Field of the invention

The invention relates to a method for assessing the distribution of a molecule of interest in a target tissue. More particularly, the invention provides a method for evaluating if a molecule of interest binds or is incorporated in at least one predetermined target tissue by comparison with at least one control compound. The method of the invention allows namely to evaluate the specificity, the adsorption, the distribution, the vectorization, the metabolism of a molecule of interest and/or its metabolites to a target tissue. The method of the invention finds its application in all domains involving the study of the behavior of a molecule of interest in a test system, namely, in one or several biological tissues. The method of the invention can be advantageously used in proteomics, lipidomics or pharmaceutics research in order to screen candidate molecules and evaluate their therapeutics or diagnostics potentials.

Background of the invention

Developing a drug, from early candidate molecule discovering up to the placing on the market of the product, is a long and costly process, involving significant human and equipment investment. Notably, clinical trials which involved human test, could take several years. The aim of these trials is to ensure the efficiency of the drug, highlight potential side effects and evaluate the safety concerns of the therapeutics.

Upstream of these clinical trials, the preclinical trials have a great importance. This is during this preclinical development that the candidate molecule is identified, selected and validated. Preclinical development specifically uses animal trials to study the pharmacology of candidate molecule. Particularly, the purpose of these pharmacological studies is to validate in-vitro and in-vivo the mechanism of action and measure the activity of candidate molecule in animal disease models. Moreover, they provide an evaluation of the candidate molecule behavior and its potential transformation in a living organism and help to establish their target organs or tissue and the toxicity doses for the model.

Clinical trials relevance is consequently related to the upstream of preclinical studies during which a large numbers of molecules, interesting at first sight, are finally discarded. Preclinical studies must permit a reliable selection, from dozens of candidate molecules, of the most promising one which can be an active ingredient in a drug formulation for the treatment of a given pathology. Poor preclinical evaluation may lead to the selection of a candidate molecule that will demonstrate its inefficiency in clinical phases, causing losses in terms of time and cost. Therefore, it is important to have reliable investigations to evaluate whether a candidate molecule acts on the right target, especially on a given tissue and does not involve side effects or be blocked by a biological barrier. For example, numerous molecules in-vitro identify as potential active compound for central nervous system (CNS) disease treatment are finally unable, during in-vivo trials, to cross blood-brain barrier and thus are totally ineffective.

Today, more than 35% of candidate molecules discarded in clinical trials have been wrongly selected in preclinical phases following incorrect evaluation of their action in target tissue.

For that reason, there is a real need of reliable methods of candidate molecule selection to ensure its high binding specificity for a given target tissue allowing the accurate evaluation of its pharmacokinetics within this tissue. There is also a need in the case of phytosanitary product development to have more data about potential toxicity or not of the product on the living organism by using a method to evaluate if a candidate molecule binds or is incorporated in at least one target tissue.

Summary of the invention In this context, the inventors developped a method to assess the targeting efficiency of a molecule of interest for a given tissue. The method of the invention allows validating the ability of this molecule to cross biological barrier(s) post administration to reach said tissue but also to assess the specificity of the molecule for said tissue. More generally, the method of the invention allows in-vivo or ex-vivo/in-vitro evaluation of biological parameters of the molecule of interest, such as its absorption, its vectorization and/or its metabolism depending on target tissue or chosen test system.

Thus, the method of the invention can be used to achieve a fast and reliable screening of molecules with a potential therapeutic effect and be able to select the one that may enter in a drug formulation to treat a given disease depending of a target tissue. The method of the invention can also be applied to verify potential side effects from the molecule of interest, for example on untargeted tissue(s) where the molecule can be bound. As well, the method of the invention can be used in proteomics, for example to screen biomarkers to select reliable diagnostic molecule(s) of a given pathology. Today, a large number of molecules have been listed and classified depending on their target tissue, their specificity or not for these tissue, etc. In the present invention, the inventors propose to use its prior knowledge to select new candidate molecules. More specifically, the method according to the invention proposes to use, as a marker, a molecule with well- known properties for the target tissue and to compare its distribution in said tissue with the distribution of the candidate molecule. Any methods allowing the in vivo, ex vivo or in vitro visualization of the distribution of a molecule within tissue can be used. Particularly, the localization of the marker and the candidate molecule can be performed on whole-animal using magnetic resonance imaging (MRI) or on tissue sections, for example, using mass spectrometry imaging (MSI) The purpose of the invention is a method for evaluating if a molecule of interest binds or is incorporated in at least one target tissue, comprising visualizing and comparing the distribution of the molecule of interest and of at least one control compound within the target tissue or on the target tissue surface of at least one animal that has previously received the molecule of interest and/or the control compound. The method of the invention can be used to evaluate the distribution of all molecules measurable using imaging techniques, especially a protein, a peptide, a lipid, an antibody, a nucleic acid, an inorganic or organic compound, etc. More specifically, the molecule of interest is a candidate molecule, consequently exogenous, which is involved or can be involved in a pharmaceutical or phytosanitary development. According to the invention, the candidate molecule also refers to a molecule with a pharmaceutical or phytosanitary potential or one of their metabolites.

The target tissue studied can be a whole organ, a specific region within an organ, a biological barrier, etc. For example, the target tissue is an organ such as a lung, an eye, a liver, a kidney, a heart, etc., or a biological barrier such as the blood-brain barrier, or a specific region of an organ, such as a tumor tissue, especially a cerebral tumor tissue, etc.

According to the invention, the control compound(s) and the molecule of interest may be previously co-administrated to the animal.

Advantageously, the concentrations of control compound(s) and molecule of interest are adapted to the studied animal model. In some embodiments, these concentrations are identical.

The administration can be performed using an enteral or parenteral route.

In a particular embodiment, the visualization and comparison of the distribution of the molecule of interest and of the control compound are performed on the surface of at least one target tissue section obtained from a previous animal tissue sampling, or tissue removal.

When mass spectrometry imaging is used, it is possible to add a step of normalization of the signals that are associated respectively to the mass spectra of the molecule of interest and to the control compound on the surface of the target tissue section. To this end, the spectral characteristic selected as the signal for the target molecule in the sample may be weighted by an extinction coefficient (TEC) specific to both the molecule and the target tissue. This weighting normalizes the signal and makes it dependent solely of the quantity of the molecule at the origin of the signal. Moreover, when a MALDI matrix is used, it is possible to weight the signal associated to the mass spectra of the target molecule and control compounds in said tissue to take into account homogeneity deposition of the MALDI matrix. Advantageously, the method of the invention is applied to a tissue from an animal wherein the molecule of interest and both a positive control compound and a negative control compound were previously administrated. The method of the invention can also be used with a tissue sample from an animal wherein the molecule of interest and an intermediate control compound were previously been administrated.

