Effects of Topical Treatment of Cannabidiol Extract in a Unique

Home , Methylglyoxal

Effects of topical treatment of cannabidiol extract in a unique manuka factor 5 manuka honey carrier on second intention wound healing on equine distal limb wounds: A preliminary study

VC McIvera, AS Tsanga, NE Symondsa, NR Perkinsb, E Uquillasa, CM Darta, LB Jeffcotta, AJ Darta* aResearch and Clinical Training Unit, University Veterinary Teaching Hospital, The University of Sydney, Camden, New South Wales, Australia. bSchool of Veterinary Science, The University of Queensland, Gatton Campus, Gatton, Queensland, Australia *Corresponding author: [email protected] Keywords equine, horse, wound healing, cannabis, cannabidiol, wounds, limb

Objective Evaluate the effect of topical 1% cannabidiol on second intention wound healing in distal limb wounds of horses

Design Experimental

Animals Six Standardbred horses

Methods A total of five 2.5cm x 2.5cm full thickness skin wounds were created on the dorsomedial aspect of the metacarpi of 6 horses. Wounds were contaminated with faeces on the day of wound creation. Each wound was then assigned to a treatment group; compounded 1% cannabidiol in unique manuka factor (UMF) 5 manuka honey, UMF5 manuka honey, UMF 20 manuka honey or saline. Each treatment was applied topically daily for a total of 42 days. Legs were bandaged and bandages were changed, daily, for 13 days post-operatively. Digital photographs of each wound were taken on day 1 then weekly for 6 weeks. Wound size, daily healing rate and total time to healing were recorded and compared statistically.

Results Irrespective of the treatment, wounds did not retract as expected in the first 7 days after wound creation. There was no difference in wound area, daily healing rate, days to complete healing between treatment groups.

This is the author manuscript accepted for publication and has undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1111/avj.12932

This article is protected by copyright. All rights reserved. Conclusions This preliminary study failed to demonstrate any difference in wound healing variables between treatment groups in this model of second intention wound healing. This was unexpected due to the established effects of UMF20 manuka honey on wound healing using the same model. This may be due to systemic effects of cannabidiol and study design. Further research into the use of cannabidiol in equine wounds is warranted.

Keywords equine, horse, wound healing, cannabidiol, manuka honey

Abbreviations CBD, cannabidiol; MGO, methylglyoxal; TGF-β, transforming growth factor β; THC tetrahydrocannabinol; UMF, unique manuka factor

In horses, wounds involving the distal limb are often left to heal by second intention due to extensive tissue loss and contamination.1-5 In the horse healing of wounds in the distal limb is characterised by a weak and protracted inflammatory phase, resulting in greater wound retraction and slower wound contraction when compared to wounds of the body.1 As a result, the horse is uniquely prone to the development of exuberant granulation tissue.1 Consequently, there have been many studies focussed on developing topical agents to improve wound healing in the horse.2-5

Honey, as a wound dressing, dates back to the ancient Egyptians,6 but fell out of favour when modern antimicrobial agents were developed.7 However, as antimicrobial resistance has developed, honey has re-emerged as a valued topical treatment for wounds. Honey is a natural product that boasts both anti- inflammatory and antimicrobial effects and it is particularly attractive because, currently, there has been no reported bacterial resistance to honey.8 Manuka honey has been the focus of much of the recent research into medicinal honeys due to it’s superior antimicrobial efficacy and quality assurance.7 Unique manuka factor (UMF) is a trademarked grading system used by producers in New Zealand that benchmarks the antimicrobial activity of manuka honey against the a standard antiseptic, phenol.7 In general a UMF rating of 0-4 has non-detectable antimicrobial activity, 5-9 has minimal antibacterial activity, 10-15 is regarded as therapeutically useful as an antimicrobial, while 16-30 has superior antimicrobial activity.7 In a number of studies, application of UMF 20 manuka honey has been shown

