Described herein is an insect model for measuring blood-brain barrier (BBB) permeability, brain uptake, and metabolism, release, or both, and concentrations and fractions of unbound drug in the brain of chemical compounds. Additionally brain uptake, metabolism and/or release of chemical compounds in vertebrate brains can be derived. The model can be used to identify and quantify metabolite(s) formed in the brain.

Investigation of brain uptake and metabolism of chemical compounds is important in drug discovery; successful central nervous system (CNS) drugs have to cross the BBB, while BBB penetration may cause unwanted side effects for peripheral acting drugs. Furthermore, CNS drugs can be transformed into CNS active chemical compounds or toxins in the brain by metabolism. In addition, the free drug hypothesis states that it is the concentration of unbound drug or toxins in the brain that is important.

Described herein is a procedure to prepare, expose with chemical compound(s), and analyse the brain of one or more insect(s) for chemical compounds, metabolites of chemical compounds or/and chemical compounds released from the exposed chemical compounds. Specifically, described is a procedure to prepare the insect brains for ex vivo studies that reduce potential disruption of the BBB and thereby leakage into the brain.

In addition, described is a procedure to be able to evaluate free concentrations or fractions of unbound drugs in the brain. The model uses insects in screening for substances with a biological effect on the brain or central nervous system and/or effect on a disease or disorder of the brain or central nervous system. It further relates to use of such insects in screening for substances that have a desired biological activity and which do not need or should not cross the blood-brain barrier.

BACKGROUND

In vertebrates the BBB consists of a single layer of microvascular endothelial cells that strictly prevent free movements of molecules from the blood into the brain and thus contributing to the homeostasis of the micro-environment. The BBB endothelial cells prevent paracellular diffusion of hydrophilic compounds by virtue of laterally transmembrane proteins, forming the tight junctions. In addition, ATP binding cassette transporters prevent the entry of the majority of invading xenobiotics by efflux mechanisms (Abbott, N.J., Ronnback, L. & Hansson, E. Astrocyte- endothelial interactions at the blood-brain barrier, Nature Reviews Neuroscience 7, 41-53 (2006)). Passage of the BBB by chemical compounds may either happen through passive diffusion or aided by membrane bound transporter proteins, usually classified as chemistry- or biology-based respectively. Neuropathogens (e.g., bacteria) may also pass the BBB but do so by disrupting the BBB integrity, which is a different approach compared to chemical compounds.

In drug research it is important to determine brain penetration both for drug candidates with CNS therapeutic potential but also for compounds that can cause CNS mediated side effects. There are two commonly accepted concepts of assessing brain barrier penetration i.e., the extent of brain exposure at steady-state and the rate of brain uptake at initial state. The extent of brain uptake is frequently measured as the fraction of the concentration measured in the brain and in plasma at steady state.

The extent models are regarded as the most relevant for discovery of CNS drugs but the use of these models are hampered by being expensive, time- and animal- consuming. Consequently, several in vitro BBB permeability models are developed to predict the in vivo behaviour of chemical compounds. However, even complex in vitro models which also include the Pgp transporter systems (Andersson, O., Hansen, S.H., Hellman, K., Olsen, L.R., Andersson, G., Badolo, L, Svenstrup, N., Nielsen, P. A. (2013) The grasshopper: A novel model for assessing vertebrate brain uptake J Pharmacol Exp Ther 346:21 1-218) seem not to meet the intricate complexity of the BBB and therefore may not describe the in vivo behaviour very well.

Recent research has shown that insects are suitable models for assessment of vertebrate BBB permeability prediction of chemical compounds in early drug discovery (Nielsen, P. A., Andersson, O., Hansen, S.H., Simonsen, K.B., Andersson, G. (2011) Models for predicting blood-brain barrier permeation. Drug Discov Today 16: 472-475). These models make use of the evolutionarily conserved essential mechanisms important in CNS protection, which operate in mammals and non-vertebrate organisms. In both insects and vertebrates there is one specific cell layer performing the barrier function and it has been shown that the insect barrier cells (glia) contain pleated septate and tight junctions nearly identical to the proteins that make up the vertebrate tight junctions (Hindle, S.J. and Bainton J. (2014). Barrier mechanisms in the Drosophila blood-brain barrier, Frontiers in Neuroscience 8 (414): 1-12). Furthermore, it has been shown that insects possess a homologue to the major ATP binding cassette (ABC) transporter (MDR/Pgp) (Mayer F et al., 2009 Evolutionary conservation of vertebrate blood-brain barrier chemoprotective mechanisms in Drosophila. J Neuroscience 29: 3538-3550).

Not only BBB passage is a prerequisite for CNS activity, during and after passage of the BBB the drug is potentially metabolized by degrading enzymes. Some CYPs are unique to the brain or much more profoundly expressed in brain than in others tissues. Neuronal CYP isoforms are expressed in defined cell populations and specific regions of the brain, e.g. in the olfactory mucosa, bulb, striatum, hypothalamus, and cortex. The neuronal CYPs most likely metabolize odorants, xenobiotics, and steroid hormones, which act specifically in these cell populations. These findings indicate that the CYP isoforms in the brain have additional and more specific functions than just drug detoxification.

Studies on the function of brain CYPs are challenging for several reasons

(Miksys, S., and Tyndale, R. F. (2013) J Psychiatry Neurosci 38, 152-163). In the whole organism, CYPs in the liver produce many of the same metabolites as those in the brain, and many of these metabolites can cross the BBB from the periphery, making relative contributions of hepatic and brain metabolism in vivo difficult to study.

The practical approaches to routinely investigating large numbers of new compounds have been to measure the amount of drug in brain and correlate that with plasma levels in vivo or the rate of BBB permeation in vitro. These approaches have their benefits, especially in the initial phase of drug discovery but as sole analyses it has led to that medicinal chemistry programs have favored compounds and classes displaying high total CNS-to-plasma concentration ratios, sometimes rendering in lipophilic drugs that dissolves well within the lipophilic brain content. However, it is generally accepted that it is the unbound drug (i.e. the free concentration of drug in the brain) that is available to exert the effect on their targets or susceptibility for biotransformations, and not the total tissue levels.

In CNS drug discovery there is a need for efficient screening of compounds aimed at targets within the CNS system. This screening is preferentially performed in insect models with intact BBB function and will contribute to a positive selection of chemical compounds penetrating the BBB and available for CNS target engagement. Especially to be able to isolate and perform experiments on the whole brain with an intact brain-membrane and functional enzymatic system gives an advantage in studying BBB penetration and other biological processes, e.g., metabolism. Optimally the models should enable identification of potential metabolic activities. Such screening comprises chemical compounds within a number of indications (e.g., pain, epilepsy, Parkinson, schizophrenia, Alzheimer, sleep disorders, anxiety, depression, eating disorders, drug abuse including smoking).

SUM MARY

Herein is provided a predictable ex vivo insect screening model to determine for example brain uptake, formation of metabolites, or release of chemical compounds. In addition, the model can be used for evaluating free concentrations and fractions of unbound drug or toxins in the brain, as well as all parameters calculated based on the measured parameters in model. The model will improve the chemical compound screening procedures/processes in drug discovery and toxicology evaluations. The methods described herein are particularly useful as the brain barrier damage is reduced compared to previous reported insect based brain-barrier screening models. In addition, the method described herein allows the brain to be intact making it possible to study biological processes such as metabolism and the formation of metabolites, in a qualitative, quantitative and distributional way, thus chemical compounds exposed to the brain, formed in the brain, or released in the brain for example by a chemical compound delivery system, can be studied in the brain as whole or in specific regions or cells thereof depending on the analytical technology used, for example how the brain is divided.

The present disclosure relates to a method comprising:

dissecting out of an insect head the insect brain comprising the compound eyes; treating the obtained brain comprising the compound eyes with a medium comprising one or more chemical compounds; and

analyzing the brain (excluding the compound eyes) and/or medium.

The method described herein is essentially a method for investigating the blood- brain barrier. The method involves treating a dissected out insect brain comprising compound eyes with a medium comprising one or more chemical compounds, then analyzing the brain (excluding the compound eyes) and/or medium.

As described further below, the method can involve analyzing the medium which is used to treat the brain, for example to determine if chemicals have leached out of the brain through the blood-brain barrier during the treating step. However, typically the method will involve analyzing the brain (either instead of or in addition to the medium), in which case the method usually involves removing the compound eyes prior to analyzing the brain.

Thus, typically the method comprises the step of removing the compound eyes (e.g. prior to analyzing the brain).

Typically, the brain has intact nerve connections to the compound eyes prior to the compound eyes being removed, e.g. during the treating step.

Thus, typically the method comprises the step of removing the compound eyes including their nerve connections prior to analyzing the brain.

Optionally, the brain additionally comprises intact antenna(s). Typically, any antenna(s) are removed prior to treating the brain comprising the compound eyes with the one or more chemical compounds in a medium.

If still present, any antenna(s) are typically removed prior to analyzing the brain. Typically, the brain is dissected out together with its neural lamella.

Typically, the brain and compound eyes comprises a neural lamella during exposure to the medium comprising one or more chemical compounds.

Typically, the neural lamella is removed prior to analyzing the brain.

Typically, the compound eyes (including their nerve connections) and neural lamella are removed prior to analyzing the brain.

Typically, the medium is a liquid medium.

Typically, the medium additionally comprises a compound that is not capable of crossing the blood-brain barrier. This can act as a control, as if this compound is found in the brain in the analyzing step, it is evidence that the blood-brain barrier was compromised during the dissecting step.

Suitable compounds that are not capable of crossing the blood-brain barrier include Evans blue dye, Lucifer yellow and atenolol.

Typically, the compound not capable of crossing the blood-brain barrier is a dye such as Evans blue dye or Lucifer yellow. Compounds which are easily detected such as dyes (including fluorescent compounds) are particularly suitable, as they can be used as a simple visual test to determine whether the blood-brain barrier may have been compromised.

Typically, the brain comprising the compound eyes is transferred from the medium comprising one or more chemical compounds to at least a second medium having a different composition than the first medium prior to removing the compound eyes. The transfer from the first medium to the second medium may be accompanied by washing the brain. Typically, the brain is treated with the medium comprising one or more chemical compounds for a period of 1 minute to 8 hours, or 1-360 minutes, or 5 to 240 minutes, or 10 to 100 minutes.

Typically, the method is carried out under ambient conditions, e.g. at a temperature between 2 and 50°C, or from 10 to 40°C, or from 15 to 30 °C, most typically about room temperature (e.g. 25°C).

The method described herein allows for the investigation of the blood-brain barrier and metabolism in the brain. The various steps can be modified in various ways, depending on what is to be investigated, for example by

- dividing the exposed brain into two or more slices, and investigating the localization and distribution of a chemical compound throughout a slice analyzing the amount of a chemical compound in the brain after exposure, to determine the rate of penetration through the blood-brain barrier into the brain

- analyzing the exposure solution after exposure to determine the rate of transfer of a chemical compound through the blood brian barrer out of the brain

analyzing the brain and/or exposure solution for related chemical compounds to investigate the metabolism of compounds in the brain and/or kinetics of chemical reactions in the brain

subjecting the live insect to one or more test compounds prior to dissecting out the brain.

The final exemplary embodiment allows the method to be used to determine the penetration of a test compound through the blood-brain barrier in an in vivo model, or investigate how a test compound affects the blood-brain barrier. Thus, the live insect could be subjected to a test compound for an extended time, for instance for at least 30 minutes, for at least 1 hour, for at least 4 hours, for at least 8 hours, for at least 12 hours, from 1 day to four weeks or more, for at least one week or the like. The exposure could be via a food source, and/or as part of the environment that surrounds the insect (such as an airbourne chemical or a waterbourne chemical contained in precipitation such as rain). In such in vivo experiments, the environment can be controlled to provide the desired level of exposure for the study, such as by periodically administering sprays containing the chemical to simulate exposure via chemicals contained in rain. After subjecting the live insect to the one or more test compounds, the brain could be dissected out in accordance with the method described herein, the brain treated with a medium comprising one or more chemical compounds, and then the brain and/or medium analysed. In such embodiments, the medium comprising the chemical compound may contain a chemical compound with low permeability through the blood-brain barrier, such that the integrity of the blood-brain barrier after subjecting to the test compound can be determined. The results can then be used to determine the penetration of a test compound in vivo, and/or the effect that the test compound has on the blood-brain barrier.

In a preferred form, the method described herein is a method of analysing and/or determining and/or localizing brain uptake, metabolism and/or release or efflux of one or more chemical compound(s) and/or metabolites thereof in an insect brain.

In a preferred form, the method described herein is a method for determining half-life, kinetics of metabolism and/or elimination and/or determination of unbound concentrations and/or free fraction of one or more chemical compound and/or metabolites thereof in the brain and/or in a medium comprising a dissected brain comprising the compound eyes (including their nerve connections).

In a preferred form, the method described herein is a method for determining unbound concentrations and/or free fraction of one or more chemical compound and/or metabolites thereof in the brain and/or in a medium comprising a dissected brain comprising the compound eyes (including their nerve connections).

In a preferred form, the method is a method for determining half-life, kinetics of metabolism and/or prodrug release and elimination and/or determination of unbound concentrations and/or free fraction of metabolites or released chemical compounds thereof in the brain and/or in a medium comprising a dissected brain comprising the compound eyes (including their nerve connections).

In a preferred form, the method is a method for determining half-life, kinetics of metabolism and/or prodrug release and elimination and/or determination of unbound concentrations and/or free fraction of metabolites or released chemical compounds thereof in the brain and/or in a medium comprising a dissected brain comprising the compound eyes (including their nerve connections)

Also disclosed is the use of the method to determine the metabolism of a chemical compound in the brain. Typically, the use is to determine the half-life or kinetics of metabolism of a chemical compound in the brain. Additionally or alternatively, the use is to identify and quantify metabolites formed from a chemical compound in the brain.

Also disclosed is the use of the method to determine the penetration of a chemical compound through the blood-brain barrier.

In such uses, the penetration could be penetration into the brain, in which case following exposure the brain is typically analysed to determine the uptake of the chemical compound, for example to screen chemical compounds as drug candidates. Alternatively, the penetration could be efflux of compounds out of the brain, in which case the solution to which the brain is exposed is typically analysed for the presence of the compound after exposure.

Also disclosed is the use of the method to determine the accumulation of a chemical compound in the brain.

Also disclosed is the use of the method to determine the distribution of a chemical compound throughout the brain.

Also disclosed is the use of the method to evaluate the free concentration or fraction of a chemical compound in the brain (i.e. the amount of a chemical compound that is not bound, such as bound by proteins).

Also disclosed is the use of the method to determine the sensitivity of insect antennae.Also disclosed is the use of the method to determine the influence insect antennae have on brain chemistry, particularly on metabolism in the brain.

Not all chemicals can cross the blood-brain barrier, and in some situations it is desirable that a chemical can cross the blood-brain barrier (such as with a drug intended to act on the brain), in which case the method can be used to screen for such compounds.

However, in other situations it may be undesirable for a compound to cross the blood-brain barrier. For instance, the chemical compound may be a drug that is not intended to act on the brain (such that the method can be used to screen whether pharmaceuticals may have unintended side effects in the brain), environmental toxins (such as pesticides or airborne pollutants), food additives etc.. The method can be used to investigate the tendancy of such compounds to cross the blood-brain barrier, and also how such compounds accumulate and are metabolized in the brain.

DESCRIPTION OF THE DRAWINGS

Figure 1 shows a schematic picture of the locust brain (compound eyes (and their connection) and lamella not included to simplify the model) and equilibrations between different compartments of drug. Free (also mentioned as unbound) and bound drug in different compartments of the drug is depicted. ICF = intra cellular fluid; ISF = interstitial fluid; Blood/buffer = medium; Protein compartment = blood, other body fluids, food & beverages, solid samples as earth and mud, gases content. Free drug (unbound) equilibrate between compartments. The C _to t,brain = total drug concentration in the brain (drug in ICF and ISF), C _un bound,brain = unbound drug concentration in the brain ISF (drug concentration in buffer/blood (buffer/blood is also mentioned as medium)). F _ub = free fraction in the brain (Cunbound,bm C _to t,b™n)- DETAILED DESCRIPTION

The method described herein finds a variety of uses, including analyzing brain uptake (i.e. penetration of chemicals through the blood-brain barrier (BBB)), analyzing localization of brain uptake (i.e. determining penetration of chemicals into the brain and distribution of chemicals within the brain following penetration of the BBB), studying the reactivity and metabolism of compounds within the brain, and studying how chemical compound affect the integrity of the blood-brain barrier. The treating and analysis steps will vary of course according to what is being investigated, but nevertheless the preferred aspects described herein will generally apply to any method described herein unless it is clear from the context that this is not the case.

For instance, in methods which investigate the uptake of chemicals across the blood-brain barrier, the analyzing step typically comprises

homogenizing or disintegrating the brain followed by determining the concentration(s) of at least one of the one or more chemicals and/or metabolites thereof in the homogenized or disintegrated brain; and/or

determining the concentration(s) of at least one of the one or more chemical compound(s) and/ or metabolites thereof in the first or second medium or any subsequent medium.

In methods which investigate the distribution/localization of chemical compounds in the brain, the analyzing step typically comprises dividing the brain in two or more slices or pieces, and then

determining the concentration(s) and/or localization of at least one of the one or more chemical compound(s) and/ or metabolites thereof in at least one of the pieces or slices of brain. Thus, also disclosed is a method of analysing and/or determining and/or localizing brain uptake, metabolism and/or release from drug delivery system of one or more chemical compound(s) and/or metabolites thereof in an insect brain.

said method comprising the steps:

dissecting out the brain comprising the compounds eyes (including their nerve connections) of the insect head;

treating the obtained brain comprising the compounds eyes with one or several chemical compounds;

removing compound eyes (including their nerve connections and optionally the neural lamella)

homogenising or disintegrating the brain or slicing the brain in two or more pieces or slices;

determining the concentration(s) of the one or more chemical compound(s) and/ or metabolites thereof in the homogenised or disintegrated brain material;

or determining the concentration(s) and/or localizing the one or more chemical compound(s) and/or metabolites thereof in the one or more of the pieces of brain material, or

determining the concentration(s) or/and localisation of one or more chemical compound(s) and/ or metabolites thereof in the in the one or more of the pieces or slices of brain material from the first or second medium or any subsequent medium, or determining the concentration(s) of one or more chemical compound(s) and/ or metabolites thereof in the first or second medium or any subsequent medium.

Additionally provided herein is a method for studying blood-brain barrier penetration, metabolism and/or one or more chemical compounds or metabolites thereof in the brain of an insect, determining and/or localizing one or more chemical compound or a metabolite thereof in an insect, said method comprising

dissecting out the brain comprising the compound eyes of the insect head;

treating the brain comprising the compound eyes, with a medium comprising one or more chemical compounds;

removing the compound eyes and neural lamella;

dividing the brain into two or more pieces;

determining the concentration(s) or distributions of the one or more chemical compound or metabolites thereof in at least one of the brain pieces.

In some embodiments the removed brain comprising the compound eyes (including their nerve connections) are transferred from the solution of one or more chemical compounds to a second solution containing no or other chemical compounds compared to the first solution.

Alternatively, material may be added to the medium to the solution that bind or sequester one or more of the chemical compounds. Example of such material could be proteins, peptides, plastics, and inorganic material such as zeolites, sand and metals. This would for example allow determination or studying of reduction or elimination of the one or more chemical compounds in the brain, metabolism and/or efflux which is important, e.g. , in clearance of central acting drugs or toxins from the brain. In addition, this will allow determination of free concentration of unbound chemical compounds or fractions of free brains concentration in the brain. In addition, properties like the brain to plasma concentration ratio (K _p ), K _puu , and V _{U braill} etc. can be estimated using the methods described herein.

