WO2020141293A1

WO2020141293A1 - Eye assay

Info

Publication number: WO2020141293A1
Application number: PCT/GB2019/050021
Authority: WO
Inventors: Robert Alastair Howie SCOTT
Original assignee: Scotthealth Ltd
Priority date: 2019-01-04
Filing date: 2019-01-04
Publication date: 2020-07-09

Abstract

The present invention relates to amethod for evaluating one or more of corneal, transconjunctival and scleral penetration of a topical preparation,comprising; a) preparing or obtaining a sample of eye tissue; b) contacting at least one portion of the eye tissue sample with a volume of a topical preparation, wherein the or each portion of eye tissue is independently selected from the group of: a portion of the cornea with epithelium intact, a portion of cornea with epithelium removed, a portion of the sclera and a portion of the conjunctiva, or any combination thereof; c) sampling at least one portion of the eye tissue sample independently selected from the group of: cornea, aqueous humour, iris, lens, vitreous body, retina, choroid, retinal pigment epithelium, sclera and optic nerve stump; and d) analysing the or each sampled portion for at least one component of the topical preparation.

Description

Eve Assay

Technical Field of the Invention

The present invention relates to a method for evaluating ocular tissue penetration of a topically-applied ocular drug, and an assay incorporating the same. Background to the Invention

Ocular drugs are preferably designed to be administered topically (e.g. eye drops), rather than by other methods such as by injection, due to the ease of preparation and ease of self-administration associated with topical formulations.

Ex vivo methods are commonly used for assessing penetration of ocular drugs. Prior art ex vivo methods for assessing the efficacy/penetration of topical ocular drug formulations generally consist of applying a drop of said formulations to the front surface of an eye, allowing it to penetrate into the eye for a certain amount of time, and then analysing the eye to determine drug penetrance. However, such methods give no information on the relative permeability of different parts of the eye (such as the cornea, sclera and conjunctiva) to the formulation as the drop is applied to the whole of the front surface of the eye.

Other methods involve excising parts of the eye, such as the cornea or a portion of the cornea, and testing them on their own. However, these methods require time- consuming preparation of the eye tissue sample, and involve at least partial destruction of the eye tissue which may result in less reliable results.

It would therefore be advantageous to provide an ex vivo method of assessing penetrance of compounds (especially ocular drugs) into ocular tissues, which requires less sample preparation, easier sample preparation and/or less invasive/destructive sample preparation than prior art methods. It would further be advantageous to provide an assay based on such methods, which may allow for optimisation of topical ocular drug formulations and/or administration protocols thereof. It is therefore an aim of the invention to overcome or at least partially mitigate at least one problem of the prior art.

Summary of the Invention

According to a first aspect of the invention there is provided a method for evaluating one or more of corneal, transconjunctival and scleral penetration of a topical preparation, comprising; a) Preparing or obtaining a sample of eye tissue;

b) contacting at least one portion of the eye tissue sample with a volume of a topical preparation, wherein the or each portion of eye tissue is independently selected from the group of: a portion of the cornea with epithelium intact, a portion of cornea with epithelium removed, a portion of the sclera and a portion of the conjunctiva, or any combination thereof; c) sampling at least one portion of the eye tissue sample independently selected from the group of: cornea, aqueous humour, iris, lens, vitreous body, retina, choroid, retinal pigment epithelium, sclera and optic nerve stump; and d) analysing the or each sampled portion for at least one component of the topical preparation.

The eye tissue may comprise porcine eye tissue. Porcine eye tissue is advantageous as porcine cornea have similar corneal permeability coefficients to the human eye.

Step a) may comprise preparing or obtaining a sample of eye tissue from a harvested eye, such as a harvested porcine eye.

Step a) may comprise dissecting an eye and/or removing unneeded tissue from an eye. The method may comprise removing unneeded extraocular and/or adnexal tissue from the eye, which may include removing the rectus muscles at their insertions on the sclera, removing the retro-orbital adipose tissue (fat), removing the conjunctiva at the limbus and/or removing a portion of the conjunctiva (e.g. trimming the conjunctiva), for example. The conjunctiva may be removed if the eye tissue sample is not going to be tested for trans-conjunctival penetration.