This article is protected by copyright. All rights reserved. to reliably enhance short and long term second intention wound healing in contaminated and uncontaminated equine, distal limb, wound models.2-5 Specifically, treated wounds retract less and develop a healthier wound bed, characterised by improved histologic wound healing variables, earlier in the healing process when compared to untreated wounds.2-4 While most of the beneficial effects of manuka honey on wound healing is attributed to methylglyoxal (MGO), evidence would suggest the antimicrobial efficacy of MGO appears to rely on the presence other known and many unknown antimicrobial and ant-inflammatory constituents within manuka honey, to reach optimal efficacy.7,9

In human medicine, there is another natural product, marijuana, which has recently ignited significant interest as a therapeutic substance for a variety of medical conditions. Marijuana (Cannabis sativa) contains more than 60 biologically active chemical agents, known as cannabinoids, that have also been shown to have potent antimicrobial and anti-inflammatory activity.10-12 Tetrahydrocannabinol (THC) is the most well known compound and is used for both medicinal and illicit purposes due to its psychoactive effects.10-12 Cannabidiol (CBD), a non- psychoactive constituent, is also found in relatively high concentrations in the plant.13 The cannabinoids act within the body on CB1 and CB2 receptors, found predominantly within neuronal and immune tissues respectively.14,15

With the aim of avoiding the psychotropic effects of marijuana, cannabidiol extract has been used selectively, and has been shown to have multiple, potentially beneficial effects, medically.16.17 Cannabidiol extract, used topically, has been shown to activate CB2 receptors in the skin to attenuate inflammation and fibrogenesis,16 as well as promoting skin re-epithelialisation in a mouse wound model.17 Similar to manuka honey, a synergism between various compounds within the marijuana plant has been described, suggesting that compounds like CBD and THC together exert a stronger effect than the individual components used alone.18 Similarly a proposed synergy between the cannabinoids and another major component of the hemp plant, the terpenoids, has been suggested.19 Terpenoids share the same precursor as the phytocannabinoids and have some therapeutic benefits in their own right.19

The purpose of this study was to investigate the potential for CBD extract to modulate second intention healing of distal limb wounds in horses. Specifically, the aim was to determine if a 1%

This article is protected by copyright. All rights reserved. CBD extract combined with UMF5 manuka honey as a carrier would deliver a beneficial effect on second intention wound healing in a contaminated distal limb wound model in horses. Including UMF 5 manuka honey as the carrier in the CBD preparation was attractive to investigate the possibility of any synergism between manuka honey and cannabidiol that would offer a novel natural product for use in equine distal limb wounds. Our hypothesis was that the combination of UMF5 manuka honey and cannabidiol would enhance second intention wound healing variables in contaminated, equine distal limb wounds.

Materials and Methods Six adult Standardbred horses (3 geldings, 3 mares aged between 3 and 10 years) without evidence of wounds or scars on their forelimbs were used in the study. The study was approved by the Institutions Animal Care and Ethics Committee. One week prior to surgery the horses were weighed, identified with a numbered collar and treated with an anthelmintic. They were housed in pairs in grass yards for the duration of the study. Lucerne hay was fed at 2.5% body weight in divided feeds daily and free access to water was available throughout the study.

Wound creation Each horse underwent a clinical examination on the morning of surgery. The dorsal aspect of both metacarpi in all horses were clipped. A 14G catheter was placed in the left jugular vein, after aseptic preparation and local anaesthesia of the site. Each horse received a single dose of phenylbutazone (2.2 mg/kg IV) and tetanus prophylaxis prior to surgery. Horses were sedated with xylazine hydrochloride (1.1 mg/kg IV) and anaesthesia was induced with diazepam (0.1 mg/kg IV) and ketamine (2.2 mg/kg). Anaesthesia was maintained using total intravenous anaesthetic agents (Triple Drip).20

All wounds were created on the same day. The skin over the metacarpi was not aseptically prepared. A total of five full thickness skin wounds (2.5 cm x 2.5 cm) were created on the dorsomedial aspect of the metacarpi (three on one forelimb, two on the contralateral forelimb). The limb to receive the three wounds was randomly assigned by blindly pulling the horse number and the treatment protocol out of a hat. The wounds were made medial to the extensor tendons and were

This article is protected by copyright. All rights reserved. evenly spaced between the carpus and the fetlock. Wounds were created by making a full thickness skin incision around a standard, flexible template using a scalpel, and the skin was removed with surgical scissors. Two grams of fresh faeces collected from a horse that was not included in the study was placed on each wound, and each wound was individually bandaged. The bandage consisted of a low adherent, absorbent dressing cut to size to prevent cross contamination (Melolin, Smith and Nephew Medical Ltd, Hull England) covered with a soft gauze roll (Easifix K, BSN Medical, Hamburg, Germany) and compressed under an elastic adhesive tape (Tensoplast Vet, BSN Medical, Hamburg, Germany). The horses were allowed to recover unassisted.

