Method of analyzing percutaneous absorption of an agent

Field of the invention The present invention relates to a method of analysis of skin permeability, and in particular a method of measurement of local percutaneous absorption of an exogenous agent.

Background of the invention In various circumstances there is a need or desire to detect an exogenous agent or sensing a level of an exogenous agent in the human or animal body. Some examples include detecting a drug or medication, measuring the uptake level of a topically applied substance or measuring the presence and/or effect of environmental agents in a subject. When studying percutaneous absorption, known methods of

measurement of skin absorption of an agent include measuring a concentration of the agent in blood, taking skin samples for analysis, in addition to various in vitro methods using skin. It is also known to calculate permeability coefficients ( K _P ) for chemical compounds based on their size (molecular weight or molecular volume) and octanol/water partition coefficient (Potts, R.O. & Guy, R.H. Pharm Res (1992) 9: 663). The mass of chemical absorbed through the skin can then be calculated

(Semple, Occup Environ Med 2004;61 :376-382) as

Us _k ⁼ Kp C A t wherein

Usk is the mass of chemical absorbed through the skin (mg) K _P is the permeability coefficient (cm-lr ¹ )

C is the concentration on the skin (mg -cm ³ )

A is the area exposed (cm ² ), and t is the duration of exposure (h).

Using a blood sample for analysis entails drawing blood from the subject, which may be more or less of an uncomfortable procedure for the subject, and normally requires a medically qualified person to perform it. Furthermore, measuring blood levels of an agent, after exposure of subject to the agent, gives a system-wide average concentration of the blood, as the agent, if present in the blood, will be instantly diluted throughout the circulatory system. This may make it difficult to relate the absorbed levels to the amount of exposure of the agent on a localized area of skin.

Taking a skin sample after exposure to an agent is an invasive procedure, which may be anything from uncomfortable to painful for the subject, and runs the risk of infection at the sample site.

An example of a method using skin samples is shown in US 2011/0112121 A1 , which discloses methods of analyzing skin penetration. The analytical method involves isolating the epidermis of sacrificed animals by heating the skin samples, taking biopsies and analyzing the content of the biopsies by LC/MS.

Skin permeability may actively be influenced by human interference in different manners. One example of external manipulation of skin permeability is ultrasound treatment, as shown in US 6,190,315 B1 , wherein is disclosed a method using ultrasound to enhance transdermal transport of drugs.

However, studying skin permeability without affecting the absorption process itself has proven a challenge. Any in vitro method of analysis, using e.g. cultured tissue or skin from animals, or synthetic membranes, will not accurately mimic the in vivo system when studying skin absorption. Such experiments are commonly used as a first step to provide a rough indication of e.g. toxicity of chemicals.

It is previously known to use so called microneedles in devices for sampling of bodily fluid. See e.g. WO2015/027093, WO2016/164208, WO2011/116388,

WO2015/187066 and WO2014/088493. Such devices may be used to sample interstitial fluid or blood, and analyzing various endogenous agents, e.g. glucose levels or levels of various biomarkers. A microneedle within such an array is provided with a capillary bore for withdrawing the interstitial fluid. Usually a number of microneedles are provided on a substrate, and by capillary action, and/or an applied suction or sub-atmospheric pressure, a sample is extracted from the skin and into a collection or analysis chamber.

With hollow microneedles a new type of pain-free general extraction method of bodily fluids can be used. The skin penetration with microneedles is perceived as minimally invasive and causes no pain, with needles too short to reach sensory nerves in the skin. It can utilize interstitial fluid, instead of blood, as measurement media, extracted through a matrix of sub-millimeter long silicon microneedles at a skin depth where no blood capillaries exist. An example of such a microneedle is disclosed in WO 2017/095321. In this publication a microneedle is disclosed having a capillary bore extending in the longitudinal direction and having an opening close to the distal end. The proximal end is connected to a substrate on which the microneedle is provided. In this example, the distal end is configured with a bevel.

Summary of the invention

Thus, there is a need for an improved method of measurement of local percutaneous absorption of an exogenous agent.

An object of the present invention is to provide a method for analyzing agents applied to the skin.

A further object of the present invention is to provide a method for determining whether a person has been exposed to an agent capable of penetrating the skin.

