METHOD FOR INVESTIGATING BIOEQUIVALENCE

The present disclosure relates to a method for investigating bioequivalence of a topically applied or applicable test substance with respect to a reference substance.

BACKGROUND OF THE DISCLOSURE

Topically Applied Drug Products (TADPs) are key in addressing consumer and public health needs. In many countries, strategies to control healthcare spending rely upon the availability and use of generic medicines. The safety and effectiveness of high-quality generic medicines is ensured through a demonstration of bioequivalence (BE). The methodology for assessing the BE of systemically absorbed drugs and for the statistical assessment of comparative systemic bioavailability (BA) based upon pharmacokinetic (PK) endpoints for drugs that were transported via the blood circulation (e g. oral, infusion, subcutaneous injection) is well established. Assessing the bioequivalence (BE) of locally acting topical dermatologic drug products, e g. in vivo, is however challenging, because of technical difficulties associated with measuring and comparing drug concentrations in the tissues of the skin. Conventional systemic pharmacokinetics (PK) studies that utilize blood draws provide an efficient approach by which to assess the BE of systemically acting drug products. However, these systemic PK studies are of limited relevance for assessing the rate and extent to which drugs become available in the skin from products administered upon the skin, i.e. from topically applied drugs, and intended to act locally in the skin.

Dermal Open Flow Microperfusion (dOFM) is a dermal sampling method that continuously collects diluted interstitial fluid (ISF) in a manner analogous to the way that blood draws collect plasma or serum from the systemic circulation. Thus, dOFM is able to assess the cutaneous PK of a topically applied drug at or near the site of action in the dermis. This and other techniques, e.g. microdialysis (MD), have been developed to assess the BE of topical dermatological generic drug products.

The present disclosure relates to an approach suitable to develop a highly standardized and most efficient clinical setup for all clinical studies needed to show cutaneous BE, i.e., the Optimal Cutaneous BE Study. SUMMARY OF THE DISCLOSURE

The present disclosure aims to provide improvements relating to the investigation of bioequivalence of a test substance, e.g. a generic drug, with respect to a reference substance, e.g. an originator drug. This aim is achieved by the subject-matter disclosed herein and, particularly, but not necessarily restricted to the subject-matter defined in the independent claims. Advantageous refinements and embodiments are set forth in the dependent claims and in the remaining disclosure.

In a first aspect, the present disclosure relates to a method for investigating bioequivalence of a topically applied or appliable test substance with respect to a reference substance ("topically appliable" expediently means that the relevant substance can be or is intended to be applied topically, e.g. for treating a certain disease), the method comprising: evaluating whether there is bioequivalence of the test substance with respect to the reference substance based on a) first application site data comprising data indicative for a dermal situation relating to the test substance or the reference substance below a first application site onto which the test substance or the reference substance has been applied, and based on b) second application site data comprising data indicative for a dermal situation relating to the test substance or the reference substance below a second application site onto which the test substance or the reference substance has been applied, wherein each of the first application site data and the second application site data comprises region data indicative for the dermal situation in a region below the respective application site, wherein at least one of the first application site data and the second application site data comprises or both of the first application site data and the second application site data comprise region data for a plurality of distinct regions below the respective application site.

Having region data from different region below the respective application site (e.g. a reference site with the reference substance or a test site with the test substance) facilitates the investigation of bioequivalence. Further, a plurality of sites may be advantageous from a statistical point of view as averages may be calculated.

In a second aspect, the present disclosure relates to a method for investigating bioequivalence of a topically applied or applicable test substance with respect of a topically applied or applicable reference substance, the method comprising evaluating whether there is bioequivalence of the test substance with respect to the reference substance based on first study data obtainable or obtained using a first study setup (e.g. a pre-study setup or pilot study setup) with one or more first study subjects, and based on second study data obtainable or obtained using a second study setup (e.g. a pivotal study setup) with one or more second study subjects, wherein the first and second study setups are different.

Using data obtained using different study setups in combination with one another has the advantage that data obtained from subjects from a pilot study may be used in combination with data from subjects from a pivotal study to investigate bioequivalence. In this way, the number of subjects required to obtain a predetermined statistical power of the bioequivalence determination may be lowered in the pivotal study as opposed to an approach in which the bioequivalence is determined only from data obtained in the pivotal study.

In a third aspect, the present disclosure comprises a method for analytically validating the method of the first and/or second aspect, the method comprising the step of performing the analytical validation using a suitable surrogate matrix.

Using a developed suitable surrogate matrix for analytical validation of the methods of the first and/or second aspect replaces the need of performing a matrix collection study to collect a real matrix and the subsequent validation study on the real matrix. This results in a overall quicker, more efficient, and more cost-effective evaluation of bioequivalence,

In a fourth aspect, the present disclosure relates to a system comprising an electronic control unit configured to control the execution of and/or to assist in executing the method of the first and/or the second and/or the third aspect of the disclosure.

According to one embodiment, the system may control and/or assist in evaluating the bioequivalence of the test substance. According to one embodiment the system may control and/or assist the determination of the first and/or second application site data, and/or of the region data of the plurality of region of the first and/or the second application site data.

According to one embodiment the system may control and/or assist the disregarding of a region and in particular of that respective region data. The system may for example perform statistical evaluation to determine which region to disregard. According to one embodiment, the system may control the execution of and/or assist in executing or evaluating the study results of the first and/or the second study setup.

The system may, for example, assist in evaluating the number of subjects required in the second study setup based on the data collected in or with the first study setup.

According to one embodiment, the system may control the execution of and/or assist in performing the analytical validation using a suitable surrogate matrix.

In a fifth aspect, the present disclosure relates to a, e.g. volatile or non-volatile, storage medium having stored thereon machine-readable instructions which, when executed by a processor of an electronic control unit, cause the electronic control unit or the system of the fourth aspect to control execution of and/or to assist in executing the method of the first and/or second and/or the third aspect of the disclosure.

In a sixth aspect, the present disclosure relates to a computer program or computer program product, which comprises machine-readable instructions which, when executed by a processor of an electronic control unit, cause the electronic control unit or the system of the fourth aspect to control the execution of and/or to assist in executing the method of the first and/or second and/or the third aspect of the disclosure.

This summary does not necessarily describe all features of the present disclosure. Other embodiments will become apparent from the following detailed description.

We note that features described in conjunction with one of the aspects do also apply for the other aspects. Also, features described in conjunction with other features (even features from one of the aspects mentioned above) may be extracted from the context they are described in.

DETAILED DESCRIPTION

Definitions

Before the present disclosure is described in detail below, it is to be understood that this disclosure is not limited to the particular methodology, protocols and reagents described herein as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present disclosure which will be limited only by the appended claims. Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art.

The term “comprise” or variations such as “comprises” or “comprising” according to the present disclosure means the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers. The term “consisting essentially of’ according to the present disclosure means the inclusion of a stated integer or group of integers, while excluding modifications or other integers which would materially affect or alter the stated integer. The term “consisting of’ or variations such as “consists of’ according to the present disclosure means the inclusion of a stated integer or group of integers and the exclusion of any other integer or group of integers.

The terms “a” and “an” and “the” and similar reference used in the context of describing the disclosure (especially in the context of the claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context.

As used herein, the term “about” indicates a certain variation from the quantitative value it precedes. In particular, the term “about” allows a ±5% variation from the quantitative value it precedes, unless otherwise indicated or inferred. The use of the term “about” also includes the specific quantitative value itself, unless explicitly stated otherwise. For example, the expression “about 80°C” allows a variation of ±4°C, thus referring to range from 76°C to 84°C.

Strategies to control healthcare spending rely upon the availability and use of generic medicines. The safety and effectiveness of high-quality generic medicines is ensured through a demonstration of bioequivalence (BE).

The United States Food and Drug Administration (FDA) recognizes the public policy benefit of generic competition but has understandably embraced safeguards to protect the public from generics that do not meet quality standards and/or are not objectively bioequivalent to the brandname (or originator) product. To be approved by the FDA, it has to be demonstrated that the generic drug product has the same active (pharmaceutical) ingredient, route of administration, dosage form and strength, and proposed labeling as the brand-name drug. In addition, it has to be demonstrated that the generic drug is “bioequivalent” to the relevant brand-name product. When acceptable information of this type is provided, the generic applicant is permitted to rely on the FDA's previous findings of safety and effectiveness for the referenced brand-name drug, and thus, in theory, the sponsor of the generic does not have to provide its own clinical studies to demonstrate the generic drug product's safety and effectiveness.