For example in some cases a discrete amount of a drug or a chemical compound is administrated to a subject to raise the concentration in blood, brain, or both. After the administration, the concentration of the drug or chemical compound will in the first phase increase in blood, brain, or both, while in a subsequent phase the concentration will decrease. This is important to simulate to be able to predict beneficial, toxicological, or both, effects of drugs and chemical compounds.

Thus, in some aspects, provided herein is a method for determining half-life, kinetics of metabolism and/or elimination and/or determination of unbound concentrations and/or free fraction in brain of one or more chemical compound and/or metabolites thereof in the brain of an insect comprising the compound eyes (including their nerve connections), said method comprising;

dissecting the brain comprising the compound eyes (including their nerve connections connections) of the insect head;

treating the brain comprising the compound eyes with a first medium comprising one or more chemical compound;

transferring the brain comprising the compound eyes from the first medium to a second medium not comprising the same composition of chemical compounds as the first solution;

removing the compound eyes and optionally the neural lamella;

homogenising or disintegrating the brain or dividing the brain in two or more pieces or slices; determining the concentration(s) of the one or more chemical compound(s) and/ or metabolites thereof in the homogenised or disintegrated brain material from the first or second medium or any subsequent medium; and/or

determining the concentration(s) and/or localisation of the one or more chemical compound(s) and/or metabolites thereof in the one or more of the pieces or slices of brain material from the first or second medium or any subsequent medium; and/or

determining the concentration(s) of the one or more chemical compound(s) and/or metabolites thereof in the first or second medium or any subsequent medium.

The present disclosure further provides a method for determining unbound concentrations and/or free fraction of one or more chemical compound and/or metabolites thereof in the brain and/or in a medium comprising a dissected insect brain comprising the compound eyes (including their nerve connections), said method comprising;

dissecting out a brain comprising the compound eyes (including their nerve connections connections) of the insect head;

treating the obtained brain comprising the compound eyes with a first medium comprising one or more chemical compound;

removing the brain from the first medium ;

arrange the brain in a second medium different from the first medium;

remove the dissected brain comprising the compound eyes (including their nerve connections), after a period of time;

homogenise the brain matter and determine the concentration(s) of the one or more chemical compound(s) and/or metabolites thereof in the homogenised or disintegrated brain matter from the first or second medium or any subsequent medium; and/or

determine concentration(s) of the one or more chemical compound(s) and/ or metabolites thereof in the first or second medium or any subsequent medium.

determining the unbound concentrations and/ or the free fraction in the brain (f _u ,brain) by measuring the total brain concentration (C _to t,bnnn) and the concentration in the medium {C(medium)), Where f _u ,brain ⁼ C( _me diu )fCtca,brain-

The present disclosure further provides a method for determining half-life, kinetics of metabolism and/or prodrug release and elimination and/or determination of unbound concentrations and/or free fraction of metabolites or released chemical compounds thereof in the brain and/or in a medium comprising a dissected insect brain comprising the compound eyes (including their nerve connections), said method comprising;

dissecting the brain comprising the compound eyes (including their nerve connections connections) of the insect head;

treating the brain comprising the compound eyes with a first medium comprising one or more chemical compound;

transferring the brain comprising the compound eyes from the first medium to a second medium not comprising the same composition of chemical compounds as the first solution;

homogenising or disintegrating the brain or dividing the brain in two or more pieces or slices;

determining the concentration(s) of the one or more chemical compound(s) and/ or metabolites thereof in the homogenised or disintegrated brain material from the first or second medium or any subsequent medium; and/or

determining the concentration(s) and/or localisation of the one or more chemical compound(s) and/or metabolites thereof in the one or more of the pieces or slices of brain material from the first or second medium or any subsequent medium; and/or

determining the concentration(s) of the one or more chemical compound(s) and/or metabolites thereof in the first or second medium or any subsequent medium.

The present disclosure further provides a method for determining half-life, kinetics of metabolism and/or prodrug release and elimination and/or determination of unbound concentrations and/or free fraction of metabolites or released chemical compounds thereof in the brain and/or in a medium comprising a dissected insect brain comprising the compound eyes (including their nerve connections), said method comprising;

dissecting the brain comprising the compound eyes (including their nerve connections connections) of the insect head;

treating the brain comprising the compound eyes with a first medium comprising one or more chemical compounds;

transferring the brain comprising the compound eyes from the first medium to a second medium not comprising the same composition of chemical compounds as the first solution;

removing the compound eyes (including their nerve connections) and the lamella; transferring the brain to a new medium comprising one or more chemical compound(s) able to react with reactive metabolites formed in the brain or a medium or chemical compounds that form chemical compounds in the brain that can react with reactive metabolites formed in the brain

homogenising or disintegrating the brain or dividing the brain in two or more pieces or slices;

determining the concentration(s) of the one or more chemical compound(s), metabolite(s) and/ or reactive metabolite products thereof in the homogenised or disintegrated brain material from the first or second medium or any subsequent medium; and/or

determining the concentration(s) and/or localisation of the one or more chemical compound(s) and/or metabolites thereof in the one or more of the pieces or slices of brain material from the first or second medium or any subsequent medium; and/or

determining the concentration(s) of the one or more chemical compound(s), metabolites and/or reactive metabolite products thereof in the first or second medium or any subsequent medium.

In some embodiments the method relates to determination of unbound concentrations and/or free fraction in the brain of one or more chemical compound and/or metabolites. In one embodiment the one or more chemical compound(s) and/or metabolites thereof originate from the first medium.

In some embodiments, the method relates to the determination of the uptake of one or more test compounds in vivo, or the determination of the effect that one or more test compounds have in vivo on the blood-brain barrier, said method comprising

exposing a live insect to one or more test compounds;

dissecting out of an insect head the insect brain comprising the compound eyes; treating the obtained brain comprising the compound eyes with a medium comprising one or more chemical compounds; and

analyzing the brain (excluding the compound eyes) and/or medium.

In said method, the exposing step typically lasts for at least 30 minutes, for at least 1 hour, for at least 4 hours, for at least 8 hours, for at least 12 hours, more suitably at least 24 hours, or at least 1 week, for example from 24 hours to 4 weeks.

The test compound may be any of the chemical compounds described herein, but preferred are the drugs, environmental toxins, food additives and nanoparticles described herein, with environmental toxins being particularly preferred. In such methods, the chemical compound used in the treating step is usually a chemical compound that is not capable of crossing the blood-brain barrier. This therefore allows the integrity of the blood-brain barrier after the in vivo exposure to be verified. Thereafter, the analyzing step can be analyzing to determine the penetration of the test compound into the brain (for example by washing then homogenizing the brain and detecting the test compound or by slicing the brain and analyzing the distribution of the test compound through the slices), or to determine the penetration of the chemical compound into the brain (such that the integrity of the blood-brain barrier after exposure to the test compound may be verified).

In one embodiment the one or more chemical compound or metabolite thereof in the brain is treated with a medium comprising chemical compound(s) for a period of 1 min. - 8 h, to ensure penetration. In some embodiments, the brain is treated with a medium comprising one or more chemical compounds for a period of 1 min to 6 hours, and in some embodiments from 5 mins to 4 hours.

These treatment times will of course vary depending on the study which is undertaken. For instance, the treatment may be with a first medium comprising a first chemical compound for a first period of 1 min. - 8 h, or from 1 min to 6 hours, or from 5 mins to 4 hours, followed by exposure to a second medium optionally containing a second chemical compound for a second period, wherein the second period is typically from 1 min. - 8 h, or from 1 min to 6 hours, or from 5 mins to 4 hours.

The second medium may be a simple buffer or the like, in which case the leaching of the first chemical compound (or metabolite thereof) following treatment with the first medium may be studied.

In some embodiments described herein the neural lamella and compound eyes remain connected to the brain when subjected to homogenization or by disintegration, e.g. by ultra sound.

One embodiment provides a method of conducting blood-brain barrier penetration, metabolism and/or chemical compounds studies of one or several chemical compounds in an insect brain.

In some aspects the brain comprising the compound eyes additionally comprises the nerve connections of the compound eyes.

In some embodiments the method comprises anesthetizing the insect and/or fixing the head of the insect prior to the dissecting the brain comprising the compound eyes. In some embodiments the antennae and/or nerve cords of the brain and compounds eyes are intact in the brain obtained from the dissection. In some embodiments the antenna(s) and/or nerve cords are removed together with the compound eyes.

Without wishing to be bound by theory, it is believed that the antennae can alter the brain chemistry upon detecting certain chemical compounds such as carbamazepine, which may impact the metabolism of that or other chemical compounds in the brain. In view of this, removing the antennae prior to exposure to the chemical compound allows the blood-brain barrier to be investigated while the brain is in normal metabolic conditions. Alternatively, the antennae can be present during exposure to the chemical compound, allowing the response of the brain upon the chemical compound being detected by the antennae to be investigated.

Thus, the method disclosed herein can be used to investigate the sensitivity of insect antennae to a chemical compound, and to investigate the influence insect antennae have on brain chemistry (such as in response to the chemical compound) and particularly on brain metabolism. In such embodiments, the method is carried out to determine the brain uptake of a chemical compound in a dissected out insect brain having antennae, and an otherwise identical insect brain without the antennae. If the brain uptake is significantly different, this can be an indication that the brain chemistry/brain metabolic conditions have been altered by the detection of the chemical compound by the antennae. Such embodiments may monitor for the presence and/or distribution of the chemical compound in the brain, and/or any metabolites thereof

In some embodiments, the method disclosed herein therefore comprises:

providing a first and a second insect head, wherein the first and second insect head are otherwise identical;

dissecting out of a first insect head the first insect brain comprising the compound eyes and antennae;

treating the obtained first brain comprising the compound eyes and antennae with a medium comprising one or more chemical compounds;

removing the compound eyes (including their nerve connections) and antennae;

analyzing the first brain to determine the concentration or localization of the one or more chemical compounds and/or metabolites thereof in the first brain; dissecting out of a second insect head the second insect brain comprising the compound eyes but excluding the antennae; treating the obtained second brain comprising the compound eyes with the medium comprising one or more chemical compounds;

removing the compound eyes (including their nerve connections); analyzing the second brain to determine the concentration or localization of the one or more chemical compounds and/or metabolites thereof in the second brain; and

determining whether the insect antennae are sensitive to the chemical compound by comparing the results from analyzing the first and second brains. In such methods, the first and second insects should be otherwise identical, meaning that they are from the same species, are the same age, size and sex, and have experienced the same environment during their lifespan.

The determining step in such methods may involve comparing the concentrations of the chemical compound in the first and second insect brains, with for instance a greater than 50% difference indicating a sensitivity, or a greater than 20% difference, or a greater than 10% difference.

In some embodiments the neural lamella is removed prior to homogenising or disintegrating the brain or dividing the brain in two or more pieces. In some embodiments the pieces are slices. In one embodiment the pieces have a size of 1-500 μηι, as possible to determine chemical compounds by mass spectrometry imaging (MSI) techniques. One example of a suitable piece is a micro-slice obtained for example by a vibratome or microtome.

In some embodiments the solution of one or more chemical compounds is removed. Additionally in some embodiments the outside of the brain is in some embodiments washed prior to and/or after removal of the brain eyes. The wash is mainly to remove compounds not having entered the brain in order to reduce the risk of contamination and generally it is the outside of the brain that is washed in order not to cause contamination.

In some embodiments albumin and/or blood plasma is comprised in the medium comprising the one or more chemical compound. This provides the option of plasma protein binding and reduces the free amount of the chemical compound available for brain penetration and brain uptake (free vs. protein bound chemical compounds).

In some embodiments the methods described herein, wherein are utilizing an intact brain comprising compound eyes (including their nerve connections) in order to maintain a functional enzymatic and metabolic activity. Analysing may for example mean comparing the ability of chemical compounds to cross the BBB. Determining may for example mean measuring the quantity of a chemical compound, or a metabolite thereof in the brain.

Those skilled in the art recognize that the embodiments described herein can be combined as long as the key steps are maintained.

In one aspect the concentration of the chemical compound is determined by LC/MS, however any other suitable technique for determination of the concentration of chemical compounds or metabolites thereof can be used as well and is within the capacity of those skilled in the art. In this respect the determination of the concentration of the chemical compound is performed by homogenizing and/or disintegrating (e.g., by ultra sound) the dissected brains (one or more brains may be pooled in one vial for analysis), and analyzing the concentration of the tested chemical compound(s) and formed chemical compounds in the homogenate by liquid chromatography with mass spectrometric detection (LC/MS) of the chemical compounds.

The concentrations and regio- and cell-specific distribution of the one or more chemical compounds or metabolites thereof may for example be determined by matrix- assisted laser desorption ionization MS imaging (MALDI-MSI) LC/MS. In this respect the localisation of the chemical compound(s) may, for example, be performed by slicing the brain into pieces such as micro-slices suitable technique such as a vibratome or microtome, and determine where the chemical compound(s), tested or/and formed, is localised in the micro-slices by matrix-assisted laser desorption ionization MS imaging (MALDI-MSI) LC/MS.

Simple visual methods can also be used to analyse the penetration of chemical compounds into the brain, particularly where the compounds show a distinctive colour or fluorescence. Thus, non-BBB permeable dyes can be used as a simple test to determine the integrity of the BBB - should the dye penetrate the brain and be visible in a brain slice, then it is an indication that the BBB integrity has been compromised. Suitable microscopic and spectroscopic means would be familiar to the skilled person, and include microscopy (including fluorescence microscopy). Suitable low cost, smartphone based sensors are described in Sensors 2015, 5, pp11653-1 1664.

Typically, the step of analyzing the brain comprises determination and/or localization of at least one of the one or more chemical compound(s) performed by liquid chromatography/mass spectrometry (LC/MS), liquid chromatography/mass spectrometry- mass spectrometry (LC/MS-MS) or matrix-assisted laser desorption ionization-mass spectrometry imaging (MALDI-MSI). In one embodiment the nerve connections to the compound eyes are intact during the exposure period while the compound eyes and neural lamella are removed before analysis of the compound concentration in the brain.

Provided herein are thus methods for determining the exposure of one or more test compounds in a brain of an insect. The concentration of the test compound (i.e. a particular chemical compound of interest) may be varied or constant during the exposure period.

In some embodiments it is the dorsal part of the insect head which is dissected out of the cuticle to consist substantially of the brain comprising the compound eyes.

In some embodiments one or more of the following may also be dissected out: neural lamella, antennae, and nerve associations. The brain comprising the compound eyes optionally including neural lamella, antennae, and nerve associations may be treated with the medium comprising the one or more test compounds. For example the number of test compounds may be 1-10 compounds. Additionally various exposure periods as well as different exposure concentrations may be used. The brain uptake of the test chemical compound(s) is determined as the concentration (amount) of the chemical compound measured in the isolated brain where neural lamella, compound eyes, antenna, and nerve connections are removed. The concentration may for example be determined quantitatively by an analytical method, such as LC/MS, LC/MS- MS, LC-chemiluminescent nitrogen detector (CLND) or HPLC. In addition, the measured chemical compound concentration in a brain may be quantified relative to the exposure concentration. The method is aimed as a test of chemical compounds as well as identification of the formation of potential metabolites of the test compound(s). In one embodiment the test compound is bioactive compounds which are studied in order to determine their ability to cross the BBB. Additionally the formation of metabolites of the bioactive molecules at defined exposure concentrations can be determined.

Provided herein is a method for testing compounds' ability to pass the BBB also at low exposure concentrations minimizing and avoiding saturation of transporter and metabolizing systems. Moreover, the high sensitivity allows quantification of low brain barrier permeating compounds at low exposure concentrations. This approach has particular relevance when testing if a given drug may pass the BBB. Thus, as described herein the method is particularly useful when the chemical compound is a bioactive molecule, especially a CNS (central nervous system) bioactive molecule or peripheral acting compound that should not penetrate the BBB and potentially causing side- effects. Described herein is a method for testing compounds' ability to pass the BBB also at high exposure concentrations to saturate all or specific transporter systems. This makes it possible to evaluate transporter systems involvement in penetration or extraction from the brain. Moreover, the high sensitivity obtained by the methods described herein allows quantification of low brain barrier permeating compounds at low exposure concentrations. This approach has particular relevance when testing if a given drug may pass the BBB. Thus, the embodiments described herein may be useful when the chemical compound is a bioactive molecule, especially a CNS bioactive molecule or peripheral acting compound that should not penetrate the BBB and potentially causing side-effects. In some aspect the methods described herein may be used to study the effect of particles, such as nanoparticles entering the brain or particles and formulations altering brain barrier uptake and/or elimination and/or metabolism of chemical compounds.

Provide herein are rational strategies for screening compounds for neurological indications and aging, as well as generating a simple system using an intact whole brain for determining a compound's brain penetration and transformations in the brain generating new active chemical compounds or toxins. Additionally provided is a rational screening of compounds in insect models mimicking BBB dysfunction as a consequence of neurological disorders and aging (using insect of different ages).

Drug discovery is a long and costly process, requiring vast amount of chemical and biological resources. The possibilities provided herein to use insects as model systems or a method have been thoroughly exploited in order to improve compound selection processes and reduce the costs during the drug discovery phase. It is contemplated that insect models and methods as provided herein can provide a better foundation than the existing in vitro models for selection of compounds for further development or to be tested in vertebrates.

Generally the disclosure is applicable to any of a drug discovery programs targeting a variety of diseases and disorders of the central nervous system, specifically neurodegenerative disorders, including but not limited to: Parkinson's Disease, Alzheimer's Disease, Huntington's Disease, meningitis, arachnoid cysts, Tourette's, diseases with motor neuron inclusions, tauopathies, corticobasal degeneration; neuropsychiatric disorders, such as: depression, bipolar disease, schizophrenia, anxiety, dementia, addiction, aggression, and ADHD; or symptoms thereof such as catalepsy, epilepsy, encephalitis, migraine, ataxia, and locked-in syndrome. Moreover, the model is applicable for drug discovery programs targeting peripheral targets where CNS driven side effects cannot be tolerated, where CNS exposure cautioning to an awareness of potential risks of side effects or screening of chemical compounds which effects on CNS functions is unknown.

Additionally provided herein is screening for chemical compounds which exert a biological effect that alters an activity or function in the central nervous system, brain or eye, whether normal or subject to a disease or disorder, as to screening for agents which exert a biological effect that is ameliorative of a sign or symptom of a disease or disorder. Described herein is also the possibility to test whether or not peripheral acting drugs, bioactive molecules and toxic agents, such as pesticides, other environmental toxins, unintentionally penetrate the brain barrier.

Following identification of a test substance with desired biological activity using a method in accordance with any aspect or embodiment described herein the test substance may be formulated into a composition comprising at least one additional component, for example a pharmaceutically acceptable vehicle, carrier or excipient.

Successfully using intact (with intact brain-barrier) isolated vertebrate brains for ex vivo studies of metabolism, release, or both, of drugs in the brain has not been reported, in fact we have studied the use of isolated whole zebrafish brain employing otherwise the same methodology described herein without success, hence expected metabolites were not formed. Whereas using in vivo zebrafish studies confirmed that expected metabolite are formed in the brain. Furthermore, by analysing the exposure/incubation medium immersing the brain(s) it is possible to qualitatively and quantitative study how the metabolites and chemical compounds are formed or released in the brain, respectively, is excreted from the brain, for example into a second medium differing from the first medium. Similarly, by first incubate the brain by a first medium of chemical compound(s) for a period of time (e.g., a predetermined time), and subsequently transfer the brain(s) to a second medium differing from the first medium (i.e. not containing the same chemical compound(s) as the first medium) it is possible to study how the chemical compounds of the first medium are excreted from the brain, for example into the second medium. And the disappearance of the chemical compound(s) and of the metabolites formed from the chemical compound(s) may thus be studied.