When step b) comprises contacting a portion of cornea with epithelium removed, step a) may comprise debriding a portion of the corneal epithelium.

Step a) may comprise irrigating the eye tissue sample to clean it and remove any debris. The eye tissue sample may be irrigated with PBS (phosphate-buffered saline), for example.

Step b) may comprise contacting a single portion of the eye tissue sample (for example only the conjunctiva), or contacting more than one portion of the eye tissue sample (for example the cornea and the sclera). In some embodiments the portion of the eye tissue may comprise two or more different areas of the eye, such as the cornea and sclera, for example, and the combined tissue portion may be used in step b).

Step b) may comprise contacting the or each portion of the eye tissue sample with at least one delivery device configured to contain the or each volume of the topical preparation. The or each delivery device may comprise a well, comprising at least one aperture through which the topical preparation may be delivered. Step b) may comprise contacting the or each portion of eye tissue with at least one well comprising a perimeter wall, such as a ring, the volume enclosed by the walls forming an aperture through the well, and then at least partially filling the interior (i.e. the aperture) of the or each well with a volume of the topical preparation. The or each well may have a diameter, length and/or width of at least and/or no more than 1 mm, 2 mm, 3 mm, 4 mm, 5 mm, 6 mm, 7 mm, 8 mm, 9 mm or 10 mm. The or each well may be a LASEK alcohol well, for example. The delivery device may be placed against the or each portion of eye tissue sample before adding the topical preparation or at least partially filling the device with the topical preparation. The or each delivery device may be held against the or each portion of eye tissue with sufficient force so as to form a liquid-tight seal between the two, such that substantially no topical preparation leaks out of the delivery device.

In embodiments wherein more than one portion of the eye tissue is contacted with the topical preparation, the method may comprise contacting each portion with volumes of the preparation via different delivery devices, e.g. in different wells, and/or may comprise contacting at least two portions with a volume of the preparation in a single well. Any number and combination of these are intended to be covered by the invention.

The method may comprise contacting at least and/or no more than 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95% of the surface area of the or each portion of the eye tissue sample. In some embodiments, the method may comprise contacting substantially 100% of the or each portion of the eye tissue sample.

The method may comprise contacting the or each portion of the eye tissue sample with the topical preparation for at least and/or no more than 5 seconds, 10 seconds, 15 seconds, 20 seconds, 30 seconds, 1 minute, 2 minutes 3, minutes, 4 minutes, 5 minutes, 10 minutes, 15 minutes, 20 minutes, 30 minutes, 40 minutes 50 minutes or 60 minutes.

In preferred embodiments, the method may comprise contacting the or each portion of the eye tissue sample for between 5 seconds and 20 minutes, preferably between 5 seconds and 15 minutes, more preferably between 5 seconds and 10 minutes, such as between 10 seconds and 10 minutes.

The above methods thus allow for very precise control over the site(s), area(s) and time(s) of contact between the topical preparation and the eye tissue sample, without the need for time-consuming and damaging tissue preparation, which cannot be achieved by prior art methods, by simply contacting the sample with a delivery device (preferably a well) on its surface.

The method may further comprise a step of incubating and/or washing the eye tissue sample after step b) but before step c). The eye tissue sample may be incubated at ambient temperatures, such as room temperature, and may be incubated for at least 5 min, 10 min, 15 min, 20 min, 30 min, 40 min, 50 min or at least 60 min, for example. The eye tissue sample may be washed with PBS, for example.

The topical preparation may comprise one or more pharmaceutical compounds and/or one or more cell penetration enhancement agent.

The or each pharmaceutical compound may comprise a drug, preferably an ocular drug, for example Lutein. The or each cell penetration enhancement agent may comprise a cell penetrating peptide, for example a cell penetrating peptide comprising polyarginine or oligoarginine.

