). From their findings, they conclude that CB1 activation leads to ERK1/2 dephosphorylation in a concentration-dependent way, which does not promote bCEC proliferation. Active CB1 would decrease cAMP levels, contributing to the inactivation of mitogen-activated protein-kinase (MAPK) and therefore acting as an antagonist in the EGF pathway responsible for cell proliferation. 12 (Figure 2A)

The claim that CB1 acts as an antagonist of EGF-induced cellular proliferation is not supported by studies conducted by Yang in 2010 and in 2013. The discrepancy in results might be explained by the use of different animal models (mice vs. cows) or a lower dosage of the synthetic CB1R agonist (1 and 10 nm vs. 10μM). 13

While CB1 regulates attractive chemotaxis of CECs, CB2 would modulate repulsive chemotaxis by activating MAPK and raising the cAMP levels. HU-308 is an example of CB2 agonist, as its antinociceptive effects were halted in CB2−/− mice. CB2 activation accelerates wound healing in injured murine corneas. By subjecting bovine corneal epithelial cells with a CB2R agonist (JWH133), Murataeva further discovered that active CB2R promotes MAPK activation, ERK phosphorylation, adenylyl cyclase activity and cAMP quantities, without affecting the proliferation of corneal epithelial cells.

This study also conflicts with the 2013 study by Yang et al., as it suggests that the presence of AEA is more important than 2-AG in the mechanism responsible for repairing corneal damage. While Yang et al. noted the presence of 2-AG and the absence of AEA in corneal wounds, Murateava found that corneal injury can be correlated with an increased amount of AEA, which is synthesized by the NAPE-PLD pathway. 14 According to Murataeva, the differences in results are mainly due to the quantity of cannabinoids used. 15

Reduction of Corneal Neovascularization 16,17

Two studies have noted a significant reduction in corneal neovascularization by inhibiting the action of CB1. Gene therapies have examined the effects of different CB1 antagonists in the angiogenesis of human, murine and rabbit corneas in vitro. Some of these CB1 inhibitors also modulate cannabinoid pathways, leading to corneal haze and fibrosis reduction, fast wound healing and corneal density augmentation.

Because of the paucity of research into CB1 antagonist’s actions, it is necessary to proceed to in vivo experiments before confirming such protective effects.

Cannabis’ Destructive Effects on the Cornea

Cannabis-Induced Corneal Opacification and Dehydration

In two feline studies, severe corneal opacities were developed at the application site after chronic topical administration of cannabinoids. Delta-9-THC delivery via osmotic minipumps had a more intense corneal opacification in cats than cannabis extracts. Both treatments lasted 9 days. On the third day, irregularities in the corneal epithelium were noticed and forecasted the corneal opacities (visible from the fourth to the sixth day). Contrastingly, cannabichromene, cannabidiol and acute application of delta-9-THC did not induce ocular toxicity in cats. Other animal experimental study has reported corneal opacification and ulceration after oral administration of synthetic cannabinoids in dogs, but not in rhesus monkeys, nor in rats. 18–21

It has been inferred that these corneal opacities were caused by a decreased corneal hydration. Active CB1 inhibits the pumping action of the corneal endothelial cells, which stops the removal of aqueous humor out of the cornea. 19,20

The activation of these receptors can therefore lead to reduced lacrimation and corneal opacification. Further experimental studies must be undertaken to validate if these ophthalmic changes also occur in humans.

CONCLUSION

Future Perspectives

While the exact mechanisms underlying corneal pain, inflammation, and toxicity are still unclear, more than half of the studies have noted cannabinoids’ therapeutic effects on the cornea. Only a few studies warn about their potential toxic properties on the eye ( Table 1 ).

Hence, the direct link between corneal endothelial cells to cannabinoids toxicity requires further validation. Studies examining patients using cannabinoids for more than a year and experimental research observing corneal endothelial cell death in vivo are required. Clinical trials attesting that cannabis’ benefits outweigh health hazards would allow the use of legal and safe cannabinoid treatments for corneal diseases.

Footnotes

Declaration of interest

The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.