In addition, by analysing the medium it is possible to evaluate concentration(s) of unbound compound(s) in the brain and fractions of unbound compound(s) in the brain by relating with concentration(s) in the whole brain. As follows, using these methods for example drug-drug interactions can be studied. Additionally, it is possible to study how different chemical compounds directly or indirectly impact each other's brain penetration, excretion and metabolism. Additionally, by using a brain comprising the compound eye less or no contribution to the final brain concentration by diffusion through potential damages in the brain barrier is obtained. Consequently provided herein is a more reliable method useful for decision-making compared to the existing insect ex vivo and in vitro models. The method may for example speed up the drug screening process and reduce the late phase attrition rate.

Described herein is a method allowing the determination of one or more chemical compound(s) preferentially penetrating the brain barrier, and hence the brain uptake, or a chemical compound formed or released in the brain, such as a metabolite of the chemical compound. The method comprises a refined dissection of the insect brain where no or at least substantially less contribution to the brain uptake should be attributed to diffusion of the one or more chemical compound(s) in and out of the brain through potential leakages in the brain barrier introduced during the dissection procedure, an example thereof is the removal of the compound eyes before treating the brain with the exposure medium.

In previous models the eyes are removed by cutting the nerve connections to eyes and antennae prior to exposure of the chemical compound. It has been found that these models cause an increased brain concentration of peripheral acting drugs (e.g., atenolol, quinidine) compared to brain concentrations obtained according to the methods described herein i.e., where compound eyes are present during the exposure phase. In contrast, central acting drugs characterized by being readily brain barrier permeating (e.g., carbamazepine) do not display any significant difference in measured brain concentrations comparing the conventional model and the methods described herein. In some embodiments the compound eyes and antennae are present during the exposure phase.

As used herein the terms "whole brain" and "intact brain" are to be understood as a brain having an intact brain barrier.

It has been discovered that maintaining the compound eyes compared with removing them reduces the brain concentration of drugs characterized by low permeation of the brain barrier (e.g., atenolol or quinidine) while high permeable drugs showed no difference in brain concentrations. It is important to be able to predict brain uptake of low permeation compounds correctly so that the permeation is real over the brain barrier and not happens because of a consequence of the methodology. On the contrary, no difference between the brain concentrations of low permeation drugs (e.g., atenolol or quinidine) was obtained when both compound eyes and antennae remain intact compared to when only compound eyes are intact. However, unexpectedly, high permeable compounds like central acting drugs (e.g., carbamazepine) show decreased brain concentration when both the antennae and the compound eyes and their connections are intact compared to studies where only the compound eyes are intact.

In some aspects maintaining the antennae and the compound eyes result in concentrations not translating as well as maintaining the compound eyes without the antennae. It has thus surprisingly been found that maintaining the compound eyes enables a method for more accurately study of chemical compounds permeating the brain barrier. As maintaining the compound eyes, their nerve connections as well as the antennae and their nerve connections or removing compound eyes, their nerve connections as well as the antennae and their nerve connections does not provide the same benefits accordingly one aspect relates to maintaining the compound eyes and their nerve connections only.

It has been discovered that maintaining the compound eyes and their nerve connections to the brain enable the study of metabolism of chemical compounds in the brain.

Described herein is a method allowing the determination of chemical compounds permeating the brain barrier only (i.e., brain uptake) or chemical compounds formed or released in the brain while less and substantially no contribution should be attributed to diffusion of chemical compounds in and out of potential disruptions of the integrity in the brain barrier introduced during the dissection procedure. The herein presented insect ex vivo model enables a method for determining brain up-take, especially at long-time exposure without disruptions in the brain barrier. Moreover, the method in some aspects provides accurate determination of the concentration of the chemical compound being taken up in the insect brain.

Provided herein is a method for determining the brain metabolism, identifying, and determining concentrations, or both, in the brain of one or more metabolites formed by a chemical compound having permeated the brain barrier. Furthermore, brain concentrations of chemical compounds, metabolites formed thereof, or released from a drug delivery system, such as nanoparticles, liposomes, permeating the brain barrier may be determined by the herein described methods. The insect ex vivo methods allows studying the metabolism and/or release of chemical compound or metabolites thereof in an intact brain with intact brain barrier integrity and functional biological systems, e.g., enzymes and drug transporters, e.g., ABC transporter system. Additionally provided herein is a method for obtaining metabolites of chemical compounds by isolating one or more metabolite(s) formed in the brain. Accordingly the methods described herein provide a further aspect by enabling the possibility to obtain metabolites of chemical compounds, according to such aspect the metabolites may for example be used for further studies such as activity, e.g. pharmacological activity, and toxicity studies.

Further provided herein is a method for obtaining concentration of unbound chemical compounds in the brain or fractions of unbound chemical compounds in the brain relating to total concentrations in the brain by measuring the concentration of the compounds in the media. This can be done either by analysing the content of the initial incubation medium at different time points or moving the brains to a different media compared to the incubation medium, and analyse the content of the medium at different time points.

Accordingly the methods described herein provide a further aspect by enabling the possibility to obtain free concentrations and free fraction in the brain (F _ub ) of chemical compounds, according to such aspect the unbound concentrations and free fractions in brain may for example be used for further studies such as activity, e.g. pharmacological activity, and toxicity studies. Methods described herein allow determination of parameters commonly used in drug discovery. This includes parameters, which commonly require studies on vertebrates and characterized by being experimentally cost and labor demanding, e.g. properties like free fraction unbound in brain (F _ub ), volume of distribution in the brain (V _{U braill} ), and unbound brain to unbound plasma concentration ratio (K _P)UU ).

Provided herein is a methodology for screening (e.g. , analysing and determining) chemical agents (i.e. , one or more chemical compound(s)) ability to penetrate the brain barrier also at low exposure concentrations. Moreover, the methodology includes the potential to identify chemical compounds formed or released in the brain from the tested chemical agent (chemical compound) and is generally useful for screening for agents developed in drug discovery programs targeting a variety of diseases and disorders, specifically neurodegenerative disorders, such as: Parkinson's Disease, Alzheimer's Disease, Huntington's Disease, diseases with motor neuron inclusions, tauopathies, corticobasal degeneration; neuropsychiatric disorders, such as: depression, bipolar disease, schizophrenia, anxiety, dementia, addiction and aggression. Moreover, applicability for drug discovery programs targeting peripheral targets where no CNS driven side effect can be tolerated is provided herein. Provided herein is the screening of agents developed in drug discovery programs targeting eating disorders and sleep disorders etc.

The terms "chemical compound" or "chemical compounds" or "chemical agents" are used interchangeably herein and refer to any compound which has a potential to be useful in drug discovery, for human medicine and/or veterinarian medicine or to be tested for toxicological effects including bioactive compounds, examples thereof are small molecules Mw (molecular weight) 16-1000, large molecules Mw>1000, macrocyclic compounds, peptides, proteins, nanoparticles, coated nanoparticles , drug delivery systems (both general and brain targeting) for example chemical compounds linked to carriers or compositions known to affect the brain uptake such as lipoproteins, acylations, polyethylenglycol (PEG), liposomes, albumin, or other molecules or compositions. As used herein the chemical compound should be able to be contained in a medium, i.e. in solution, as a suspension, or emulsion etc.

Typically, the chemical compound is a drug, an environmental toxin, a food additive, a protein, a nanopartide, a bacterium, a virus, a parasite, an antibody, or RNA.

By "environmental toxin" is meant a chemical that is used in agriculture or industry that is generally understood to be detrimental to flora and fauna, and/or carcinogenic. Such toxins include those intentially released into the environment such as insecticides, pesticides, fertilizers and the like, those that are released as airborne pollutants or in effluent from domestic or industrial settings such as surfactants; polychlorinated biphenyls (PCBs); plasticizers such as bisphenol-A; heavy metals like arsenic, lead, mercury, aluminium and cadmium; mould and fungal toxins; volatile organic compounds; dioxins; and combustion products such as soot (including diesel particulate matter from automobiles).

Bacteria, viruses and pasasites are also of interest. Thus, as used herein, the term "chemical compound" is understood to include bacteria, viruses and parasites unless it is otherwise clear from the context. The method provides a simple methodology to investigate how more complex entities such as bacteria, viruses and parasites interact, affect and traverse the blood-brain barrier.

Likewise, nanoparticles such as metal nanoparticles, plastic microbeads and nanoparticles from combustion such as soot particles are also of interest, and as used herein the term "chemical compound" should be considered to encompase nanoparticles. Of particular interest are small RNA molecules which are known as small interfering RNA (siRNA), or sometimes referred to as short interfering RNA or silencing RNA. These are typically 20-25 base pairs in length. siRNAs operate within the RNA interference pathway and interfer with the expression of specific genes with complementary nucleotide sequences by degrading mRNA after transcription, resulting in no translation.

In some embodiments, the chemical compound is a drug, an environmental toxin, a food additive, a protein, a nanoparticle, an antibody, or RNA.

In some embodiments, the chemical compound is a drug, an environmental toxin, or a food additive.

In some embodiments, the chemical compound is a drug.

In some embodiments, the chemical compound is a central nervous system active chemical compound or delivery system of a central nervous system active chemical compound.

In some embodiments, the chemical compound is a material that binds or sequesters compounds that may cross the blood-brain barrier from the brain to the medium, such as proteins, peptides, plastics, and inorganic material such as zeolites, sand and metals.

Used herein is the term "medium". In particular the term is used in connection with medium comprising one or more chemical compound(s) wherein the medium is allowing the one or more chemical compound(s) to contact the brain. Suitable media are solutions, i.e., a solution comprising one or more chemical compound(s), a solvent comprising one or more chemical compound(s), a suspension, emulsion a gas comprising one or more chemical compound(s). One example presented herein is a liquid medium comprising one or more chemical compound(s). Another example is blood from mammals, humans, non/human primates and rodents.

The term "bioactive compound" is a compound that potentially has an effect on a living organism, tissue or cell. Bioactive compounds can have positive and/or negative influence on health or environment. Bioactive compounds can be found in plants, animals or are compounds that do not occur naturally in nature and are synthetically produced.

The term "drug" (e.g., chemical compound) refers to a compound, for example a small molecule, when absorbed into the body of a living organism, alters normal bodily function. More specifically, a drug is a compound, e.g. a chemical or biological compound that may be used in the treatment, cure, prevention, or diagnosis of disease or used to otherwise to enhance physical or mental well-being. One sub-group of drugs that may be useful to study in the models and methods described herein are psychoactive drugs, which are chemical compounds that cross the BBB and act primarily upon the central nervous system where they alter brain functions, resulting in changes in perception, mood, consciousness, cognition and behaviour. The term "drug" may be used for regulatory approved drugs as well as drugs not being regulatory approved.

The medium (i.e. the first, second and/or subsequent medium) may additionally contain an efflux or influx inhibitor.

By "efflux inhibitor" is meant a compound that inhibits the proteins that enable efflux of compounds across the blood-brain barrier out of the brain. Efflux inhibitors therefre increase the brain uptake of chemical compounds.

By "influx inhibitor" is meant a compound that inhibits the proteins that enable influx of compounds across the blood-brain barrier into the brain. Influx inhibitors therefre decrease the brain uptake of chemical compounds.

Typical efflux inhibitors include BCRP, CYP3A and/or Pgp inhibitors such as itraconazole, lopinavir, ritonavir, clarithromycin, ketoconazole, indinavir, conivaptan, verapamil, reytrhomycin, diltiazem, donedarone, quinidine, ranolazine, amiodarone, felodipine, azithromycin, voriconazole, nefazodone, cyclosporine, cimetidine, Ko143, dioxin, and cimetidine.

Typical non-Pgp inhibitors are selected from voriconazole, nefazodone, and cimetidine.

Suitable efflux inhibitors are selected from itraconazole, lopinavir, ritonavir, clarithromycin, ketoconazole, indinavir, conivaptan, verapamil, reytrhomycin, diltiazem, donedarone, quinidine, ranolazine, amiodarone, felodipine, and azithromycin.

The medium (i.e. the first, second and/or subsequent medium) may additionally contain an inhibitor of metabolic enzyme activity.

By "inhibitor of metabolic enzyme activity" is meant a compound that inhibits the activity of enzymes active in the metabolism.

Exemplary enzymes that are active in the metabolism include cytochrome P450 enzymes (CYP) such as CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1 , and CYP3A4/5. Examples of inhibitors of each of these enzymes are set out in the Table below: CYP Inhibitor Ki (μΜ)

1A2 furafylline 0.6-0.73

a -naphihoflavone 0.01

2A6 tranylcypromine 0.02-0.2

methoxsaien 0.01-0.2 pilocarpine 4

tryptamine 1.7

2B6 3-isopropeny!-3-methy! diamantane (4) 2.2

2-isopropeny!-2-methyi adamaniane (4) 5.3

sertraline 3.2

phencyciidine 10

triethy!enethiophosphoramide (thiotepa) 4.8

clopidogrel 0.5

ticlopidine 0.2

2C8 montelukast

quercetin 1.1

trimethoprim 32

gemfibrozil 69-75 rosiglitazone 5.6

piogiitazone 1.7

2C9 sulfaphenazole 0.3

fluconazole 7

fluvoxamine 6.4-19 fluoxetine 18-41

2C19 ticlopidine 1.2

nootkatone 0.5

2D6 quinidine 0.027-0.4

12E 1 d i β thy id i t h i o ca r b a m a t e ^" 978-34

ciomethiazoie i

dialiyidisulfide 150

3A4/5 ketoconazole 0.0037- 0.18

itraconazole 0.27, 2.3 azamulin 17

tro!eandomycin 10

verapamil 24

Such inhibitors can be used in the medium as part of a study of the brain metabolism, for instance. As part of these studies, the substrates for the relevant metabolic enzymes may also be present. Exemplary substrates for each of the CYP metabolism enzymes are indicated in the Table below:

nifedipine oxidation 5.1- 47

In addition to inhibiting relevant metabolic enzymes, it may be desirable to augment their activity using a metabolic enzyme inducer which may be included in the medium (i.e. the first, second or subsequent medium). This could be done as a positive control, or to provide a larger differential in enzyme activity to ensure that measurable results can be obtained within the desired experimental timeframe.

Examples of enzyme activity inducers for a number of CYP enzymes are shown in the Table below: CYP in Vitro Inducer Recommende d Conce itratiors | Fold !ndt ctiors as Positive Controls {μΜ} of the Po siisve Co ntrois 1 in Enzym e Activity

1A2 omeprazole 25-100 1 14-24

lansoprazole 10 I io

2B6 phenobarbital 500-1000 I 5-Ϊ0

^~ 2C8 ^~" rifampin _

2C9 rifampin 10 I 3,7

2C19 rifampin 10 ! 20

3A4 rifampin 10-50 ! 4-31

Examplary inducers and inhibitors are listed on the FDA website, available at: http://www.fda.gov/Drugs/DevelopmentApprovalProcess/Developm entResource s/DruglnteractionsLabeling/ucm080499.htm

Examples of insects that may be used are: (Taxonomy according to: Djurens Varld, Ed B. Hanstrom; Forlagshuset Norden AB, Malmo, 1964)

Order Suborder/family Comment

Dictyoptera Blattoidea Cockroach

Mantoidea

Orthoptera Grylloidea Crickets

Acridoidea Grasshoppers

Cheleutoptera Stick insects

Lepidoptera Moths

Hymenoptera Formicoidea Ants

Vespoidea Wasps

Bee like

Apoidea

Hymenopterans

Bombinae Bumble-bees

Apine Proper bees

Odonata Dragonflies

Diptera Nematocera Mosquitos

Flies E.g

Brachycera

Drosophila

In some embodiments the insect species is selected from the group consisting of Blattoidea, Acridoidea, Cheleutoptera, Brachycera and Lepidoptera. One example is Acridoidea (Locusta migratoria and Schistocera gregaria). Additional examples of insects are: Order Suborder/family Comment

Ephemerida Mayflies

Plecoptera

Dermoptera Forficuloidea Earwigs

Homoptera Cicadinea Cicadas

Aphidine Plant-louse

Heteroptera Hemipteran

Coleoptera Beetles

Trichoptera Caddis fly

In some aspect there are benefits to use large insects, such as the migratoty locust, Locusta migratoria and the desert locust, Schistocera gregaria or cockroach where it is feasible to dissect the brain and to make quantitative brain concentration measurements. Moreover, larger brains are practical when slicing.

In some embodiments, the insect is a locust or cockroach, in some emodiments the insect is a locust, such as a migratoty locust, Locusta migratoria or the desert locust, Schistocera gregaria.

The locust has been used to develop screening models to determine brain uptake of different therapeutic drugs and compare this model with existing literature data from conventional in vivo or in situ vertebrate studies.

EXAMPLES

Example 1

The following experiments use insects selected from the order Acridoidea and specifically Locusta migratoria and Schistocera gregaria. The insects were obtained from local or commercial animal suppliers or bred in-house. The grasshoppers were reared under crowded conditions at 28° C and a 12: 12 dark:light photocycle and fed fresh grass or Chinese cabbage and wheat bran ad libitum. Animals used are adult males or females between two to four weeks after adult emergence. A cut is made through the frontal part of the locust head and the brain is dissected out such that its connections to the compound eyes and optionally antennae are intact. The dissected brain (comprising compound eyes) is placed in a well of a microtiter plate containing the test solution, such as the substance of interest and 4.2% bovine serum albumin. After various times of exposure the preparation is washed in saline and the brain is dissected under microscope with fine forceps. The neural lamella surrounding the brain, the compound eyes and antennae (if present) are removed in saline and the brain is then ultrasonic dissected (UD) in saline, centrifuged and the supernatant frozen until analyses. Drug concentration is analysed by HPLC, LC/MSMS or other methods. Alternatively, after removal of the neural lamella, the compound eyes and antennae (if present), the brain is then freezed and micro-sliced using a vibratome or microtome. The chemical compounds concentrations and localisations are analysed in the micro- slices by matrix-assisted laser desorption ionization MS imaging (MALDI-MSI) or other methods.

Further details are provided herein below.

EXAMPLE 1-A (atenolol with eyes and 15 minutes)

A cut is made through the frontal part of the locust head. The brain and its nerve connections to the compound eyes, antenna, and ocelli are removed from its cuticle shell. The brain preparation is dissected in locust buffer such antenna and ocelli are removed while neural lamella and compound eyes remain on the brain matter. The brain matter is put into a microtiter well containing 250 μΙ of a 3 μΜ atenolol solution heated to 30°C in a block thermostat. After 15 minutes exposure time the brain is removed from the well and the neural lamella and compound eyes are removed in ice cold locust buffer. The brain is washed in ice cold locust buffer. Brains from two animals are placed in a test tube containing 150 μΙ of a protein precipitation solution and sonicated. The test tubes are centrifuged at 10 000 xg for 5 minutes at 4°C. 100 μΙ of the supernatants are transferred to analysis vials and analysed by LC/MSMS.. The average brain uptake was measured to be below 0.21 pmol/brain and the standard deviation was 0.02 pmol/brain.

The average brain uptake was 0.38 pmol/brain and the standard deviation was

0.05 pmol/brain when prior ex vivo insect model technology is used, i.e., when compound eyes are removed before test solution exposure.

EXAMPLE 1-B (atenolol with eyes and 45 minutes)

A cut is made through the frontal part of the locust head. The brain and its nerve connections to the compound eyes, antenna, and ocelli are removed from its cuticle shell. The brain preparation is dissected in locust buffer such that antenna and ocelli are removed while neural lamella and compound eyes remain on the brain matter. The brain matter is put into a microtiter well containing 250 μΙ of a 3 μΜ atenolol solution heated to 30°C in a block thermostat. After 45 minutes exposure time the brain is removed from the well and the neural lamella and compound eyes are removed in ice cold locust buffer. The brain is washed in ice cold locust buffer. . Brains from two animals are placed in a test tube containing 150 μΙ of a protein precipitation solution and sonicated. The test tubes are centrifuged at 10 000 xg for 5 minutes at 4°C. 100 μΙ of the supernatants are transferred to analysis vials and analysed by LC/MSMS. The average brain uptake was 0.21 pmol/brain and the standard deviation was 0.01 pmol/brain.