“Bioequivalence”, as used herein, is a term used in pharmacokinetics to assess the expected in vivo biological equivalence of two proprietary preparations of a drug (e.g. test substance and reference substance). If two products or substances are said to be bioequivalent it means that they would be expected to be, for all intents and purposes, the same, e.g. have the same (therapeutic) effect. Specifically, two pharmaceutical products may be bioequivalent if they are pharmaceutically equivalent or pharmaceutical alternatives and their bioavailabilities, in terms of rate (Cmax and Tmax) and extent of absorption (area under the curve, AUC), after administration of the same molar dose under the same conditions, are similar to such a degree that their effects can be expected to be essentially the same.

Defined in a different way, bioequivalence is the absence of a significant difference in the rate and extent to which the active ingredient or active moiety in pharmaceutical equivalents or pharmaceutical alternatives becomes available at the site of drug action when administered at the same molar dose under similar conditions in an appropriately designed study.

It is preferred that bioequivalence is defined as having been achieved if the 90% confidence interval (CI) of the mean maximum drug concentration (Cmax), and the area under the timeconcentration curve (AUC) of the generic formulation of a drug (test substance) relative to a reference brand-name drug (reference substance) is within a range of about 80% to 125%, e.g. 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, or 125%. Preferably, the generic formulation of a drug (test substance) relative to a reference brand-name drug (reference substance) is within a range of about 70% to 135%.

More preferably, the generic formulation of a drug (test substance) relative to a reference brandname drug (reference substance) is within a range of about 75% to 130%.

Even more preferably, the generic formulation of a drug (test substance) relative to a reference brand-name drug (reference substance) is within a range of about 80% to 125%.

In general, to determine bioequivalence between two products such as a potentially to-be- marketed generic product (test substance) and a commercially-available brand-name product (reference substance), pharmacokinetic studies are conducted whereby each of the preparations is administered in a cross-over study to volunteer subjects, generally healthy individuals but occasionally in patients.

Described herein is specifically the determination of the bioequivalence of an active pharmaceutical ingredient (API) and/or dermal drug, especially a topically applied or appliable drug substance.

The term "dermal drug" as used herein refers to an active substance intended for treatment of skin diseases. The term "dermal", as used herein, refers to the stratum corneum, the epidermis and the dermis.

The term “bioavailability” as used herein refers to the rate and extent to which a drug (test substance) relative to a reference brand-name drug (reference substance) becomes available at the site of action. For topical/dermal drugs, the site of action may be the stratum corneum, the epidermis, the dermis or deeper tissues. However, transdermal administration has a barrier, the stratum corneum (SC), the top layer of the skin, provides the main barrier of the skin. The skin wall contains lipid bilayers through which drugs must pass. The SC barrier is not easy to overcome. For example, the permeability of the intestine wall i s much higher compared to the SC in the skin. There have always been challenges to delivering the therapeutic amounts of drugs to targets. Bioavailability in dermal drug delivery is mostly limited by instability of the compound and by the skin barrier.

In the methods of the present disclosure, pharmacokinetic parameters for determining bioequivalence can be measured. “Pharmacokinetics” (abbreviated as “PK”), as used herein, is to determine the fate of substances administered externally to a living organism. In practice, a PK study is applied mainly to drug substances, though in principle it concerns itself with all manner of compounds delivered externally to an organism. Pharmacokinetics is often studied in conjunction with pharmacodynamics. Pharmacodynamics explores what a drug does to the body, whereas pharmacokinetics explores the concentration profile in different body parts and what the body does to the drug. Pharmacokinetics includes the study of the mechanisms of absorption and distribution of an administered drug, the rate at which a drug action begins and the duration of the effect, the chemical changes of the substance in the body (e.g. metabolism by enzymes) and the effects and routes of excretion of the metabolites of the drug. Pharmacokinetics is divided into several areas which include the extent and rate of absorption, distribution, metabolism and/or excretion. Pharmacokinetics describes how the body affects a specific drug after administration. Pharmacokinetic properties of drugs may be affected by elements such as the site of administration, the concentration in which the drug is administered and the dosage form (immediate or controlled release for example). These may affect the absorption rate. An important pharmacokinetic parameter is the biological half-life or elimination half-life of a substance, which is the time it takes for a substance to lose half of its pharmacologic or physiologic activity.

For a pharmacokinetic comparison, at least one pharmacokinetic parameter selected from the group consisting of absorption rate constant, bioavailability, apparent volume of distribution, steady-state volume of distribution, unbound fraction, rate of elimination, clearance, renal clearance, metabolic clearance, fraction excreted unchanged, elimination rate constant, biologic half-life, maximum concentration (Cmax), area under the concentration vs. time curve (AUC) and toxicity, preferably dermal toxicity, is assessed.

The term “microdialysis (MD)”, as used herein, refers to a minimally-invasive sampling technique that is used for continuous measurement of free, unbound analyte concentrations in the extracellular fluid of virtually any tissue. Analytes may include endogenous molecules (e.g. hormones) to assess their biochemical functions in the body, or exogenous compounds (e.g. drugs) to determine their distribution within the body. The microdialysis technique requires the insertion of a small dialysis catheter (also referred to as dialysis tube) into the tissue of interest. The dialysis tube is designed to mimic a capillary and consists of a shaft with a semipermeable hollow fiber membrane at its tip, which is connected to inlet and outlet tubing. The probe is continuously streamed with an aqueous solution (dialysis solution) comprising additives... Once inserted into the tissue or (body)fluid of interest, small solutes can cross the semipermeable membrane by passive diffusion. The direction of the analyte flow is determined by the respective concentration gradient and allows the usage of microdialysis probes as sampling as well as delivery tools. The solution leaving the probe (dialysate) is collected at certain time intervals for analysis. Described herein is specifically the microdialysis (MD) of the skin. The drug is preferably an active pharmaceutical ingredient (API) or dermal drug.

The term “dermal microdialysis (dMD)”, as used herein, is a versatile sampling technique that can be used to recover soluble endogenous and exogenous molecules from the extracellular compartment of the dermis Due to its minimally invasive character, dermal microdialysis can be applied in both clinical and preclinical settings. To perform dermal microdialysis dialysis tubes having a lumen and a semipermeable membrane are inserted into the tissue, e.g. the dermis or the subcutis, and perfused at a low speed with a physiological salt solution (dialysis solution). Endogenous or exogenous molecules soluble in the extracellular fluid, specifically intestinal dermal fluid, diffuse into the dialysis tube and are collected in small vials for analysis. The duration and timing of the collected dialysate samples allows kinetic evaluation of the events occurring in the tissue.

Specific placement of probes into the dermal layer opens the way for studies of inflammatory events most prominently driven by that part of the skin. Dermal microdialysis can also be applied in drug discovery or pharmacokinetic/pharmacodynamic (PK/PD) studies and in the study of percutaneous penetration of potentially harmful exogenous agents from the environment.

In addition, dermal microdialysis can be applied for bioequivalence studies. In this case, the dialysis tube allows the diffusion of the drug (test substance) or reference brand-name drug (reference substance) from the dermal intestinal fluid to a dialysis solution comprised in its lumen.

The term “Open Flow Microperfusion (OFM)”, as used herein, refers to a probe-based method that is used to evaluate the pharmacokinetics and pharmacodynamics of drugs directly in tissue. The use of the OFM probes allows small amounts of extracellular fluid to be collected from target tissues for analysis.

The Open Flow Microperfusion technique requires the insertion of a small perfusion catheter (also referred to as perfusion tube) into the tissue of interest. The perfusion tube is designed as a tube with (macroscopic) openings.