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CBD Won’t Reduce IOP

Cannabidiol (CBD) oil may be “on trend” along the reclaimed-wood paneled shelves of spiritual-healing apothecaries across the country, but its impact on intraocular pressure (IOP) may be less than ideal. Although the cannabis plant—from which CBD is derived—is popularly believed to lower pressure, the particular chemical extract connected to that effect may actually be rendered powerless by application of CBD oil, according to newly published research.

Investigators affiliated with the Gill Center for Biomolecular Science and the Department of Psychological and Brain Sciences at Indiana University say their study, which appears in the December Investigative Ophthalmology & Visual Science, shows that, while Δ9-tetrahydrocannabinol (THC) can lower IOP, CBD can actually interfere with that effect. THC, the team explained, affects IOP by activating two receptors—CB1 and GPR18—and that this response was much stronger in the male mice tested than the females. But the focus of the investigation was to understand the impact of the cannabis plant’s less psychoactive element: CBD. Interestingly, the CBD acted as a negative allosteric modulator at CB1, essentially raising IOP and cancelling out any of the benefits achieved from THC.

“The regulation of ocular pressure by THC and CBD is more complex than previously appreciated,” the authors explain. “THC acts via a combination of CB1 and GPR18 receptors in a sex-dependent manner, while CBD can both raise IOP and interfere with the effects of THC. The potential of CBD to elevate ocular pressure should be evaluated further as a potential deleterious side effect, particularly with long-term use.”

Cellular Physiology & Biochemistry

International Journal of Experimental Cellular Physiology, Biochemistry and Pharmacology

Corresponding Author: Zhao-Hui Song

Department of Pharmacology and Toxicology, University of Louisville School of Medicine, Louisville, Kentucky, 40292 (USA)
Tel. +1-502-852-5160, Fax +1-502-852-7868, E-Mail [email protected]

Cannabidiol Signaling in the Eye and Its Potential as an Ocular Therapeutic Agent

Alyssa Aebersold Max Duff Lucy Sloan Zhao-Hui Song

Department of Pharmacology and Toxicology, University of Louisville School of Medicine, Louisville, Kentucky, USA

Key Words Cannabidiol • Molecular target • Mechanism of action • Therapeutic potential • Ocular pharmacology

Abstract Cannabidiol (CBD), the major non-intoxicating constituent of Cannabis sativa, has gained recent attention due to its putative therapeutic uses for a wide variety of diseases. CBD was discovered in the 1940s and its structure fully characterized in the 1960s. However, for many years most research efforts related to cannabis derived chemicals have focused on D9-tetrahydrocannabinol (THC). In contrast to THC, the lack of intoxicating psychoactivity associated with CBD highlights the potential of this cannabinoid for clinical drug development. This review details in vitro and in vivo studies of CBD related to the eye, the therapeutic potential of cannabidiol for various ocular conditions, and molecular targets and mechanisms for CBD-induced ocular effects. In addition, challenges of CBD applications for clinical ocular therapeutics and future directions are discussed.

Introduction

A brief history of cannabis
Cannabis sativa is a plant species that includes both cannabis and hemp. It first appeared in Chinese medical texts around 2000 years ago [1]. Records from Britain indicate that cannabis was brought from Egypt by Napoleon’s troops in the early 1800s [2]. Shortly thereafter, hemp was introduced to Western medicine when in 1840, a hemp tincture from ground plant matter was reported to be an effective treatment for convulsive disorders and tetanus [3]. By 1851, a cannabis extract was included in the 3 rd edition of the Unites States Pharmacopoeia and readily available in American pharmacies [4, 5].
In 1913, however, cannabis was made illegal in California due to a wide-spread anti-narcotics campaign [5]. Cannabis became federally illegal when Harry Anslinger from California introduced the Marijuana Tax Act of 1937 banning the sale and use of cannabis nationally [5, 6]. A negative stigma continued to develop in the US around cannabis, then associated with narcotics, that culminated with the Controlled Substances Act (CSA) of 1970, which classified cannabis and cannabinoids as Schedule I with no recognized medical use [7]. Recently, America is witnessing a revival in the popularity of cannabis, both medically and recreationally. In 1996, California was the first state to legalize cannabis for medical use and more states have followed California in recent years [8]. To date, 16 states and Washington D.C. have legalized both medical and recreational cannabis with an additional 26 states legalizing medical cannabis at varying degrees. Moreover, the Agricultural Acts of 2014 and 2018 removed hemp from the list of controlled substances and redefined industrial hemp as cannabis containing less than 0.3% THC [9, 10]. As a result of the recent wave of recreational and medical cannabis legalization, in conjunction with the end to the prohibition of hemp, cannabis research is quickly expanding.