The average brain uptake was 0.38 pmol/brain and the standard deviation was 0.12 pmol/brain when prior ex vivo insect model technology is used, i.e., compound eyes removed before test solution exposure.

Comparison with brain uptake obtained using prior ex vivo insect technology shows that there is lower brain uptake of the low brain barrier permeable substance atenolol when brains with the connections to the compound eyes remain on the brain matter are exposed to atenolol. This shows that there are no or less leakage in the new dissection method used in example 1-A and 1-B.

EXAMPLE 1-C (quinidine with eyes and 15 minutes)

A cut is made through the frontal part of the locust head. The brain and its nerve connections to the compound eyes, antenna, and ocelli are removed from its cuticle shell. The brain preparation is dissected in locust buffer such that antenna and ocelli are removed while neural lamella and compound eyes remain on the brain matter. The brain matter is put into a microtiter well containing 250 μΙ of a 3 μΜ quinidine solution heated to 30°C in a block thermostat. After 15 minutes exposure time the brain is removed from the well and the neural lamella and compound eyes are removed in ice cold locust buffer. The brain is washed in ice cold locust buffer. . Brains from two animals are placed in a test tube containing 150 μΙ of a protein precipitation solution and sonicated. The test tubes are centrifuged at 10 000 xg for 5 minutes at 4°C. 100 μΙ of the supernatants are transferred to analysis vials and analysed by LC/MSMS. The average brain uptake was 1.54 pmol/brain and the standard deviation was 0.13 pmol/brain.

The average brain uptake was 2.70 pmol/brain and the standard deviation was 0.35 pmol/brain when prior ex vivo insect model technology is used, i.e., compound eyes removed before test solution exposure. EXAMPLE 1-D (quinidine with eyes and 45 minutes)

A cut is made through the frontal part of the locust head. The brain and its nerve connections to the compound eyes, antenna, and ocelli are removed from its cuticle shell. The brain preparation is dissected in locust buffer such that antenna and ocelli are removed while neural lamella and compound eyes remain on the brain matter. The brain matter is put into a microtiter well containing 250 μΙ of a 3 μΜ quinidine solution heated to 30°C in a block thermostat. After 45 minutes exposure time the brain is removed from the well and the neural lamella and compound eyes are removed in ice cold locust buffer. The brain is washed in ice cold locust buffer. . Brains from two animals are placed in a test tube containing 150 μΙ of a protein precipitation solution and sonicated. The test tubes are centrifuged at 10 000 xg for 5 minutes at 4°C. 100 μΙ of the supernatants are transferred to analysis vials and analysed by LC/MSMS. The average brain uptake was 4.14 pmol/brain and the standard deviation was 1.05 pmol/brain.

The average brain uptake was 4.64 pmol/brain and the standard deviation was

0.75 pmol/brain when prior ex vivo insect model technology is used, i.e., compound eyes removed before test solution exposure.

Comparison with brain uptake obtained using prior ex vivo insect technology shows that there is lower brain uptake after 15 minutes of the Pgp substrate quinidine when brains with the connections to the compound eyes remain on the brain matter are exposed to the test compound.

EXAMPLE 1-E (3 μΜ quinidine and 25 μΜ verapamil with eyes and 15 minutes) A cut is made through the frontal part of the locust head. The brain and its nerve connections to the compound eyes, antenna, and ocelli are removed from its cuticle shell. The brain preparation is dissected in locust buffer such that antenna and ocelli are removed while neural lamella and compound eyes remain on the brain matter. The brain matter is put into a microtiter well containing 250 μΙ of a 3 μΜ quinidine and 25 μΜ verapamil solution heated to 30°C in a block thermostat. After 15 minutes exposure time the brain is removed from the well and the neural lamella and compound eyes are removed in ice cold locust buffer. The brain is washed in ice cold locust buffer. . Brains from two animals are placed in a test tube containing 150 μΙ of a protein precipitation solution and sonicated. The test tubes are centrifuged at 10 000 xg for 5 minutes at 4°C. 100 μΙ of the supernatants are transferred to analysis vials and analysed by LC/MSMS. The average brain uptake was 2.95 pmol/brain and the standard deviation was 0.57 pmol/brain.

The average brain uptake was 4.64 pmol/brain and the standard deviation was 0.83 pmol/brain when prior ex vivo insect model technology is used, i.e., compound eyes removed before test solution exposure.

EXAMPLE 1-F (3 μΜ quinidine and 25 μΜ verapamil with eyes and 45 minutes) A cut is made through the frontal part of the locust head. The brain and its nerve connections to the compound eyes, antenna, and ocelli are removed from its cuticle shell. The brain preparation is dissected in locust buffer such that antenna and ocelli are removed while neural lamella remains on the brain matter. The brain matter is put into a microtiter well containing 250 μΙ of a 3 μΜ quinidine and 25 μΜ verapamil solution heated to 30°C in a block thermostat. After 45 minutes exposure time the brain is removed from the well and the neural lamella and compound eyes are removed in ice cold locust buffer. The brain is washed in ice cold locust buffer. . Brains from two animals are placed in a test tube containing 150 μΙ of a protein precipitation solution and sonicated. The test tubes are centrifuged at 10 000 xg for 5 minutes at 4°C. 100 μΙ of the supernatants are transferred to analysis vials and analysed by LC/MSMS. The average brain uptake was 8.14 pmol/brain and the standard deviation was 0.65 pmol/brain.

The average brain uptake was 11.83 pmol/brain and the standard deviation was 2.16 pmol/brain when prior ex vivo insect model technology is used, i.e., compound eyes removed before test solution exposure.

In example 1-E and 1-F quinidine is co-administered with the Pgp inhibitor verapamil, which blocks the efflux of quinidine out of the brain. Pgp blockage increase the brain uptake and this is seen when data from example 1-C and 1-D is compared with example 1-E and 1-F. Comparison of the data obtained in example E with brain uptake obtained using prior ex vivo insect technology shows that there is lower brain uptake after 15 minutes of the Pgp substrate quinidine when the connections to the compound eyes remain on the brain matter exposed to the test compound and verapamil. Contrasting the brain uptake measured using quinidine without Pgp inhibitor (example 1-D) there is lower uptake of quinidine co-administered with verapamil after 45 minutes when the new dissection method is used, i.e., example 1-D and 1-F). This can be explained by the fact that in addition to Pgp blockage there is brain barrier leakage when using prior technology while only Pgp blockage is present in example 1- F. Moreover, the data shows significant saturation of the Pgp transporter after 45 minutes.

EXAMPLE 1-G (carbamazepine with eyes and 15 minutes)

A cut is made through the frontal part of the locust head. The brain and its nerve connections to the compound eyes, antenna, and ocelli are removed from its cuticle shell. The brain preparation is dissected in locust buffer such antenna and ocelli are removed while neural lamella and compound eyes remain on the brain matter. The brain matter is put into a microtiter well containing 250 μΙ of a 3 μΜ carbamazepine solution heated to 30°C in a block thermostat. After 15 minutes exposure time the brain is removed from the well and the neural lamella and compound eyes are removed in ice cold locust buffer. The brain is washed in ice cold locust buffer. . Brains from two animals are placed in a test tube containing 150 μΙ of a protein precipitation solution and sonicated. The test tubes are centrifuged at 10 000 xg for 5 minutes at 4°C. 100 μΙ of the supernatants are transferred to analysis vials and analysed by LC/MSMS. The average brain uptake was 8.70 pmol/brain and the standard deviation was 0.58 pmol/brain.

The average brain uptake was 9.27 pmol/brain and the standard deviation was 0.38 pmol/brain when prior ex vivo insect model technology is used, i.e., compound eyes removed before test solution exposure.

EXAMPLE 1-H (carbamazepine with eyes and 45 minutes)

A cut is made through the frontal part of the locust head. The brain and its nerve connections to the compound eyes, antenna, and ocelli are removed from its cuticle shell. The brain preparation is dissected in locust buffer such that antenna and ocelli are removed while neural lamella and compound eyes remain on the brain matter. The brain matter is put into a microtiter well containing 250 μΙ of a 3 μΜ carbamazepine solution heated to 30°C in a block thermostat. After 45 minutes exposure time the brain is removed from the well and the neural lamella and compound eyes are removed in ice cold locust buffer. The brain is washed in ice cold locust buffer. . Brains from two animals are placed in a test tube containing 150 μΙ of a protein precipitation solution and sonicated. The test tubes are centrifuged at 10 000 xg for 5 minutes at 4°C. 100 μΙ of the supernatants are transferred to analysis vials and analysed by LC/MSMS. The average brain uptake was 15.40 pmol/brain and the standard deviation was 0.15 pmol/brain. The average brain uptake was 15.38 pmol/brain and the standard deviation was 0.57 pmol/brain when prior ex vivo insect model technology is used, i.e., compound eyes removed before test solution exposure.

Comparison with brain uptake obtained using prior ex vivo insect technology shows that there is no difference between the brain uptake obtained for the high brain barrier permeable compound, carbamazepine, using brain matters with or without compound eyes. This shows that compounds which easily permeate the brain barrier mainly permeate the brain barrier and only to a lower extent enter the brain via potential leakages in the brain barrier.

The brain matter used in examples 1-A - 1-H has larger brain exposure surface than the brain matter used in prior insect ex vivo technology and increased permeability surface give rise to increased brain uptake. However, this is not the case comparing the brain uptake obtained with present and prior ex vivo insect technology. For the low permeability compound the brain uptake is significantly lower when the present technology is used while the brain uptake of the brain permeable compound carbamazepine is the same using prior and present technologies. This shows that prior technologies may introduce a leakage in the brain barrier allowing atenolol to enter the brain. EXAMPLE 1-1 (risperidone with eyes and 15 minutes)

A cut is made through the frontal part of the locust head. The brain and its nerve connections to the compound eyes, antenna, and ocelli are removed from its cuticle shell. The brain preparation is dissected in locust buffer such that antenna and ocelli are removed while neural lamella and compound eyes remain on the brain matter. The brain matter is put into a microtiter well containing 250 μΙ of a 3 μΜ risperidone solution heated to 30°C in a block thermostat. After 15 minutes exposure time the brain is removed from the well and the neural lamella and compound eyes are removed in ice cold locust buffer. The brain is washed in ice cold locust buffer. One brain is placed in a test tube containing 150 μΙ of a protein precipitation solution and sonicated. The test tubes are centrifuged at 10 000 xg for 5 minutes at 4°C. 100 μΙ of the supernatants are transferred to analysis vials and analysed by LC/MSMS. The average brain uptake using males was 3.05 pmol/brain and the standard deviation was 0.88 pmol/brain.

The average brain uptake using females was 3.45 pmol/brain and the standard deviation was 0.18 pmol/brain. EXAMPLE 1-J (risperidone with eyes and 45 minutes)

A cut is made through the frontal part of the locust head. The brain and its nerve connections to the compound eyes, antenna, and ocelli are removed from its cuticle shell. The brain preparation is dissected in locust buffer such that antenna and ocelli are removed while neural lamella and compound eyes remain on the brain matter. The brain matter is put into a microtiter well containing 250 μΙ of a 3 μΜ risperidone solution heated to 30°C in a block thermostat. After 45 minutes exposure time the brain is removed from the well and the neural lamella and compound eyes are removed in ice cold locust buffer. The brain is washed in ice cold locust buffer. One brain is placed in a test tube containing 150 μΙ of a protein precipitation solution and sonicated. The test tubes are centrifuged at 10 000 xg for 5 minutes at 4°C. 100 μΙ of the supernatants are transferred to analysis vials and analysed by LC/MSMS.. The average brain uptake using males was 6.82 pmol/brain and the standard deviation was 0.30 pmol/brain.

The average brain uptake using females was 6.93 pmol/brain and the standard deviation was 1.19 pmol/brain.

EXAMPLE 1-K (trazodone with eyes and 15 minutes)

A cut is made through the frontal part of the locust head. The brain and its nerve connections to the compound eyes, antenna, and ocelli are removed from its cuticle shell. The brain preparation is dissected in locust buffer such that antenna and ocelli are removed while neural lamella and compound eyes remain on the brain matter. The brain matter is put into a microtiter well containing 250 μΙ of a 3 μΜ trazodone solution heated to 30°C in a block thermostat. After 15 minutes exposure time the brain is removed from the well and the neural lamella and compound eyes are removed in ice cold locust buffer. The brain is washed in ice cold locust buffer. One brain is placed in a test tube containing 150 μΙ of a protein precipitation solution and sonicated. The test tubes are centrifuged at 10 000 xg for 5 minutes at 4°C. 100 μΙ of the supernatants are transferred to analysis vials and analysed by LC/MSMS. The average brain uptake using males was 2.45 pmol/brain and the standard deviation was 0.26 pmol/brain.

The average brain uptake using females was 2.06 pmol/brain and the standard deviation was 0.56 pmol/brain.

The data obtained in example J - K show despite using a model without any leakage in the brain barrier the increased brain exposure surface introduced as described herein allows brain uptake measurements on single brains. EXAMPLE 1-L (carbamazepine with eyes no antennae and 45 minutes)

A cut is made through the frontal part of the locust head. The brain and its nerve connections to the compound eyes, antenna, and ocelli are removed from its cuticle shell. The brain preparation is dissected in locust buffer such that antenna and ocelli are removed while neural lamella and compound eyes remain on the brain matter. The brain matter is put into a microtiter well containing 250 μΙ of a 3 μΜ carbamazepine solution heated to 30°C in a block thermostat. After 45 minutes exposure time the brain is removed from the well and the neural lamella and compound eyes are removed in ice cold locust buffer. The brain is washed in ice cold locust buffer. Brains from two animals are placed in a test tube containing 150 μΙ of a protein precipitation solution and sonicated. The test tubes are centrifuged at 10 000 xg for 5 minutes at 4°C. 100 μΙ of the supernatants are transferred to analysis vials and analysed by LC/MSMS. The average brain uptake was 15.60 pmol/brain and the standard deviation was 1.13 pmol/brain.

EXAMPLE 1-M (carbamazepine with eyes and antennae and 45 minutes) A cut is made through the frontal part of the locust head. The brain and its nerve connections to the compound eyes, antenna, and ocelli are removed from its cuticle shell. The brain preparation is washed in locust buffer. The brain matter is put into a microtiter well containing 250 μΙ of a 3 μΜ carbamazepine solution heated to 30°C in a block thermostat. After 45 minutes exposure time the brain is removed from the well and the neural lamella, antennae, and compound eyes are removed in ice cold locust buffer. The brain is washed in ice cold locust buffer. Brains from two animals are placed in a test tube containing 150 μΙ of a protein precipitation solution and sonicated. The test tubes are centrifuged at 10 000 xg for 5 minutes at 4°C. 100 μΙ of the supernatants are transferred to analysis vials and analysed by LC/MSMS. The average brain uptake was 8.51 pmol/brain and the standard deviation was 0.97 pmol/brain.

EXAMPLE 1-N (atenolol with eyes no antennae and 45 minutes)

A cut is made through the frontal part of the locust head. The brain and its nerve connections to the compound eyes, antenna, and ocelli are removed from its cuticle shell. The brain preparation is dissected in locust buffer such that antenna and ocelli are removed while neural lamella and compound eyes remain on the brain matter. The brain matter is put into a microtiter well containing 250 μΙ of a 3 μΜ atenolol solution heated to 30°C in a block thermostat. After 45 minutes exposure time the brain is removed from the well and the neural lamella and compound eyes are removed in ice cold locust buffer. The brain is washed in ice cold locust buffer. Brains from two animals are placed in a test tube containing 150 μΙ of a protein precipitation solution and sonicated. The test tubes are centrifuged at 10 000 xg for 5 minutes at 4°C. 100 μΙ of the supernatants are transferred to analysis vials and analysed by LC/MSMS. The average brain uptake was 0.35 pmol/brain and the standard deviation was 0.07 pmol/brain.

EXAMPLE 1-0 (atenolol with eyes and antennae and 45 minutes)

A cut is made through the frontal part of the locust head. The brain and its nerve connections to the compound eyes, antenna, and ocelli are removed from its cuticle shell. The brain preparation is washed in locust buffer. The brain matter is put into a microtiter well containing 250 μΙ of a 3 μΜ atenolol solution heated to 30°C in a block thermostat. After 45 minutes exposure time the brain is removed from the well and the neural lamella, antennae, and compound eyes are removed in ice cold locust buffer. The brain is washed in ice cold locust buffer. Brains from two animals are placed in a test tube containing 150 μΙ of a protein precipitation solution and sonicated. The test tubes are centrifuged at 10 000 xg for 5 minutes at 4°C. 100 μΙ of the supernatants are transferred to analysis vials and analysed by LC/MSMS. The average brain uptake was 0.33 pmol/brain and the standard deviation was 0.02 pmol/brain.

Example 1-L - 1-0 show when brains dissected such both nerve connections to the eyes and antennae are intact there is a reduced brain concentration of compounds readily permeable while brain concentrations of compounds with low brain barrier permeability are at the same level as the brain dissection with only the nerve connections to the eyes intact.

A summary of the Example 1 is provided in the following table:

Uptake Uptake

Example Active(s) Exposure Antennae (with eyes) (no eyes)

(pmol/brain) (pmol/brain)

1 -A 3 μΜ Atenolol 15 mins No <0.21 ±0.02 0.38±0.05

1 -B 3 μΜ Atenolol 45 mins No 0.21 ±0.01 0.38±0.12

1 -C 3 μΜ Quinidine 15 mins No 1 .54±0.13 2.70±0.35

1 -D 3 μΜ Quinidine 45 mins No 4.14±1 .05 4.64±0.75

3 μΜ Quinidine /

1 -E 15 mins No 2.95±0.57 4.64±0.83

25 μΜ verapamil

3 μΜ Quinidine /

1 -F 45 mins No 8.14±0.65 1 1.63±2.16

25 μΜ verapamil

3 μΜ

1 -G 15 mins No 8.70±0.58 9.27±0.38

Carbamazepine

3 μΜ

1 -H 45 mins No 15.40±0.15 15.38±0.57

Carbamazepine

1 -1 3 μΜ Risperidone 15 mins No 3.05±0.88 3.45±0.18

1 -J 3 μΜ Risperidone 45 mins No 6.82±0.30 6.93±1 .19

1 -K 3 μΜ Trazodone 15 mins No 2.45±0.26 2.06±0.56

3 μΜ

1 -L 45 mins No 15.60±1.13

Carbamazepine

3 μΜ

1 -M 45 mins Yes 8.51 ±0.97

Carbamazepine

1 -N 3 μΜ Atenolol 45 mins No 0.35±0.07

1 -0 3 μΜ Atenolol 45 mins Yes 0.33±0.02

Example 2 - METABOLISM STUDIES

EXAMPLE 2-P (Clozapine metabolites to N-desmetylclozapine and Clozapine N-oxide, 15 minutes)

A cut is made through the frontal part of the locust head. The brain and its nerve connections to the compound eyes, antenna, and ocelli are removed from its cuticle shell. The brain preparation is dissected in locust buffer such antenna and ocelli are removed while neural lamella and compound eyes remain on the brain matter. The brain matter is put into a microtiter well containing 250 μΙ of a 3 μΜ Clozapine solution heated to 30°C in a block thermostat. After 15 minutes exposure time the brain is removed from the well and the neural lamella and compound eyes are removed in ice cold locust buffer. The brain is washed in ice-cold locust buffer. Brains from two animals are placed in a test tube containing 150 μΙ of a protein precipitation solution containing 50% MeOH/2% v/w ZnS0 ₄ and sonicated. The test tubes are centrifuged at 10 000 xg for 5 minutes at 4°C. 100 μΙ supernatant is transferred to the analysis vials and analysed by LC/MSMS. The average concentration measured by LC/MSMS was 51.2 nM for Clozapine and 68.3 nM for the metabolite N-Desmethylclozapine. Conversion of measured LC/MSMS concentration can be done by assuming that each brain has a volume of 1.6 μΙ and since two brains are diluted in 150 μΙ, the brain concentration for each brain can be estimated by multiplying the measured concentration by 150 μΙ divided by 2 times 1.6 μΙ. The obtained average brain uptake was 2.4 μΜ of Clozapine and in addition 3.2 μΜ of the metabolite N-Desmethylclozapine and 50 nM of the metabolite Clozapine N-oxide was formed. In addition, analyzing the solution immersing the brain 240 nM of Clozapine N-oxide and 110 nM of N-Desmethylclozapine were detected.