Preferentially, the visualization step to obtain the distribution of the molecule of interest is achieved using an imaging technique, notably using mass spectrometry imaging and especially MALDI imaging.

According to the invention, it is possible to evaluate the specificity of a molecule of interest for the target tissue by comparing the distribution of said molecule within the target tissue with the distribution of said molecule of interest in at least one other untargeted tissue. According to the invention, it is possible to evaluate the tissue penetration ratio of the molecule of interest for the target tissue by comparing the distribution of said molecule within the target tissue with the distribution of a control compound within same target tissue.

The method of the invention also allows to calculate a tissue targeting coefficient of the molecule of interest for the targeting tissue that takes into account the specificity of the molecule for said tissue and the tissue penetration ratio of said molecule in said tissue.

In a particular embodiment, the method of the invention can comprise a step of evaluation of kinetics elimination of the molecule of interest in the target tissue that compares the distribution of said molecule into at least two sections of the target tissue that were previously obtained by sampling, at different times (tl) and (t2), animals that were previously administered with the molecule of interest and the control compound.

The invention also concerns a computer-readable data medium comprising computer- executable instructions suited to enable a computer system to execute the comparison of the molecule of interest distribution with one control compound distribution according to the inventive method.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1: Schematic representation of the distribution of three molecules of interest Ml, M2 and M3 and of two control compounds, positive M+ and negative M-, obtained during mass spectrometry imaging experiment on tissue sections comprising the target tissue (Tl) and a peripheral tissue, non-targeted. The specificity of each molecules of interest is assessed by comparing the distribution (spectral signature) of the molecules of interest with the distribution (spectral signature) of the positive (M+) and negative (M-) control compounds, in said target and non-target tissues.

Figure 2: Schematic representation of the distribution of the three molecules of interest Ml, M2 and M3 and of the two control compounds, positive (M+) and negative (M-), obtained during mass spectrometry imaging experiment on tissue sections comprising the target tissue (Tl) and a peripheral tissue, non-targeted. The similarity and/or dissimilarity of the distributions of each molecules of interest compared to the control compounds distributions allows appreciating spatial similarities of the distribution for each molecule within the target tissue. The tissue penetration ratio for the molecules of interest is evaluated by comparing intensity maps of said molecules of interest and control compounds related to the amount of control compounds in said target tissue. Figure 3: Images related to histological staining (Fig. 3A), molecular distribution (Fig. 3B) and schematic view (Fig. 3C) of a mouse kidney tissue section wherein olanzapine (molecule of interest Mc), posaconazole (positive control compound M+) and methsuximide (negative control compound M-) have previously been injected. Molecular image clearly shows two different areas (Fig 3B) respectively related to positive control compound (center of Fig. 3B) and negative control compound (all around the positive control area on Fig. 3B). It permits to easily delimit the target tissue i.e. the median region of the kidney, or medulla (Tissue 1 in the center of Fig 3C) from non-target tissue i.e. the external region of kidney, or renal cortex (Peripheral Tissue 2 of Fig. 3C).

Figure 4: Mass spectra of olanzapine (A), methusuximide (B) and posaconazole (C) corresponding respectively to discrete image coordinate at the level of high olanzapine intensity tissue area (A), of non-target tissue (B) and target tissue (C). Isotopic pattern of each ion corresponding to said molecules are also presented (see inserts above each corresponding mass spectra).

Figure 5: Visualization on a mouse kidney tissue section of the distribution of posaconazole (positive control compound M+) at the level of medulla region (left picture on Fig. 5), of olanzapine (molecule of interest Mc) in the whole kidney (center picture on Fig.5), of methsuximide (negative control compound M-) at the level of renal cortex (right picture on Fig. 5) using mass spectrometry imaging. Dashed-lines indicate peripheral and median regions of kidney. Figure 6: Overlay of the distributions, on mouse kidney tissue section images, of the molecule of interest, Mc, and the positive control compound, M+, (left picture on Fig. 6) ; of the molecule of interest Mc and the negative control compound, M-, (center picture on Fig. 6) and of the two control compounds M+ and M- (right picture on Fig. 6). The precise evaluation of similarity/dissimilarity of these distributions is easily obtained using successive overlay images.

Figure 7: Schematic representation of a strategy to assess agonist' affinity efficiency according to an embodiment of the method of the invention in the case of a receptor occupancy study.

Figure 8: Schematic representation of a strategy to assess agonist' affinity efficiency according to another embodiment of the method of the invention in the case of a receptor occupancy study.

DETAILED DESCRIPTION OF THE INVENTION

The method of the invention is based on the comparison of the behavior in a specific target tissue of a control compound which has already known compartmental properties for said tissue, with a molecule of interest which has unknown compartmental properties for said tissue and that have to be studied. After administration of these molecules to an animal and by comparing the distribution of these molecules in the animal target tissue, it is possible to validate the significance or non-significance of the presence of the molecule of interest within said tissue, but also its tissue targeting specificity, its tissue penetration ratio, the relative or absolute amount in said tissue, as well as its metabolism, regulation, etc.

Depending on the molecule of interest and the target tissue, it is also possible to evaluate the best route of administration, to verify the good vectorization of the molecule in the organism or to validate its ability to cross through biological barrier to reach target tissue. Choice of control compounds

Upstream to the study of biological parameters of the molecule of interest, it is important to choose with high precision the control compounds with predetermined specificity for a target tissue that will serve as reference.

A control compound is a molecule whom properties to a target tissue, i.e. its biodisponibility (ability to target a tissue), its vectorization (presence/concentration in the target tissue/ability to cross biological barrier) and/or its regulation (behavior of the molecule inside the target tissue), are already known. The control compound is usually a compound exogenous from the target tissue studied. Thus, a "positive control compound" is a molecule known to be specifically distributed in the target tissue after administration to the animal chose as study model. Preferentially, in order to help further data explanation, a positive control compound is not localized/distributed in the non-target tissues adjacent to the target tissue. Conversely, a "negative control compound" is a molecule known to be missing from the target tissue post injection to the animal. According to the invention, it is also possible to use an "intermediary" control compound highlighting intermediary properties for the target tissue, compared to positive and negative control compounds. Notably, an intermediary control compound can be a molecule targeting two different tissues with the same or changed ratio (for example 60/40, 70/30, etc.). Hereinafter, "control molecule", "control" or "marker" can be used indifferently to designate a "control compound".