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Cannabidiol
Cannabidiol (CBD) is one of over 120 chemicals produced by the Cannabis sativa plant termed phytocannabinoids [11, 12]. There are potentially more, as 21 previously unknown cannabinoids were recently identified [13]. The two most abundant phytocannabinoids in cannabis are psychoactive and intoxicating D9-tetrahydrocannabinol (THC) and non-intoxicating CBD.
CBD was first isolated in the 1940 and its structure and stereochemistry fully determined in 1963 [14, 15]. CBD and THC are both derived from cannabigerolic acid [16]. Although the structure of CBD was discovered before THC [15, 17], THC had been the major focus of research related to cannabis and cannabinoids. This focus is driven, in part, by the activity of THC at the canonical cannabinoid receptors, CB1 and CB2. However, there are many targets for cannabinoids other than CB1 and CB2. For example, CBD has upwards of 65 known targets consisting of receptors, enzymes, ion channels and transient receptor potential (TRP) channels [18].

Cannabinoids in pharmaceuticals
Cannabinoid containing drugs are approved for medical use in the USA and other countries. The drugs differ in their formulation and indicated uses. Dronabinol (Marinol) was the first cannabinoid-containing medicine approved by the FDA in 1985. It is a soft gel capsule containing synthetic THC [19]. Syndros is an oral solution of dronabinol [20]. Cesamet (nabilone) is the third synthetic cannabinoid drug approved by the FDA in May of 2006 [21]. All three are prescribed for anorexia associated with weight loss in AIDS patients and nausea/vomiting in cancer patients [19-22]. While plant-derived THC is a Schedule I substance, Marinol is listed under Schedule III and Cesamet and Syndros are controlled under Schedule II [19-21].
Epidiolex is an oil formulation of CBD approved by the FDA in June of 2018 for treatment of Lennox-Gastaut syndrome and Dravet syndrome, two rare and severe forms of pediatric epilepsy [23]. In July of 2020, it was approved for treating seizures in a rare genetic disease, tuberous sclerosis complex (TSC) [24]. Epidiolex is the only FDA approved drug containing a compound directly derived from cannabis. It was originally classified as schedule V, but is no longer a controlled substance as the FDA deemed it safe and effective for treatment of the aforementioned conditions [25]. Sativex is a 1:1 alcohol solution of THC and CBD administered as an oromucosal spray that is approved in 25 countries for the treatment of pain and spasticity in multiple sclerosis patients [26]. Despite its approval in other countries, Sativex is not yet approved by the FDA in the US.

Research on cannabidiol
CBD, through a variety of mechanisms and targets, has numerous potential therapeutic uses for a plethora of conditions. The assertion of potential therapeutic actions of CBD is based on pre-clinical data, limited clinical data and ongoing human clinical trials. Pre-clinical studies show that CBD has antioxidant [27, 28] anti-inflammatory [27], anti-
convulsant [29, 30], neuroprotective [31], and anti-cancer properties [32]. CBD also shows potential as a therapeutic agent in cardiovascular [33], neurological, and neuropsychiatric disorders [26]. The completed clinical trials involve CBD use in epilepsy and seizures disorders (21 trials), general pain and pain associated disorders (19 trials), drug abuse and use disorders (14 trials), other neurologic conditions (4 trials) and psychiatric conditions (11 trials). In addition, there are currently 85 active clinical trials in the United States containing CBD (including Epidiolex and Sativex) on clinicaltrials.gov.
Over the past two decades, multiple studies have investigated the therapeutic potentials of CBD in the eye. There are several published reviews of cannabinoids for treatment of glaucoma [34, 35], and retinal disorders [36, 37]. Nevertheless, there are currently no reviews that focus solely on CBD for ocular conditions. In this review, we aim to fill the gap in literature with a focus on CBD ocular pharmacology. We will discuss therapeutic potentials of CBD for ocular conditions, ocular molecular targets for CBD, and mechanisms of actions of CBD in the eye.