EXAMPLE 2-Q (3 μΜ Clozapine and 25 μΜ verapamil and 15 minutes)

A cut is made through the frontal part of the locust head. The brain and its nerve connections to the compound eyes, antenna, and ocelli are removed from its cuticle shell. The brain preparation is dissected in locust buffer such that antenna and ocelli are removed while neural lamella remains on the brain matter. The brain matter is put into a microtiter well containing 250 μΙ of a 3 μΜ Clozapine and 25 μΜ verapamil solution heated to 30°C in a block thermostat. After 45 minutes exposure time the brain is removed from the well and the neural lamella and compound eyes are removed in ice cold locust buffer. The brain is washed in ice cold locust buffer. Brains from two animals are placed in a test tube containing 150 μΙ of a protein precipitation solution and sonicated. The test tubes are centrifuged at 10 000 xg for 5 minutes at 4°C. 100 μΙ of the supernatants are transferred to analysis vials and analysed by LC/MSMS. The average brain uptake was 4.6 μΜ Clozapine brain concentration and 2.7 μΜ of N- desmethylclozapine is formed.

EXAMPLE 2-R (Clozapine metabolites to N-Desmetylclozapine and Clozapine N-Oxide, 45 minutes exposure)

A cut is made through the frontal part of the locust head. The brain and its nerve connections to the compound eyes, antenna, and ocelli are removed from its cuticle shell. The brain preparation is dissected in locust buffer such antenna and ocelli are removed while neural lamella and compound eyes remain on the brain matter. The brain matter is put into a microtiter well containing 250 μΙ of a 3 μΜ Clozapine solution heated to 30°C in a block thermostat. After 45 minutes exposure time the brain is removed from the well and the neural lamella and compound eyes are removed in ice cold locust buffer. The brain is washed in ice cold locust buffer. Brains from two animals are placed in a test tube containing 150 μΙ of a protein precipitation solution and sonicated. The test tubes are centrifuged at 10 000 xg for 5 minutes at 4°C. 100 μΙ of the supernatants are transferred to analysis vials and analysed by LC/MSMS. The average brain uptake was 9.5 μΜ of Clozapine in addition 7.7 μΜ of the metabolite N- Desmethylclozapine and 134 nM of the metabolite clozapine N-oxide was formed. In addition, analyzing the solution immersing the brain 300 nM of Clozapine N-oxide and 1 μΜ of N-Desmethylclozapine were detected.

Examples 2-P - 2-R show the use of the described model to determine drug metabolites in the brain. Clozapine is often referred to as the gold standard for the treatment of schizophrenia but is underutilized due to safety concerns. However, clozapine's therapeutic mechanism is eluding researchers. Recently, two companies (Merck and ACADIA) published on what they thought to be the drug's underlying mechanism of action. In the body, clozapine is transformed in a 40-80% yield to N- Desmethylclozapine, a metabolite speculated to be clozapine's therapeutic active molecule potentially without its side effects.

CYP1A2 and, to a lesser extent, CYP3A4, are involved in the demethylation of clozapine-forming N-DES in human liver (CYP2D6 is another CYP involved in the metabolism of clozapine). We tested Clozapine 3 μΜ, alone or in combination with verapamil (25 μΜ), a substrate of CYP3A4. Clozapine was demethylated in the locust brain and formed N-Desmethylclozapine to roughly 60%, which corroborated very well with the human data. The competitive inhibition of CYP3A4 with verapamil reduced overall metabolism of clozapine and, subsequently formation of N-Desmethylclozapine (35%). Thus Clozapine is an excellent prodrug of N-Desmethylclozapine.

Table 1. Showing the results when the procedures in examples 2-P - 2-R were used to study three more compounds

The following compounds were tested

N-PCAP1 : 8-chloro-4-methyl-1 1-(piperazin-1-yl)-5H-dibenzo[b,e][1 ,4]diazepine 0-PCAP3: 8-chloro-4-methyl-11-(piperazin-1-yl)dibenzo[b,f][1 ,4]oxazepine

N-Desmethylclozapine

(NDMC): 8-chloro-11-(piperazin-1-yl)-5H-dibenzo[b,e][1 ,4]diazepine Sample PCAP1 PCAP3 N-Desmethylclozapine

Alone 3.8 μΜ 8.0 μΜ 3.8 μΜ

+ Verap 6.6 μΜ 1 1.8 μΜ 5.4 μΜ

The three compounds showed BBB penetration and decreased metabolism in the presence of verapamil. The main metabolite for PCAPs 1 and 3 is the oxidation of the benzylic position (results not shown). The greater concentration in the brain of PCAP3 than PCAP1 and N-Desmethylclozapine depends mainly on the exchange of (NH) to (O), which reduces the bond-donating properties of the molecules. This follows the theories very well.

EXAMPLE 2-S (Clozapine metabolites to N-desmetylclozapine and their distribution in brain, 15 and 45 minutes)

A cut is made through the frontal part of the locust head. The brain and its nerve connections to the compound eyes, antenna, and ocelli are removed from its cuticle shell. The brain preparation is dissected in locust buffer such antenna and ocelli are removed while neural lamella and compound eyes remain on the brain matter. The brain matter is put into a microtiter well containing 250 μΙ of a 3 μΜ Clozapine solution heated to 30°C in a block thermostat. After 15 or 45 minutes exposure time the brains are removed from the well and the neural lamella and compound eyes are removed in ice cold locust buffer. The brain is washed in ice cold locust buffer. Brains are fast-cold in a freezer after the exposure time to stop metabolism to - 80 °C. The brains are micro-sliced to 10 μηι, and analyzed with using matrix-assisted laser desorption ionization MS imaging (MALDI-MSI), Figures 1 and 2.

Analysing the chemical compounds distribution by matrix-assisted laser desorption ionization MS imaging (MALDI-MSI). After 15 minutes exposure the distribution, the test agent (clozapine) and the metabolite are mainly distributed at the edges of the brain, after 45 minutes the both chemical compounds are located in the whole brain, it is also clearly showed that clozapine is metabolised to N- desmethylclozapine in specific regions in the brain.

The data obtained in the abovementioned examples show that compounds which readily permeate the brain barrier mainly permeate the brain barrier and only to a lower extent enter the brain via potential leakages in the brain barrier. For the low permeability compound (atenolol) the brain uptake is significantly lower when the present technology is used while the brain uptake of the brain permeable compound carbamazepine is the same using prior and present technologies. This shows that prior technologies likely introduce a leakage in the brain barrier allowing atenolol and other low permeable compounds to enter the brain.

Contrasting the brain uptake measured using quinidine without Pgp inhibitor there is lower uptake of quinidine co-administered with verapamil after 45 minutes when the new dissection method is used. This can be explained by the fact that in addition to Pgp blockage there is brain barrier leakage when using prior technology while only Pgp blockage is present when the present technology is used. Moreover, the data shows significant saturation of the Pgp transporter after 45 minutes

Surprisingly when brains are dissected while maintaining the nerve connections to the eyes and the antennae intact there is a reduced brain concentration of compounds readily permeable while brain concentrations of compounds with low brain barrier permeability are at the same level as the brain dissection with only the nerve connections to the eyes intact. Although unpredictable this indicates that removing the antennae does not create leakage (enhanced permeability of low permeability compounds) similar to when the compound eyes are removed. It is important that the permeability is due to permeation cross the brain barrier and not do so because of the methodology used. Even more remarkable is the reduction in brain concentrations of high permeable compounds (e.g., carbamazepine) when the antennae remains attached to the brain. Thus, unexpectedly maintaining the antennae and their nerve connections gives unrealistic low concentrations of the chemical compounds.

Described herein is a completely dissected brain comprising the compounds eyes (and their nerve connections) that still have functional brain barrier and enzymatic activity for evaluating brain barrier penetration and metabolism. Especially the latter is difficult to achieve in vivo administration which often generates confounding results by peripheral metabolism, generating peripheral metabolites that are permeating the brain barrier. Dissecting the mammal brain, you need to analyse different part of the brain due to its size, the brain barrier is disrupted and the enzymatic system is not predictable stable enough, thus the combination of brain barrier penetration and brain metabolism is one example of a benefit described herein. For example, clozapine was demethylated in the locust brain and formed N-desmethylclozapine which corroborated very well with fate of clozapine in humans. The inhibition of CYP3A4 with verapamil reduced overall metabolism of clozapine and, subsequently formation of N- desmethylclozapine. The data obtained with the present method showed decreased metabolism in the presence of verapamil. Moreover, the new method can be used to interpret why concentrations in the brain of PCAP3 are higher than PCAP1 and N- desmethylclozapine, which mainly depends on the exchange of (NH) to (O), which reduces the bond-donating properties of the molecules. This follows the theories of brain barrier penetration very well, increasing the number of hydrogen donators decreases permeability.

Moreover the data obtained using MALDI-MSI showed that the new method is useful to determine metabolism and distribution of test agents and their metabolites in the brain. After 15 minutes exposure, clozapine and its metabolite are mainly distributed at the edges of the brain, after 45 minutes the both chemical compounds are located in the whole brain, it is also clearly showed that clozapine is metabolized to N- desmethylclozapine in specific regions in the brain.

Example 3 - Metabolic Studies of Clozapine

Here, we report the initial studies comparing pharmacokinetic data using the locust model and data extracted from the literature for some reference compounds, with special emphasis on clozapine and its metabolites /V-desmethylclozapine (NDMC) and clozapine /V-oxide (CNO) (Scheme 1).

Scheme 1. Metabolic pathways of clozapine ³

N-Desmethylclozapine

a) Structures of clozapine, clozapine /V-oxide (CNO) and /V-desmethylclozapine

(NDMC). CYPs involved in each pathway are indicated, the major ones in bold.

RESULTS AND DISCUSSION

Method in short. In the locust ex vivo model the brain is removed from the insect and placed in a well containing the test compound of interest. After exposure, the concentration in the brain is measured by LC-MS/MS by homogenizing the brain followed by centrifugation. The absence of a vascular system in insects makes the ex vivo model independent of blood flow through the brain. Thus, the insect based ex vivo brain penetration, efflux, and metabolism screening model uses controlled 'in vitro like' exposure conditions allowing direct comparison of chemical compounds. Although the brain has no vascular system, but because of the small size of the locust brain (1.5x1.5x0.8 mm), the distance to reach the center of the brain is about 400 μηι, making up for the vascular system.

The clozapine and metabolites story. In 2003-2004, Merck & Co and ACADIA Pharmaceuticals, independently suggested that NDMC the major metabolite of clozapine was behind the unique efficacy of clozapine to improve cognition and negative symptoms in the treatment of patients with schizophrenia. The metabolite NDMC is detected in concentrations from 20-150% of that observed for clozapine in humans. One difference between clozapine and NDMC is the activity at muscarinic receptors, whereas clozapine is a competitive antagonist, NDMC is an agonist at muscarinic receptor Ml NDMC was evaluated in a phase lib trial for the treatment of schizophrenia. Neither dose of NDMC demonstrated improved efficacy as compared to placebo. However, peripheral dose dependent side effects were noted, preventing the evaluation at higher doses, thus the NDMC hypothesis remains to be tested. In theory replacing the /V-methyl with hydrogen, which adds a hydrogen bond donor would theoretically decrease the blood-brain barrier penetration, and could be the reason for NDMCs lack of efficacy in the trial. Clozapine /V-oxide is another major clozapine metabolite in humans, but is also used as a research tool as an inert ligand in chemical genetics using the Designer Receptors Exclusively Activated by Designer Drugs (DREADD) technology.

Clozapine, NDMC and CNO were incubated with the isolated locust brain and the brain content was analyzed for clozapine, NDMC and CNO (Table 1). In addition, test solutions and actual concentrations of the compounds in the wells after experiments were measured. Test solutions were analyzed to confirm initial concentrations and to exclude contaminations by the other two compounds. Atenolol that is not readily penetrating the brain barrier was included as quality control reference in the experiments to secure that the experimental procedure did not damaging the brain barrier (results not shown). The test compounds test solutions used in this study are made as 1 μΜ and 3 μΜ solutions, but the actual concentrations measured in the well after the experiments varied. This is commonly seen for several of compounds tested in the ex vivo model. In-house studies have shown that the reduced well concentration most likely is due to the compounds sticking to the wall in the well. This is a common problem, which in general is more pronounced for more lipophilic compounds.

Table 2. Analysis of clozapine, NDMC and CNO. ^a

a) Values represent the mean ± SD of three independent experiments (n = 3), two locust brains pooled in each experiment, from one male and one female. NDMC = N-Desmethylclozapine, CNO = Clozapine N-oxide As seen from the ex vivo studies in Table 2, clozapine passes the locust brain barrier and concentrations of 0.1 μΜ or above are obtained at all conditions used in this study. Over time from 15 minutes to 45 minutes, three times increased incubation time, the clozapine brain concentration increases approximately three times and increasing the well concentrations of clozapine, using 1 and 3 μΜ test solutions, respectively, leads to at least a 10-fold increase of the clozapine brain concentration. Thus, the brain concentration increases more than linear upon increasing exposure concentration.

In addition to clozapine we investigated if it was possible to identify the two major clozapine metabolites, NDMC and CNO. Both these metabolites were identified in all of the studies where the brains were incubated with clozapine. In addition, the two metabolites were found in the well solutions in all studies where the brains have been incubated with clozapine. This despite only clozapine was added to the test solution used in the study, corroborating with the analysis of the test solution only clozapine was present. As the volume of the test solution in the well is about 150 times larger relative to the insect brain volume (250 μΙ versus 1.6 μΙ) large quantities may have crossed the brain barrier from the brain and out in the well solution. In particular, at 1 μΜ and 15 minutes incubation the amount of CNO in the brain is below detection limit (<0.02 μΜ) while the CNO concentration in the well 0.04 μΜ. Thus, despite low CNO concentrations are detected in the brain a substantial CNO amounts are formed in the brain and transferred across the barrier to the well solution.

NDMC the main metabolite is present in higher concentrations than clozapine itself. After 15 minutes incubation at 1 μΜ, the NDMC brain concentration reaches levels more than 20 times higher than the clozapine brain concentration. After 45 minutes at 1 μΜ the NDMC level is around 10 times higher than clozapine. Increasing the exposure concentration from 1 to 3 μΜ reduces the difference in brain concentration between clozapine and NDMC. After 15 minutes incubation at 3 μΜ the NDMC level is approximately three times higher than clozapine and after 45 minutes the NDMC concentration is approximately twice that of clozapine. This indicates that the metabolism of clozapine to NDMC is concentration dependent and influences the difference in brain concentrations between clozapine and NDMC. Interesting, plasma concentration of clozapine in patients is between 1-3 μΜ.

Like CNO, NDMC is found in both the brains and in the well solutions. Thus, NDMC produced in the brain crosses the brain barrier and is detected in the wells. The NDMC concentrations measured in the wells after 15 or 45 minute at 1 or 3 μΜ are around 0.1 μΜ or below. Despite the NDMC concentrations found in the brain are 100- fold higher than the CNO brain concentrations, the well concentrations of the two compounds are of similar magnitudes. Although, significant amounts of both CNO and NDMC cross the barrier in order to reach concentrations in the solutions around 0.1 μΜ, it is likely that CNO is actively transported from the brain while well concentrations of NDMC origins from passive diffusion.

Incubation of the brains with NDMC shows that the compound crosses the brain barrier. Neither CNO nor clozapine is detected in the brains or wells after NDMC exposure. Interestingly, at 1 μΜ the concentration of NDMC in the brain is lower than NDMC concentration obtained when the brains are incubated with clozapine. This despite the well concentration of NDMC is higher than clozapine. This observation indicates that clozapine is more prone to cross the brain barrier than NDMC and in addition the conversion of clozapine is fast at 1 μΜ. NDMC is not converted to CNO or clozapine suggesting that the conversion of clozapine to NDMC is an irreversible process, which is in line with results from humans and rodents.

Treatment of the brains by CNO shows that CNO is less prone to permeate the brain barrier and only low concentrations of CNO are detected in the brain. Despite the low brain concentration of CNO, clear brain levels of NDMC are reached in each of the four experimental settings while the well concentrations are all below the detection limit. In contrast to NDMC, clozapine is only detected at the highest used concentration (3 μΜ) and after 45 minutes incubation.

The data in Table 2 suggest that there is equilibrium between the inter- conversion of clozapine and CNO while the formation of NDMC from clozapine is irreversible, thus NDMC acting as a metabolic sink. This could explain why The CNO well concentration is high when the brains are incubated in 3 μΜ clozapine. At this concentration the conversion of clozapine to NDMC has started to reach a maximum and CNO may be transported out of the brain before reverting to clozapine. Thus, the data suggests that NDMC is a product of metabolism of clozapine and indirectly of CNO. Reversible metabolism of clozapine and CNO in schizophrenic patients and guinea-pigs has been described. However, in mice no significant back-transformation to clozapine was found in plasma when injecting 3 μΜ CNO i.p., however latter study did not analyze the concentration of formed NDMC which might be the end product in a fast interconversion. In fact, Loffler et al. (2012) raised a concern that some of the effects of CNO using the DREADD technology could be mediated by the conversion of CNO to clozapine. In the seminal reference stated to show high brain barrier penetration of CNO, the biodistribution of carbon-1 1 labeled clozapine and CNO was determined. Ten minutes after injection of labeled clozapine and CNO in the mice tail vein, clozapine showed a 24-fold higher brain concentration than CNO and at 60 minutes post injection the cerebral concentration of both compounds was almost identical, the latter is mainly due to a substantial reduction of clozapine concentration and not because of an increase of CNO concentration. The study also showed a differentiated regional distribution of clozapine and CNO in the brain. A caveat is that the procedure used in the study neither discriminate between brain uptake of CNO and the amount CNO formed in the brain from clozapine and vice versa, nor does it take into account of peripheral metabolism as it measures total radioactivity of all C11- labelled compounds permeating the brain barrier. This makes it difficult to interpret the interesting time dependent slight increase of CNO concentration in the brain, which could be a slow uptake of CNO or any other C1 1-labelled compound formed peripherally (e.g., clozapine).

Moreover, the data show that of the three compounds studied, clozapine is the one that most readily passes the brain barrier. As a matter of fact, at 1 μΜ treatment with clozapine render in that higher or equal amounts of NDMC are reached in the brain by exposing the brains to clozapine compared with exposure with NDMC directly. Thus, clozapine might be a good NDMC prodrug, reducing peripheral exposure overall by using a lower dose of the active drug.

Example 4 - Clozapine Interaction Studies.

Next we turned to look at interaction studies by co-administration of clozapine (3 μΜ) with verapamil (25 μΜ), fluvoxamine (25 μΜ), or both, after 45 minutes incubation and thereafter analyzing the concentrations of clozapine, NDMC, and CNO in brains and wells (Table 3).

