WO2013057401A1

WO2013057401A1 - Device for studying the permeability of artificial or synthetic biological membranes

Info

Publication number: WO2013057401A1
Application number: PCT/FR2012/052149
Authority: WO
Inventors: Damien SALMON; Fabrice Pirot; Laura RODRIGUEZ; Karine Padois; Céline MOCH; Françoise FALSON; Christine Pivot
Original assignee: Universite Claude Bernard Lyon 1; Hospices Civils De Lyon
Priority date: 2011-10-10
Filing date: 2012-09-25
Publication date: 2013-04-25
Also published as: FR2981156A1; FR2981156B1

Abstract

Diffusion cell comprising a donor compartment intended to contain a composition of chemical compounds to be assayed and a receiver compartment intended to contain a receiver medium collecting the quantity of chemical compound having passed through the artificial or synthetic biological membrane, intended to be separated by an artificial or synthetic biological membrane, characterised in that: • - the receiver compartment is embodied in the form of a multi-well plate, • - the donor compartment is embodied in the form of a plurality of individual compartments whereof the number is equal to the number of wells, • - the membrane is divided into as many fragments as there are wells, Method of measuring the diffusion of a molecule through a biological membrane implementing the cell and use of a thermoreversible receiver medium.

Description

DEVICE FOR STUDIING THE PERMEABILITY OF BIOLOGICAL, ARTIFICIAL OR SYNTHETIC MEMBRANES

The invention relates to the pharmaceutical, medical, veterinary and cosmetic fields.

It relates to a device for studying the permeability of biological, artificial or synthetic membranes to chemical compounds, hereinafter referred to as "diffusion cell". In the remainder of the description and in the claims, the following terms mean:

- biological membranes: isolated tissues (e.g., epithelium, endothelium, connective tissue) or samples of human, animal or plant organs (e.g., skin, stomach, intestine, bladder, lungs);

artificial membrane: a thin layer of chemically or physically modified natural material (e.g., cellulose ethers);

synthetic membrane: a thin layer of material obtained by chemical synthesis (e.g., polysiloxanes, polytetrafluoroethylene).

Each of these membranes has a different permeability and selectivity to chemical compounds. The study of membrane permeability to chemical compounds is a key step for the development of human or veterinary health products, and cosmetics. Such a study is also required for different types of consumer products under the REACH European Regulation. In fact, these permeability studies make it possible to estimate, in part, the tissue pharmacokinetics (i.e., absorption, distribution, elimination) of a chemical compound. In these studies, one or more chemical compounds of interest are dispersed in liquid, semi-solid or solid formulations or vehicles and then contacted with the membrane surface. They then undergo penetration (i.e., transport in the membrane) and permeation (i.e., transport through the membrane). The determination of penetration and permeation characterizes the permeability of a biological, artificial or synthetic membrane to a chemical compound.

As already mentioned, the devices used for the in vitro or ex vivo study of the membrane permeability are called diffusion cell (CDD). Different types of CDD are currently described according to their geometry. The fixed-term contracts include donor compartment and a recipient compartment separated by a biological, artificial or synthetic membrane. The entire device can be positioned vertically (so-called Franz CDD) or horizontally (eg, CDD called Ussing chamber). The recipient compartments are filled with a liquid (eg, solution of electrolytes or colloidal) whose composition is adapted according to the properties of the chemical compound (or permeant) to be studied. Thus, the study of the permeability of a chemical compound exhibiting marked hydrophobicity requires the addition to the receiving liquid of adjuvants such as:

(i) co-solvents (e.g., ethanol, methanol, polyethylene glycols),

(ii) surfactants (e.g., polysorbates, poloxamers),

(iii) macro molecules (e.g., serum albumin, hydroxyethylstarch).

In order to seal the device, a clamp is placed between the donor and recipient compartments. The membrane placed between the two compartments held by the clamp acts as an O-ring for the entire device. An example of Franz's CDD is shown in Figure 1.

In addition, in order to approach the physiological conditions, the CDD recipient compartment is maintained at a constant temperature of 37 ° C. The temperature on the surface of the membrane facing the donor compartment is then close to 33 ° C. Depending on the intended applications, the donor compartment can be kept at a constant temperature. The geometry of the donor compartment is suitable for depositing liquid, semi-solid and solid formulations. The end of the filing procedure coincides with the start of the permeability study.

At a regular time interval, an aliquot of the receptor solution is removed by means of a syringe, and then replaced with an equivalent volume of the receiving solution, in order to maintain a constant volume in the recipient compartment. At the end of the treatment period, the donor and receiver compartments are emptied. The permeate is extracted from the membrane by a suitable mechanical or chemical treatment.