The choice of the control compounds is related to the target tissue studied using the method of the invention. For example, the diazepam is known to be localized in the brain after injection whereas its distribution is highly limited in the kidney. This molecule can consequently be used as a positive control compound for the brain as well as a negative control compound for the kidney. Similarly, the olanzapine is localized in the brain whereas it is not in the lung. The olanzapine can consequently be used as a positive control compound for the brain as well as a negative control compound for the lung. The molecule properties can also be dependent to the animal model studied. Thus, for a given study, the properties of the molecule of interest and control compounds are preferably always taken into account for the target tissue of the same animal model.

Advantageously, for the study of a given molecule of interest for a given target tissue, at least one positive control compound and one negative control compound are used to compare the distribution of the molecule of interest to the distribution of these two control compounds. Naturally, it also is possible to use only one control compound. In this case, a positive control compound is preferred.

To help the further understanding of the molecules distribution in the target tissue, control molecules which can be easily differentiated from the molecule of interest are advantageously used. For example, in the case of a mass spectrometry imaging study, control compounds with mass to charge ratio far away from the mass to charge ratio of the molecule of interest are advantageously chosen. As well, control compounds and the molecule of interest can be labeled before the administration, for instance using different fluorescent markers.

To facilitate and optimize the choice of control molecules and to make it possible the method automation, all informations available on molecules which potentially can be used as control compounds are advantageously listed in a database. These data may be derived from the literature (scientific papers, patents...), from previous pharmaceutical study, etc. Examples of data which can be inserted in this database are listed in the table below for specific molecules and tissues.

Table 1 : Examples of data to take into account in the choice of positive and negative control compounds depending on tissues or organs.

Target Tissue Controls +/- m/z

Brain + : Olanzapine 313

- : Atenolol 267

Kidney + : Oxaliplatin 398

- : Diazepam 285 Liver + : Propranolol 260

- : Floroquinolone 402

Lung + : Tiotropium 432

- : Olanzapine 313

+ : positive control ; - : negative control ; m/z : masse to charge ratio

Administration of control and target molecules:

Upon control(s) selection achieved, the control and target molecules have to be administrated to the animal used as study model.

Depending on the desired study, the animal model can change. The skilled person knows which animal model is well adapted depending on target tissue, molecule of interest, biological properties to evaluate, etc. For example, in the case of pre-clinical trials on a candidate molecule requiring the sacrifice of the animal, non-human mammals such as rodents (mice, rats, rabbits, hamster, etc..) are preferentially used. Others non-human mammals can be used, especially monkeys, dogs, etc. In some cases, when the target tissues can be obtained using a simple biopsy, it could be interesting to use a human mammal as animal model. It is also possible to use others animal models such as fishes, insects, for instance to study the impact of a molecule on the environment or a particular ecologic medium.

According to an embodiment, after administration of the molecule of interest and control compound(s) to the non-human animal, said animal is sacrificed. According to the invention, the method is advantageously performed ex-vivo and/or in-vitro.

According to the invention and in a general terms, all administration route of the controls and target molecules can be used, such as enteral route (i.e. drug administration by the digestion process of a gastrointestinal tract) or parenteral route (i.e. other route of administration than by the gastrointestinal tract). For example, the molecules can be administrated by different routes such as epicutaneous, epidural, intra-arterial, intravenous, subcutaneous (with a specific localization), intra-cardiac, intra-cavernous inject, intra- cerebral, intradermal, intramuscular, intra-osseous infusion, intra-peritoneal, intra-thecal, intra-vesical, intra-vitreal, nasal, oral, rectal or intra-vaginal.

The administration route can be chosen depending on the molecule of interest, the tissue targeted by the method, etc... The method of the invention can also permit to select the most adapted route of administration. Indeed, the method of the invention allows evaluating the ability of a molecule of interest to cross a biological barrier to reach the target tissue. In this case, experiments are advantageously performed in parallels using same conditions (i.e.: same control compounds, same concentrations) only the route of administration being changed. According the invention, it is possible to deliver to the same animal several molecules of interest to simultaneously study the potential of these molecules.

In a particular embodiment, the molecule of interest and control compound(s) are co- administrated to the animal, i.e. injected by means of a same medium including all said molecules. Thus, it is certain that the administration of all molecules took place at the same time (tO) to the animal. Otherwise, it is possible to administrate these molecules independently of one another, or the molecule of interest independently of the control compounds. In this case, the molecules are preferentially administrated to the animal at the same time (TO), for example thanks to multiples simultaneous injections.

In another embodiment, the control compound can be administrated to an animal different from the animal that is administrated with the molecule of interest. For example, it is possible to administrate the positive control molecule to a first animal, the negative control compound to a second one and the molecule of interest to a third one. However, because of the biological variability between animals, these experimentations are preferably performed in parallel in order to decrease this variability. In a specific embodiment, the same dose of the molecule of interest and of each control compound is administrated to the animal. Thus, the evaluation step of the penetration ratio can be easily achieved further.

In some cases, it may be interesting to administrate different doses, or concentrations, depending on molecules, to take into account their biological specificities for the animal model studied. Advantageously, the properties of the control compound(s) in the animal model are already known so that the skilled person knows if doses must be adapted. It is also possible to adapt the dose of the molecule of interest, for example, by com parison with an analog compound (with structural similarities) whom properties in said animal model have been already assessed.

Blood-brain barrier study case

The method of the invention can also be used to select candidate molecules in pharmaceutical development of drugs targeting the central nervous system as well as specific histological region in the brain, especially tumor tissue. I ndeed, such molecules must be able, after administration (for example via injection) to the patient, to cross the blood- brain barrier (BBB). This physiological barrier, mainly composed of endothelial cells lining the blood capillaries, separates the bloodstream from the cerebrospinal fluid. The BBB protects the brain from pathogenic agents, toxins and hormones circulating in the blood, but may also limit the penetration of some drugs. The evaluation of the BBB permeability to candidate molecules is a critical factor of efficiency measurement of a potential related treatment.

Advantageously, in such case, the method of the invention is performed using mass spectrometry imaging (MSI), widely used to study central nervous system from its first development. The continuous improvement of spatial resolutions available during MSI experiment gives the access to fine histological and biological structure as the BBB can be.