Verapamil is a L-type calcium channel blocker, it has been used in the treatment of hypertension, angina pectoris, cardiac arrhythmia and recently cluster headaches and as preventive medication for migraine, and it is on the World Health Organizations list of Essential Medicines, the most important medications needed in a basic health system. Verapamil is also an inhibitor of drug efflux pump proteins such as Pgp, and CYP3A, especially CYP3A4 Ki between 3-6 μΜ. As described earlier a homolog to Pgp has been identified in the locust brain and a functional homolog of the mammalian CYP3A4 has also been identified in locusts. Fluvoxamine functions as a selective serotonin reuptake inhibitor with antidepressant properties. Administration of fluvoxamine to patients receiving clozapine therapy may increase the steady-state serum concentrations of clozapine by a factor of 5 to 10. In rats, fluvoxamine is reported to inhibit CYP1A2 and 2C19 with Ki values of 0.041 and 0.087 μΜ, respectively. The Ki values for CYP2C9 and 2D6 were 2.2 and 4.9 μΜ, respectively, whereas the Ki for CY3A4 was 24 μΜ. Verapamil has also been reported to have an intermitted Pgp inhibition. Thus, Verapamil and Fluvoxamine moderately inhibit the two major CYPs responsible for the formation of CNO and DMC and Pgp.

Table 3. interactions studies with clozapine and verapamil, fluvoxamine, or both. ³ Test compd Clozapine

Verapamil Fluvoxamine NDMC (μΜ) CNO (μΜ) (3 μΜ) (μΜ)

Clozapine (brain) — — 5.60 ± 1 .2 9.4 ± 0.08 0.18 ± 0.02

Well 1 .28 ± 0.10 0.1 ± 0.02 0.06 ± 0.01

Clozapine (brain) 25 μΜ — 19.0 ± 1 .7 5.6 ± 2.00 0.14 ± 0.04

Well 1 .80 ± 0.10 0.05 ± 0.02 0.01 ± 0.00

Clozapine (brain) — 25 μΜ 16.0 ± 2.3 1 1 .6 ± 1 .71 0.45 ± 0.01

Well 1 .90 ± 0.68 0.09 ± 0.05 0.05 ± 0.01

Clozapine (brain) 25 μΜ 25 μΜ 17.4 ± 0.9 6.0 ± 1 .5 0.20 ± 0.04

Well 1 .70 ± 0.62 0.05 ± 0.01 0.02 ± 0.00 a) 45 minutes incubation. Values represent the mean ± SD of three independent experiments (n = 3), two locust brains pooled in each experiment, from one male and one female. NDMC = N- Desmethylclozapine, CNO = Clozapine N-oxide

Co-administration of verapamil with clozapine increases the brain concentration of clozapine 3-4 times compared to the concentration obtained without any verapamil co-administration. Similar comparison of the NDMC brain concentrations show that the formation of NDMC is reduced to almost half the concentration when verapamil is co- administered. Thus, despite higher clozapine brain concentration less NDMC is produced suggesting that the metabolism of clozapine to NDMC is reduced by verapamil. Co-administration with verapamil does not play any significant role on the CNO brain concentrations. However, the CNO well concentrations are higher without verapamil co-administration suggesting that verapamil reduces the production of CNO. Alternatively, since verapamil is a known to block or reduce efflux of transporter substrates it may be speculated that verapamil reduces efflux of CNO out of the brain. Co-administration with fluvoxamine, results in high brain concentrations of clozapine and CNO. The brain and well concentrations of clozapine reach same levels as when verapamil is co-added.

No change in NDMC levels is seen with or without fluvoxamine coadministration, despite that the clozapine concentration is increased three fold, we read this as a slight inhibition in NDMC formation. Unexpectedly, addition of fluvoxamine increases the CNO brain concentration or the relative concentration compared with clozapine remains as without fluvoxamine. Using human liver microsomes fluvoxamine is reported to reduce the formation of CNO from clozapine with 10-50%. However, an explanation might be that the metabolism of CNO in the brain is also inhibited (e.g., the back transformation to clozapine) and Pgp is moderately inhibited. Interestingly, fluoxetine which has a similar CYP and Pgp profile as fluvoxamine, was reported to increase the concentration of clozapine, CNO and NDMC when co-administrated with clozapine reaching levels of 15 μΜ and leading fatal outcomes. Whereas the well concentration remains at the same level as when no inhibitor is added the relative CNO concentration in the well is reduced with fluvoxamine which might be because of a moderate inhibition of the Pgp.

When adding both fluvoxamine and verapamil the concentrations of clozapine, NDMC, and CNO are at the levels reached when verapamil is added alone, i.e., high clozapine, low NDMC and CNO. The results in Table 3 imply fast metabolism of clozapine by one or more pathways and these may be inhibited by verapamil. The main metabolite is NDMC, which at 1 μΜ reaches brain levels higher than those reached by clozapine itself. Fluvoxamine inhibits metabolism of clozapine and have a slight impact on the pathway producing NDMC. Generally, the diffusion of metabolites out of the brain seems relative low. However, as the solution volume in the well is large the actual amounts of metabolites effluxed in moles are large.

Example 5 - Risperidone, Fluoxetine, Haloperidol, and Citalopram.

To test if drugs other than clozapine are metabolized in the locust brain we investigated the brain penetration, efflux and metabolism of the central acting drugs risperidone, fluoxetine, haloperidol, and citalopram. Moreover, we pre-selected one known major metabolite based on commercial availability of the metabolites of each of the CNS acting compounds and investigated if it was possible to identify and quantify them, see Scheme 2.

Scheme 2. Structures of central acting drugs and one of their metabolites.

N-desmethylcitalopram

In the peripheral, risperidone is metabolized by CYP2D6 and CYP3A4 to 9-OH risperidone and both compound are substrates for Pgp. As seen from Table 4 risperidone and 9-OH risperidone are detected in the locust brain when risperidone alone is added to the well. Moreover, the brain concentrations of risperidone increase whereas 9-OH risperidone concentrations are decreased when risperidone is co- incubated with verapamil. This indicates that verapamil reduces efflux of risperidone by interacting with the Pgp and at the same time inhibits conversion of risperidone to 9-OH risperidone leading to higher risperidone brain concentrations. Support to this is given by comparing the data obtained after 15 and 45 minutes exposure. When the exposure time is extended from 15 to 45 minutes both risperidone and 9-OH risperidone brain concentrations increases. However, when risperidone and verapamil is added to the well, only the risperidone brain concentration gets higher while the 9-OH risperidone concentration remains the same. Treatment of the brain with 9-OH risperidone show that the compound permeates the brain barrier and significant amounts are measured in the brain homogenates. The brain uptake of 9-OH risperidone increases when the exposure time is increased from 15 for 45 minutes. Moreover, 9-OH risperidone increases when the compound is co-administered with verapamil. This supports that 9- OH risperidone is an efflux substrate and that verapamil inhibits this efflux.

Table 4. Analysis of risperidone and 9-OH risperidone. ³

a) Values represent the mean ± SD of three independent experiments (n = 3), two locust brains pooled in each experiment, from one male and one female. Exposing the brains to fluoxetine alone shows that fluoxetine permeate the brain barrier (Table 5, entries 1-4). However, both fluoxetine and norfluoxetine are detected in the locust brain. Co-administration of fluoxetine with verapamil increases the brain concentration of fluoxetine and slightly reduces the concentration norfluoxetine. The concentrations of fluoxetine and norfluoxetine are very high compared to risperidone. In addition, norfluoxetine show no indication of being a Pgp substrate, as it is not detected in the wells. This is seen in previous locust studies where fluoxetine is characterized by very high brain concentrations.

Table 5. Analysis of fluoxetine, citalopram and haloperidol. ³

a) Values represent the mean ± SD of three independent experiments (n = 3), two locust brains pooled in each experiment, from one male and one female, b) Incubation 45 minutes c) Metabolites: Norfluoxetine (entries 1 -4), /V-Desmethylcitalopram (entries 5-8), 3-(4-Fluorobenzoyl)propionic acid (entries 9-12).

Citalopram is metabolized by both CYP2C19 and CYP3A4 to the major metabolites /V-desmethylcitalopram, and Λ/,/V-didesmethylcitalopram. Exposing the insect brain to citalopram for 45 minutes shows that the compound enters the brain and /V-desmethylcitalopram is formed in concentrations approximately 2-3 times higher than the brain concentrations than that reached for the parent compound. Co-administration of citalopram with verapamil increases the citalopram brain concentration more than 5 times whereas /V-desmethylcitalopram concentration only increases approximately 3 times, rendering in an equal concentration of the both. This suggests that citalopram is a CYP3A4, Pgp substrate or both, which is reflecting the case in mammals. Minor concentrations of the formed metabolite /V-desmethylcitalopram is found in the wells, which do not increase with an increasing concentration in the brain indicating a slight inhibition of efflux in the present of verapamil. Thus, the relative high increase of N- desmethylcitalopram in the present of verapamil could be because of inhibition of efflux and further metabolism forming A/./V-didesmethylcitalopram.

Analyzing haloperidol showed that it passes the brain barrier and that significant brain concentrations are detected in the brain. No significant brain concentrations of the metabolite 3-(4-fluorobenzoyl)propionic acid is detected. However, the measured levels in the wells of the metabolite are around 0.25 μΜ. This indicates that 3-(4- fluorobenzoyl)propionic acid is formed in the brain and subsequently efficiently transported out. Co-incubation of haloperidol with verapamil increases the brain concentration of haloperidol almost 3-times while the 3-(4-fluorobenzoyl)propionic acid metabolite is detected in the wells but below detection level in the brain. Well concentrations remained the same despite an increase of haloperidol levels which indicates that verapamil inhibit the formation of 3-(4-fluorobenzoyl)propionic acid, the efflux, or both. These findings support the observations done in mammals where CYP1A2 and CYP3A4 is known to metabolize haloperidol forming 3-(4- fluorobenzoyl)propionic acid. After intramuscular administration, plasma contained 3-(4- fluorobenzoyl)propionic acid in comparable concentrations to haloperidol. However, only haloperidol was found in rat's brain, no 3-(4-fluorobenzoyl)propionic acid was detected. In contrast, using brain homogenate in in vitro studies showed that haloperidol is converted into 3-(4-fluorobenzoyl)propionic acid. In the latter study the metabolite is trapped in the homogenate, thus taken together, these findings suggest that haloperidol in mammals is converted to 3-(4-fluorobenzoyl)propionic acid in the brain and that this metabolite is efficiently transported out of the brain and excreted peripherally (see Figure 1).

Example 6 - Free concentration of drug in the brain.

The practical approaches to routinely investigating large numbers of new compounds have been to measure the amount of drug in brain and correlate that with plasma levels in vivo or the rate of BBB permeation in vitro. These approaches have their benefits, especially in the initial phase of drug discovery but as sole analyses it has led to that medicinal chemistry programs have favored compounds and classes displaying high total CNS-to-plasma concentration ratios, sometimes rendering in lipophilic drugs that dissolves well within the lipophilic brain content. However, it is generally accepted that it is the unbound drug that is available to exert the effect on their targets or susceptibility for biotransformations, and not the total tissue levels.

Furthermore, a basic assumption in pharmacokinetics is that unbound drug concentrations are equal on both sides of a physiological membrane at steady state, this may not be the case for brain tissue since there are active efflux and influx processes both at the BBB (free drug plasma and free drug interstitial fluid (!SF)) and intercellular (free drug ISF and intracellular fluid). The comparative importance of unbound drug concentrations in different brain compartments (ISF) or (ICF) will depend on where the relevant receptors are situated. If the drug in question is actively transported across the cell membrane or blood-brain barrier, brain ISF concentrations could be expected to differ from brain ICF concentrations and unbound drug concentrations in blood.

Although, the only method of directly measuring concentration of unbound drug in the brain ISF (C _u ,braimsF) is microdialysis, caution must be taken for regional variations in brain. Unfortunately, the utility of this method in early drug discovery programs is limited by the complexity, cost and time requirements. Alternative methods to microdialysis have used free fraction unbound in brain (F _ub ) and volume of distribution in the brain (V _{U braill} ), using brain homogenate equilibrium dialysis method and brain slice uptake technique, respectively. Both V _{U braill} and F _ub describe much the same property to estimate and relate C _{U bra} inisF to whole brain concentrations (A _braill ). However, homogenization of the brain in the homogenate equilibrium dialysis changes the brain tissue binding properties and might lead to inherent errors in the results.

As an alternative the brain slice method has been brought forward as a model with more physiological basis, preserving much of the complex cellular integrity, including cellular barriers and intact circuitry, and as a result conserving functionality, resulting in an in vitro environment more comparable to the in vivo brain than seen in the homogenate method. The key assumption of the experiment is that, at equilibrium, the free drug concentration in the brain slice ISF or ECF is equal to the drug concentration in the buffer in the surrounding media. Although having a more physiological basis, the slice method does not include a viable brain barrier and no metabolic activity has been reported.

Hence, the locust Bpem model could potentially be an alternative, complement, or both, to the brain slice and homogenate models as well as to in vivo studies. For example, in our earlier study, formation of the metabolite 3-(4-fluorobenzoyl)propionic acid from haloperidol agreed when combining two independent studies, one using in vivo analysis of brain concentrations, and the other in vitro analysis of brain homogenate, and that at first sight these studies might have been viewed as contradicting each other, either alone did not show both the formation of the metabolite and the subsequent efflux.

The key assumption in this set-up of the whole brain model is that the unbound brain to unbound plasma concentration ratio (K _P)UU ) equals one at steady state. Assuming that compounds cross the brain barrier only by passive diffusion and that the free drug concentration ISF is equal to the drug concentration in the well.

Kp _t uu ~ 1 C _u ,brainlSF ~ C _u ,Plasma /well (1)

This assumption is obviously not always true, however studies have shown that extended time closedown energy demanding processes, e.g., active transport, without affecting the brain barrier integrity. Hence, this model will have three phases: 1) Brain uptake, measuring brain barrier permeation rate, 2) Elimination phase with active metabolic pathways and active transporters, and 3) Equilibration phase, with passive diffusion over the brain barrier. Another approach would be to use selective molecules to inhibit specific biological functions (CYPs and Pgp), as in previous examples with verapamil.

Drugs are normally administered in a bolus dose, generating and initial high drug levels in plasma and brain that is time dependency declining. To mimic this, we incubated the brains with drug for 45 minutes thereafter the brains were transferred to a blank buffer solution, and analyzed after additional 30, 60, and 90 minutes (Table 6). The incubation time (45 minutes) was based on previous studies showing that 15 minutes is not enough for clozapine to be fully distributed in the whole locust brain (result not shown).

Table 6. Time dependent depletion studies of clozapine, CNO and NDMC. ^a

a) Values represent the mean ± SD of three independent experiments (n = 3), two locust brains pooled in each experiment, from one male and one female, b) Time after 45 minutes of incubation with clozapine.

After 45 minutes incubation with 1 μΜ clozapine solution, the same trend as in the previous study was seen, NDMC was formed in high amounts and CNO was detected in the wells but not in the brains. Subsequent incubations in a blank buffer gave after 30 minutes 4-5 fold reduction of clozapine (from 1 ,78 to 0.42 μΜ) and an 1 μΜ increase of the metabolite NDMC, no CNO was detected, neither in brain nor in wells. After additional 30 minutes, no clozapine was detectable while the amount of NDMC increased still, a slight decrease in the concentration of NDMC was seen after 90 minutes of incubation in a blank buffer. Incubation with 3 μΜ clozapine followed the same trend as with 1 μΜ, with the expected higher concentrations overall. Metabolism is an energy consuming process and the decrease of metabolism could be related to the fact that all energy is consumed especially when analyzing the 60 and 90 minutes incubations in blank buffer. However, the reduction of clozapine over time in brain corroborate with what was seen in the PK study in mice using 1 1 -C labeled clozapine.

The initial study had a relative short equilibration time, 90 minutes, which may question if the steady state was reached, both the brain homogenate assay and brain slice assay use longer equilibration times between 3-5 hours. Analyzing the brain concentrations only it looks like NDMC is a more stable compound than clozapine in the locust brain. However, the free fraction of drug, F _ub is 0.06 and 0.008, for clozapine and NDMC, respectively, clozapine has a larger free fraction compared with NDMC which correlate with previous studies in rodents using microdialysis. The F _ub is reported to be species-independent, this was verified to encompass the locust brain as well in a correlation study with rodent F _ub (mice and rats) using the brain homogenate model.

Fub = Ctot., brain I C _u ,brainlSF (2)

This makes also clozapine more available to activate GPCRs and susceptible for biotransformations compared to NDMC. In addition, from the table it might be viewed that CNO have a large free fraction of drug only confined to the ISF, this may be the case, which would explain why CNO works in the DREADD technology, low permeation still gives a high free fraction of drug. However previous studies with verapamil and fluvoxamine showed on a potential efflux mechanism via Pgp.

In the insect brain models the brain is viable and surrounded by an intact brain barrier with active transporter proteins, and active metabolism. By incubating a brain of an insect with the compound of interest the brain is loaded with the compound. Subsequent transfer of the brain to a well containing pure buffer, i.e., a blank medium allows equilibrium to be reached across the brain barrier. In these studies the brain barrier is intact and the active transport and metabolism is only being abridged due to shortage of energy. The intact brain barrier, active transport across the brain barrier, and metabolism is likely influencing the equilibration time after transfer of the brain to a blank medium. The effect of the brain barrier integrity has been studied by loading the brain for 45 minutes with a drug substance. Before transferring the brain to a blank medium the brain is split in two pieces of about equal size and thereafter placed in the blank medium. The brain and the well concentrations are measured at different time points and compared with similar studies using an intact brain. Effect on the equilibrium time of active transport and metabolism is studied by adding verapamil to the blank medium.

The experiments are executed by cutting through the frontal part of the head of the locust. The brain comprising neural lamella and connections to compound eyes, but not antennae is dissected out of the cuticle in locust buffer and put into a microtiter well containing 250 μΙ of the test solution heated to 30°C in a block thermostat. After 45 minutes exposure time (i.e., the loading phase) the brain comprising the neural lamella and connections to the compound eyes is transferred from the well and washed. The brain comprising the neural lamella and connections to the compound eyes is put into a blank solution +/- verapamil (blank solution with and/or without verapamil) for different amounts of time for further equilibration. Some brains are divided in the middle before being put into blank solutions (no verapamil). Brains from two animals are put into 37.5 μΙ Millipore water. At the same time 100 μΙ samples are taken from each of the wells corresponding to each sample with brains (in total 200 μΙ from two wells) and put into 200 μΙ 40% ACN (acetonitrile) (25 μΙ + 25 μΙ + 950 μΙ 20% ACN for exposure solution). 1 12.5 μΙ of 100% ACN is added to the test tubes containing brains and the brains are sonicated and all test tubes centrifuged at 10 000 xg for 5 minutes at 4°C. 50 μΙ of the supernatants (brains) are transferred to analysis vials and diluted with 150 μΙ Millipore water. 200 μΙ of the well samples are transferred to analysis vials without being diluted.

The compounds used in this study include carbamazepine, risperidone, and clozapine and the reported data in table 7-9 are well concentrations and concentrations in the brain. Table 7 shows the results obtained when carbamazepine is used as test substance. As expected the brain concentration of carbamazepine reduces with time while the well concentration increases when the brain after the loading phase is placed in a blank medium. After 120 minutes in the blank medium equilibrium is reached. At 120 minutes the fraction unbound in the brain is 14.83% in the intact brain while it is estimated to be 16.47% in brains divided in two. F _ub is 17.37% when verapamil is added to the blank medium. In general, only small differences in well and brain concentrations between the three tests, i.e. intact brain, disintegrated brain (divided), and intact brain in blank medium containing verapail are seen.

Table 7. Brains loaded with 3μΜ carbamazepine in 45 minutes, i.e. total brain concentration equal 9.31 ±0.1 1 μΜ. Intact or divided brains are subsequently placed in wells containing blank medium or blank medium containing verapamil.

When brains after a loading phase of 45 minutes in 3 μΜ risperidone are transferred to a blank medium the brain concentration decreases rapidly during the first 120 minutes. In the period 120 to 240 minutes there is only a slight decrease in the brain concentration (Table 8). The well concentration display initial increase followed by a decrease. Both the well and the brain concentrations are higher when verapamil is added to the blank medium. This is likely explained by the fact that verapamil is known to reduce metabolism and efflux. By using the presented method the free fraction in the brain is estimated to be 16.41 % using divided brains and 12.39% using intact brains and verapamil in the blank solution. When intact brains are used F _ub is estimated to be 20.06%. While F _ub estimated by using either the split brains or intact brains placed in blank with verapamil is almost constant after 120 minutes the F _ub obtained using the intact brain matter continues to increase even after 120 minutes. This may be explained as an effect of metabolism and efflux, which are two events both taking place in intact brains placed in blank medium without verpamil.