The probe is continuously streamed with an aqueous solution (perfusion solution) that closely resembles the (ionic) composition of the surrounding tissue fluid at a low flow rate. Once inserted into the tissue or (body)fluid of interest, small solutes can cross the permeable membrane by passive diffusion. The solution leaving the probe (perfusate) is collected at certain time intervals for analysis. Described herein is specifically the Open Flow Microperfusion (OFM) of the skin. The drug is preferably an active pharmaceutical ingredient (API) and/or dermal drug.

The term “dermal Open Flow Microperfusion (dOFM)”, as used herein, is a versatile sampling technique that allows the investigation of transport of drugs in the tissue, e.g. in the dermis, and their penetration into the tissue, e.g. into dermis, after local, topical and/or systemic application. Dermal Open Flow Microperfusion can be used for conducting of tissue-specific pharmacokinetic (PK) and pharmacodynamic (PD) studies of drugs, for the assessment of (dermal) bioavailability, or for the evaluation of topical generic products, which need to demonstrate bioequivalence to the reference listed drug product to obtain market approval. In particular, dOFM facilitates a continuous assessment of the in vivo cutaneous kinetics of topically administered drugs directly in the tissue, e.g. the dermis, in human subjects. dOFM can assess the intradermal drug concentrations by sampling the dermal interstitial fluid for a predetermined time, e.g. for up to 48 h. The dOFM approach allows a direct PK approach for BE assessment in the dermis. In addition, dOFM allows a direct comparison of two different topical drugs in the same subject and avoids therefore high intrasubject variability.

As mentioned above, dOFM can be applied for bioequivalence studies. In this case, the perfusion tube allows the perfusion of the drug (test substance) or reference brand-name drug (reference substance) from the dermal intestinal fluid to a perfusion solution comprised in its lumen.

The term “dialysate”, as used herein, refers to the liquid obtained from/being the result of microdialysis, particularly dermal microdialysis (dMD). The dialysate described herein comprises a test substance or reference substance obtained from an individual treated on its skin with the test substance or reference substance. The dialysate is obtained from the individual via a dialysis tube embedded/inserted into the tissue underlying the skin of said individual. The dialysis tube comprises a lumen and a semipermeable membrane. The test substance or reference substance applied to the skin penetrates the skin and enters the dermal interstitial fluid that surrounds cells and tissue underlying the skin. The dialysis tube allows the diffusion of the test substance or reference substance from the dermal intestinal fluid (via the semi-permeable membrane) into a dialysis solution comprised in its lumen.

The term “perfusate”, as used herein, refers to the liquid obtained from/being the result of Open Flow Microperfusion (OFM), particularly dermal Open Flow Microperfusion (dOFM). The perfusate described herein comprises a test substance or reference substance obtained from an individual treated on its skin with the test substance or reference substance. The perfusate is obtained from the individual via a perfusion tube embedded/inserted into the tissue underlying the skin of said individual. The perfusion tube comprises a tube with (macroscopic) openings. The test substance or reference substance applied to the skin penetrates the skin and enters the dermal interstitial fluid that surrounds cells and tissue underlying the skin. The perfusion tube allows the diffusion of the test substance or reference substance from the dermal intestinal fluid (via the permeable membrane) into a perfusion solution comprised in its lumen.

The term “test substance”, as used herein, refers to a substance or mixture administered or added to a test system in a study, which substance or mixture is used to develop data. In the context of the present disclosure, pharmacokinetic parameters of a test substance are determined. In the context of the present disclosure, the test substance is applied to the skin of a subject. Preferably, the test substance is an active pharmaceutical ingredient (API) or a dermal drug.

More preferably, the test substance is a generic drug, particularly a generic dermal drug.

The term “reference substance”, as used herein, refers to a reference substance or mixture administered or added to a test system in a study, which is other than the test substance for the purpose of establishing a basis for comparison with the test substance for known chemical or biological measurements. In the context of the present disclosure, pharmacokinetic parameters of a reference substance are determined. In the context of the present disclosure, the reference substance is applied to the skin of a subject. Preferably, the reference substance is an active pharmaceutical ingredient (API) or a dermal drug.

More preferably, the reference substance is a reference brand-name drug, particularly a reference brand-name dermal drug.

As mentioned above, the United States Food and Drug Administration (FDA) recognizes the public policy benefit of generic competition but has understandably embraced safeguards to protect the public from generics that do not meet quality standards and/or are not objectively bioequivalent to the brand-name product. To be approved by the FDA, it has to be demonstrated that the generic drug product (test substance) has the same active ingredient, route of administration, dosage form and strength, and proposed labeling as the brand-name drug (reference substance). In addition, it has to be demonstrated that the generic drug is “bioequivalent” to the relevant brand-name product. When acceptable information of this type is provided, the generic applicant is permitted to rely on the FDA's previous findings of safety and effectiveness for the referenced brand-name drug, and thus, in theory, the sponsor of the generic does not have to provide its own clinical studies to demonstrate the generic drug product's safety and effectiveness.

The term “generic drug”, as used herein, refers to a pharmaceutical drug that contains the same chemical substance as a drug that was protected by chemical patents. Generic drugs are allowed for sale after the patents on the original drugs expire. Because the active chemical substance is the same, the medical profile of generics is believed to be equivalent in performance. A generic drug has the same active pharmaceutical ingredient (AIP) as the original, but it may differ in some characteristics such as the manufacturing process, formulation, excipients, colour, taste, and packaging.

Although they may not be associated with a particular company, generic drugs are usually subject to government regulations in the countries in which they are used. They are labelled with the name of the manufacturer and a generic non-proprietary name of the drug. A generic drug must contain the same active ingredient as the original brand-name formulation. The U.S. Food and Drug Administration (FDA) requires generics to be identical to or within an acceptable bioequivalent range of their brand-name counterparts, with respect to pharmacokinetic and/or pharmadynamic properties.

Embodiments of the disclosure

The present disclosure will now be further described. In the following passages, different aspects of the disclosure are explained in more detail. Each embodiment or aspect so defined or explained may be combined with any other aspect or aspects unless clearly indicated to the contrary. In particular, any feature indicated as being preferred or advantageous may be combined with any other feature or features indicated as being preferred or advantageous, unless clearly indicated to the contrary.

The present inventors have proposed a method for determining or investigating bioequivalence between a test substance and a reference substance.

Thus, in a first aspect, the present disclosure relates to a method for investigating bioequivalence of a topically applied or appliable test substance with respect to a reference substance, wherein the method comprises: evaluating whether there is bioequivalence of the test substance with respect to the reference substance based on a) first application site data comprising data indicative for a dermal situation relating to the test substance or the reference substance below a first application site onto which the test substance or the reference substance has been applied, and based on b) second application site data comprising data indicative for a dermal situation relating to the test substance or the reference substance below a second application site onto which the test substance or the reference substance has been applied, wherein each of the first application site data and the second application site data comprises region data indicative for the dermal situation in a region below the respective application site, wherein at least one of the first application site data and the second application site data comprises or both of the first application site data and the second application site data comprise region data for a plurality of distinct regions below the respective application site.

Each of the distinct regions may have its associated, e g. unique, region data.

Topically applied or appliable substances can exist in many forms and can comprise for example ointments, gels, creams, lotions, solutions, suspensions, patches for transdermal delivery, foams, and shampoos, but are not limited to these. An example of an application site on which to test a topical substance (e.g. the test substance and/or the reference substance) may be a portion of skin surface of a subject, e.g. a surface on a thigh of a subject, a surface on an arm of a subject, a surface on the back of a subject or a surface on any other skin portion of a subject.

The size of a single application site can be in particular between 5.5 cm ² and 15.5 cm ² e.g. between 7.5 cm ² and 12.5 cm ² or between 9.5 cm ² and 10.5 cm ² .

The size of an application site with two dOFM probes can for example be ca. 2.2 cm x 2.5 cm (ca. 5.5 cm ² ).

The size of an application site with four dOFM probes can for example be ca. 4.2 cm x 2.5 cm (ca. 10.5 cm ² ).

The size of an application site with six dOFM probes can for example be ca. 2.2 cm x 2.5 cm (ca. 15.5 cm ² ) An example of a region below an application site, may be for example the epidermis and/or the dermis and/or the adipose tissue of the skin located underneath the application site. The plurality of distinct regions can be different regions of the dermis below an application site. The regions of the plurality of distinct regions underneath a single application size can be horizontally distributed along the surface of the application site.