Another object of the present invention is to provide a method of measurement of local percutaneous absorption of an agent with minimal effect on the absorption process itself.

Yet another object is to provide a method of measurement of local percutaneous absorption of an agent with minimal effect on the metabolism and effect of the absorbed agent. The above-mentioned objects are achieved by the present invention according to the independent claim. Preferred embodiments are set forth in the dependent claims.

The present disclosure relates to a method of analyzing local percutaneous absorption of an agent, characterized by the steps a) applying the agent to the skin of a subject b) extracting interstitial fluid from the skin using at least one hollow microneedle; c) determining a presence of the agent in the extracted interstitial fluid, wherein the presence of the agent in the extracted interstitial fluid indicates local percutaneous absorption of the agent. In one embodiment of the invention the determination of the presence of the agent in the extracted interstitial fluid further comprises determining a concentration of the agent in the extracted interstitial fluid.

In one embodiment of the invention, the method further comprises a step of calculating a ratio of the amount of agent in the interstitial fluid in relation to the amount of agent applied to the skin surface.

In one embodiment of the invention a skin surface area through which the extraction is performed, is essentially within a skin surface area where the agent is applied.

In one aspect, the invention relates to a method of analyzing the ability of a helper agent to modify local percutaneous absorption of an agent comprising performing the method according to the above in the presence and absence of said helper agent, respectively, and comparing the results.

Further, a method for determining whether a person has been exposed to an agent capable of penetrating the skin, comprising steps of extracting interstitial fluid from the skin of a subject with at least one hollow microneedle, and determining a presence of the agent in the interstitial fluid is disclosed.

In a further aspect, the invention relates to the use of at least one microneedle in a method according to the invention. Detailed description

As mentioned above, known methods of analyzing concentration of agents in interstitial fluid focus on analysis of endogenous agents, or substances transported to the interstitial fluid via the bloodstream. Known methods for analyzing local percutaneous absorption of agents are commonly invasive, painful and/or require time-consuming steps and resources.

The inventor has realized that by using extraction of interstitial fluid with using hollow microneedles, a method of analyzing the ability of an agent to penetrate a skin's outer layer is provided which mitigates the problems of prior art. Thus a method is provided comprising the steps of a) applying an agent to the skin of a subject b) extracting interstitial fluid from the skin using at least one hollow

microneedle; c) determining a presence of the agent in the interstitial fluid. This method is based on the realization that the presence and/or amount of an exogenous agent in the interstitial fluid will directly correlate to the amount absorbed by the skin after application of, or direct exposure to, the agent on a skin surface.

An agent to be determined in the present invention may be a chemical compound that is capable of being absorbed through the human or animal skin. Notably, the use of the above method, and in particular the use of at least one hollow microneedle to extract interstitial fluid from the skin, has minimal effect on the absorption process itself. The extremely small size of the needles barely affects the skin surface, and the small amount of interstitial fluid extracted is a very small volume compared to the total volume of the interstitial fluid in the sample area of the skin. Thus, the disclosed method provides an analysis method which may be performed during ongoing administration of the agent to the patient, and will not effectively influence the absorption or the uptake and metabolism of the agent. The step of determining a presence of the agent in the interstitial fluid may comprise determining a concentration of the agent in the interstitial fluid. Depending on the nature of the agent, various methods of detection and measurement may be used.

After determining the concentration of the agent in the interstitial fluid, a ratio of the amount of agent in the interstitial fluid in relation to the amount of agent applied to the skin surface may be calculated. Thus, a measurement of the agent’s ability to penetrate the skin’s outer layers is obtained, as well as the agent’s efficacy in penetration of the skin.

Preferably, the extraction of the interstitial fluid is performed within a skin surface area that is essentially within a skin surface area where the agent is applied.

Thereby, a calculated ratio directly correlates to the local application of, or exposure to, the agent on the skin surface.

Such a method may have many applicable uses. One example is within drug discovery or the cosmetics industry, wherein an active, or passive, substance’s ability to penetrate skin needs to be quickly and easily assessed.