The method of the invention allows obtaining a BBB model of study using positive control compound of BBB crossing (for example the diazepam) and negative control compound of BBB crossing (for example the atenolol). These markers, after administration to the animal, are followed by MSI directly on the tissue section near blood vessels. Thus, it is possible to evaluate the target efficiency of the molecule of interest, such as anticancer drug, for instance for the treatment of a multiform glioblastoma, by comparing the distribution of the molecule of interest (or their metabolites) with the distribution of the positive and/or negative markers in said tissue. Adsorption Case Study: Receptor-Occupancy Example In pharmacology, the receptor occupancy is defined by the capacity of an agonist (a small or large molecule such as a drug) to bind to a specific receptor or enzyme within biological tissue. This binding involves a functional response in the organism that leads to an efficient treatment of a pathology or disease. Different mechanisms can be used to achieve the binding, for example covalent linkage, ionic interaction, Van der Waals force, hydrogen binding, etc. The affinity of an agonist to a receptor can be evaluated in order to choose the best drug candidate for said receptor, exhibiting for instance a high degree of binding and full efficacy of action. A competitive binding can occur that involves for instance an agonist and an antagonist which have different affinity to a same receptor. In this case there is a competition between the two different molecules because the receptor can be bound to only one molecule at a time.

Advantageously, the method of the invention can be used to assess this competition directly within tissue sections, using MSI or any molecular imaging technique in order to evaluate the affinity of a molecule of interest (the agonist) to a specific receptor or enzyme by comparison with a positive marker (the antagonist) with a high binding affinity.

According to an embodiment, after the administration of the molecule of interest to the biological model, and sampling, the target tissue can be washed using a solution containing the antagonist (the positive marker) and let incubating. Alternatively, the experiment can be performed using a co-administration of the molecules (agonist and antagonist). The positive marker (the competitive antagonist) will compete with the molecule of interest (agonist) which is already bound to the receptor. Then, it is possible using MSI to follow the agonist and the antagonist on dosed tissue section. The efficiency of the candidate can be evaluated by comparing the intensity and localization of the two competitive molecules on resulting molecular images. Different parameters can be monitored, such as the agonist/antagonist concentrations, the incubation time, etc. The loca lization of the receptor itself on the tissue section can be assessed, depending on its ionization potential, especially for high molecular species. Different strategies can be applied to detect the receptor or enzymes on tissue (digestion, labeling, Tag-Mass...). For instance, different strategies can be applied to assess receptor occupancy, by combining in/ex vivo protocol. For example, it is possible to incubate the agonist and the antagonist on tissue section as well as to administer both compounds at the same time, etc. There is no limitation for performing these kinds of evaluation using molecular imaging.

Adduct ion forms

For mass spectrometry imaging experiment, all markers or target molecules can be detected as protonated [M+H] ⁺ or deprotonated [M-H] ^" for positive or negative ionization mode respectively. Nevertheless, they can be detected on mass spectrum as an adduct form with different counter ion. For example, it might be potassium [M+K] ⁺ , sodium [M+Na] ⁺ , chloride [M+CI] ^" , etc... This list is not exhaustive.

Target tissue preparation The method of the invention allows visualizing a molecule of interest in at least one given target tissue of interest.

In the context of the invention, the term « tissue » refers to a set of functional grouped cells. The target tissue can be a set of similar cells with same origins, an organ, a part of an organ, a specific region of an organ with, optionally, multi-cells assemblies. For example, the target tissue can be a tumor localized within an organ.

According to the visualization technique used, it can be necessary or useful to perform prior preparation steps on said tissue.

According to animal model and/or target tissue chosen, it is possible to perform an ex-vivo analysis, for example on a tissue section. In that case, the tissue or a tissue sample is sampled at a given time post administration (tl). The sampling can be a simple biopsy, especially when the animal is a human mammal. In the case of a non-human animal, the sacrifice of the animal can be performed before sam pling.

It is also possible in some case to perform in-vivo analysis on the living whole animal.

In a particular embodiment, the analysis can be achieved on a tissue section. I n this case, the tissue section can be obtained from fresh tissue, frozen tissue, or fixed/embedded tissue, for example with paraffin. All means suitable for obtaining thin tissue sections, as a few micrometers thick, can be used If necessary, the tissue sections can receive a pretreatment, especially depending on molecules to be detected, the analytical technique, etc... Thus, it is possible to use chemical or biochemical agents on tissue sections to optimize the detection of the molecule of interest and control compounds. For example, it is possible to use solvents and or detergents to permit the detection of define classes of molecule or improve the direct extraction of molecules from tissue. As well, it is possible to use specific enzymes capable of cleaving peptides or proteins, in order to target for example, digest fragments which have the same localization and/or amount on tissue as the parent molecule. It is also possible to perform antibody labeling (coupled or not with a tag), on tissue sections, or to use fluorescence labeled molecules or radioactivity to allow the detection of the molecule of interest and control compounds.

It is also possible to change the animal model used and/or the target tissue and/or the tissue section in order to modify their abilities to bind or incorporate the molecule of interest. Thus, this treatment can include a chemical or biological modification of animal model and/or target tissue and/or tissue section which permits to increase or inhibit the penetration or targeting ability of a molecule of interest for a given target tissue. This treatment can be performed previously, subsequently or simultaneously to the administration of the molecule of interest and/or control compound. For example, in the case of BBB, there is some efflux transporters in the barrier which are able to eject the molecules crossing the BBB. The effect of these transporters can be modulated (decreased or suppressed) using inhibitors or genetic modification, as a "knock-out", on the gene or the gene expression of said transporters.

If mass spectrometry imaging requiring a matrix is used to study tissue section, and notably, MALDI or ME-SIMS (Matrix Enhanced Secondary Ion Mass Spectrometry), the said matrix is advantageously adapted to the molecule of interest. For instance, the choice can take into account the mass range covered. The skilled person knows, from existing liquid or solid matrices, which one can be used depending on studied molecules and/or target tissue. Similarly, all deposition method of the matrix can be used, especially manual spraying, automatic spraying, sublimation, sieving and micro spotting. Visualization of molecular distribution and generated data treatment The step of distribution visualization of the molecules of interest and control compounds can be performed using any techniques allowing the accurate identification and visualization, in vivo or ex vivo, of molecules within a tissue.

Notably, in the case of in vivo analysis, it is possible to use a tomographic technique such as the magnetic resonance imaging (MRI), the autoradiography, the positron emission tomography (PET), the mono-photon emission tomography, etc.

In the case of the analysis of a tissue section, it is possible to use mass spectrometry imaging related techniques such as MALDI imaging (Matrix-Assisted Laser Desorption/lonization), LDI (Laser Desorption/lonization), DESI (Desorption by Electrospray), LESA (Liquid Extraction Surface Analysis), LAESI (Laser Ablation Electrospray Ionization), DART (Direct Analysis in Real Time), SIMS (Secondary ion mass spectrometry) JEDI (Jet Desorption Electrospray Ionization), in combination with different kinds of mass analyzer as TOF (Time of flight), Orbitrap, FTICR (Fourier Transform Ion Cyclotron Resonance), quadruple (simple or triple), etc... It is also possible to use fluorescence, immunohisto-staining or chemistry, etc. As a general rule, all techniques allowing the visualization of molecule on the surface of a tissue section can be used.