Results

Therapeutic potentials of cannabidiol for ocular conditions
CBD is recognized for its antioxidant, anti-inflammatory and neuroprotective properties. In this section, we discuss the observed effects of CBD in ocular tissues and its indication for ocular disorders. Specifically, we will discuss studies of CBD in corneal inflammation and pain, endotoxin-induced inflammation, excitotoxicity, diabetic retinopathy, and intraocular pressure (Table 1 and Table 2).

Corneal Inflammation and pain
The cornea is a thin and avascular tissue that is innervated by sensory nerves. When corneal damage occurs due to infection, surgery, or trauma, it can develop into corneal neuropathic pain characterized by hyperalgesia, chronic and debilitating pain, and inflammation [38, 39]. The inflammatory response to corneal damage leads to the production of proinflammatory cytokines, recruitment of leukocytes, release of pain-producing neuropeptides, and neovascularization (NV) in the cornea [38, 39].
In a recent study, CBD was found to be anti-nociceptive and anti-inflammatory in a mouse model of corneal hyperalgesia [39] (Table 1). Mice with silver nitrate cauterized corneas that treated with CBD showed lower pain scores in capsaicin pain challenges, indicating an antinociceptive effect of CBD. Moreover, CBD treated corneas showed less corneal neutrophil infiltration which is indicative of a CBD-induced anti-inflammatory effect. Lastly, WAY100635, a 5HT1A antagonist, blocked the effects of CBD, suggesting that the anti-inflammatory and anti-nociceptive effects are likely mediated through activation of the serotonin 5HT1A receptor [39]. This study highlights CBD as a potential therapeutic for corneal pain and inflammation.

Endotoxin-induced inflammation
The mammalian retina contains three distinct glia cells types: Müller cells, astrocytes, and microglia. Microglial cells are the resident macrophages of the retina and play important roles in retinal homeostasis [40]. Activation of microglial cells induces the release of pro-inflammatory cytokines, such as IL-1b and TNFα, instigating an inflammatory response. Prolonged microglial activation and chronic inflammation contribute to disease pathology and retinal degeneration [40].
The degree of microglial activation may relate to the severity of injury. In vitro and in vivo treatment with lipopolysaccharide (LPS), an endotoxin from bacteria, is used to study inflammation through activated microglia [41]. Extracellular adenosine can function as an endogenous anti-inflammatory agent suppressing immune cell responses. For example, adenosine inhibits pro-inflammatory cytokine expression such as TNFα [42]. However, the anti-inflammatory effects of adenosine are short, as it is rapidly taken up by adjacent cells. Inhibitors of adenosine uptake may enhance the adenosine signaling and endogenous
activity [43]. CBD has been shown to decrease TNFα expression and inhibit equilibrative nucleoside transporter 1 (ENT1) reuptake of adenosine in LPS treated primary microglia cells and retinas from LPS treated rats [44] (Table 1). The effect of CBD is primarily mediated through the activation of the A2A receptor, the most abundant adenosine receptor in the rat retina, as a result of CBD-induced inhibition of adenosine reuptake [44]. These results suggest that CBD may be a good anti-inflammatory agent for endotoxin-induced retinal damage.

Excitotoxicity
Excitotoxicity is implicated in glaucoma as a result of elevated levels of the excitatory neurotransmitter glutamate in the retina [45, 46]. Over-stimulation of a glutamate receptor, such as the N-methyl-D-aspartate (NMDA) receptor, a sodium and calcium permeable channel, results in excess intracellular calcium. Increased intracellular calcium is cytotoxic, as well as induces release of more glutamate, cellular swelling, and eventually cell death [47, 48]. The process of excitotoxicity also involves the activation of nitric oxide (NO) synthase and accumulation of NO and superoxide. Overproduction of these oxygen species produces oxidative stress leading to lipid peroxidation, mitochondrial dysfunction, DNA damage and eventually, cell death [46, 49].
One method to measure oxidative stress is through peroxynitrite/nitrotyrosine formation and lipid peroxidation [50]. Peroxynitrite is a product of a superoxide reaction primarily responsible for oxide- and superoxide-dependent cytotoxicity. It is highly unstable, highly reactive and difficult to measure, therefore the presence of peroxynitrite is measured by levels of nitrotyrosine which is formed by nitration of protein-bound tyrosine [50].
In a rat model of neurotoxicity, intravitreal injection of NMDA induces nitrite/nitrate accumulation, lipid peroxidation, nitrotyrosine production, apoptosis, and inner retinal neuronal loss [51]. CBD treatment decreased levels of peroxynitrite/nitrotyrosine production, prevented neurotoxicity, and lowered the amount of apoptosis (Table 1). The neuroprotective effect of CBD was dependent upon blockage of nitrotyrosine formation [51]. The retinal antioxidant and neuroprotective effects of CBD in the rat model of retinal excitotoxicity suggest that it may be beneficial as a neuroprotectant for the treatment of ocular conditions such as glaucoma.