Wound treatments Bandages were removed 24 h after wound creation and the faeces rinsed off the wounds using sterile saline. The wounds on the leg with two wounds were treated daily with either 2ml of UMF5 manuka honey (carrier), (Comvita Australia Pty Ltd, Brisbane, Australia) or 1% CBD extract (equivalent of 10mg CBD) in 2 ml of UMF5 manuka honey (CBD). The wounds on the leg with 3 wounds were treated with 2ml of 0.9% sterile saline (saline), 2ml of UMF 20 manuka honey (UMF20), (Comvita Australia Pty Ltd, Brisbane, Australia), or 2ml of UMF5 manuka honey. The 1% CBD extract was mixed with a carrier (UMF 5 manuka honey) to provide a similar treatment volume to optimise contact with the wound. The treatment allocation on each leg was predetermined so that treatments on each leg were evenly distributed across all wounds and all horses. Treatments were randomly assigned by pulling the horse identification and the assigned treatment protocol for each leg randomly out of a hat. After treatment, wounds were individually bandaged, as described, to prevent mixing of treatments. Wounds were bandaged after wound creation and for a total of 13 days and bandaged were replaced daily. Treatment was applied daily for 12 days under a bandage. On day 13 bandages were removed and wounds were left open and treated daily for a total of 42 days.

Wound measurements Digital photographs of the wound were obtained on days 1, 7, 14, 21, 28, 35, 42 after wound creation and wounds were measured using image analysis software (Image J, US National Institutes of Health, Bethedsa, USA). A 2.5 x 2.5 cm template (6.25 cm2) was placed next to the wound as a

This article is protected by copyright. All rights reserved. standard reference (Figure 1). The area of the wounds was measured three times and the average calculated to produce a single measure for each wound for each measurement day. The area of the granulation tissue, not including the advancing edge of epithelium on each side of the granulation bed, was measured. Overall time to complete healing was recorded. Wounds were considered completely healed when granulation tissue was no longer visible. Overall rate of wound healing (cm2/day) was determined by dividing the wound area on day 1 by the total days to complete healing.

Statistical analysis

A general linear model was used to analyse the data, with each horse having multiple measurements over time after wound creation. The model included fixed effects of treatment (saline, carrier, UMF20, CBD), day (1, 7, 14, 21, 28, 35, 42), leg (left, right), position on leg (top, middle, bottom) and the interaction involving treatment and day. A random effect was entered for each horse to account for correlations between measurements made in the same horse. Residuals form the final model were inspected to check model assumptions.

Summary outcome measures were derived at the level of each wound for each horse to represent total days to complete healing, and overall healing rate (change in wound area per day from day 1 to complete healing). Separate general linear models were used to analyse these outcomes using the same approach as described above.

Significant effects in the main model were then subjected to pairwise comparisons of estimated marginal means as follow-up tests, using standard error terms derived from the general linear model. All follow-up tests associated with significant unadjusted p-values were then adjusted using the False Discovery Rate approach to correct for the risk of elevated type I error due to multiple comparisons.21

The analysis was first run with wounds treated with the carrier (UMF 5 manuka honey) on the separate legs included as separate treatments. Initially this was run with the wounds treated with the carrier identified as left or right. The analysis was then subsequently run with the wounds treated by

This article is protected by copyright. All rights reserved. the carrier being identified as ipsilateral or contralateral to the wound treated with CBD. These analyses were run to determine if there was any local effect of the CBD on healing. These analyses showed no difference in mean wound area on any treatment day between any of the treatment groups so the mean wound area for all the carrier wounds were combined for the final analysis.

Analyses were performed in STATA (Stata Data Analysis and Statistical Software, StateCorp LP, College Station, TX, US) using an alpha threshold of 0.05.