Table 8. Brains loaded with 3μΜ risperidone in 45 minutes, i.e. total brain concentration equal 6.94±0.38 μΜ. Intact or divided brains are subsequently placed in wells containing blank medium or blank medium containing verapamil.

In order to investigate the effect of metabolism in the presented method clozapine was used as test compound. In this study the free brain concentration of clozapine and its metabolite NDMC was measured. The data in Table 9 shows that clozapine is metabolized and NDMC is being produced during the equilibration phase, i.e. the clozapine brain concentration decreases during the entire experiment while _iI DMC _S oa _p z _i

NDMC increases. Interestingly, there is more clozapine in the brain when verapamil is added to the blank solution while the NDMC concentration is lowest when verapamil is added to the blank solution. This is in agreement with the assumption that verapamil reduces the metabolism of clozapine to NDMC. Low well concentrations of clozapine and NDMC are seen for all brain, however, the well clozapine concentrations display initial increase followed by a decrease. NDMC in the well increases during the entire experiment.

The high brain and low well concentrations give rise to calculated low free fractions in the brain, clozapine being around 3.5% after 4 hours when verapamil is absent while F _ub is around in the presence of verapamil. F _ub for NDMC is estimated to be around 1 .0%. This corroborate with studies using brain homogenate and microdialysis.

Table 9. Brains loaded with 3μΜ clozapine in 45 minutes, i.e. total brain concentration equal 12,81 ±2.37μΜ. Intact or divided brains are subsequently placed in wells containing blank medium or blank medium containing verapamil. Concentrations and F _ub of clozapine (upper part of the table) and its metabolite NDMC (upper part of the table) is measured in the brain and well at different time points.

Intact Split brain Intact brain in verapamil

Time Brain (μΜ) Well (μΜ) F _u b Brain (μΜ) Well (μΜ) F _u b Brain (μΜ) Well (μΜ) F _u b

(%) (%) (%)

15 6.31 ±1 .59 0.07±0.01 1 .03 7.31 ±1 .79 0.07±0.02 0,97 9.38±1 .14 0.08±0.01 0.90

30 5.50±0.87 0.07±0.00 1 .36 5.81 ±0.82 0.07±0.01 1 ,20 9.06±1 .33 0.12±0.01 1 .32

60 3.50±0.47 0.08±0.01 2.21 3.56±0.65 0.07±0.01 2,10 10.06±1 .89 0.13±0.01 1 .26 o

120 2.31 ±0.47 0.06±0.01 2.68 4.13±1 .90 0.07±0.01 1 ,70 6.56±0.65 0.13±0.01 1 .99

240 1 .27±0.37 0.04±0.00 3.47 1 .26±0.34 0.05±0.01 3,61 5.75±1 .90 0.10±0.03 1 .82

15 10.63±0.85 0.03±0.01 0.32 12.56±2.45 0.02±0.01 0,14 10.06±1 .88 0.03±0.0.00 0.33

30 10.94±4.33 0.05±0.01 0.43 1 1 .69±1 .50 0.04±0.01 0,33 9.56±1 .79 0.04±0.01 0.46

60 10.50±1 .42 0.07±0.01 0.69 12.38±1 .53 0.07±0.07 0,54 9.50±1 .52 0.03±0.02 0.33

120 8.69±3.29 0.09±0.02 1 .08 12.88±0.60 0.09±0.01 0,70 8.38±1 .22 0.09+0.01 1 .10

240 1 1 .13±1 .80 0.12±0.01 1 .10 12.75±2.45 0.12±0.03 0,95 8.50±0.85 0.1 1 ±0.01 1 .27 To verify the F _ub model, additional compounds were tested. In this study the brains were exposed to the test compounds for 45 minutes. The brains were then removed from the well and either a) transferred to a well containing blank buffer medium or b) lamella and eyes were dissected followed by division of the brain and then the divided brains were placed in wells containing blank buffer medium. After 240 minutes the brains were washed. The content of the compounds were measured in the brains and in the medium. The data obtained are shown in Table 10.

Table 10

The data in Table 10 show that the ranking of the F _ub measured in the insect model reflect what is seen for rodents and as a matter of fact the ranking F _ub obtain in insects is identical to that obtained in rodents. These studies show that the insect brain model is useful to estimate the free fraction in the brain. In addition, the model can be used to investigate equilibration time and potential metabolism. The locust model enables the efficient study of compounds and the their metabolites simultaneously. The model is useful to determine the free fraction in the brain, metabolites and the equilibration time and these properties are affected by the brain barrier integrity and co-substances like verapamil which influence the metabolism and transporter proteins.

In the experiments the brain matters may be prepared in different ways. Examples on preparation of brain matters include experiments where a cut is made through the frontal part of the head the locust separating the brain and its nerve connections to the compound eyes, antenna and ocelli still in its cuticle shell. The brain is dissected in locust buffer removing the neural lamella and connections to compound eyes. The brain is put into a microtiter well containing 250 μΙ of the test solution heated to 30°C in a block thermostat. After 45 minutes exposure time (i.e. the loading phase) the brain is removed from the well and the neural lamella dissected and washed in ice cold locust buffer or the brain is washed and put into a blank solution +/- verapamil for different amounts of time. Some brains are divided in the middle before being put into blank solutions (no Verapamil). Brains from two animals are put into 37.5 μΙ Millipore water. At the same time 100 μΙ samples are taken from each of the wells corresponding to each sample with brains (in total 200 μΙ from two wells) and put into 200 μΙ 40% ACN (25 μΙ + 25 μΙ + 950 μΙ 20% ACN for exposure solution). 1 12.5 μΙ of 100% ACN is added to the test tubes containing brains and the brains are sonicated and all test tubes centrifuged at 10 000 xg for 5 minutes at 4°C. 50 μΙ of the supernatants (brains) are transferred to analysis vials and diluted with 150 μΙ Millipore water. 200 μΙ of the well samples are transferred to analysis vials without being diluted.

The brain barrier integrity may be conserved or disrupted in the brain matter used to determine the free fraction in the brain and the time to reach equilibrium. The brain barrier may be disrupted by dividing or splitting the brain, chemical treatments, by hyper-osmotic pressure e.g. by using mannitol, ultra-sonic means, or other means whereby the brain barrier is disrupted.

Example 7 - Metabolic-ID studies of clozapine and midazolam.

In the early drug discovery phase the metabolites for at potential drug candidate are generally not known. Therefore, more traditional metabolic-IDs studies were performed. Clozapine and midazolam were selected as test compounds to enable comparison with available literature, as most of their metabolites are known, at least the ones formed in the periphery.

A cut is made through the frontal part of the locust head. The brain comprising the neural lamella and compound eyes (including their nerve connections) is dissected of the insect head. The brain preparation is dissected in locust buffer such that antenna and ocelli are removed while neural lamella and compound eyes remains on the brain matter. Prepared brains were incubated separately in clozapine and midazolam at concentrations 1 , 3 and 10 μΜ. Brains (from two grasshoppers) were combined and mixed with 40 μΙ of acetonitrile. The brain samples were ultrasonicated for 20 min, centrifuged and the supernatants were collected for the analysis. The LC/MS data obtained from clozapine, midazolam, and their metabolites are summarized in the supplementary information. All the detected metabolites were tentatively identified according to the accurate mass data, retention times, and high resolution fragment ion data. The metabolite structures profiles are expressed as a percentage of each metabolite from total LC/MS peak area, assuming identical LC/MS response for metabolites and parent compounds, which probably is not the case, but still generally used as a rough measure.

Clozapine, totally 18 metabolites (M1 - M18) were observed. Four oxidated/hydroxylated metabolites were observed, two with reaction sites in chlorobenzodiazepine (M1 , M2) and two in /V-methylpiperazine (M3, M4). Also, a further hydrogenation of M1/M2 in chlorobenzodiazepine (M5) was observed, as well as a combination of M1/M2 and M3/M4 with a further dehydrogenation (M6). Yet, phase I metabolites formed via N-demethylation (M7), a loss of C2H2 (M8), combination of M7 and M8, (M9) were observed. The conjugative metabolism consisted of piperazine methylation (M10), further acetylation of M7 (M 14), glucoside conjugations directly to clozapine (M12, M13) or further to M7 (M15, M16), nitrile formation (M17, M18) to piperazine, and direct S-glutathione conjugation (M1 1). The reaction mechanism of M1 1 probably includes epoxide intermediate in benzodiazepine-ring-system.

NDMC (M7) is the most abundant clozapine metabolite (53% at 1 μΜ) compared to the parent clozapine (43 %), and direct glucoside conjugation (M13) the second most abundant metabolite (2%). CNO (M3) the third largest metabolite formed in 0.7% relative clozapine. Metabolites M2, M3, M5, M7, M8, M10, M12 and M13 were observed in both brain and incubation well samples, while the metabolites M 1 , M4, M6, M9, and M14 - M18 were not observed in any of the incubation well samples. The relative abundance of metabolites with respect to clozapine was decreasing as a function of test concentration, suggesting saturation of the metabolic enzymes.

Except for M5, M10, and M14, the identified metabolites have been found in earlier studies. M10 is an addition of CH2, corresponding to adding a methyl to the piperazine and is detected in minor amount 0.01 % at the two highest concentrations only. M 10 could be formed by trapping of a one carbon fragment (formaldehyde), if this is an experimental artifact or areal result is not clear, the formaldehyde could be because the demethylation of clozapine to NDMC. M5 is two subsequent oxidation first a hydroxylation and followed by an oxidation, this not an unlikely metabolite as the primary oxidations products (M1 and M2) might be trapped in the brain compartment and susceptible for further transformations, this can be noted as these metabolites are not readily detected in the wells. M14 is a conjugation, the /V-acylation of NDMC. Interestingly, the incubations were quenched by acetonitrile in large excess, acetonitrile is known to form cyanide as a metabolite that could react with reactive metabolic intermediates. In fact, M17 and M18, two nitrile metabolites were formed which corroborates with the metabolites identified by metabolic trapping experiment using potassium cyanide.

Although one metabolite from direct S-glutathione conjugation was identified (M1 1), several glutathione metabolites are reported to be formed via a reactive nitrenium intermediate. After further inspection of the chromatograms three additional LC/MS peaks with exact mass fitting to S-glutatione conjugates were observed, but their abundance was very low and no supporting MS/MS data was obtained. In addition, similar very minor LC/MS peak with intensity close to detection limit was observed for a m/z fitting to exact mass of GSH-conjugation replacing chlorine atom (m/z 598.2442), as well as replacement of chlorine by S-thiomethyl (m/z 339.1638) but no confirming MS/MS data was obtained.

Midazolam, 14 metabolites were observed. The known hydroxymetabolites 4- hydroxymidazolam (M1) and 1-hydroxymidazolam (M2) were observed, the combination of the two oxidation sites in M5, although the structure of latter not definitely determined. A number of additional oxidation/hydroxylation products alone or in combination with a reduction in methylimidazole were found. Two metabolites from glucoside conjugations were found one directly to midazolam (M13) in 2% and the second in minor amounts of 0.05% relative midazolam after hydroxylation and hydrogenation (M14).

Almost all of the observed metabolites were present in both brain and incubation well samples in all test concentrations. Similarly to clozapine, the relative abundance of metabolites with respect to midazolam was decreasing as a function of incubation concentration, suggesting saturation of enzymatic reactions. However, less than 10% of the midazolam was metabolized compared with > 55% for clozapine, thus a longer incubation time would give a more pronounced metabolism.

Interestingly, the metabolism of midazolam was investigated by Olsen et a/, in vivo in locusts in order to evaluate the presence of an enzyme with functionality similar to human CYP3A4/5. Midazolam was injected in the locust, and the feces was collected and analyzed for midazolam metabolites. Hydroxylated metabolites of midazolam identical to human metabolites were detected in locusts and the apparent affinities (Km values) were in the same range as reported in humans, in addition, the formation of bydroxylated metabolites could successfully be inhibited by co-administration of ketoconazoie, a known CYP3A4/5 inhibitor. Besides phase I metabolites, the main metabolites were a number of glucose and glucose-phosphate conjugated metabolites. This differentiates the metabolic profiles in the brain and periphery, contrary to the periphery one phase II metabolite was found in the brain in a relative amount comparable with the individual hydroxylated metabolites. However, six metabolites found in brain had undergone a reduction in the methylimidazole. These metabolites have not been identified previously, if these metabolites are brain specific or produced because of species differences remains to be evaluated.

The LC/MS data obtained from clozapine, midazolam, and their metabolites are summarised in Tables 10 and 1 1 , and ion chromatograms are shown in the Appendices I and II. All the detected metabolites were tentatively identified according to the accurate mass data, retention times, and high resolution fragment ion data. The tentative identifications for the metabolite structures are shown in the Schemes 3 - 4. The metabolite profiles in all analysed samples, are shown in Schemes 5 and 6.

For clozapine, totally 18 metabolites (M1 - M18) were observed. Four oxidated/hydroxylated metabolites were observed, two with reaction sites in chlorobenzodiazepine (M1 , M2) and two in methylpiperazine (M3, M4). Also, a further hydrogenation of M1/M2 in chlorobenzodiazepine (M5) was observed, as well as a combination of M1/M2 and M3/M4 with a further dehydrogenation (ketone formation) in methylpiperazine (M6). The fragment ion data for M6 is however not unambiguous, and alternatively it may be that the dehydrogenation is not in methylpiperazine but rather as a quinone-imine formation to benzodiazepine (after hydroxylation to carbon adjacent to chlorine-binding carbon). Yet, phase I metabolites formed via N-demethylation (M7), a loss of C ₂ H ₂ (M8), combination of M7 and M8, (M9) were observed.

The conjugative metabolism consisted of piperazine methylation (M10), further acetylation of M7 (M14), glucoside conjugations directly to clozapine (M12, M13) or further to M7 (M15, M16),nitrile formation (M17, M18) to piperazine, and direct S- glutathione conjugation (M1 1). The reaction mechanism of M11 probably includes epoxide intermediate in benzodiazepine-ring-system, and it is worth noticing that also three additional LC/MS peaks with exact mass fitting to S-glutatione conjugates were observed, but their abundance was very low and no supporting MS/MS data was obtained (see M1 1 ion chromatogram in Appendix I). In addition, similar very minor LC/MS peak with intensity close to detection limit was observed for a m/z fitting to exact mass of GSH-conjugation replacing chlorine atom (m/z 598.2442), as well as replacement of chlorine by S-thiomethyl (m/z 339.1638) but no confirming MS/MS data was obtained. Formation mechanism of M17 - M18 is bit unclear; typically this reaction is known to occur for iminium-type reactive metabolites after trapping/stabilizing with cyanide.

N-demethylation (M7) was clearly the most abundant clozapine metabolite, and direct glucoside conjugation (M13) the second most abundant metabolite for clozapine in grasshopper brain. Both M7 and M13 were also observed in the samples collected from wells after the incubation, although the relative amounts were significantly lower compared to the actual brain samples. Also metabolites M2, M3, M5, M8, M 10 and M12 were observed in both brain and incubation well samples, while the metabolites M1 , M4, M6, M9, and M14 - M 18 were not observed in any of the incubation well samples. The relative abundance of metabolites with respect to clozapine was decreasing as a function of test concentration, suggesting saturation of the metabolic enzymes.

For midazolam, 14 metabolites were observed. Well known hydroxymetabolites 4-hydroxymidazolam (M1) and 1-hydroxymidazolam (M2)) were observed, together with metabolites hydroxylated/oxidated in methylimidazole (M3, M4). Identification of M2 was confirmed by using in-house standard for 1-hydroxymidazolam. Methylimidazole was the site of most of the other metabolic reactions observed, including further hydrogenation of M1 (M7), further hydrogenation of M2/M3/M4 (M8), further oxidation and hydrogenation of M2/M3/M4 (M9, M10, M1 1), and hydroxylation in chlorodihydrobenzodiazepine with further hydrogenation in methylimidazole (M6). In addition, dioxidated metabolite M5 (site not known) was observed, as well as reductive defluorination (M12), and glucoside conjugations directly to midazolam (M13) and after hydroxylation and hydrogenation (M7, M8 or M9) (M14).

The metabolite profile for midazolam did not show very clear main metabolites.

However, the glucoside conjugation M13, together with hydroxylated/oxidated M 1 - M3, oxidated and hydrogenated M6 and M7, and dioxidated and hydrogenated M9 - M1 1 were the most abundant metabolites observed in grasshopper brain, their relative abundances changing a bit as a function of incubation concentrations. Almost all of the observed metabolites were present in both brain and incubation well samples in all test concentrations, although the amounts were significantly lower in well samples compared to the actual brain samples. Only M5 and M14 were not observed in any of the incubation well samples and M8 was observed in all other samples but not in 1 μΜ incubation well samples. Similarly to clozapine, the relative abundance of metabolites with respect to midazolam was decreasing as a function of incubation concentration, suggesting saturation of enzymatic reactions.

Scheme 3. The suggested metabolic pathways for the observed Clozapine metabolites.

Scheme 5. MS/MS fragment ion identification for Clozapine

m/z 255 m/z 206 m/z 192 (radical) (radical) (radical)

+H = m/z 95 Table 10. Metabolite profile for clozapine in incubations with 1 , 3 and 10 μΜ initial concentration with grasshopper brain. Most abundant metabolites are denoted *.

% of combined LC/MS peak area in each sample (parent + metabolites = 100%)

Cloza

Sample M1 M2 M3 M4 M5 M6 M7 M8 M9 M10 M11 M12 M13 M14 M15 M16 M17 M18

-pine

1 (1 μΜ brains 1 +2) 44.2 - 0.01 0.62 - 0.18 - 52.6* 0.15 0.17 - 0.01 0.07 1 .58* 0.1 1 0.02 0.24 0.03 0.40

2 (1 μΜ brains 3+4) 42.3 - 0.004 0.55 - 0.15 - 54.7* 0.13 0.14 - 0.002 0.09 1 .73* 0.05 0.02 0.09 0.01 0.28

3 (1 μΜ brains 5+6) 44.3 - 0.01 0.92 - 0.20 - 50.9* 0.14 0.17 - - 0.21 2.77* 0.08 0.05 0.20 0.02 0.28

4 (1 μΜ ννβΙΙ 1 +2) 93.6 - - 0.50 - 0.12 - 4.72* - - - - - 1 .06* - - - - -

5 (1 μΜ ννβΙΙ 3+4) 95.6 - - 0.48 - 0.06 - 3.41 * - - - - - 0.49 - - - - -

6 (1 μΜ ννβΙΙ 5+6) 94.3 - - 0.56 - 0.12 - 4.20* - - - - 0.01 0.79 - - - - -

7 (3 μΜ brains 1 +2) 63.5 0.01 0.04 0.87 0.01 0.38 0.01 32.1 * 0.15 0.12 0.01 0.02 0.15 2.02* 0.31 0.02 0.06 0.23 0.37

8 (3 μΜ brains 3+4) 58.5 0.01 0.03 0.97 0.01 0.36 0.01 35.2* 0.17 0.16 0.01 0.02 0.19 2.56* 0.63 0.03 0.12 0.99 0.30

9 (3 μΜ brains 5+6) 61 .4 0.01 0.03 0.71 0.01 0.26 0.01 35.0* 0.17 0.15 0.01 0.01 0.05 1 .84* 0.06 0.01 0.13 0.03 0.39

10 (3 μΜ well 1 +2) 96.9 - - 0.35 - 0.1 1 - 2.06* - - 0.002 0.02 0.56 - - - - -

1 1 (3 μΜ well 3+4) 96.0 - - 0.40 - 0.14 - 2.76* - - 0.004 0.02 0.67 - - - - -

12 (3 μΜ well 5+6) 95.9 - - 0.45 - 0.1 1 - 2.90* - - - 0.01 0.65 - - - - -

13 (10 μΜ brains 1 +2) 78.2 0.02 0.04 0.47 0.01 0.36 0.03 14.7* 0.10 0.03 0.01 0.02 0.59 4.28* 0.35 0.04 0.09 0.51 0.29

14 (10 μΜ brains 3+4) 79.1 0.02 0.04 0.48 0.02 0.39 0.03 15.8* 0.13 0.04 0.02 0.02 0.22 2.67* 0.29 0.01 0.07 0.60 0.37

15 (10 μΜ brains 5+6) 77.9 0.02 0.04 0.50 0.01 0.31 0.03 17.8* 0.13 0.05 0.01 0.02 0.22 2.09* 0.24 0.01 0.07 0.49 0.37

16 (10 μΜ well 1 +2) 97.3 - 0.00 0.29 - 0.09 - 1 .63* 0.01 - 0.01 0.001 0.05 0.65 - - - - -

17 (10 μΜ well 3+4) 97.2 - 0.01 0.26 - 0.1 1 - 1 .72* 0.01 - 0.01 0.005 0.03 0.65 - - - - -

18 (10 μΜ well 5+6) 97.9 - 0.00 0.26 - 0.08 - 1 .24* 0.01 - 0.01 0.001 0.02 0.45 - - - - -

Table 11. Metabolite profile for midazolam in incubations with 1 , 3 and 10 μΜ initial concentration with grasshopper brain. Most abundant metabolites are denoted *.