Diabetic retinopathy
Globally, diabetic retinopathy is a major cause of vision loss. Oxidative stress, caused by reactive oxygen species, is one of the main factors in diabetic retinopathy progression [52].
The retina is particularly sensitive to reactive oxygen species because it is the most metabolically active tissue in the body and therefore easily affected by diabetes [52]. Diabetic retinopathy is characterized by retinal hypoxia, increased retinal vascular permeability, and retinal angiogenesis [52, 53]. These processes cause the death of inner retinal and ganglion cells and ultimately, vision loss.
Inflammation is another important component in diabetic retinopathy. Hyperglycemia triggers the release of proinflammatory cytokines such as vascular endothelial growth factor (VEGF), Intercellular adhesion molecule-1 (ICAM-1), and Tumor necrosis factor α (TNFα) [54, 55]. The elevation of these proinflammatory cytokines further facilitates pathologic changes in diabetic retinopathy as a result of neovascularization by VEGF, leukocyte adhesion and transmigration by ICAM-1 and further release of cytokines by TNFα [53-55]. Research shows that VEGF, ICAM-1, and TNFα are upstream regulators of proinflammatory and oxidative stress pathways which activate p38 MAP kinase [55]. Activation of p38 MAP kinase has been reported in diabetic retinas in high glucose conditions and is implicated in retinal ganglion cell apoptotic death [56-58]. In addition, p38 MAP kinase activity is linked to vascular hyperpermeability in diabetic retinas [55].
One report assessed the therapeutic potential of CBD in a streptozotocin-induced diabetic rat model through measurement of oxidative stress and proinflammatory cytokines [58] (Table 1). The antibiotic streptozotocin (produced by Streptomyces achromogens) induces type 1 diabetes through partial destruction of the pancreatic β cells after a single injection. In the streptozotocin-induced diabetic rat model there were increases in oxidative stress, retinal neuronal cell death, and vascular hyperpermeability associated with increased levels of VEGF, ICAM-1, TNFα, and activation of p38 MAP kinase [58]. Importantly, CBD decreased reactive oxygen species (ROS) formation, suppressed VEGF, ICAM-1, and TNF-a expression, and prevented activation of p38 MAP kinase [58]. Taken together, these findings suggest that CBD is a potential therapeutic agent for diabetic retinopathy capable of protecting against inflammation, retinal neuronal cell death, and preservation of the blood-retinal barrier.