Results All horses were clinically healthy throughout the trial. There was no evidence lameness or swelling and no wounds developed exuberant granulation tissue. There was no difference in wound area between any of wounds on day 1 following wound creation before treatment commenced. The general patterns of healing were similar across all treatment groups (Figure 2). There were no differences in the daily mean wound area between any of the treatments on any of the treatment days.

There was no change in wound area for any of the wounds, irrespective of treatment, between days 1 and 7. After day 7, there were significant differences in mean wound area over time within each treatment group. The daily, mean wound area for days 14 to 42 was significantly smaller than day 1 and 7 means in all treatment groups (P < 0.05). In all treatment groups, daily mean wound area for days 21 to 42 were smaller than the day 14 mean (P < 0.05). Daily mean wound area for days 14 to 42 were significantly smaller than the mean wound area on day 7 (P < 0.05) for all treatment groups. Beyond day 21 the rate of reduction in wound area appeared to slow and comparisons of mean wound area within any one treatment group produced significant results in some treatment groups and non-significant results in other treatment groups.

There was no difference in the mean (± SE) overall healing rate (cm2/day) between any of the treatment groups (saline: 0.108 ± 6.5; carrier: 0.113 ± 0.006; UMF 20: 0.100 ± 0.008; CBD: 0.111 ± 0.008). There was no difference in the mean (± SE) total days to healing between any of the treatment groups (saline: 84.9 ± 0.008; carrier: 83.2 ± 5.0; UMF 20: 88.5 ± 6.8; CBD: 84.4 ± 6.7).

Discussion There were no differences between the measured wound healing variables from wounds treated with the different treatment regimens in this study. This was unexpected because a series of recent studies have shown UMF 20 manuka honey consistently improves measured wound healing variables when applied, daily, to contaminated or uncontaminated, surgically created, experimental, distal limb wounds left to heal by second intention in horses, when compared to control wounds.2-5 In these studies, wounds treated with UMF 20 manuka honey consistently retracted less than untreated wounds.2,3 The authors suggested the presence and efficiency of myofibroblasts in a mature granulation bed in the first 7 days after wound creation in treated wounds, limits wound retraction when compared to untreated wounds.2,3 While the most obvious benefit of application of UMF 20 manuka honey is seen in the first 7 days after wound creation, improved overall healing times have also been reported.3,5

The same contaminated wound healing model was used in the present study.2,3 The mean wound area on day 1 before treatment commenced was consistent across all treatment groups. This was expected and consistent with the results of previous studies.2,3,5 The mean wound area in all treatment groups was then expected to increase between day 1 to day 7.2,3,5 However, there was no change in the mean wound area irrespective of treatment. . Specifically, wounds treated with saline were expected to retract more than wounds treated with UMF20 manuka honey,2,3 but the results of the current study showed that the pattern throughout healing was the same for all treatment groups with no apparent effect of treatment.

There are few likely explanations for the unexpected pattern of healing in the current study.2-5 One possibility is that there was a systemic effect of the topical CBD. CBD is suggested to have potential anti-inflammatory12,22, antioxidant23 and analgesic properties24 as well as potent antibacterial effects.11,25 It would appear the activation of CB2 receptors in cells such as lymphocytes, monocytes, macrophages, fibroblasts and keratinocytes, amongst others,26-29 suppress inflammation by reducing expression of various cytokines including transforming growth factor β (TGF‐β), transforming growth factor α, interleukin-1 and insulin-like growth factor-1.30 In most

This article is protected by copyright. All rights reserved. species, TGF‐β has the broadest spectrum of activity in wound healing and is directly associated with the regulation of the fibrotic response.31,32 In horses, the different isoforms of TGF‐β interact to play a pivotal role in orchestrating wound healing.33-35 TGF-β1 peaks soon after wounding while TGF-β3 peaks 7-9 days later and modulates the fibrotic response.33-35 While the process of second‐ intention wound healing in the horse is complex and not completely understood, the concentrations and temporal expression patterns of these two cytokines within the wound appear to be essential for the normal progression of wound healing, particularly in wounds on the distal limb.33-35 It is believed in horses, and in particular in distal limb wounds, an imbalance in the expression of TGF β 1 and 3 can cause a weak and prolonged inflammatory response that leads to delayed wound healing.1