Protocols used in Example 7

Experimental preparation: Desert locusts, Schistocerca gregaria (L), used in the present study were obtained from a commercial animal wholesaler (Petra Aqua, Prague, Czech Republic). At arrival the locusts were housed under crowded conditions in ELC insect gages supplied by Small-life supplies (Great Britain) and adapted to a 10: 14 hour dark/light cycle with a temperature of 25°C to 34°C depending on locust proximity to the light bulb. The animals were fed Chinese cabbage and wheat bran ad libitum. All experiments were carried out 2 to 3 weeks after adult emergence. The insect brains used in the ex vivo experiments were dissected by cutting off the frontal part of the head, above the esophagus. The brain, comprising compound eyes (including their nerve connections) of the insect head and with the neural lamella was dissected free from fat and tissue using fine forceps, and placed in a microwell plate (96U Microwell; Nunc) containing 250 μΙ of the compound of interest dissolved in an insect buffer.

Chemicals: All drugs used in the insect ex vivo studies and HEPES were purchased from Sigma-Aldrich (Stockholm, Sweden). All other chemicals were analytical reagent grade. The test compounds were purchased from Sigma-Aldrich (Stockholm, Sweden).

Test solution preparation: Stock solutions were prepared by dissolving the test compounds in DMSO. Final test solution concentrations were obtained by diluting stock solutions with insect buffer [NaCI 147 mM, KCI 10 mM, CaCI ₂ 4 mM, NaOH 3 mM, and HEPES 10 mM pH 7.2].

Experiments are performed by incubating brains in a solution of insect buffer and test compound(s). The ex vivo studies are performed in triplicate tests with two brains pooled in each test tube (i.e., a total of six brains in each experiment) as described in previous publications. Each brain was transferred to a well containing 250 μΙ of a solution containing the compound of interest diluted in insect buffer. The brains were removed from the compound solutions after fixed times of exposure, the neural lamellas and compound eyes were dissected in ice cold insect buffer and washed twice in ice cold insect buffer.

Exemplary Method: A cut is made through the frontal part of the head separating the brain and its nerve connections to the compound eyes, antenna and ocelli still in its cuticle shell. The brain is dissected in locust buffer (observe: with neural lamella and connections to compound eyes) and put into a microtiter well containing 250 μΙ of the test solution heated to 30°C in a block thermostat. After 15 or 45 minutes exposure time (i.e., the loading phase) the brain comprising the neural lamella and connections to the compound eyes is transferred from the well and washed. The brain comprising the neural lamella and connections to the compound eyes is put into a blank solution +/- verapamil for different amounts of time for further equilibration. Some brains are divided in the middle before being put into blank solutions (no Verapamil). Brains from two animals are put into 37.5 μΙ Millipore water. At the same time 100 μΙ samples are taken from each of the wells corresponding to each sample with brains (in total 200 μΙ from two wells) and put into 200 μΙ 40% ACN (25 μΙ + 25 μΙ + 950 μΙ 20% ACN for exposure solution). 112.5 μΙ of 100% ACN is added to the test tubes containing brains and the brains are sonicated and all test tubes centrifuged at 10 000 xg for 5 minutes at 4°C. 50 μΙ of the supernatants (brains) are transferred to analysis vials and diluted with 150 μΙ Millipore water. 200 μΙ of the well samples are transferred to analysis vials without being diluted.

The Method provides the following

- The first 15 and 45 minutes of exposure gives a measure of permeability rate of compounds, and production of metabolites in the brain and efflux of metabolites in the well to determine potential efflux mechanisms and concentration.

- After 45 minutes, in blank solution without exposure, gives an estimate of the elimination rate of the parent compound and metabolites. Analysing well concentration and compare with the concentration in the brain at any given time point, gives both free concentration in the brain, total concentration in the brain and free fraction in the brain. Depending on time point it is a measure with and without functional biological system.

- Adding Verapamil or any other chemical compound it is possible to evaluate drug- drug interactions, and how that impact drug uptake, metabolism and efflux. Other examples are BCRP inhibitor Ko-143, Mrp-1 and 2, CYP inhibitors.

By dividing the brain in the middle, or any other analogous method to disrupt the brain barrier integrity it is possible to circumvent the brain barrier and compare with an intact brain barrier, for example to evaluate free brain concentrations, total brain concentrations and free fraction in the brain.

At equilibrium the unbound concentration in the brain is equal to the concentration in the well and transfer of the brain from the sample solution to a blank medium, allows estimation of the unbound brain concentration when equilibrium is reached. The unbound concentration in the brain can thus be estimated either by using the intact brain, with and without additional chemicals or by using a brain which is divided and the brain barrier circumvented. Method A is useful method for estimating various properties needed in drug research:

• The first time points includes a viable brain with brain barrier integrity, an active metabolic brain, and transporter proteins such as Pgp and BCRP.

• The later time points can describe a viable brain as well as an energy depleted brain, with metabolic and transporter system not functionally optimally, but with the brain barrier integrity fully intact. • To expedite energy consumption to generate an energy depleted brain, chemical compound(s) can be added to the medium that make use of transporter system (e.g., Pgp) and/or metabolism

• To alter the energy consumption in the brain, the temperature in the experimental setup can be adjusted. To reduce the energy consuming reactions in the brain the temperature can be reduced while increasing temperature may expedite energy consumption leading to depletion of the biochemical energy storage in the brain

• Destroying the brain barrier integrity by cutting the brain in half or with any other brain barrier integrity disrupting technology, the unbound concentration in the brain without impact of the brain barrier can be measured similar to the brain slice methodology.

Independently combining 1 to 5 of these five set-up(s) of the model different properties of the compound can be calculated. These properties can be used themselves or used to estimate different parameters used in the drug discovery process.

Ctot,brain = total chemical compound(s) concentration in the brain (drug concentration in ICF and lSF)

C _{U b} rain = unbound (free) chemical compound concentration in the brain ISF (drug concentration measured medium)

F _ub (free fraction, brain)= F _ub = free fraction in the brain (C _un bound,brain/Ct ₀ t,brain)

C _U piasma = unbound chemical compound(s) concentration plasma = chemical compound(s) that is not bound to tissue and proteins in the plasma

C(buffer) ⁼ C( _m edium) ⁼ C(biood/buffer)= is the concentration of chemical compound(s) in the immersing medium or buffer in the well, if proteins are present as in blood it is the free concentration in the well.

Pgp = ProMDR1= chemical compound transporter efflux transporter (efflux pump) across brain barrier from brain to periphery

At steady state: Kpuu = C _Ut brainlC _UtPlasma

When KpUU=1 gives

fu, brain— F _U b~ C _U) brainlCtot,brain ~ C( _me dium)l Ctot,brain~ C _u (blood/buffer)l Ctot,brain

Vu,brain ~ k ^" \IF _u b ~ Ctot,brainl 'C (medium)- Ctot,brain/C _u (blood/buffer)

V _{U b} rain can be used to calculate

C _u ,ceii= brain intracellular unbound chemical compound concentrations = C _u ,p _/asma *Kpuu*Kpuu,cell

unbound concentrations chemical compounds in plasma

Receptor occupancy (RO) can be calculated using, C _to t,brain either from in vivo studies or ex vivo studies using the present model, F _ub can be calculated with the locust model described herein using the following equation: RO=(C _u, , _ra , (C _u, , _{ra n} + KD))*100%; where RO represents the percentages of receptors occupied in relation to the unbound concentration of chemical compound(s) at the target (C _u ,brain) and the potency of the drug (KD)

By comparing concentrations in brains and medium at different time points (early and late), metabolic and transporter systems impact on catabolic and efflux rates, and C _to t,brain and C _U nbound,brain be analysed.

F _ub for different studies can be compared for example: F _ub in the verapamil studies divided by the F _ub intact brain give an estimate on the involvement of Pgp and metabolism. Thus, F _ub (verapamil)/ F _ub = unity: No or little impact of verapamil (Pgp, metabolism), whereas F _ub (verapamil)/ F _ub < 1 or F _ub (verapamil)/ F _ub > 1 : transporters, metabolism, or both, have an influence on brain concentrations.

Other compounds than verapamil can be used for example Ko-143 can be used to study the efflux pump BCRP. Other pump inhibitors can be used to study specific efflux and influx pumps. In addition, CYP specific inhibitors and inducers can be used to study metabolism by specific CYPs. Several of these compounds can be combined to simultaneously analyze several transports and CYPs.

Steady state ratio of unbound brain to unbound plasma drug concentrations (K _P)UU ) is considered to be probably the most relevant measure of blood-brain barrier function. K _PiUU can be assessed by using the model presented here to estimate C _unb0U nd,brain and the well concentration before moving the brain to the blank media. Kpuu expresses the free concentration in the brain divided by the free concentration in plasma at equilibrium, i.e., Kp,uu=C _U nbound,b in/Cunbound,piasma- Thus, by using the model describe here where compounds solubilized in plasma (e.g., human or rodent) or plasma mimicking solutions it is possible to measure C _un bound,bmin and C _un bound,piasma and hereby K _p>uu .

Alternatively, C unbound, plasma can be measured using an alternative method for the specific species of interest, human, rodent or other mammalian. This data can be combined with the data using C _un bound, brain or F _U b using the model described herein. This allow estimation of Kpuu.

Different medium can be used in the experiment, the brains can directly be immersed with blood and/or other body fluids (e.g., from the CSF, the lymphatich system, intracellular matrix, brain matter), for example from humans or other mammalian species to evaluate, detect and analyze compounds that can penetrate the brain barrier into the brain and their metabolism in the brain or that interrupt the brain-barrier to increase passage of other chemical compounds and their metabolites. This can be used for evaluate CNS active and harmful compounds, including nanoparticles, gaseous chemical compounds immersed in a media (e.g., smoke and diesel particles), environmental chemical compounds and food including beverages. When said human or mammalian have been exposed to an environment containing chemical compounds, both on purpose or by accident. The mediums blood or other body fluids can originate from that a mammalian have been exposed or/and administrated a chemical compound(s), the body fluid have thereafter been collected by a syringe, dialysis, and/or biopsy and used as medium in the experiment. Alternatively, the body fluid(s) can be collected by syringe, dialysis, and/or biopsy and thereafter is a chemical compound(s) added to the body fluid and used in the experiment as a medium. The medium can originate from that a mammalian have been exposed or/and administrated a chemical compound(s), the body fluid(s) have thereafter been collected by a syringe, dialysis, and/or biopsy and an additional chemical compound(s) is added to the body fluid (used as medium in the experiment). An example of the latter is to expose a mammalian to a compounds collect he body sample an add additional chemical compound(s) to evaluate how these impact the brain uptake, metabolism and/or transporter mechanism of the chemical compound first exposed to the mammalian.

The medium can be exposed to the insect brain by using in-line dialysis, thus an apparatus can be connected directly to live mammalian (e.g., human, non-human primates, rodents, dog) to extract the body fluid (e.g., blood) so that the body fluid passes an immersing the insect brain in a loop that is connected back into the mammalian (e.g., human, non-human primates, rodents, dog). This loop can be applied both externally (outside the body) and internally ( in the body). Alternatively, the insect brain can be implanted in situ in a mammalian (e.g., human, non-human primates, rodents, dog) and after a specific exposure time the insect brain can extracted and analyzed to evaluate, detect analyze compounds that can penetrate the brain barrier into the brain and their metabolism in the brain or that interrupt the brain-barrier to increase passage of other chemical compounds and or impact their metabolism. This can be used for evaluate CNS active and harmful compounds, including nanoparticles, gaseous chemical compounds immersed in a media (e.g., smoke and diesel particles), environmental chemical compounds and food including beverages., without terminating the individual followed by dissection of the brain. Moreover, this method allows real-time monitoring of the effects of the brain and on the brain of chemical compounds.

Chemical compounds in environmental fluids and food can be exposed to the brain by using in-line exposure, thus an apparatus can be connected to the environmental fluid (e.g., water, earth, and/or air) or food so that the fluid is immersing the brain or immersing the brain in situ. The brain can also be exposed to the body fluid environmental chemical compounds, food using a microfluid device (lab-on-chip), fluid device and/or flow reactors allowing real-time monitoring. The V _Utbrain reflects the distribution of the drug inside the brain, i.e. drug bound to tissue plus drug inside the brain divided by the unbound drug concentration in the brain. C _un bound,brain can be measured using the current model where the brain is transferred to the blank medium. In this model C _un bound,brain is equal C _un bound,weii- The drug concentration inside the brain is equal C _to t- Thus, V _{U b} rain can be estimated through the parameters measured using the model presented here, i.e. In this model the brain barrier is intact and effect of the barrier is included this system.

A common way to assess V _U)br ain is the brain slice method. In this method a brain slice is placed in a well and at equilibrium C _un bound,brain is measured as the well concentration. Thus, in this method the brain barrier effect is circumvented. In Method A we describe a method using an insect brain and where the brain is split when transferred to the blank medium. In this system the brain barrier integrity is circumvented as in the brain slice method.

Trapping reactive metabolites

In the metabolic ID study clozapine we showed that it is possible to add a nucleophile or a nucleophile precursor to the assay to trap reactive intermediates that are formed, e.g. acetonitrile was added to the intact brain, that was subsequently metabolized to a reactive cyano nucleophile to trap two metabolites formed from clozapine. The metabolites formed had characteristic nitrile substitution. Adding the cyano directly to a brain with an intact brain barrier is problematic due to low permeability of the nucleophile.

Example 8 - Comparative Study of Model Systems

This Example provides a comparative study of the various model systems available to investigate penetration through the blood-brain barrier.

Method: A cut is made through the frontal part of the head separating the brain and its nerve connections to the compound eyes, antenna and ocelli still in its cuticle shell. The brain is dissected in locust buffer:

1. with neural lamella and connections to compound eyes +/- antennae

2. with neural lamella and without compound eyes or antennae

3. without neural lamella and without compound eyes or antennae

4. The brain remains in the cuticle shell including antennae

and put into a microtitre well containing 250 μΙ of the test solution heated to 30°C in a block thermostat. After 45 minutes exposure time the brain is removed from the well and the neural lamella, eyes and antennae dissected and washed in ice cold locust buffer. Brains from two animals are put into 37.5 μΙ Millipore water. At the same time 25 μΙ samples are taken from each of the wells corresponding to each sample with brains (in total 50 μΙ from two wells) and put into 950 μΙ 20% ACN. 1 12.5 μΙ of 100% ACN is added to the test tubes containing brains and the brains are sonicated and all test tubes centrifuged at 10 000 xg for 5 minutes at 4°C. 50 μΙ of the supernatants (brains) are transferred to analysis vials and diluted with 150 μΙ Millipore water. 200 μΙ of the well samples are transferred to analysis vials without being diluted.

Locust buffer prepared: 20/1-2016

Locust species/age: Schistocerca gregaria, adult, delivered from Petra Aqua 16/2-2016 Test compounds:

Atenolol 3 μΜ

Carbamazepine 3 μΜ

nd = not detectable, where the measured levels were below the detection limit The results show that under conditions 3 (i.e. without neural lamella), the BBB is compromised and atenolol penetration is high. The results for carbamazepine under conditions 3 are also the highest measurement, but the difference is not as noticeable as the penetration of this compound through an in-tact BB is high to begin with.

The results under conditions 4 show that the measurable penetration is much lower when the brain remains in the insect cuticle shell during exposure.

The results from 1 + and 1- show that the presence of the antennae can strongly influence the results obtained. This allows the method disclosed herein to be used to determine the sensitivity of insect antennae to a chemical compound. The presence or absence of compound eyes also has an effect, with conditions 2 giving results that are approaching those of the brain without neural lamella.

The data from Examples 1-A to 1-0, and 8 show that compounds with low blood-brain barrier permeability (such as atenolol) and compounds that are actively transported out from the brain (such as quinidine and quinidine+verapamil, Examples 1-C to 1-F) display significantly lower brain uptake when the compound eyes are retained on the brain matter during exposure in accordance with the methods of the disclosure (e.g. conditions 1- from Example 8) compared to previous methods where the compound eyes are removed before exposure (e.g., conditions 2 and 3 in Example 8). However, for compounds that readily permeate the blood-brain barrier (e.g., carbamazepine, Example 1-L to 1-M), the brain uptake with and without the compound eyes is similar.

The fact that compounds with low BBB permeability enter the brain when the compound eyes are removed (e.g., Example 1-A and 1-B) demonstrates that removal of the compound eyes may compromise the integrity of the blood-brain barrier, introducing a possible leakage. However, as shown by Example 1-G and 1-H, the leakage does not significantly affect the total brain concentration of compounds such as carbamazepine that readily permeate the blood- brain barrier (e.g. Example 1G and 1 H). Thus, removal of the compound eyes introduces leakage in the brain barrier which affects the total brain concentrations of compounds with low blood-brain permeability, while readily permeable compounds may already permeate the blood- brain barrier at higher rates than facilitated by diffusion through the holes introduced by removal of the compound eyes.

In addition, the results from conditions 1+ and 1- in Example 8 show that a similar leakage is not introduced when the antenna are removed. Thus, atenolol is not detected (concentrations below detection limit) independent of whether or not the antennae are intact. In view of this, the observation on atenolol using test conditions 2 in Example 8 (brain and neural lamella, excluding compound eyes and antennae) can be attributed to the leakage caused by removal of the compound eyes.

Surprisingly, the antennae affect the brain concentrations of the compound with high BBB permability. Thus, retaining the antennae during the exposure phase decreases the concentrations in the brain to unrealistically low levels of the highly permeable compound carbamazepine. As seen before, introducing a leakage by removing the eyes had very little effect on carbamazepine (c.f. conditions 2 compared to conditions 1-), which excludes the possibility that the higher concentration seen without antennae depends on a leakage caused due to removal of the antennae. Thus, we have found that the conditions 1- in Example 8 best predict compound's permeability both for the compounds with low permeability and for compounds with high permeability.

The Examples show that the methods described herein provide a useful methodology to investigate various aspects of the brain, including the penetration of chemicals through the blood-brain barrier, how chemicals affect the integrity of the blood-brain barrier, and how chemicals are distributed and metabolized in the brain. Dissecting out the insect brain in combination with the compound eyes ensures the blood-brain barrier remains in tact during further in vitro studies, providing improved methodologies for studying the properties of the blood-brain barrier and brain.