In the case of a mass spectrometry analysis, several detection modes can be used as direct mass spectrometry (MS) or tandem mass spectrometry (Msn, MRM, SRM...). Experimental parameters such as mass range, laser fluency are fixed to optimize target detection in terms of intensity, sensitivity and resolution. Thus, the acquisition of mass spectra is performed to obtain a signal. From the mass spectrum, it is possible to have access to useful data for target molecules study. For data treatment, different spectral characteristic can be used as the peak intensity on mass spectrum, the signal to noise ratio (S/N), the peak area, etc...Of course, for a given study, the same spectral characteristics are used to analyze the distribution of the molecule of interest and control compounds.

Results can be obtained from the same tissue section or from different sections of said tissue. In some cases, it may be preferable to use as much tissue sections as molecules studied. According to the invention, the distribution of the molecule of interest is directly visualized on the target tissue surface, or inside the tissue during a whole-animal or whole-organ analysis. To validate these results, the distribution of the molecule of interest is compared with positive and/or negative control compounds distribution. Thus, one avoids taking into consideration non-significant results.

By comparing the distribution of the molecule of interest in a target tissue with another non- target tissue, for example an adjacent tissue, it is possible to confirm if the molecule is specifically distributed in said target tissue rather than adjacent tissue. For instance, if the molecule is distributed both in the target tissue and the adjacent tissue, this molecule is not a good candidate for a treatment targeting specifically/exclusively said target tissue.

By comparing the distribution of the molecule of interest in the target tissue with another non-target tissue, and/or the distribution of the molecule of interest with the distribution of the positive control, it is possible to evaluate the penetration ratio or the relative amount of the molecule of interest in the target tissue. The biodisponibility of the molecule of interest, especially the proportion of said molecule reaching the target tissue compared to the administrated dose, can also be assessed by an accurate way using known absolute quantification techniques.

The invention also permits to provide a targeting coefficient to the molecule of interest for the given target tissue. This targeting coefficient takes into account the specificity of the molecule of interest for said target tissue and the penetration ratio of this molecule in the target tissue. Thus, it is possible to list the tissue targeting coefficient of several molecules, for example candidate molecules for a therapeutic treatment targeting a given tissue. These targeting coefficients might be quickly compared to select the most adapted candidate molecule. The method of the invention permits also to evaluate the kinetics of elimination or the metabolism of the molecule in said tissue. For this purpose, same molecules (interest and control) are administrated at same doses, to several identical animals at the same start time (tO). Animals are then sacrificed at different times (tl to tn). Then, the distribution of the molecule of interest is compared directly on tissue sections of the target tissue sampled from each animal to have access to the scalable distribution of the molecule of interest between tO and tn.

Normalization of the signal

During an mass spectrometry imaging experiment, it can be important to normalize mass spectra related to each molecule to take into account the Tissue Extinction Coefficient (TEC). Additionally, a normalization step can be useful to take into consideration matrix effect when a mass spectrometry imaging requiring a matrix compound is used to visualize the molecules.

More particularly, a given molecule at a given concentration does not emit a signal of the same intensity depending on the tissue in which it is detected. Similarly, two different molecules at an identical concentration in a given tissue have different signal intensities. According to the nature of the tissue and/or the localization of the molecule in said tissue, a loss or a gain of intensity of the molecule signal can be observed compared to its signal on an inert sample support or to the signal of a standard molecule. This tissue extinction coefficient can be calculated for each molecules (and each target tissue), and consequently used to weight the signal obtained for each molecules.

Similarly, when the tissue section studied is covered with a matrix compound, it can induce a loss of signal intensity related to the molecule of interest. To prevent this fact, it is possible to calculate the extinction coefficient related to this matrix effect and use it to weight the signal obtained for each molecules studied on the analyzed tissue section.

Taking into account TEC and/or matrix effect and corresponding normalization of signal intensity allows obtaining reliable signals related to the real concentration of each molecule, independently of each kinds of tissue and/or matrix compound used. A direct quantification of the molecules from mass spectrometry normalized results is then possible. The document WO2012/126873 relates to a method to calculate the tissue extinction coefficient and the matrix effect coefficient. It further discloses how to take into account these coefficients for the detection of a molecule within a tissue. This method can be advantageously be implemented with the method of the invention to normalize the signals obtained from the molecule of interest and control compounds. Otherwise or in addition, others calculation methods allowing such normalization of mass spectra related to studied molecules signal can be used.

Advantageously, the normalization step of the signal is performed before the comparison of molecules distributions. Mass Spectrometry Imaging Analysis

The method of the invention can be easily implemented with mass spectrometry imaging experiment. In this case, the peak intensity, peak area or signal to noise ratio of each molecule of interest and control compounds are visualized and compared.

The implementation of the analysis step of the distribution of the molecule of interest according to the invention is illustrated below, in general terms, based on the use of MSI on a tissue section that comprises the target tissue and an adjacent non-target tissue surrounding said tissue. Spectral characteristics, especially peak intensities of three molecules of interest (Ml, M2 and M3) as well as one positive control compound (M+) and one negative control compound (M-) are studied. a) Distribution analysis of the molecule of interest within the target tissue

The figure 1 shows schematically the intensities obtained using mass spectrometry imaging of the target tissue, for the three molecules of interest (Ml, M2 and M3), the control molecules M+ and M-. These intensities reflect their distribution within the tissue.

From the spectral data directly visualized on the tissue sections, it is possible to confirm the presence or absence of each molecule of interest within the target tissue. Moreover, by comparing the spectral characteristics of the molecules of interest and control compounds, it is easy to confirm if this distribution is significant or not.

To simplify the results, the different tissue areas are define as two concentric squares, the target tissue corresponding to the center square (second picture column on Figure 1). The values indicated in the small squares on the picture correspond to the normalized intensity values of each molecule. The mean of these normalized values is calculated for each molecule, for the target tissue (global spectral signature of region Tl) and for the adjacent tissue (global spectral signature of region T-Tl). To evaluate the action efficiency of the molecule of interest on target tissue, its action is weighted by taking into account its presence or absence in non-target tissue section, i.e. the adjacent tissue noted T. For this, one links mean intensities of the molecule of interest in and outside the target tissue to the mean intensities of the positive and negative control compounds in the same tissues.

