A major concern when developing any topical formulation is the rate and extent that the active ingredient/s and/or the vehicle (carrier agent) will penetrate into and through the skin. Extensive research shows that transdermal drug penetration will vary substantially between different species, meaning that a formulation developed for humans will have a different profile of how much drug moves through then skin and how quickly, than to other animals such as horses. These findings strongly suggest that topical medications intended for use in horses must be developed with an understanding of the kinetics and characteristics of drug penetration through equine skin. However, to date, there is very little basic information of this nature available.

Inadequate knowledge of topical drug delivery can adversely affect animals and owners following use of topical formulations. Too much drug moving through the skin can lead to toxic effects while too little drug can render the formulation ineffective. Intentional or inadvertent application of creams, sprays and gels to a horse’s skin prior or during competition can lead to detectable levels of drugs in the blood or urine of the animal, which may infringe the rules and lead to penalties. Drug penetration through skin may also be exacerbated if the skin is damaged, either by physical trauma or by cleaning agents, again leading to higher systemic drug levels.

This report is intended to address the deficiency of basic knowledge of transdermal drug penetration in the horse. A centre of expertise was established both to undertake this research and to provide advice to interested stakeholders, including industry, horse owners and trainers, stewards and regulators.

Models were developed and validated, particularly in vitro techniques, to characterise the effects of site of application, vehicle formulation and changes in the integrity of the skin surface, such as skin damage or disease, on transdermal drug penetration. This knowledge will be used to develop more effective and safer topical preparations for use in the horse.

Background
The skin is the largest organ of the body, accounting for more than 10% of body mass. It has important protective and homeostatic roles and is generally being regarded as a critical protective barrier to the external environment. Extensive studies have shown that the skin is more complex than merely a barrier which becomes apparent when agents are applied to the skin either deliberately or accidentally. The extent of absorption through the epidermis, dermis and systemically becomes important when we consider that drugs are applied to the skin for (i) local effects (e.g. corticosteroids for dermatitis); (ii) transport through the skin for systemic effects (e.g. fentanyl, nicotine, oestradiol and testosterone patches); (iii) surface effects (e.g. sunscreens and anti-infectives); (iv) to deeper target tissues (e.g. non-steroidal anti-inflammatory agents (NSAIDs) for muscle inflammation); and (v) accidental exposure (e.g. solvents in the work place, agricultural chemical, or allergens).

Investigation of human skin has revealed that the major resistance to drug penetration is the outermost layer, the stratum corneum. Several theories have been proposed for drug passage through the stratum corneum into the viable epidermis and dermis, including the “bricks and mortar” theory, representing keratinocytes held together by a lipid bilayer. However, differences in skin thickness, density of on the same individual display different pharmacokinetics of percutaneous drug penetration. More importantly, extrapolation of percutaneous penetration data between species is not practical and use of products in veterinary practice without consideration of these interspecies variations is risky.

In this report, we examine the current knowledge of transdermal drug penetration and specifically investigate factors affecting drug movement through equine skin.

Aims/Objectives
The specific aims of this project were as follows:

• to develop in vitro methodologies to measure drug movement through equine skin
• to measure the regional differences (e.g. thorax vs axilla vs lower leg) in drug penetration
• to measure transdermal penetration of drugs of interest to the equine industry, including nonsteroidal anti-inflammatory drugs (NSAIDs), corticosteroids and analgesics (e.g. fentanyl)
• to determine the effects of vehicles (e.g. propylene glycol) on penetration of drugs in the horse
• to determine the effects of skin damage (e.g. rashes) and altered microenvironment (skin cleansers) on transdermal drug movement in the horse.

This knowledge will specifically benefit the following: Owners and trainers of horses; Pharmaceutical companies when developing topical formulations for the horse; Veterinarians; Regulators, stewards and officials associated with equine competition.

Methods used
The primary techniques used were in vitro models involving Franz-type diffusion cells. Skin can be harvested from horses and stored before applying it to a diffusion cell, meaning that several individual horse skins can be studied under the same conditions. Since the stratum corneum is the primary barrier to drug penetration, diffusion cells permit direct and accurate measurement of how much and how quickly any particular drug can move through skin. Diffusion cells also negate the effects of cutaneous blood flow characteristic of in vivo models, so regional differences and the effects of vehicles can be studied without the confounding effect of alterations in blood flow.

A number of different drugs commonly used in equine medicine were studied in this project, including hydrocortisone, fentanyl (an opioid analgesic), methylsalicylate (Dencorub®, an anti-inflammatory medication) and testosterone. Analytical techniques were also developed for each of these drugs and their metabolites using high pressure liquid chromatography (HPLC).

A final model of in vivo transdermal drug penetration was developed to link in vitro findings to in vivo situations. Microdialysis, where a semi-permeable probe is placed under the skin to measure drug penetrating through skin, was successfully trialled using a dog model. Further trials of this novel technique in the horse are continuing.

Results/Key findings
It was found that regional differences in the penetration of drugs through equine skin, with hydrocortisone and methylsalicylate penetrating faster and to a greater extent through skin from the leg, which is useful if applying these drugs for anti-inflammatory activity to the legs of horses.

Conversely, fentanyl from a commercial TTS system (patch) penetrates better through thorax and groin regions and application of fentanyl patches to upper body regions in the horse is recommended when analgesia is required.

The results showed significant effects of the vehicle on the transdermal penetration of testosterone in the horse, with vehicles containing ethanol or propylene glycol (both commonly found in topical creams and gels) associated with greater drug amounts moving through the skin.

An important finding was the effect of perturbations to the skin surface, affecting the outermost layer, the stratum corneum. These were created using common clinical approaches to treating skin, including shaving, application then removal of adhesive tape and cleaning skin with chlorhexidine (either aqueous or dissolved in alcohol). All these treatments significantly enhanced the penetration of methylsalicylate through skin.

Implications for relevant stakeholders for
Any stakeholder in the equine industry, from trainers, owners, veterinarians, officials and regulators should be aware of the potential from any substance applied to skin to pass through this outermost barrier. They should also be aware that there are significant species differences between how much and how fast a particular formulation will penetrate skin, meaning that a formulation developed and registered for one species will be unlikely to have the same effect and safety margin when applied to another species, including the horse. This has important implications for using drugs with potential toxic effects or low margins of safety, where small changes in uptake of drugs applied topically should be avoided. Equally, stakeholders entering horses into competition should be aware of the potential for altered appearance of drugs and metabolites in the body following topical application which may lead to infringement of the rules of competition.

Stakeholders should be mindful of many of the findings in this report when applying topical drugs to the horse. There are regional differences, meaning that a formulation applied to one part of the body may have a different level of effect if applied elsewhere. Different formulations of the same drug can have significant effects on how much of the drug actually penetrates through the skin and is available for whatever purpose it was designed. Most importantly, changes in the integrity of the skin, such as rashes, abrasions, skin disease or the use of agents to clean the skin, can substantially alter the amount and rate of active drug uptake.

Importantly, stakeholders should also be aware that the basic knowledge of equine topical drug formulation is increasing, particularly with the current project, with more drugs being produced specifically for horses. Topical drugs are easy to apply and have many other advantages, meaning that the potential to treat a range of medical conditions, such as joint pain, skin conditions and cutaneous pests (i.e. fleas, biting flies), is increasing. Better understanding of transdermal drug penetration in the horse will lead to more products that are more effective for topical application to horses.