The or each cell penetration enhancement agent may comprise a coating, a nanoemulsion and/or nanospheres (such as nanospheres modified with a peptide such as deslorelin or glycyrrhizin).

The or each cell penetration enhancement agent may comprise a penetration enhancer (also called sorption promoters or accelerants). Penetration enhancers penetrate into tissues and reversibly decrease barrier resistance. The or each penetration enhancer may comprise: sulphoxides (such as dimethylsulphoxide, DMSO); Azones (e.g. laurocapram); pyrrolidones (for example 2-pyrrolidone, 2P); alcohols and alkanols (ethanol, or decanol); glycols (for example propylene glycol, PG); surfactants; glutaric or citric anhydride; and terpenes.

The or each cell penetration enhancement agent may comprise active fragments of molecules and/or molecules which have had their charge modified to improve tissue penetration.

Potential sites and modes of action identified for penetration enhancers include the intercellular lipid matrix in which the accelerants may disrupt the packing motif, desmosomal connections between epithelial cells, or altering metabolic activity within the ocular barrier tissues, or exert an influence on the thermodynamic activity/solubility of the drug in its vehicle.

The concentration of the or each pharmaceutical compound and/or the or each cell penetration enhancement agent in the topical preparation may be at least and/or no more than 0.01 mg/mL, 0.02 mg/mL, 0.03 mg/mL, 0.04 mg/mL, 0.05 mg/mL, 0.10 mg/mL, 0.20 mg/mL, 0.30 mg/mL, 0.40 mg/mL, 0.50 mg/mL, 1.0 mg/mL, 2.0 mg/mL, 3.0 mg/mL, 4.0 mg/mL, 5.0 mg/mL or 10 mg/mL.

In preferred embodiments, the concentration of the or each pharmaceutical compound and/or the or each cell penetration enhancement agent in the topical preparation may be between 0.01 mg/mL and 5.0 mg/mL, more preferably between 0.05 mg/mL and 5.0 mg/mL. Step c) may comprise sampling any number of portions of the ocular tissue, for example two, three, four or more portions, or all portions from the sample.

Step c) may comprise at least one of the following steps: removing at least one portion (e.g. extraction or excision); freeze-thawing the or each portion; and homogenising the or each portion. In preferred embodiments, step c) comprises performing all three of the above-mentioned steps.

Step c) may comprise sampling the or each portion separately or sampling at least two portions together. At least two portions may be sampled together for example by removing, freeze-thawing and/or homogenising each portion together, or by removing and freeze-thawing each portion separately and then either homogenising them together or homogenising them separately and combining the homogenised portions.

Step c) may comprise collecting a sample of the aqueous humour without dissecting or substantially cutting the tissue. The aqueous humour may be collected with a syringe, for example, which may for example be inserted into the aqueous humour through the corneal limbus.

Step c) may comprise collecting a sample of the aqueous humour by freezing the eye tissue sample and dissecting out at least a portion of the solid frozen aqueous humour.

Step c) may comprise a step of centrifuging the or each sampled portion to separate the sample by size.

Step d) may comprise analysing the or each portion with high performance liquid chromatography (HPLC), gas chromatography (GC) and/or mass spectrometry (MS). Step d) may comprise analysing the or each portion with liquid chromatography- mass spectrometry (LC-MS).

Step d) may comprise qualitative analysis (e.g. analysing for the presence of at least one component of the topical preparation) and/or quantitative analysis (e.g. analysing for the amount, proportion, mass and/or concentration of at least one component of the topical preparation, for example) At least one component to be analysed for in step d) may comprise a pharmaceutical compound, a drug, an ocular drug (for example Lutein), a cell penetration enhancement agent, a cell penetrating peptide (for example polyarginine or oligoarginine containing cell penetrating peptides), a coating, a nanoemulsion and/or nanospheres (such as nanospheres modified with a peptide such as deslorelin or glycyrrhizin).