In particular, the plurality of distinct regions can be a plurality of distinct region under a single application site, e.g. regions for which dOFMs profiles are obtained or can be obtained via probes. An application site can have more than one dOFM profiles or probes. In particular each dOFM profile or probe of an application site can define one region of the plurality of regions. The dOFM probes in an application site can be distributed horizontally, e.g. on substantially the same plane, with respect to each other, e.g. along the application site. We note that the samples for obtaining region data can not only be obtained by dOFM or dMD. Other sampling techniques are also suitable for this purpose, e.g. tape stripping or other continuous or discontinuous sampling techniques, some of which are discussed below. Hence the plurality of distinct regions can be a plurality of distinct regions under a single application site, e.g. regions for which a plurality of samples is obtained or can be obtained through one or more different sampling techniques.

The regions of the plurality of distinct regions can be arranged at the same depth (e.g. same distance from the skin's surface) with respect to each other. As such the region data of each region of the plurality of region data may represent data captured at the same skin depth for each region. However, if the data is obtained using probes introduced into the skin, the depth of the regions may vary between probes of the same sites and/or different sites. Other sampling methods may entail variations in depth as well.

The number of dOFM profiles or probes for one application site can correspond to the number of regions of the plurality of regions and the total number of dOFM profiles can correspond to the total number of regions of the plurality of regions. An application site with four dOFM probes can therefore correspond to the plurality of regions comprising four regions. Vice versa an application site with four regions might correspond to an application site with four dOFM probes.

Similarly, the number of different samples obtained through one or more different sampling techniques can correspond to the number of regions of the plurality of regions. An application site with four tape strips for tape stripping can therefore correspond to a plurality of regions comprising four regions. An application site with four dOFM probes and four tape strips can therefore correspond to a plurality of regions comprising eight regions.

The application site data and the region data can comprise data indicative for the dermal pharmacokinetics of the relevant substance or an ingredient thereof and/or indicative for the dermal concentration of the relevant substance or an ingredient thereof for that application site (e g. application site data) or more specifically in that region under a specific application site (e g. region data). The region data may comprise data indicative for the concentration of the relevant substance or an ingredient thereof (e.g. the active ingredient).

According to one embodiment, for evaluating the bioequivalence, at least one region from the plurality of distinct regions is disregarded. Especially, although the region may comprise data which could be used for investigating the bioequivalence, the data of that specific region may be disregarded as this may enhance the statistical power as the variations in the data which is evaluated may be reduced.

According to one embodiment the region data indicative for the dermal situation in the disregarded region is removed from the associated application site data, i.e. from the first application site data or the second application site data, to provide modified application site data, wherein the modified application site data is used for determining whether there is bioequivalence.

According to one embodiment the at least one region which is disregarded is selected so as to homogenize region data, e.g. the distance or depth of the regions, sampling or sample weight obtained in the regions, local dermal blood flow in the regions, number of hairs in the regions and/or similar, of the remaining regions associated with the first and second application sites where region data for the remaining regions is used for the evaluation of the bioequivalence based on the first and second application site data.

In other words, the disregarded region most significantly contributes or is responsible for the inhomogeneity in the region data(set) for the first and/or second application sites. In the case of dermal blood flow for example, a high flow results in lighter concentrations values, such that a reasonable usage of the data may not be possible. Disregarding that potential region data comprising the dermal blood flow, would hence result in a more homogenous evaluation. Similarly, the number of hairs in the region may compromise the data set of the region comprising e g. the excessive hairs. Using this data would thereby compromise the evaluation method by bringing some degree of inhomogeneity. Disregarding this region thereby results in a more efficient and correct evaluation of the bioequivalence of the substances.

In particular, applying statistical methods on the collected region data gives an insight which data can cause inhomogeneous results. For example, the statistical model may show that the region comprising excessive follicles does not have enough information or not enough "clean" data to be useful for the performance of a valuable statistical analysis on the whole application site data. As such that specific region data may be disregarded and the modified application site data (e.g. the one without the disregarded region data) used.

Bioequivalence is determined by comparison of dermal PK profiles of test and reference drug products (substances) on application sites (e.g. per thigh). Dermal PK profiles are influenced by sampling factors like probe depth. By harmonizing probe depth of dOFM probes of test and reference application sites (e.g. per thigh), variation is decreased and the statistical power of bioequivalence assessment is increased.

Disregarding at least one region and in particular disregarding the region data of the disregarded region has therefore the advantage of providing a more homogenized set of application site data resulting in statistically more significant evaluations of bioequivalence between the test substance and a reference substance. Disregarding one region may be necessary and/or advantageous if for example the concentration and/or the volume of the sample of that region is too low to be considered significative for further evaluations. Also, if for example one region is deeper than others, meaning that its distance from the skin's surface (i.e. the depth) is greater than the one for the other regions (or deviates the most from an average depth), the region can be disregarded.

Furthermore, because only a subset of the region data available per application site, e.g. region data for just one region, is disregarded it is not necessary anymore do disregard a whole application site (e.g. a whole thigh of a subject) and as such disregard a whole subject. As such, less subjects are required for the studies, resulting in quicker, more efficient, and more cost- effective evaluation of bioequivalence. According to one embodiment the number of regions for which region data is available for the first and second application sites is different. This could for example be the case if one application site comprises more dOFM probes than the other application site, resulting in more regions for which region data is available. For example, a pilot study might require or be conducted with a different number of dOFM probes than a pivotal study conducted for the same test substance.

According to another embodiment at least one region is disregarded for the first and second application sites when evaluating bioequivalence. For example, if one region of the first application site and one region of the second application site have too low concentration values for the sample or too low sample weight, both regions might be disregarded. The reason for disregarding a region from a first application site and the reason for disregarding a region from a second application site does not have to be the same. For example, one region may be disregarded from a first application site because of the number of hairs in the regions is homogenized in this way, while another region of the second application site might be disregarded, because its sample volume is too low or the depth is not homogenized.

According to another embodiment at least one region is disregarded for only one of the first and second application sites. According to one embodiment the number of disregarded regions is equal to the difference in the number of regions, for which region data is available, between the first and second application sites.

If for example the first application site has four regions and the second application site only has three regions, only one region is disregarded and the region is comprised in the first application site. If the first application has e.g. four regions and the second application site instead has e.g. three regions, then according to this embodiment, the modified first application site data would only comprise three regions, and the other region would be disregarded from the first application data.

According to one embodiment the number of disregarded regions for one application site is equal to the difference in the number of regions, for which region data is available, between the one application site and an application site with modified application site data.

If for example a first application site has 5 regions, and a second application site has 4 regions, however one region data was already disregarded from the second site beforehand because of apparent inhomogeneity with the other regions, then according to this embodiment, two region data (e.g. the difference between five and three) from the first application site may be disregarded.

According to another embodiment the number of regions for which region data is available for the first and second application sites is equal. Also, in this example disregarding of region data may be necessary and/or advantageous if it homogenizes the region data for the remaining regions. Hence in such a situation a region of the first application site and/or a region of the second application site may be disregarded.

According to one embodiment the number of regions considered for the bioequivalence evaluation associated with the first application site and the number of regions considered for the bioequivalence evaluation associated with the second application site are equal. The number of regions considered for the bioequivalence evaluation associated with the first and/or with the second application site may depend on various factors such as the amount of dOFM probes for the application site, the amount of useable region data for each region of the plurality of regions, and/or the homogeneity of the region data and/or similar factors.

According to one embodiment the number of regions considered for the bioequivalence evaluation associated with the first application site and the number of regions considered for the bioequivalence evaluation associated with the second application site are different.

In such a case a so-called unbalanced approach, such as an unbalanced scaled average bioequivalence approach (SABE) can be used.

For dOFM clinical studies the balanced SABE was usually used, implying however the necessity of an identical number of regions e g. dOFM profiles or probes in both application sites on the same thigh. If one dOFM PK profile per thigh is lost, the entire thigh will have to be excluded from bioequivalence calculation. The use of unbalanced SABE enhances statistical power as the loss of one region data, e g. one dOFM profile or probe, will not automatically exclude the affected thigh from bioequivalence determination. This will reduce the loss of thighs and therefore reduce the number of subj ects in a study.