As illustrated figure 1, the M2 molecule highlights a high intensity on the whole tissue (target and non-target tissue) and is globally unspecific to target tissue Tl. Conversely, Ml molecule exhibits a high intensity in the target tissue Tl solely and is almost non-detected in the adjacent tissue T. Its distribution is rather similar to the M + molecule. Thus, one can evaluate a first factor Fl representative of the specificity of the molecule of interest distribution in the target tissue, using as reference value, the normalized intensities of the peaks on mass spectra of each molecule of interest, in and outside the target tissue, which are compared with the normalized intensities of the peaks of the positive and negative control compounds in and outside the target tissue. To simplify the calculation, the factor Fl of the positive control compound is considered as 100%, and the factor Fl of the negative control compound is 0%.

Table 2 below lists the intensity results obtained for each molecule. Table 2: Summary of mean intensity values per molecules and tissue area

The Fl factor allows ranking the molecules of interest depending on their specificity for the target tissue.

Based on this factor solely, it is already possible to sort the molecules of interest depending on their tissue targeting specificity. Thus, in the present case, the M3 molecule, with less than 25% of spectral efficiency, can be eliminated from the candidate molecules for a treatment targeting tissue Tl. b) Analysis of the part of the molecule of interest in the target tissue

According the invention, it is also possible to evaluate the proportion of the molecule of interest reaching the target tissue relative to the administrated dose, and compared to the positive control compound.

More specifically, as illustrated figure 2, the method of the invention allows to evaluate the similarities and dissimilarities of the molecule of interest distribution compared to the positive control compound in the target tissue.

Spatial similarities of the molecules distribution are taking into account to evaluate the specific action of each one on the target tissue. For example, form the intensity map obtained for each molecule (column 1 and 2 on figure 2), the standard deviation, or the variance of the molecule of interest value, is calculated compared to the value of the positive control compound for each position (i.e. for each square having a x.y _j position).

The standard deviation can be calculated according to the mathematical equation below: a _Mi : Standard deviation of the molecule of interest Mi compared to positive marker M ⁺

X _Mi : Intensity value of the molecule of interest Mi at (x,, y _j ) coordinates

X _M+ : Intensity value of positive control compound M+ at (x,, y _j ) coordinates

(X _M ) : Mean intensities of the molecule of interest and the positive control compound at y _j ) coordinates From these standard deviation values, the method of the invention allows calculating a second factor F2 highlighting the penetration ratio of the molecule of interest in the target tissue. The F2 factor corresponds to the percentage of similarity between the distribution of the molecule of interest and the positive control compound (F2 = 100%). Generally speaking, the F2 factor of the negative control compound is 0%. By comparing the standard deviation of the molecule of interest with positive control compound (and eventually of the negative control compound), it is possible to evaluate the F2 coefficient for said molecule of interest.

These factor F2 values obtained for each molecules of interest M l, M2 and M3 are disclosed on figure 2. More specifically, this experiment allows viewing that molecule M l exhibits a high recovery percentage with the positive control compound M+. Regarding the molecule M2, even if M2 is detected in the target tissue area, it doesn't exhibit a distribution similar to the positive control compound M+. And, M3 distribution is similar to the negative control compound one. Its recovery percentage with M+ is almost zero. c) Targeting Coefficient calculation of a molecule of interest for a given tissue

According to the invention, it is possible to calculate a targeting coefficient of a given molecule of interest for a given target tissue. This targeting coefficient reflects the specificity of said molecule for said tissue, as well as its spatial distribution (or penetration ratio) in said tissue.

For example, the targeting coefficient corresponds to the mean of the values obtained for Fl and F2 factors for said molecule in said tissue. Targeting coefficients (or targeting percentage) are reported in table 3 below for each molecules of interest Ml, M2 and M3. Thus, it is possible to rank the molecules of interest depending on their action more or less efficient and more or less specific of said tissue.

Table 3 : Summary of the targeting coefficients of the molecules of interest and control compounds for the target tissue Tl Fl (%) F2 (%) % Targeting

M+ 100% 100% 100%

M- 0% 0% 0%

Ml 90% 75% 83%

M2 61% 48% 54%

M3 22% 0% 11%

In the present case, the Ml molecule is the best candidate for the target tissue Tl because it has a more targeted and specific action than M2 molecule. The M3 molecule, as previously assessed, is not a good candidate molecule for tissue Tl.

EXAMPLES

The method of the invention will now be described in further detail using specific examples and the figures presented above. These examples are given for illustrative purposes only and by no means restrict the scope of the invention. Of course, in an almost identical manner an imaging device other that MALDI can be used, such as, for example, the following sources: SIMS, DESI, DIOS, ICP, MALDI microscope, SNOM, SMALDI, LA-ICP, ESI (liquid extraction on tissue), MILDI, JEDI, ELDI, etc.

EXAMPLE 1: EVALUATION OF OLANZAPINE BINDING IN HISTOLOGICAL REGIONS OF KIDNEY

In example 1, the method of the invention is used to evaluate the distribution of the olanzapine in mouse kidney and to study its targeting to specific region of the kidney.

Material & Methods

Hydroxycinnamic acid (CHCA) from Sigma Aldrich (Saint-Quentin Fallavier, France),

Trifluoroacetic acid (TFA) from Sigma Aldrich Acetonitrile/DMSO/Water from Sigma Aldrich

Olanzapine from Lilly Research Laboratories (Eli Lilly and Co, Indianapolis, IN)

Positive control compound: Posaconazole from Sigma Aldrich

Negative control compound: Methsuximide from Sigma Aldrich

Delimitation of target tissue

Target tissue, hereinafter "Tissue 1", comprises the medulla and renal calices.

Non-target adjacent tissue, hereinafter "Tissue 2", is also considered and comprises the renal cortex region.

Molecule of interest, positive & negative control compounds selection

The olanzapine is a drug used in the treatment of certain forms of schizophrenia and bipolar disorders. It is one of the most commonly used antipsychotics. It belongs to the class of thienobenzodiazepine. Olanzapine is already known as a molecule targeting the brain. Pharmacokinetic studies have demonstrated that olanzapine was quickly absorbed by the organism and especially accumulated in the liver, spleen or kidney. Moreover, the distribution of olanzapine is highly specific to some kinds of tissue within the same organ allowing, for kidney example, a prominent differentiation of cortex and medulla region. In this example, the inventors have studied the tissue targeting properties of olanzapine within kidney. Two positive and negative control compounds are selected for their properties to selectively target tissues 1 and 2 respectively.

The posaconazole, known as specifically binding tissue 1 and not tissue 2, is chosen as positive control compound.

The methsuximide, known as specifically binding tissue 2 and not tissue 1, is chosen as negative control compound.