The method may further comprise a step of removing any remnant topical preparation from the or each delivery device after step b) and after or (preferably) before step c). The method may further comprise a step of analysing any remnant topical preparation, for example measuring its volume and/or analysing its composition, such as concentrations of the or each component, for example. By measuring the concentration and volume of the or each component in any remnant topical preparation, it is possible to calculate the mass of the or each component which did not leave the delivery device and was therefore not taken up into the ocular tissues. This therefore provides a simple method of measuring the total uptake of the or each component into the ocular tissues.

According to a second aspect of the invention there is provided a method for performing an ex vivo assay for evaluating one or more of corneal, transconjunctival and scleral penetration of a topical preparation, comprising performing the method of the first aspect of the invention on a plurality of eye tissue samples, and varying at least one parameter selected from: topical preparation composition; the or each portion of the eye tissue sample contacted with the topical preparation; and the length of time that the topical preparation is in contact with the or each portion of the eye tissue sample.

Varying the topical preparation composition may comprise varying one or more of the following: the pharmaceutical compound(s), for example drug(s) (such as ocular drug(s)), present in the preparation; the number of different pharmaceutical compounds; the concentration of the pharmaceutical compound(s); the cell penetration enhancement agent(s), for example cell penetrating peptide(s), present in the preparation; the number of cell penetration enhancement agents; and the concentration of the cell penetration enhancement agent(s).

Detailed Description of the Invention In order that the invention may be more clearly understood one or more embodiments thereof will now be described, by way of example only, with reference to the accompanying drawings, of which:

Figure 1 illustrates a LASEK alcohol well suitable for use in embodiments of the methods of the invention

Figure 2 is a table summarising an embodiment of the method of the second aspect of the invention

Figure 3 is a table summarising the different portions of the eye tissue from each eye tissue sample of Figure 3 to be sampled at each time point in an embodiment of the method of the second aspect of the invention.

Figure 1 illustrates a delivery device in the form of a LASEK alcohol well 100 suitable for use in embodiments of the methods of the invention. The LASEK alcohol well 100 comprises a handle portion 101 and a tip portion 102. The tip portion 102 comprises a circular metal ring 103 comprising an aperture 104 therethrough. In use, the metal ring 103 is located over the appropriate portion(s) of the eye tissue sample and pressed down with sufficient force to form a liquid-tight seal between the surface of the eye tissue sample and the metal ring 103. The aperture 104 and ring 103 when placed on the portion of eye tissue sample thus forms a“well” with the eye tissue sample forming the bottom of the well, onto which a topical preparation can be applied. A known volume of a topical preparation is then placed in the well formed within the ring 103 and the LASEK alcohol well 100 maintained in position for the desired amount of time, in order to keep the topical preparation in contact with the portion of the eye tissue sample within the well for said desired amount of time. In some embodiments, after the desired time has elapsed, any remnant topical preparation may be removed from the well, for example with a pipette, before the LASEK alcohol well 100 is removed.

Figure 2 is a table summarising an embodiment of the method of the second aspect of the invention. In this embodiment, a number of different embodiments of the method of the first aspect of the invention are performed wherein the portion of the eye contacted with the topical preparation (“Group” column), the topical preparation composition (“Topical Preparation Components” column) and the length of time that the topical preparation is in contact with the portion of the eye tissue sample (“Application time” column) are varied. Six eyes are used for each combination of parameters. As can be seen from Figure 2, PBS is used as a control.

Figure 3 is a table summarising the different portions from each eye tissue sample of Figure 3 sampled to be sampled at each time point in an embodiment of the method of the second aspect of the invention. As can be seen in Figure 3, various portions (“Sample Taken” column) of each eye within each group at each time point are sampled and analysed.

The methods illustrated by Figures 2 and 3 thus allow very precise results to be obtained with regard to the penetration properties of components within the topical preparation through specific portions of an eye, while requiring less sample preparation, easier sample preparation and/or less invasive/destructive sample preparation than prior art methods.

The one or more embodiments are described above by way of example only. Many variations are possible without departing from the scope of protection afforded by the appended claims.