According to one embodiment the number of distinct regions for the first and/or the second application site is greater than or equal to one of the following values: 1, 2, 3. According to one embodiment the number of distinct regions for the first and/or the second application site is less than or equal to one of the following values: 8, 7, 6, 5, 4.

According to one embodiment the number of distinct regions of which region data is contained in the modified application site data is greater than or equal to one of the following values: 2, 3, 4, 5, 6.

According to a further embodiment, for evaluating whether there is bioequivalence, application site data associated with one of the first or second application sites is compared with application site data associated with the other one of the first and second application sites. In particular, the application site data for either the first or the second application site, or both, might be a modified application data of the first application site and/or of the second application site or both. Region data for at least one region of a plurality of regions associated with one of the application sites may already have been disregarded.

The substance in the first application site and the substance in the second application site may then be bioequivalent if they are pharmaceutically equivalent or pharmaceutical alternatives and their bioavailabilities, in terms of rate (Cmax and Tmax) and concentration-time curve (area under the curve, AUC), after administration of the same molar dose under substantially the same conditions, are similar to such a degree that their effects can be expected to be essentially the same.

According to one embodiment the first application site and the second application site are application sites on the same subject. The subject can be a person. The subject can be a healthy person or an unhealthy person, i.e. a patient, in particular one with a disease that may be treated by and/or with and/or through the reference substance and/or potentially the test substance. Having the first and the second application site arranged on the same subject, e.g. on the same body part of the subject, has the advantage of minimizing statistical variability and hence requiring in total a lower number of subjects for the test phase.

However, according to one embodiment the first application site and the second application site are envisaged to be sites on different subjects. This may be advantageous, if site availability on a subject is limited. However, it is expected that this will increase the variability According to one embodiment, the test substance has been applied onto one of the first and second application sites and the reference substance has been applied onto the other one of the first and second application sites.

According to one embodiment the respective application site data comprises data on the dermal concentration of an active pharmaceutical ingredient or ingredients of the substance (test or reference substance), which has been applied onto that application site.

According to one embodiment the respective region data comprises data on the dermal concentration of an active pharmaceutical ingredient of the substance (test or reference substance) in the respective region below the application site, onto which the substance has been applied.

According to one embodiment the first application site and the second application site are on the same body part, e.g. on the same thigh, and/or on different subjects or the same subject.

Clinical dermal PK profiles seem for example to be influenced by the position on the thigh. Although in cutaneous bioequivalence studies, test and reference sites are randomized and no bias is seen in bioequivalence determination, an optimized positioning of application sites on the skin region e g. on the thighs, may decrease variation and increase statistical power

According to one embodiment the first application site and the second application site are on different body parts of the same body part type, e.g. on the same subject or on different subjects, one application site on the left side of the body (e.g. left thigh) and one application site on the right side of the body (e g. right thigh).

According to one embodiment the first application site and the second application site are on the same body part, e.g. on the same subject or on different subjects, and wherein a third application site and a fourth application site are on another body part of the same body part type, e.g. on the same subject or on different subjects.

According to one embodiment application site data for the third and/or fourth application sites is considered for evaluating whether there is bioequivalence, e.g. along with the application site data for the first and/or second application site. According to one embodiment the third and/or fourth application site can be on different body parts (e.g. on the back and/or on the arm) of the subject as the first and/or second application site (e g. on the thigh).

According to one embodiment the first application site and the second application site form an application site group with one of the application sites of the application site group being a test site onto which the test substance has been applied and the other one of the application sites being a reference site onto which the reference substance has been applied.

According to a further embodiment the third application site and the fourth application site form an application site group with one of the application sites being a test site onto which the test substance has been applied and the other one of the application sites being a reference site onto which the reference substance has been applied.

Comparison of the different application site groups and, consequently, evaluation of the bioequivalence of a test product and a reference product can be obtained with any permutation of groups. For example, the first and the second application site can be compared with the third and fourth application side (e.g. the first application site group compared with a second application site group) and/or with a fifth and sixth application site (e.g. a third application site group comprising the fifth and the sixth application site).

According to one embodiment the application site groups are configured such that the test and reference sites per application site group are oriented identically relative to the body part on which the application sites of the application site group are arranged or are oriented differently, e.g. oppositely, relative to the body part on which the application sites of the application site group are arranged.

The first application site may for example be on a right thigh of a subject and the second application site may be on a left thigh of the subject, and the first application site may comprise the reference substance while the second application site may comprise the test substance.

According to one embodiment the application site groups are orientated differently, e.g. oppositely, to each other. A first application site group may for example comprise a first application site and a second application site both being arranged on the left thigh and comprising the test subject. A second application group may then comprise a third application site and a fourth application site, both being arranged on the right thigh and comprising a test substance.

According to one embodiment the application site data and/or the region data is obtained or obtainable at least partly using an invasive and/or a non-invasive measurement method.

According to one embodiment, the measurement method comprises ultrasound depth measurement, e.g. ultrasound depth measurement of a dOFM probe depth.

According to one embodiment the measurement method comprises a sampling method.

According to one embodiment the sampling method is suitable to obtain dermal samples. Dermal samples are not limited to skin samples and can encompasses for example also dermal fluids or more generally bodily fluids.

According to one embodiment the sampling method is a continuous or non-continuous sampling method.

According to one embodiment the samples are samples comprising contributions originating from the interstitial fluid below the application site.

According to one embodiment the sampling method comprises dOFM (= dermal open flow microperfusion) or dMD (= dermal microdialysis).

According to one embodiment, the sampling method comprises Raman spectroscopy.

According to one embodiment, the sampling method comprises tape stripping.

In a tape stripping process, after topical application and penetration of the substance (e.g. the test and/or the reference substance), the cell layers of the stratum corneum are removed from the same skin area using adhesive films. The tape strips contain the amount of skin and the corresponding amount of the penetrated formulation, which can be evaluated for bioequivalence.

Tape stripping the uppermost stratum corneum layers for e.g. the investigation of comparative concentration versus depth profiles between topical products has been demonstrated to be an effective sampling method. According to one embodiment the sampling method utilizes one or more dermal probes positioned below the application site, wherein each probe defines one region.

According to one embodiment the respective application site data and/or the respective region data, e.g. of the region associated with this application site, comprises any one of, any arbitrarily selected plurality of or all of:

- a distance of the region from or depth of the region below the exterior surface of the skin in the application site with which the region is associated;

- sample data obtainable or obtained from a plurality of samples taken at different times from the region and/or the application site, wherein the sample data comprises any one of, any arbitrarily selected plurality of or all of:

- sample weight;

- a sample identifier for identifying the sample, e g. indicative for the time when the sample was taken;

- data indicative for the dermal pharmacokinetics of the relevant substance which has been applied to the application site or an ingredient thereof and/or for the dermal concentration of the relevant substance or an ingredient thereof;

- information indicative for the position of the application site on the body, e.g. the body part and/or the side of the body part;

- information indicative for the position of the application site within an application site group;

- a group identifier for identifying the application site group of which the application site is part;

- a group position identifier for identifying the position of the application site within the application site group of which the application site is part;

- a region identifier for identifying the region;

- a subject identifier for identifying the subject which has the application site;

- a substance identifier for identifying the substance applied to the application site, e.g. test substance or reference substance.

The depth of the region might be relevant region data as it may give information on how deep a test substance and/or a reference substance might penetrate. Homogenization of depth data is useful in order to have comparable data between regions and between application sites.

Also collecting data at different time intervals is useful in order to evaluate the continuous effect of the application of a substance (e g. the test substance or the reference substance) on an application site over time. The dermal pharmacokinetics data is for example useful to analyses the effect the tissue under the application site has on the substance administered under that application site.

The various identifiers are also useful in order to collect and maintain a clear data set without losing information regarding the type, the location and/or the substance that has been evaluated.

This data is particularly useful in determining bioequivalence between a test substance and a reference substance.