Intraocular pressure
An estimated 3 million Americans have glaucoma, a major cause of irreversible blindness with no cure [59, 60]. Even with therapeutic intervention, approximately 10% of those diagnosed still experience vision loss [59, 60]. Although not always elevated, intraocular pressure (IOP) is currently the only treatable factor of the disease. Drug therapies such as prostaglandin analogs, b-adrenergic antagonists, cholinergic agonists, α2-adrenergic agonists, and carbonic anhydrase inhibitors are used either independently or in combination, to reduce ocular pressure [61, 62]. In 2018, the FDA approved Rhopressa, a Rho kinase inhibitor, as a novel IOP-lowering drug [61, 62]. These IOP-lowering drugs work to decrease aqueous humor production in the ciliary body and/or increase aqueous humor drainage through the trabecular meshwork or the uveoscleral pathway [61, 62]. For patients that do not respond to the above drugs or drug combinations, or patients developed tolerance to existing drugs, novel medications are needed to lower IOP and to prevent future optic nerve damage and vision loss associated with glaucoma.
THC is well documented and is consistently shown to decrease IOP [63-69]. However, the effect of CBD on IOP is much more controversial (Table 2). So far, nine independent reports have published regarding the effects of CBD on IOP: Four reports indicate that CBD has no effect on IOP [70-73], three reports demonstrate that CBD decreases IOP [74-76], and two reports show an CBD-induced increase in IOP [63, 64].
A recently published study in mice showed an increase in IOP at 1 and 4 hours post topical application of CBD at a 5 mM dose [63]. Interestingly, CBD significantly decreased IOP 1 hour post treatment in CB1 knockout mice and the effect is attributed to GPR18 activation [63]. This article was cited by the American academy of ophthalmology with the headline “CBD oil may worsen glaucoma” [77]. Altogether, the literature does not conclusively show whether CBD increases, decreases, or causes no change to IOP.

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Molecular targets and mechanisms for CBD-induced ocular effects
CBD has numerous targets in different categories such as G protein-coupled receptors, enzymes, nuclear receptors, ligand-gated ion channels, transient receptor potential (TRP) channels, and potentially others [18]. Many of these CBD targets are expressed in the eye. This section of the review focuses on the effects of CBD on these molecular targets in the eye (Table 3). It is important to point out that systemic CBD administration may result in CBD metabolites that act through molecular mechanisms different from those of CBD itself.

Serotonin receptor
Thapa et al. showed that CBD can reduce the pain score and neutrophil infiltration in mice after corneal cauterization and capsaicin challenge and this effect is mediated, in part, by 5-HT1A agonism [39] (Table 3). The hypoalgesic and anti-inflammatory effects of CBD seen in wild-type mice were still present in CB2 knockout mice, as well as CB2 knockout mice pretreated with AM251, a CB1 selective antagonist. These results suggest that the hypoalgesic and anti-inflammatory effects of CBD are not mediated by CB1 or CB2 receptors. Moreover, the effects of CBD were blocked in wild-type mice treated with WAY100635, a 5-HT1A receptor antagonist. These data demonstrate that the effect of CBD on corneal hyperalgesia inflammation is mediated by 5-HT1A agonism [39]. In support of the findings of Thapa et al. in the eye [39], CBD has been shown to be a 5-HT1A agonist in other tissues as well [78, 79].

Equilibrative nucleoside transporter 1 and A2A adenosine receptor
CBD has been shown to inhibit TNF-α response to LPS stimulation by inhibiting adenosine reuptake in retinal microglia via adenosine equilibrative nucleoside transporter 1 (ENT1) [44] (Table 3). Cells that were pre-treated with CBD showed inhibition of LPS-induced release of TNF-α. The inhibition of TNF-α release was not further enhanced nor inhibited by pretreatment of NBMPR, an ENT1 selective inhibitor [44]. These results suggest that CBD competes with NBMPR for ENT1. Furthermore, CBD inhibited TNF-α in the presence of A1A adenosine antagonist CPX, whereas the effect of CBD was blocked by pre-treatment with A2A adenosine receptor antagonist ZM241385. When CBD and adenosine were co-administered, TNF-α release was greatly reduced showing a synergistic effect that is greater than when either compound was administered alone [44]. In sum, Liou et al. showed that CBD inhibits adenosine reuptake through ENT1, which indirectly causes the enhanced activation of A2A adenosine receptor and reduction of TNF-α release [44]. The effects of CBD on ENT1 and adenosine receptors are corroborated by reports in rat and mouse striatal terminals [80] and in EOC-20 murine microglial cells [81].