Permeate assays in the receptor phase aliquots and membrane extracts are used to determine permeate permeability parameters (ie, permeability coefficient, mass of chemical compound diffusing across the membrane). per unit of time and surface or transmembrane flow) and the membrane distribution (eg, tissue distribution in epithelium) of the permeate.

Firstly, the use of an aqueous solution in the recipient compartment is likely to induce:

(i) hyperhydration of biological membranes,

(ii) extraction of endogenous compounds from biological membranes,

(iii) microbial growth during the study,

(iv) the migration of electrolytes, co-solvents, and surfactants into biological membranes which may modify the initial permeability of the membranes,

(v) the formation of air bubbles against the membrane during heating of the aqueous solution.

To solve this problem, the document Levintova, Y., F.M. Plakogiannis, and R.A. Bellantone, "An improved method for measuring skin permeability thai controls excess hydration of skin using modified Franz diffusion cells." Int J Pharm, 2011 discloses a modified Franz CDD in which the donor and optionally recipient compartments contain a gelled solution. More specifically, the solution contains 0.5% by weight of Methocel®. It is obtained by mixing methocel® in powder form with water at 80-90 ° C., at which temperature the mixture is liquid. Below this temperature, the mixture gels. Thus, at the time of sample taking to calculate the permeability of the membrane, said sample is in gelled form. It follows that the sampling operation as such is not only delicate but also very complicated. In addition, the production of CDD comprising blown glass elements has a significant economic cost. In addition, and above all, the monocompartmental structure of Franz's cell makes it necessary to take periodic aliquots of receptors at regular intervals and to replace them with an equivalent volume, which makes handling time long and often imprecise.

US-A-6 043 027 and US 2005/0063862A1 are known from diffusion cells whose donor and recipient compartments are in the form of multi-well plates, the membrane being sandwiched between the two plates. This configuration requires in particular to have a membrane of large area then that all the wells do not necessarily need to be used for a given manipulation. Furthermore, the analysis of the recipient medium in each of the wells can be performed only once the permeant deposit in each of the wells has been completed. In other words, the problem to be solved by the invention is to develop a receiving medium and a diffusion cell does not have the disadvantages mentioned above.

Thus, the new receiving medium is advantageously no longer in liquid form but in semi-solid form during the migration of the substance to be assayed and in liquid form at the time of sampling, when this is carried out at room temperature. Such a medium is called "thermoreversible". A semi-solid medium is a medium whose mechanical properties (elasticity, plasticity, deformation) and rheology (viscosity, flow) are intermediate between those of a liquid and a solid.

The semi-solid medium during the migration phase of the substance to be assayed does not cause hyperhydration of the membrane and therefore does not alter its permeability. The heating at 37 ° C of the semi-solid medium does not give rise to the formation of gas bubbles between the medium and the membrane. The use of a semi-solid medium simplifies the design of the CDD by eliminating the sampling devices and the need for mechanical agitation of the receiving fluid. In addition it is no longer necessary to use clamps. Furthermore, the migration of adjuvants to the membrane is advantageously reduced by the rheological properties of the receiving medium and the adjuvant-gelling gel molecular interactions.

As already stated, the preferred medium of the invention has the advantage of being liquid at the time of the assay as such (the assay is performed at room temperature). The assay can thus be done by high performance liquid chromatography (HPLC) or any other technique known to those skilled in the art.

In other words, the subject of the invention is first of all a thermoreversible receiving medium intended to be used in a diffusion cell, ie a receiving medium in semi-solid form at a temperature of between 30 and 40.degree. ° C, preferably between 33 and 35 ° C and in liquid form at room temperature (20 to 25 ° C). The subject of the invention is also a diffusion cell comprising, separated by a biological, artificial or synthetic membrane, a donor compartment comprising a composition of chemical compound (s) to be assayed and a recipient compartment intended to collect the quantity of chemical compounds that have passed through the biological, artificial or synthetic membrane.

The diffusion cell is characterized in that the recipient compartment contains a receiving medium in semi-solid form at a temperature between 30 and 40 ° C, preferably between 33 and 35 ° C and in liquid form at room temperature (20 to 25 ° C). VS).

The semisolid receiver medium according to the invention is defined by the ^7th Edition of the European Pharmacopoeia as a cream, a gel, an ointment or a paste. According to the invention, the semi-solid medium is a gel, that is to say a polymeric matrix forming a three-dimensional network in a liquid medium at a temperature between 30 and 40 ° C. It is more specifically a mixture of solvent and gelling polymer. The polymer or the combination of polymers used (also called gelling agents) represent between 1% and 30% of the total weight of the gel. This gel, of a hydrophilic (ie, hydrogel) or lipophilic (ie, oleogel) nature, has a composition adapted to the physicochemical characteristics of the permeant, in particular by the addition of adjuvants.