Animal preparation

Wild Type mouse of Swiss strain weighing 25-40g from Charles River was used. Olanzapine, positive and negative compounds taken up in 0.9% NaCI solution were administrated by oral route at a concentration of 8 mg/kg. Mouse was sacrificed by C02 asphyxiation. Kidneys were then removed and plunged into 100% isopentane solution cooled by liquid nitrogen for rapid freezing. Finally, kidneys were stored at -80 °C.

Preparation of samples for mass spectrometry imaging Kidney was sectioned into 10 μιη-thick layers (sagittal sections) using a Microm HM560 (Thermo Scientific, France) cooled at -21 °C. The sections were then deposited on conductive ITO (indium tin oxide) slides (Delta Technology, USA). Finally, the sections were keeping in the cryostat chamber 30 min for cryodrying and then placed in a desiccator for 30 minutes.

A CHCA matrix was used for the analysis of all kidney tissues sections. This matrix was prepared at a concentration of 10 mg/ml in acetonitrile/water+0.1% TFA (6:1, v/v). The matrix solution was deposited using the SunCollect spraying system (SunChrom, Germany) following optimised protocol.

MALDI imgge gcguisition The images were obtained using an AutoFlex Speed MALDI-TOF mass spectrometer (Bruker Daltonics, Bremen, Germany) equipped with a Smartbeam II laser. The data was generated in positive reflectron mode. A total of 500 spectra were obtained for each spot with a 1000 Hz laser frequency and a 200 x 200 μιη ² image spatial resolution on a mass range of 100 to 1000 Da. The Flexlmaging version 4.0 software was used to reconstruct the images and Quantinetix 1.6 software (ImaBiotech, Loos, France) permits the extraction of spectral intensities of ions corresponding to each studied molecule.

Localization of the molecule of interest and control compounds

Firstly, it is necessary to performed imaging mass spectrometry experiments to localize ions of interest within tissues (Figure 3) Images were achieved on whole kidney. Spatial resolution used was 200 μιη which permits to easily distinguish renal sub-structures. The figure 3 shows a kidney tissue section highlighting its different histological areas (Fig. 3A). The sections have been taken on the middle of the kidney.

From this, it is possible to accurately observe the target tissue. Notably, Figure 3B shows the overlay distributions of positive and negative control compounds as well as the molecule of interest, the olanzapine. It clearly shows the distinction between tissue 1 (center area of the tissue section) and tissue 2 (peripheral area of tissue section) as presented in the schematic figure 3C.

These results allow evaluating the efficiency of histological region targeted by the positive and negative control compounds. It is also important to highlight the molecule of interest within the tissue, which is the case in the present case.

The Figure 4 displays three mass spectra corresponding to the histological areas of interest, the tissue 1 for positive control compound (m/z 204, Fig. 4C), the tissue 2 for negative control compound (m/z 725, Fig. 4B) and a high olanzapine concentrated area (m/z 313, Fig. 4A).

Results

Fl Factor Calculation

In order to calculate Fl which is related to the specificity of spatial distribution of the molecule of interest within target tissue, one needs first to extract all intensities of the molecules on interest and control compounds from mass spectrometric image (MS image).

The Figure 5 allows visualizing separately the distribution related to positive and negative control compounds as well as olanzapine.

Polychromatic representation gives information related to relative intensities of ions of interest. The maximum value of each scale is different and corresponds to highest intensity value of each separate ion on MS image.

Each histological regions of interest are delimited on the molecular images and illustrated on figure 4 and 5 with dashed lines. The imaging data treatment software, Quantinetix, is used to extract relatives' intensities of each ions of interest and control compound for each position (or voxels) within each drawn region.

MS images on Figure 5 illustrate:

- Positive control compound (M+) distribution at the level of target tissue 1 (Molecular image on the left) ;

- Negative control compound (M-) distribution at the level of target tissue 2 (Molecular image on the right) ;

- Olanzapine distribution at the level of target tissue Tl et non-target tissue T2 (Center molecular image) The next step is to normalize the whole dataset in order to compare them. Highest intensity related to each ion is used to normalize the intensities per position. One calculates mean normalized intensities for each ions and tissue types.

By definition, one considers that positive control compound factor Fl is equal to 100% whereas negative control compound factor Fl is equal to 0%. The results obtained for the olanzapine are reported below in table 4.

Table 4: Summary of mean intensity and Fl factor values per molecules and tissue area.

Mean normalized

intensity per tissue

Tl Fi (%)

M ⁺ 0.281 0.055 100%

M ^" 0.062 0.241 0%

M _c 0.161 0.117 52.2%

Tl : target tissue ; T2 : non target tissue ; Mc : Olanzapine ; M+ : Posaconazole; M- :

Methsuximide Olanzapine Fl factor reflects the average efficiency of target molecule from a spectral and intensity point of view in studied tissues Tl and T2. A Fl value of 50% describes a global equilibrium of relative concentration of olanzapine in both tissues in comparison with positive and negative compounds. F2 Factor Calculation

The second step of tissue targeting evaluation takes into account spatial aspect of olanzapine distribution in the kidney compare with control compounds.

F2 factor allows comparing the spatial distribution of control compounds with the molecule of interest that provides information about their similarities or dissimilarities at the level of target tissue.

To visualize these properties on MS images, the overlay of olanzapine and control compounds distribution is shown in Figure 6. It allows comparing the distribution of olanzapine in target tissue (Tl) with adjacent non-target tissue (T2).

F2 factor calculation is performed using normalized intensity values from previous Fl factor evaluation. Position per position, one assesses standard deviation of intensity values of the molecule of interest and the negative control with positive control one at the level of target tissue (Tl). Then, average values of standard deviation are calculated for the olanzapine and negative control compound.

By definition, positive control compound factor F2 is considered equal to 100% whereas negative control compound factor F2 is equal to 0%.

Table 5: Summary of Fl and F2 factors values and of global targeting coefficient (%Tg) per molecules for target tissue.

Ti Fl(%) F2(%) %Tg

M ⁺ 100% 100% 100%

M ^" 0% 0% 0%

M _c 52.2% 27% 40% Tl : target tissue ; Mc : Olanzapine ; M+ : Posaconazole; M- : Methsuximide

F2 factor for the olanzapine is evaluated at 27% that reflects dissimilarity with positive control distribution in target tissue. This is especially highlighted in Figure 6 where the distribution of olanzapine is mainly localized on the right half section of the medulla (center picture on Fig 6), whereas positive control compound is homogenously distributed in tissue 1 (right picture on Fig 6).

Summary

The olanzapine shows a mean affinity for the medulla region. The results obtained with the method permit to conclude that olanzapine has a mean targeting behavior for one kinds of tissue in kidney instead of another, and is preferentially bound to a limited area in the medulla region.