According to a second aspect of the disclosure a method for investigating bioequivalence of a topically applied or appliable test substance with respect to a reference substance is provided. The method comprises: evaluating whether there is bioequivalence of the test substance with respect to the reference substance based on first study data obtainable or obtained using a first study setup (e g. a pre-study setup or pilot study setup) with one or more first study subjects, and based on second study data obtainable or obtained using a second study setup (e.g. a pivotal study setup) with one or more second study subjects, wherein the first and second study setups are different.

The first study setup and a second study setup might be two different and independently managed studies. According to one embodiment the first and the second study setup are part of a main study (e.g. branches or arms of that study).

According to one embodiment for evaluating whether there is bioequivalence for the test substance relative to the reference substance, first study data obtainable or obtained from the first study setup and second study data obtainable or obtained from the second study setup are evaluated in a combined evaluation. In particular the combined evaluation is such that the results and data information obtained in the first study setup are used and/or incorporated in the second study setup in order to maximize efficiencies and effectivity of the second study setup and consequently of the whole main study.

According to one embodiment the first study setup and/or the second study setup comprise:

- an application site for the test substance, i.e. a test site, to obtain application site data relating to the test substance after a test dose of the test substance has been applied to the application site, - an application site for the reference substance, i.e. a reference site, to obtain application site data relating to the reference substance after a reference dose of the reference substance has been applied to the application site.

In particular according to one embodiment the first study setup comprises:

- an application site, i.e. a sensitivity site, for applying the reference substance in a lower dose than the reference dose or for applying a validation substance which is known to be nonbioequivalent to the test substance to obtain application site data relating to the substance which has been applied onto the application site.

Alternatively, according to one embodiment the first study setup comprises:

- an application site, i.e. a sensitivity site, for applying the reference substance in a higher dose than the reference dose or for applying a validation substance which is known to be nonbioequivalent to the test substance to obtain application site data relating to the substance which has been applied onto the application site.

According to one embodiment, the first study setup comprises an application site, i.e. a sensitivity site, for applying sensitivity substance which is known to be non-bioequivalent to the test substance, to obtain an application site data relating to the substance which was applied on the application site.

According to one embodiment the first study setup was randomized such as to randomize if the sensitivity substance was applied in a higher dose than the reference dose, in a lower dose than reference does, or as a non-bioequivalent added to the reference dose. Through this randomization tempering is greatly reduced.

In particular according to one embodiment, the combined evaluation of the first study setup and the second study setup can be achieved by performing case studies with at least two arms (e.g. branches) of a main study, the two arms using different study arm setups.

A first arm for example will comprise a study setup with a first number of subjects. The first arm can comprise for example verifying that the selected dose for the test substance and the reference substance is in the sensitivity range of skin penetration (low and high dose showing lower and higher dermal Cmax and AUC) and can include a knowingly non-bioequivalent product of identical strength to show discriminative power of dOFM setup. In a second arm of the study, multiple regions, e.g. dOFM probes, per application site can be used and one of the multiple regions, e.g. one dOFM probe, and in particular the region data of the one of the multiple regions, can be excluded, e.g. disregarded, from the analysis (see above). Exclusion of one region data per application site allows for harmonization of application sites for test and reference drug product.

These can be compared by SABE based on the most influencing factor e.g. probe depth or sample weight. Decision on the most influencing factor can be based on region data collected from the first arm (e.g. the first study setup).

In particular according to an embodiment, based on the data obtained in the first study setup the number of subjects required for the second study setup can be evaluated.

According to one embodiment the second study setup has less application sites than the first study setup.

According to one embodiment, in the first study setup and/or in the second study setup, the application site data comprises data indicative for a dermal situation relating to the substance below the application site onto which the substance has been applied.

According to one embodiment in the first study setup and/or in the second study setup, the application site data comprises region data indicative for the dermal situation in at least one region below the respective application site.

According to one embodiment, in the first study setup, the number of regions for the test site and the reference site is greater than one. According to one embodiment the first study setup, the number of regions for the test site and the reference site is greater than two, e.g. three. According to one embodiment in the first study setup, the number of regions for the test site and/or the reference site is less than or equal to one of the following values: 8, 7, 6, 5, 4, 3.

According to one embodiment in the first study setup, the number of regions for the sensitivity site is less than the number of regions for the test site and/or the reference site. According to one embodiment the number of regions for the test site and/or the reference site in the second study setup is greater than the number of regions for the test site and/or the reference site in the first study setup. According to one embodiment the number of regions for the test site and/or the reference site in the second study setup is greater than two or greater than three.

According to one embodiment, in the second study setup, the number of regions for the test site and/or the reference site is less than or equal to one of the following values: 12, 11, 10, 9, 8, 7, 6, 5, 4.

According to one embodiment, the number of first study subjects is less than the number of second study subjects. According to an alternative embodiment the number of first study subject is greater than the number of second study subject. According to an even further embodiment the number of first study subject is equal to the number of second study subjects.

According to one embodiment, wherein the number of first study subjects and the number of second study subjects is greater than one. According to one embodiment, the first study setup is used for a number of first study subjects which is less than or equal to one of the following values: 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4.

According to one embodiment, the first study setup is used for a number of first study subjects which is greater than or equal to one of the following values: 1, 2, 3, 4.

According to one embodiment, the second study setup is used for a number of second study subjects which is less than or equal to one of the following values: 50, 40, 30, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8.

According to one embodiment, wherein the second study setup is used for a number of second study subjects which is greater than or equal to one of the following values: 1, 2, 3, 4, 5, 6, 7, 8. According to one embodiment, using the first study setup, region data for a number of different regions is obtained, the number of regions being greater than or equal to one of the following values: 10, 15, 20, 25, 26, 27, 28, 29, 30. This number of regions is considered in total with respect to al study subjects. According to one embodiment, using the first study setup, region data for a number of different regions is obtained, the number of regions being less than or equal to one of the following values: 50, 45, 40, 35, 34, 33, 32, 31, 30.

According to one embodiment, based on first study data for the first study subjects, a number is derived for the number of second study subjects for which the second study setup should be used, e g. to ensure that the bioequivalence evaluation has sufficient statistical power and/or that the number of second study subjects is minimized.

According to one embodiment deriving the number of subjects for the second study setup is done through an iterative method.

By minimizing the number of subjects required in the second study setup based on the data and information of the first study setup, is clearly advantages as it permits to minimize the overall number of subjects in the main study setup and hence the related expense required for the test phase as well as the time required for the test phase. A such, the product might hit the market earlier and help people who require the alternative medicine and which cannot permit themselves the medicament without money.

According to one embodiment the number of second study subjects is calculated by performing a power calculation using the first study data. In particular based on the data available in the first study setup, a statistical method can be developed to determine what the number (n) of subjects should be in the second study setup in order to obtain statistical significative or significant data.

According to one embodiment the power calculation can be made using the following formula:

With where Z _fc is the Z (standard normal) statistics for the kth-percentile. In the case that the PK variable deviates from a normal distribution, Equation (1) should be adapted by replacing Z statistics by t statistics.

The letter n corresponds to the number of subjects to be determined. The letter a corresponds to the accepted error. The false negative rate is the proportion of positive instances that were erroneously reported as negative and is referred to in statistics by the letter . The “power” of the study then is equal to (1 - P) and is the probability of failing to detect a difference when actually there is a difference, o is the standard deviation (estimated) and A the difference in effect of two interventions which is required (estimated effect size).

According to one embodiment the number is determined such that the number of second study subjects is minimized while ensuring a predetermined statistical power (e.g. the one according to Formula (1)) for the bioequivalence evaluation based on the first and second study data.

According to one embodiment the method to calculate the number of subjects required in the second study setup comprises an iterative method, comprising empirical data collected in the first study setup.

According to one embodiment the choice of subjects for the study, e.g. for the first and/or the second study setup, is made based on an analysis and/or selection of one or more screening parameters.

According to a further embodiment the screening parameters may be or may comprise: type of skin (e.g. skin color), age, gender, pre-illnesses, blood flow, weight, transepidermal water loss (TEWL), thickness of the stratum corneum, number of follicles in the relevant area. The subjects for the study or studies may be selected such that variation in one or more of the screening parameters within the subjects used to conduct the study is minimized. For example, one or more screening parameters for study subjects from the first study setup may be considered to select study subjects for the second study setup from candidates for the second study setup.