CB1 and GPR18
CB1 is a well-established cannabinoid receptor and CBD has been shown to be a negative allosteric modulator of CB1 [82]. CB1 is expressed in the anterior of the eye in the ciliary and corneal epithelium and trabecular meshwork, as well as the posterior of the eye in the retina [83, 84]. GPR18 is a recently identified putative cannabinoid receptor and researchers have shown that GPR18 is activated by N-arachidonoyl glycine, a carboxylic metabolite of the endocannabinoid anandamide [85]. GPR18 was further characterized in 2012 when anandamide and THC, in addition to N-arachidonoyl glycine, were shown to stimulate GPR18-mediated ERK1/2 phosphorylation [86]. Furthermore, CBD was shown to be a biased agonist for GPR18 in 2014 [87]. GPR18 is widely expressed in the ocular tissues, specifically in the ciliary and corneal epithelium, trabecular meshwork, and retina [88, 89].
To date a single paper has reported on the effect of CBD at both CB1 and GPR18 receptors in the eye (Table 3). Miller et al. showed that CBD increases IOP in wild-type mice but decreases IOP in CB1 knockout mice [63]. No CBD effect on IOP was seen in CB1 knockout mice pretreated with O-1918, a GPR18 antagonist. This report highlights that CBD has independent actions both on CB1 as a negative allosteric modulator to raise IOP and on GPR18 as an agonist to lower IOP [63].

GPR55
GPR55 is an orphan receptor activated by lysophosphatidylinositol (LPI) [90]. GPR55 is frequently referred to as a putative cannabinoid receptor because it is activated by phytocannabinoids, endocannabinoids, and synthetic cannabinoids [91]. CBD has been shown to be a GPR55 antagonist [91].
One group studied the involvement of GPR55 in the retina during development [92]. Growth cones are regions of developing neurites which facilitate axon growth by extending actin filaments into filopodia. Filopodia guide the growth cone in response to chemical or electrical stimulus. Cherif et al. found that GPR55 is expressed in growth cones during development, and its activity regulates morphology and growth [92]. Mouse embryonic neurons from GPR55 knockout mice showed smaller growth cones, fewer filopodia, and decreased outgrowth compared to neurons from wild type mice. Furthermore, retinal ganglion cells from wild type mice treated with GPR55 agonists LPI and O-1602 showed increased growth cone size and filopodia number and demonstrated chemoattraction. In contrast, CBD, a GPR55 antagonist, decreased growth cone size and filopodia number, and induced chemorepulsion (Table 3). GPR55 ligands had no effects in embryonic neurons from GPR55 knockout mice [92]. These data suggest that CBD inhibits growth cone activity and axonal growth in the retinain this experimental model.

TRPV Channels
Transient receptor potential (TRP) ion channels are trans-membrane proteins involved in a wide range of chemical and physical sensations including smell, taste, vision, temperature, and pressure [93]. CBD has been shown to be an agonist of TRPV 1, 2, 3, 4 and TRPA1 [94, 95]. TRPV channels are implicated in the activation and desensitization of inflammatory processes and chronic pain [96, 97]. Therefore, CBD may be a desirable therapeutic for chronic pain because it can activate and desensitize the TRPV channels [94, 95].
One group investigated the calcium influx activity of TRPV2 channel activity in porcine retinal pigment epithelial (RPE) cells [98]. They found that CBD strongly increased intracellular Ca 2+ levels (Table 3). In the presence of TRPV2 channel inhibitor SKF96365, CBD-mediated Ca 2+ intracellular increase was partially blocked [98]. These data suggest that CBD modulation of Ca 2+ involves TRPV2, as well as other TRPV channels that are not blocked by SKF96365.
Another study looked at TRPV2 channel regulation in ARPE-19, a human RPE cell line [99]. ARPE-19 cells preincubated with CBD demonstrated a 3-fold increase in current density, an effect that was blocked by SKF96365 (Table 3). CBD also increased membrane conductance and TRPV2 surface expression. TRPV2 are heat sensitive ion channels and heat further increased the CBD mediated increase in membrane conductance. Furthermore, the PI3 kinase inhibitor LY294002 abolished the effect of CBD on membrane conductance and surface expression. These data led to the conclusion that CBD acts through activation of TRPV2 and a PI3 kinase dependent pathway to increase cell surface expression of TRPV2 channels [99].

Discussion

Challenges of using CBD as an ocular therapeutic agent
There are several challenges for practical applications of CBD as an ocular therapeutic agent. Some of these challenges include poor bioavailability, difficulty in topical delivery, and short duration of action.