Claims

1) A method for evaluating one or more of corneal, transconjunctival and scleral penetration of a topical preparation, comprising; a) preparing or obtaining a sample of eye tissue; b) contacting at least one portion of the eye tissue sample with a volume of a topical preparation, wherein the or each portion of eye tissue is independently selected from the group of: a portion of the cornea with epithelium intact, a portion of cornea with epithelium removed, a portion of the sclera and a portion of the conjunctiva, or any combination thereof; c) sampling at least one portion of the eye tissue sample independently selected from the group of: cornea, aqueous humour, iris, lens, vitreous body, retina, choroid, retinal pigment epithelium, sclera and optic nerve stump; and d) analysing the or each sampled portion for at least one component of the topical preparation 2) A method as claimed in claim 1, wherein the eye tissue comprises porcine eye tissue.

3) A method as claimed in claim 1 or 2, wherein step a) comprises dissecting an eye and/or removing unneeded tissue from an eye.

4) A method as claimed in any preceding claim comprising contacting a portion of cornea with epithelium removed, wherein step a) comprises debriding a portion of the corneal epithelium.

5) A method as claimed in any preceding claim, wherein step b) comprises contacting substantially a single portion of the eye tissue sample.

6) A method as claimed in any one of claim 1 to 4, wherein step b) comprises contacting more than one portion of the eye tissue sample. 7) A method as claimed in any preceding claim, wherein step b) comprises contacting the or each portion of eye tissue with at least one delivery device containing the topical preparation.

8) A method as claimed in claim 7 when dependent on claim 6, wherein step d) comprises contacting more than one portion of the eye tissue sample with the same delivery device.

9) A method as claimed in claim 7 or 8, wherein the delivery device comprises a ring for placing on the portion of the eye tissue to form a well.

10) A method as claimed in any preceding claim, wherein the topical preparation comprises one or more pharmaceutical compounds.

11) A method as claimed in claim 10, wherein at least one pharmaceutical compound comprises an ocular drug.

12) A method as claimed in claim 11, wherein the at least one ocular drug comprises Lutein.

13) A method as claimed in any preceding claim, wherein the topical preparation comprises one or more cell penetration enhancement agents.

14) A method as claimed in claim 13, wherein at least one cell penetration enhancement agent comprises a cell penetrating peptide.

15) A method as claimed in claim 14, wherein the at least one cell penetrating peptide comprises polyarginine or oligoarginine.

16) A method as claimed in any preceding claim, wherein step c) comprises removing at least one portion of the eye tissue sample, freeze-thawing the or each portion and homogenising the or each portion.

17) A method as claimed in any preceding claim where step c) comprises sampling the aqueous humour, wherein the aqueous humour is sampled without dissecting or substantially cutting the tissue. 18) A method as claimed in claim 7 or 8, or any one of claims 9 to 17 when dependent on claims 7 or 8, wherein the method further comprises a step of removing any remnant topical preparation from the or each delivery device and analysing it, after step b). 19) A method for performing an ex vivo assay for evaluating one or more of corneal, transconjunctival and scleral penetration of a topical preparation, comprising performing the method as claimed in any one of claims 1 to 18 on a plurality of eye tissue samples, and varying at least one parameter selected from: topical preparation composition; the or each portion of the eye tissue sample contacted with the topical preparation; and the length of time that the topical preparation is in contact with the or each portion of the eye tissue sample.

20) A method as claimed in claim 19, wherein varying the topical preparation composition comprises varying one or more of the following: the or each pharmaceutical compound present in the preparation; the number of different pharmaceutical compounds; the concentration of the or each pharmaceutical compound; the or each cell penetration enhancement agent, for example cell penetrating peptide, present in the preparation; the number of cell penetration enhancement agents; and the concentration of the or each cell penetration enhancement agent. 21) A method as claimed in claim 20, wherein the or each pharmaceutical compound comprises an ocular drug and/or the or each cell penetration enhancement agent comprises a cell penetrating peptide.