EXAMPLE 2: Adsorption Calculation

The figure 7 explains the methodology of adsorption of a molecule on tissue using the example of receptor occupancy process. By this way, it is possible to measure the occupancy efficiency of the receptor by the drug (agonist) administered to the animal compared with an antagonist molecule. In this example, all the steps of the methodology are described, the dosed tissue analysis alone, the washing step with the antagonist solution of the control tissue and the combining analysis dosed/washing on administrated sample. No negative marker is required for this kind of experiment. In this example, the competitive binding of two benzodiazepine compounds, the Diazepam (the agonist) and the Lorazepam (the antagonist) which target the same receptor in the brain (GABA) are evaluated. The Lorazepam has a higher affinity for this receptor which is well established. That's why the well-known Lorazepam is used to study the affinity and efficiency of Diazepam to bind receptors in brain. The distribution of the agonist (Diazepam) and the antagonist (Lorazepam) in a brain of a rat is studied by MALDI mass spectrometry imaging. Of course, in an almost identical manner an imaging device other that MALDI can be used, such as, for example, the following sources: SIMS, DESI, DIOS, ICP, MALDI microscope, SNOM, SMALDI, LA-ICP, ESI (liquid extraction on tissue), MILDI, JEDI, ELDI, etc.

Materials and methods Materials

- 2,5-Dihydroxybenzoic acid (DHB) (Sigma-Aldrich, Saint-Quentin Fallavier, France)

- Trifluoroacetic acid (TFA) (Sigma-Aldrich)

- Methanol (Sigma-Aldrich)

- Diazepam (LGC Standard)

- Lorazepam (LGC Standard)

Animals

Wild Type Rat weighing 35-40g were used. Diazepam taken up in 0.9% NaCI solution was injected by intravenous route at a concentration of 15 mg/kg. Animals (dosed and control rats) were sacrificed by C0 ₂ asphyxiation. Brains were then removed and plunged into 100% isopentane solution cooled by liquid nitrogen for rapid freezing. Finally, brains were stored at -80 °C.

Preparation of samples for mass spectrometry

Brain (control and dosed tissues) were sectioned into 10 μιη-thick layers (coronal section) using a Microm HM560 (Thermo Scientific, France) cooled at -20 °C. The sections were then deposited on conductive ITO (indium tin oxide) slides (Delta Technology, USA). Finally, the sections were keeping in the cryostat chamber 15 min for cryodrying and then placed in a desiccator for 20 minutes.

Washing/incubating step with antagonist solution The antagonist solution was prepared from a stock solution of Lorazepam at 1 mg/ml in methanol (100%). The final concentration of the washing solution was fixed at 100 ριηοΙ/μί in Methanol/water+0.1%TFA (7:3). The concentration of the antagonist is a crucial factor for receptor occupancy that improves the binding capacity of the molecule. 20 μί of the solution was spot on tissue section (one dosed and on control sample) and then incubated during 1 hour at 37°C in a box inside the incubator Incu-line (VWR, France). A dosed brain section without washing was also put in the incubator.

After incubation, tissues are washed using 2 steps to remove unbounded molecule from the tissue, firstly with a solution of Methanol/water+0.1%TFA (7:3) corresponding to antagonist washing solution. Secondly, the tissue is washed using only water solution (lOmL) and then placed into desiccator for 15 min.

Preparation for acquisition by MALDI imaging

A DHB matrix was used for the analysis of all brain tissues sections with or without washing with Lorazepam solution. This matrix was prepared at a concentration of 40 mg/ml in methanol/water+0.1% TFA (1:1, v/v). The matrix solution was deposited using the SunCollect spraying system (SunChrome, Germany).

MALDI image acquisition

The images were obtained using an AutoFlex Speed MALDI-TOF mass spectrometer (Bruker Daltonics, Bremen, Germany) equipped with a Smartbeam laser. The data was generated in positive reflectron mode. A total of 700 spectra were obtained for each spot with a 1000 Hz laser frequency and a 150 x 150 μιη ² image spatial resolution on a mass range of O to 1000 Da. The Flexlmaging version 4.0 software was used to reconstruct the images.

Results

The figure 8 shows images obtain by MSI on the different conditions and following adsorption evaluation methodology.

The brain dosed with the agonist without washing highlights a specific localization of the diazepam (m/z 285) at the level of white matter of the brain. The second step involving the washing of a control tissue section with Lorazepam solution allows observing the Lorazepam related ion (m/z 321) on the brain section, especia lly in the white matter. Obviously, no detection of Lorazepam or Diazepam was observed respectively on the dosed brain section without washing and on the control brain section with washing step. These results give some information on the receptor binding sites of the two benzodiazepam species, i.e. the white matter. Finally, the last experiment combines the Diazepam dosed tissue with the Lorazepam washing step. This image is further used to calculate the efficiency and the affinity of Diazepam according to Lorazepam response. In summary, the experience provides:

^■ The distribution of the target molecule (Mc), the Diazepam

^■ The distribution of the positive marker (M+), the Lorazepam

^■ The targeted area (Tl), the white matter (receptor binding site) and the untargeted area (T2), the rest of the brain

Fl Factor; Intensity Factor

In order to calculate the factor 1 which is related to spectral parameters of the target molecule at the level of the targeted tissue, one needs firstly to extract the intensity of Diazepam and Lorazepam ions in specific area of the brain.

Then the methodology is applied to calculate Fl on the dataset.

Table 6: Summary of mean intensity values and corresponding Fl factor per molecule and tissue

Normalized mean

intensity/Tissue

Tl Fi (%)

M ⁺ 0.821 0.21 100%

M _c 0.692 0.117 85.1%

Tl : Targeted Area ; T2 : untargeted area ; Mc : Diazepam ; M+ : Lorazepam

F2 Factor; Spatial Factor

The second step of the affinity assessment of Diazepam takes into account its spatial distribution at the level of the targeted area versus positive marker localization. The F2 factor allows comparing the fine localization of the agonist and the antagonist in the white matter region and gives information about their similarity or dissimilarity.

Table 7: Summary of Fl and F2 values and corresponding targeting coefficient (%Tar) for Diazepam on target area (Receptor)

Tl : Targeted Area ; Mc : Diazepam ; M+ : Lorazepam

The F2 factor is equal to 72.5% that traduced a high similarity of the diazepam a lorazepam distribution in targeted area (receptor site). The diazepam is highly localized the white matter of the brain as well as its antagonist the lorazepam.

Conclusion

In conclusion, the two factors can be combined to estimate the global targeting factor of the agonist as presented in table 7. The diazepam shows a high affinity for the receptor site of benzodiazepine regarding the positive marker studied. The targeting of the diazepam is highly efficient for the white matter region of the brain.