If there was a systemic effect of CBD in this study, it would have been expected to have a similar influence on the progress of wound healing in all wounds. Based on the reported properties of CBD the potential effects of topical application in a contaminated equine wound healing model would be expected to be associated with it’s antimicrobial and anti-inflammatory effects. The wound healing model used in the current study was originally developed to try and more effectively replicate naturally occurring wounds than aseptic, surgical models used in earlier studies.2-5 The purpose of applying faeces was to replicate wound contamination. However, while this has been shown to increase aerobic and anerobic counts on the wounds, bacterial counts only remain elevated for 24 h.4 Nevertheless, the application of faeces for 24 h has been shown to be sufficient to affect the pattern of healing compared to non-contaminated wounds.3 Wounds treated with faeces retract further, but heal faster than un-contaminated wounds.3 This suggests the application of faeces for 24 h may stimulate the initial inflammatory response in equine distal limb wounds and enhance the wound healing process without the negative effects of persistent or overwhelming infection often associated with naturally occurring distal limb wounds.3 If CBD was to have a systemic effect in this model, it is very likely the reported anti-inflammatory and antimicrobial properties would have reduced wound retraction in all wounds in the first 7 days after healing which is consistent with the results reported in the current study.

This article is protected by copyright. All rights reserved. When designing the experiment, we had considered there could be systemic and regional effects of CBD following topical application. However, the available evidence suggested any systemic effect was likely to have been very small and unlikely to affect wound healing. A study investigating the transdermal effects of CBD applied topically for treatment of pain and inflammation in a rodent, adjuvant-induced, monoarthritis model in 260-280 g rats found daily doses of 6.2 mg and 62.3 mg CBD applied topically for 4 days were effective, while lower doses were not.36 While daily doses of 0.6 and 3.1 mg were not therapeutically effective, they were absorbed systemically and revealed a dose related, linear, pharmacokinetic correlation when the 6.2 mg dose was included.36 When the higher dose of 62.3 mg was included in the pharmokinetic correlation, it did not follow this linear relationship.36 The authors proposed that transdermal absorption could become saturated when higher doses and higher volumes of medication were applied, particularly if these doses were applied to a confined area.36 The authors also postulated that application of CBD to the skin overlying the affected joint would potentially increase the local CBD concentrations and enhance local efficacy without systemic involvement.36 In the current equine wound study, wounds treated with CBD received 10 mg of CBD topically, daily which was considered a modest dose that would have local effects without therapeutic, systemic effects. However, the current study applied the CBD to open wounds rather than transdermally, so it is possible systemic absorption of the CBD through an open wound may have been higher and less restricted by area in this study compared to transdermal application in the rat. Nevertheless, given doses of 0.6 and 3.1 mg in 260-280 g rats applied topically to the skin were absorbed systemically but not therapeutically effective,36 it was considered a dose of 10 mg in a 500 kg horse was unlikely to deliver systemic concentrations that would be therapeutically effective in the current study. There have been no studies investigating CBD in horses so it is possible the effects of CBD or the sensitivity of CBD receptors are different in horses than rodents.36

While the current study design did not anticipate a systemic effect, it did account for the possibility of local effects of CBD, for any potential effects of UMF5 as the carrier, and for any interaction between CBD and UMF5, on wound healing. Alone, UMF 5 manuka honey was considered unlikely to have any effect on wound healing based on a previous study that showed UMF 5 manuka honey had no effect on wound healing variables in an uncontaminated would healing model

This article is protected by copyright. All rights reserved. in horses.5 Furthermore, previous reports that suggest UMF 5 manuka honey is generally not recommended for therapeutic use as an antibacterial agent because the known active constituent methylglyoxal is found in low concentrations.5,7,37 Nevertheless, UMF 5 was considered an attractive carrier for the CBD in this preliminary study because different varietals of honey contain many unidentified bioactive components, that combined, contribute to therapeutic benefits of the specific varietal, and even batch, of a specific honey varietal.5,37 Trademarked manuka honey is the only honey where there is an attempt to provide a reliable measure of bioactivity but this is solely with respect to the honey’s antimicrobial properties.5 While most of the beneficial effects of manuka honey on wound healing are currently attributed to MGO, evidence would suggest the antimicrobial efficacy of MGO appears to rely on the presence other known, and many unknown, antimicrobial and ant-inflammatory constituents within the honey, to reach optimal efficacy.7,9 Synergism between the various compounds within the marijuana plant has also been described, suggesting that compounds like CBD and THC together exert a stronger effect than the individual components used alone.18 It is not unreasonable to speculate that the bioactive components of different natural products may have the potential to have synergistic effects when combined.