According to one embodiment parameter values for the different screening parameters are determined. One parameter value could for example be the value of the thickness of the stratum corneum or the color of the skin. As such, a parameter value for the thickness of the stratum corneum is determined for each subject of a subject population, e.g. for each subject in the first study setup and for the candidate subjects for the subjects of or for the second study setup.

According to one embodiment the subj ects for the first and/or second study setup are chosen such as to have homogenous parameter values for the one or more screening parameters.

Determining the parameter values for one or more different screening parameters for a subject population and choosing the subjects based on the parameter values of the one or more screening parameters allows or facilitates homogeneity in the parameter value distribution over the subjects. This may result in an improved determination of bioequivalence due to homogeneous study setups and/or in a smaller number of subjects required, e.g. for the second study setup.

In particular this procedure allows to choose (the most appropriate) subjects for the second study setup based on the parameter values of the (one or more) screening parameter of the subjects of the first study setup.

If, for example, the subjects of the first study setup had a homogenous distribution in parameter values for the thickness of the stratum corneum it is advantageous to choose study subject for the second study group which have a similar homogeneous distribution in parameter values for the thickness of the stratum corneum.

The parameter values of the screening parameters do not necessarily have to be numerical values. The number of follicles in a relevant area could for example be represented by the parameter values "low", "intermediate" and "high".

Using one or more screening parameters to homogenize study subject population, e.g. in terms of skin characteristics, may facilitate obtaining a homogeneous subject population for the study or studies. This can increase the statistical power of cutaneous bioequivalence evaluation and/or may decrease the number of subjects needed, e.g. the one for the second study (e.g. the main or pivotal study), for achieving a predetermined statistical power, e.g. 90% (or 0.9) or more or 95% (or 0.95) or more.

In a third aspect, the present disclosure comprises a method for analytically validating the methods and their embodiments described above. The method comprises the step of performing the analytical validation using a suitable surrogate matrix.

In order to show bioequivalence of a topically applied or applicable test substance with respect to a reference substance different clinical study are usually required. One of these studies comprises a matrix collection study which samples dermal interstitial fluid (ISF) as real matrix to be used for analytical method validation. Such a study however is time consuming, complex and cost intensive and usually leads to longer overall studies. As such performing the analytical validation of the above described methods for investigating bioequivalence and their embodiments by performing the analytical validation using a suitable surrogate matrix, replaces the need of a matrix collection study and of the subsequent analytical validation on the real matrix.

Through the use of a suitable surrogate matrix, instead of performing a matrix collection study, the overall study of bioequivalence is therefore quicker, more efficient, and more cost-effective.

According to one embodiment the surrogate matrix comprises perfusate and pooled human serum.

According to one embodiment the suitable surrogate matrix is based on a chemical characterization of undiluted dermal interstitial fluid (ISF).

According to one embodiment the chemical characterization of undiluted interstitial fluid comprises analytical quantification of at least one of or all of electrolytes (e.g. Na+, Ca2+, Mg2+, C1-), metabolomics profile (e.g. amino acids, lipids,) and abundant proteins (e.g. albumin, alpha- 1 -glycoprotein, immunoglobulins) in the undiluted ISF.

According to one embodiment the above described methods for investigating bioequivalence and their embodiments (e.g. first and second aspect of the present disclosure) comprise the first step of analytically validating the method by using a suitable surrogate matrix, respectively.

In a fourth aspect the present disclosure relates to a system comprising an electronic control unit configured to control the execution of and/or to assist in executing the methods and their embodiments described above.

In a fifth aspect, the disclosure relates to a, e.g. volatile or non-volatile, storage medium having stored thereon machine-readable instructions which, when executed by a processor of an electronic control unit, cause the electronic control unit or the system to control execution of and/or to assist in executing the methods and their embodiments described above.

In a sixth aspect, the disclosure relates to a computer program or computer program product, which comprises machine-readable instructions which, when executed by a processor of an electronic control unit, cause the electronic control unit or the system to control the execution of and/or to assist in executing the methods and their embodiments described above.

BRIEF DESCRIPTION OF THE FIGURES

The present disclosure is further illustrated by the following Figures and examples, however, without being restricted thereto.

Figure la: shows a schema of dermal open flow microperfusion (dOFM).

Figure lb: shows a schematic simplified view of dOFM.

Figure 2: shows an exemplary schematic presentation of application sites and sampling schedule for a main study setup.

EXAMPLES

The present disclosure is further illustrated by the following examples, however, without being restricted thereto.

Recently, dermal open-flow microperfusion (dOFM) has been identified as a potential approach to assess the BA/BE of topical products. It facilitates a continuous assessment of the in vivo cutaneous kinetics of topically administered drugs directly in the dermis in human subjects. dOFM can assess the intradermal drug concentrations by sampling the dermal interstitial fluid for up to 48 h (Figure la). The dOFM approach allows a direct PK approach for BE assessment in the dermis. Reference is particularly made to “Open Flow Microperfusion as a Dermal Pharmacokinetic Approach to Evaluate Topical Bioequivalence” by M. Bodenlenz et al., Clin Pharmacokinet (2017) 56:91-98 (DOI 10.1007/s40262-016-0442-z) in which the detailed functionality of the dOFM approach is described.

The present disclosure is however not limited to dOFM sampling techniques and others, such as e.g. dMD, tape stripping or other sampling techniques are also encompassed.

Figure la shows an exemplary dOFM probe inserted into a tissue of interest. The tissue, in this example is a skin portion of a thigh of a subject, however it might be any other skin portion of a subject, such as, e.g. an arm portion and/or a back portion. The skin portion comprises different layers, such as the epidermis LI (the outermost layer) comprising the stratum comeum Lla, and the dermis L2. The dermis is in this case the target tissue layer. On the epidermis an application site 20 is allocated in which either the reference substance or the test substance is applied.

A small perfusion catheter 12 is inserted from the epidermis into the body up to the tissue of interest. A physiological solution 11 (also called perfusion solution) which can be stored in a reservoir 10 is sent through the catheter into the tissue layer. The dOFM probe comprises an open exchange area 14 formed as a tube portion with (macroscopic) openings, which allows unrestricted exchange of compounds between the solution and the interstitial fluid, i.e. the fluid found in the space around cells. This exchange of compounds between the probe’s solution and the surrounding interstitial fluid (ISF) is driven by convection and diffusion, and occurs non- selectively in either direction. The sample (e.g. the mixture of perfusion solution with ISF) is then collected, e.g. pumped at another end of the perfusion catheter. A schematic simplified representation is given in Figure lb.

According to the example in Figure la, the diameter of the catheter and in particular of the open exchange area is ca. 0.5. mm, but can vary depending on necessity. The insertion length of each dOFM probe is ca. 30 mm, but can again vary depending on subject and necessity.

This system permits a continuous retrieval of sample after different time intervals, such as e.g. 1 hours, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 12 hours and even up to 36 hours to 48 hours.

Figure 2 shows a schematic presentation of application sites and sampling schedule. The process in Figure 2, comprises three different arms (e.g. Arm #0, Arm #1, Arm #2) corresponding to three study setups.

In this example, the skin portion of a subject is chosen to be a skin portion on a thigh of the subject. Each "badge" on the tights corresponds to an application site. In Arm #0, the left thigh 102 and the right thigh 104, each comprise four application sites, numbered with 1 to 8.

The distribution of application sites and substances in the different body parts in Arm #0 is given by following table:

As can be seen in this exemplary Arm #0 each application site 1-8 comprises two regions 100a, 100b The regions according to this example are represented by dOFM probes through which perfusate can flow as described with reference to Figure la and lb. The example is however not limited to dOFM probes and other method of sampling such as tape stripping or other methods disclosed herein can be used.

The size of an application site with two dOFM probes can be ca. 2.2 cm x 2.5 cm (ca. 5.5 cm ² )

According to this example each application site has the same number of regions.

In the Arm #0 of this example on each thigh, the reference product is applied at three different doses (intermediate dose - application site 3 and 8, low dose - application site 1 and 6, and high dose - application site 2 and 7) and the non-equi valent product on application site 4 and 5.