Bioavailability
An FDA approved drug containing CBD is administered orally [23]. However, oral administration is inefficient due to poor bioavailability of CBD. Low bioavailability of CBD requires that it to be administered at high doses to achieve therapeutic effects. However, a consequence of high dosing is an increase in adverse side effects [100, 101]. The potential adverse effects of CBD include drowsiness, dry mouth, reduced appetite, nausea, and gastrointestinal issues. The most notable serious adverse side effects of CBD are abnormal liver function tests (elevated liver enzymes) [102].
One major factor contributing to the poor bioavailability of CBD is its extensive first pass metabolism [100, 101]. Another factor limiting CBD bioavailability is its hydrophobicity. The chemical structure of CBD contains aromatic rings and an aliphatic side chain, which make it a highly hydrophobic molecule. The hydrophobicity of CBD limits its solubility in water and makes diffusion across aqueous barriers a rate limiting step for diffusion and absorption [100, 101].

Topical delivery
Therapeutic treatments for ocular conditions are frequently administered orally or topically to the eye. Extensive first pass metabolism of CBD prevents a significant amount of drug reaching the eye from oral administration. Therefore, topical administration of CBD is desired.
Developing an ocular topical delivery system is a difficult task. The eye contains sophisticated protective mechanisms and physical barriers to prevent foreign material from entering, which includes the multi-layered cornea and pre-corneal tear film. The alternating lipophilic and hydrophilic nature of the cornea makes ocular drug delivery exceptionally difficult. As a result, less than 5% of drugs applied topically enter the eye [103-105].
CBD is highly hydrophobic and insoluble in water. Studies applying CBD topically used non-aqueous vehicles for delivery [39, 63, 75]. Previously, CBD was topically delivered to the eye in mineral oil, sesame oil, soybean oil, and a soya oil/water emulsion, Tocrisolve [39, 63, 75]. In one report, CBD delivered in mineral oil produced an IOP-lowering effect whilst the effect is absent in sesame oil [75]. With Tocrisolve as a vehicle, Miller et al. [63] demonstrated that a high dose of CBD increases IOP in wildtype mice, but decreases IOP in CB1 knockout mice. This indicates that at large doses CBD produces off-target effects which are detrimental. Since CBD has at least 65 targets [18], off-target effects of CBD at high doses are very likely. These results highlight a critical need for a vehicle with high ocular permeation to administer CBD in a therapeutically relevant dose.

Duration of action
Another difficulty associated with CBD as an ocular therapeutic is its short duration of action, e.g., in lowering IOP. One report indicated CBD decreased IOP 1-2 hours after topical application to rabbit eyes, and IOP-lowering effects of CBD lasted for up to 5 hours after intravenous administration [75]. In another study, CBD required constant infusion via minipump to induce a decrease in IOP [76]. A short duration of action implies that CBD needs to be applied multiple times throughout the day to maintain therapeutic effects. However, patient compliance will be worsened with frequent dosing. In contrast, greater patient compliance is observed in prescribed medications with once daily dosing [106].

Future directions
So far, CBD has been studied preclinically for its therapeutic potentials in glaucoma, diabetic retinopathy, and corneal injury. Considering its anti-inflammatory, antioxidant, and neuroprotective properties, in the future it would be worthwhile to explore the potential of CBD in treating other ocular conditions, such as uveitis and age-related macular degeneration.
It is important to elucidate the mechanisms of action of CBD in the eye. As highlighted in this review, there are multiple molecular targets of CBD in the eye. Understanding which targets are responsible for the therapeutic and adverse effects of CBD is critical for its effective and safe use as an ocular therapeutic agent.
Finally, in the future, solving the puzzles of dosing and proper formulation for efficient, prolonged topical delivery will usher CBD forth for numerous potential ocular therapeutic indications.

Acknowledgements

The authors acknowledge the support of Department of Pharmacology and Toxicology, University of Louisville School of Medicine.

Author Contributions
AA and MD wrote the initial versions of the review. LS and ZHS edited and finalized the manuscript.

Funding Sources
While writing this manuscript, AA is supported in part by NIH grant T32 ES011564; MD is supported in part by NIH grant R25 CA134283; LS is supported in part by University of Louisville Integrated Programs in Biomedical Sciences (IPIBS) Fellowship; and ZHS is supported in part by NIH grant EY030186.

Statement of Ethics
The authors have no ethical conflicts to disclose.