Notably, any known means of application of, or exposure to, an agent on a skin surface may be used. Non-limiting examples may be applying a powder, solid, patch, paste, cream, emulsion, solution, suspension, gel, gas, aerosol or spray comprising the agent, or immersion in a fluid comprising the agent. The method above may also be used for analyzing the ability of a helper agent to modify the ability of an agent to penetrate a skin's outer layer. This may be performed by executing the method as described above in the presence and absence of said helper agent, respectively, and comparing the results.

In another aspect, a method for determining whether a subject has been exposed to an agent capable of penetrating the skin is provided, the method comprising the steps a) extracting interstitial fluid from the skin of a subject with at least one hollow microneedle; b) determining a presence of the agent in the interstitial fluid. This method is based on the same realization as previously described, i.e. that the presence of an exogenous agent in the interstitial fluid will directly correlate to the amount absorbed by the skin after exposure of the skin surface to the agent.

After determining the concentration of the agent in the interstitial fluid, a ratio of the amount of agent in the interstitial fluid in relation to the amount of agent applied to the skin surface may be calculated. Here, a measurement of the agent’s presence will indicate the subject’s exposure to the agent known to penetrate the skin’s outer layers.

Such a method would be useful in e.g. environments where it is essential to test subjects who have been exposed to e.g. airborne toxic substances.

Various hollow microneedles may be used for extraction of interstitial fluid. While a single hollow microneedle may suffice or even be desirable in a number of embodiments of the invention, it is also contemplated to use a plurality of

microneedles. The plurality of microneedles may be arranged in an array. Non- limiting examples of arrangements of microneedles may be those disclosed in WO2015/187066, WO2014/088493, or WO 2017/095321. For example, an array of hollow microneedles may be arranged in a regular pattern on a miniature chip. The microneedles may comprise an elongated body extending from a distal end with a bevel to a proximal end on the substrate along a longitudinal axis; the elongated body comprising a capillary bore extending in a longitudinal direction thereof and defining a fluid path, the proximal end being integrally connected with the substrate and the capillary bore being in fluid communication with a fluid channel of the substrate, as taught in WO2017/095321.

In use, a microneedle or plurality of microneedles may be pressed against the skin of a subject and the tips of the microneedles will penetrate into the outer layers of the skin by only a minimal depth, such that interstitial fluid may be sampled, but not deep enough to reach blood vessels or even sensory nerves. Interstitial fluid may be drawn into the microneedles by capillary suction, and/or applied suction, and may be collected for sampling in an underlying collection chamber. Example

Analysis of skin penetration by diclofenac in gel

A skin site of interest with a specific surface area is cleaned using water and soap and allowed to dry. A dose of diclofenac 1.16% topical gel (Voltaren ^® , comprising 11.6 mg diclofenac diethylamine per gram gel) is measured and applied on the chosen skin site according to the manufacturer’s instructions.

The topical gel is left in place for a specific time, e.g. one hour, before the excess gel is removed and the skin site is again cleaned with water and soap. One or more microneedles are applied to the skin site for extraction of interstitial fluid through microneedles onto a filter paper

Molecules are extracted from filter paper via elution in e.g. acetonitrile in a test vial (i.e. the filter paper is immersed into a solvent suitable for sample preparation of the molecules of interest for LC-MS analysis, e.g. combinations of acetic acid,

acetonitrile, chloroform, methanol, water, and more (Sample Preparation

Fundamentals For Chromatography, Agilent Technologies, Inc. 2013).

The purified liquid is injected into an LC-MS according to a suitable analysis protocol, e.g. as a second mobile phase carrying the sample, preceded by a washing step and a first mobile phase containing water. The separation column should be chosen according to the separation required. For diclofenac analysis, a reverse-phase Thermo Fisher Accucore RP-MS 2.6 pm, 50 x 2.1 mm could provide appropriate separation (Jones, 2012, ThermoFisher Scientific Application Note 20569).

The amount of diclofenac in the analysed interstitial fluid is compared to the total dose diclofenac applied to the skin to assess diclofenac’s ability to penetrate the skin. The present invention is not limited to the above-described preferred embodiments. Various alternatives, modifications and equivalents may be used as encompassed by the appended claims. Therefore, the above embodiments should not be taken as limiting the scope of the invention, which is defined by the appending claims. Prior art disclosures cited herein are incorporated by reference in their entirety.