In this example of Arm #0 the application sites are arranged differently to each other between one thigh and the other thigh, i.e. in the left thigh the non-equivalent substance is applied on an application site arranged at the right end of the row of application sites (site 4) as seen from left to right, while on the right thigh the non-equivalent substance is applied on the first application site (site 5) as seen from left to right.

However, the disclosure is not limited to this and any other order of application site is envisaged.

From each probe one pre-dosing sample (one hour before dosing) and 9 post-dosing samples (in hourly in two hourly intervals) can be taken and per time-interval the sample from each application site are pooled together. Arm #0 corresponds to the parameter study, which informs the design of the clinical setup of the pilot and pivotal study in terms of drug amount applied for reference and test as well as low and high dose for sensitivity analysis, drug residence time on skin, dermal sampling time period, samples to be analyzed according to appropriate PK endpoints, dermal API concentrations for analytical method development and validation prior to the pilot study.

Arm #0, however can be skipped if PK modelling is precise enough to allow the omission of the parameter study. Alternative, the PK modelling might be precise enough to at least minimize the number of subjects required for the parameter study. For example, according to the example of Figure 2, only two subjects were required for assessing the parameter study.

In order to analytically validate the method for investigating bioequivalence a matrix collection study is usually performed after the parameter study (e.g. Arm #0) and before the pilot staudy and the pivotal study.

In this example the matrix collection study however is not done. Instead an analytical validation study using a suitable surrogate matrix is performed (not shown in the Figures).

The suitable surrogate matrix is obtained by chemical characterization of undiluted interstitial fluid comprising the analytical quantification of at least one of or all of electrolytes (e.g. Na+, Ca2+, Mg2+, C1-), metabolomics profile (e.g. amino acids, lipids,) and abundant proteins (e g. albumin, alpha- 1 -glycoprotein, immunoglobulins) in the undiluted ISF.

Arm #1 in figure 2 corresponds to the pilot study. In Arm #1 it is verified that the selected dose for test and reference substance is in the sensitivity range of skin penetration (low and high dose showing lower and higher dermal Cmax and AUC) and includes the non-bioequivalent product of identical strength to show discriminative power of the dOFM setup.

In Arm #1 of this example each subject has 4 application sites on each thigh, whereby in two of these application sites 3 to 6, three regions and in the remaining two application sites 1-2 and 7- 8, only one region is used.

The distribution of application sites and substances in the different body parts in Arm #1 is given by following table:

The number of regions per application site in Arm #1 can vary. For example, the number of regions for the test site and the reference site can be greater than one, greater than two, (e.g. three). The number of regions for the test site and/or the reference site can also be less than or equal to one of the following values: 8, 7, 6, 5, 4, 3

In this example, application site 4 and 6 comprise the test product. Application sites 3 and 5, the reference product in an intermediate dose and in application sites 1-2 and 7-8 a high dose /low dose of the reference product or a non-equivalent product is applied. The distribution of reference product or non-equivalent product in application site 1-2 and 7-8 is randomized.

As can be seen in this example of Arm #1 the randomized substances (see table above) on the left thigh are applied on application sites arranged at the left of the start seen from left to right and in the right thigh at the end. The reference substances instead is applied on an application site arranged on the left of the application site comprising the test substances on each thigh.

However, the disclosure is not limited to this and any other order of application sites is envisaged.

The preferred distance between the different application sites is 1.5 cm, however also ranges between 1.0 and 1.8 cm can be used. The distance between application sites is however not limited to these values and can encompass also greater distances, e.g. at a beginning of a leg and at the end of a leg.

In particular the different application sites can be homogenously distributed between each other, e.g. at a same distance from each other. The different application sites however can also be non- homogenously distributed between each other, e.g. at different distances from each other. For example a first application site may be positioned at 1.5 cm distance from a second application site, while a third application site may be positioned at 1.8 cm from the second application site.

The data from Arm #1 is then used to determine how many subjects will become necessary for study setup two (e.g. Arm #2). An iterative method can be used to set the number of subjects required in the following arm. In this way a statistically relevant but minimized number of subjects for Arm #2 (the pivotal arm) can be assessed.

Arm #2 corresponds to the pivotal study, hence to the study which compares the test substance with the reference substance.

The distribution of application sites and substances in the different body parts in Arm #2 is given by following table:

In Arm #2 of this example each subject has two application sites (e.g. 1 and 2, 3 and 4) on each thigh comprising four regions (e.g. four dOFMs probes) per application site. On each thigh, the reference product is applied on one application site and the test substance on the other application site. From each probe one pre-dosing sample (one hour before dosing) and 9 postdosing samples (in hourly in two hourly intervals) are taken.

The size of an application site with four dOFM probes can be ca. 4.2 cm x 2.5 cm (ca. 10.5 cm ² ). If an application site has six dOFM probes it could for example have a size of ca. 6.2 cm x 2.5 cm (ca. 15.5 cm ² ).

As can be seen in this example of Arm #1 on the left thigh the reference substance is applied in an application site arranged before the application site with the test substance seen from left to right, while on the right thigh it is the opposite, i.e. the application site with the test substances is arranged before the application site with the reference substance.

However, the disclosure is not limited to this and any other order of application sites is envisaged.

The sampling schedules shown in the figure to the sampling schedule for the different dOFM probes in the various application sites of the different Arms.

In line with the present disclosure, some region data may be disregarded if the application site data is not enough harmonized. Reasons for disregarding region data can be different, such as the region being arranged at a different height than the other regions, one region comprising more follicles or an excessive blood flow in one region. Region data may be disregarded from just one application site or for several application site (e.g. for the second application site).

The comparison between the reference drug and the test drug is then evaluate in the light of the modified application site data, i.e. the application site data without the disregarded region data. The two application site data in one subject are then compared with each other for the sake of the evaluation.

In this example the number of regions per application site in Arm #2 is equal. However, it might also be different and one application site might comprise more or less regions than the other application site.

The region data can for example comprise data on the dermal concentration of an active pharmaceutical ingredient of the substance (test or reference substance) in the region below the application site, onto which the substance has been applied.

In particular, the first and second application sites, 1 and 2, may form a first application site group. The third and fourth applications items, 3 and 5 may form a second application site group. In this example both application site groups are on the same type of body part, however the disclosure is not limited to these, and the two application sites group may be on the same subject but on different body parts.

The application site data and the region site data collected in this example, can comprise any one of, any arbitrarily selected plurality of or all of:

- a distance of the region from or depth of the region below the exterior surface of the skin in the application site with which the region is associated;

- sample data obtainable or obtained from a plurality of samples taken at different times from the region and/or the application site, wherein the sample data comprises any one of, any arbitrarily selected plurality of or all of:

- sample weight;

- a sample identifier for identifying the sample, e g. indicative for the time when the sample was taken;

- data indicative for the dermal pharmacokinetics of the relevant substance which has been applied to the application site or an ingredient thereof and/or for the dermal concentration of the relevant substance or an ingredient thereof;

- information indicative for the position of the application site on the body, e.g. the body part and/or the side of the body part;

- information indicative for the position of the application site within an application site group;

- a group identifier for identifying the application site group of which the application site is part;

- a group position identifier for identifying the position of the application site within the application site group of which the application site is part;

- a region identifier for identifying the region;

- a subject identifier for identifying the subject which has the application site;

- a substance identifier for identifying the substance applied to the application site, e.g. test substance or reference substance.

Before initiating Arm #0, Arm #1, or Arm#2 (e.g. if Arm #0 is left out) a screening process can occur, in which subjects are screened with respect to screening parameters to determine the subjects for the study setups. Determining values for the screening parameter helps in particular in obtaining a homogenized group of subjects for the study setups.

Screening parameters can comprise any one of, any arbitrarily selected plurality of or all of : type of skin, age, gender, pre-illnesses, blood flow, weight, transepidermal water loss (TEWL), thickness of the stratum corneum. Hence a group of subjects is chosen who have homogenized skin characteristics based on one or some or all of the above parameters.

According to a further example, an electronic control unit is provided which is configured to control the execution of and/or to assist in executing the above method according to figure 2.