On the leg with 2 wounds in the current study, one wound was treated with UMF 5 manuka honey while the other wound was treated with 1% CBD in a carrier of UMF 5 manuka honey. On the contralateral leg, 3 wounds were created, and wounds were treated with UMF 20 as a positive control, saline as a negative control or UMF 5 manuka honey. The assumption in the study design was there would be no systemic effects of the CBD. However, the study design did accommodate the potential for a regional effect of CBD on adjacent wounds on the same leg. If indeed there was no systemic or regional effect of CBD on adjacent wounds, it was expected there would be no difference in the healing variables between the UMF 5 manuka honey treated wounds on each leg. However, if there was no systemic or regional effect, we would have still expected to see the beneficial effects of UMF 20 manuka honey treatment identified in previous wound healing studies, particularly given the study used a contaminated wound model.2-5 Specifically all wounds were expected to retract in the first 7 days after wound creation, while UMF 20 wounds were expected to retract less than saline treated wounds and potentially heal faster when treated daily for 42 days.3,5 Any beneficial effect of topically applied 1% CBD combined in a carrier of UMF 5 manuka honey

This article is protected by copyright. All rights reserved. was expected to have been seen as an improvement in wound healing variables in the CBD treated wounds when compared to the wounds on the contralateral leg treated with UMF 5 alone and wounds treated with saline. Furthermore, and in the absence of a regional effect, the healing variables in wounds treated with 1% CBD in a carrier of UMF 5 manuka honey would have been expected to be improved compared to the wounds treated with UMF 5 manuka honey alone on the same leg. However, there were no differences and in fact the wound healing variables for all wounds, despite treatment, were not different. While the study was well designed to to evaluate the effects of 1% CBD in UMF 5 manuka honey against the known effects of a positive and negative control, the design did not anticipate the possibility a systemic effect.

The same experimental model has been used successfully in previous studies and reliably showed a repeatable pattern of healing that was not replicated in the current study. The study methods including the brand of honey, age and breed of animals and techniques for wound creation, wound measurement and statistical analysis were all consistent with previous studies.2-5 While it is possible there may have been other unknown factors responsible for the unexpected results in this study, a systemic effect of CBD seems most likely. A further study into the effects of CBD on equine wound healing that accommodates potential systemic effects of CBD would need to be developed to validate this assumption. However, any further studies would be complicated by the inability to use each horse as it’s own control, so any study would need a larger study population to account for differences in wound healing between horses and would add another layer of complexity and cost to the experimental model.

In this pilot study we were unable to demonstrate an effect of 1% CBD in a carrier of UMF 5 manuka honey applied topically for 42 days on wound healing variables in a second intention healing contaminated equine distal limb wound model. The results of the study suggest there was a systemic effect of CBD in this model and further studies may be warranted.

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Figure 1. Wounds on the left forelimb of horse 1 on day 42 after wound creation. The standard 2.5 cm x 2.5 cm template that was used as a reference to accurately measure the area of the healing wound using digital pictures (A). The wound has a central area of healthy granulation tissue (B) surrounded by a pale pink region of advancing epithelium (C).

Figure 2. Days post wound creation versus wound area in a contaminated model of wound healing in the equine metacarpus. Treatment groups included unique manuka factor 20 manuka honey (UMF 20), saline control (saline), 1% cannabidiol extract in a carrier of unique manuka factor 5 manuka honey (CBD) and unique manuka factor 5 manuka honey (carrier). The plots include bars which represent 95% confidence intervals around the mean wound areas of each treatment group on the days of measurement (days 1, 7, 14,21, 28, 35, and 42). There was no difference in the pattern of healing between any of the treatment groups.