As already stated, the migration of adjuvants to the membrane is advantageously reduced by the rheological properties of the recipient medium and the adjuvant-gelling gel molecular interactions. Also, the viscosity of the receiving medium should be such that no flow is observed during the duration of the experiment, i.e. 24h, even after storage at + 4 ° C or -18 ° C.

Advantageously, the receiving medium has a viscosity similar to that of the dermis when it is in semi-solid form within the meaning of the invention.

In a preferred embodiment, the semi-solid medium is a gel comprising as a gelling agent, a poloxamer or a mixture of poloxamers. In an improved embodiment, the poloxamer is poloxamer 407, which further allows the lipophilic molecules to be solubilized chemically without the addition of a specific adjuvant. In other words, by itself, poloxamer makes it possible to obtain a saturating concentration of lipophilic permeants greater than that obtained in water.

The invention also relates to an improved diffusion cell.

In a first embodiment, the cell of the invention is in the form of a unitary device. Such a cell can be described as "monocompartmental" in that it comprises only a recipient compartment associated with a single donor compartment.

According to the invention, the donor and recipient compartments are tubular and associated by securing means.

In a first embodiment, the securing means are in the form of a male thread arranged at the base of the donor compartment intended to cooperate with a corresponding female thread, arranged at the upper end of the receiving compartment. Advantageously, each compartment has, at the thread, an air outlet orifice. In practice, the membrane is held between the two compartments by the thread, which ensures the sealing of the device during assembly.

In an alternative embodiment, the securing means are in the form of a so-called "sealing" clamping ring which adjusts to the donor and recipient compartments either by sliding or by means of threads. Advantageously, the ring has at least one orifice to allow the evacuation of air at the time of assembly. In general, a membrane area of less than 2.5 cm ² is sufficient.

In a second embodiment, the diffusion cell is multi-compartmental in that it contains several individual donor compartments and as many individual recipient compartments, each separated by a separate individual membrane. The subject of the invention is thus a diffusion cell comprising, intended to be separated by a biological, artificial or synthetic membrane, a donor compartment intended to contain a composition of chemical compound (s) to be assayed and a recipient compartment intended to to contain a receiving medium collecting the amount of chemical compound that has passed through the biological, artificial or synthetic membrane.

This cell is characterized in that:

the recipient compartment is in the form of a multi-well plate,

the donor compartment is in the form of a plurality of individual compartments whose number is equal to the number of wells,

the membrane is divided into as many fragments as wells,

and in that it furthermore has means for positioning and joining the membrane fragments between each donor compartment and each well. Such a configuration makes it possible to have a well per dosage and thus no longer have to reintroduce medium during handling. In addition, since the donor compartments are individualized, it is possible to use only one part of the plate for a given test molecule and the other part for another molecule. Similarly, each compartment cooperating with a separate well through a separate membrane, it is possible to test multiple membranes for the same molecule. Finally, the structure of the cell makes it possible to perform assays on a well at any time of the manipulation without having to wait for the filling of all the donor compartments. Advantageously, the donor compartments are in tubular form and the positioning and securing means of the membrane are in the form of a ring.

According to another characteristic, the outer diameters of the donor and inner compartments of the rings are such that the donor compartment slidably cooperates with the ring. In this way and in practice, each membrane sample is sandwiched between the outer wall of the donor compartment and the inner wall of the ring. Similarly, to prevent contact between the membrane and the bottom of the well, the ring has an outside diameter less than the inside diameter of the well and its depth is less than that of the well. Advantageously and in this case, the ring is provided in its upper part with a support flange on the edge of the well.

Advantageously, each donor compartment is provided with an individual lid, advantageously screwable. The lid optionally has in its bottom, an activated carbon filter. The invention also relates to a method for measuring the diffusion of a molecule through a biological, artificial or synthetic membrane implementing the multicompartmental cell previously described according to which:

all or some of the wells are filled with a receiving medium,

each membrane fragment is positioned and solidified between each donor compartment and each well of the multiwell plate,

all or part of the donor compartments of the test molecule are filled, if necessary, the lid of the donor compartments is closed,

the content of the recipient medium is analyzed into the molecule to be tested. The receiving medium may be a standard medium, which is at least liquid at the experimental temperature, that is, between 20 and 37 ° C.

It may also be a thermoreversible semi-solid medium within the meaning of the invention or not.

When it is in semi-solid form, the medium has the form of a gel, hydrophilic nature (ie, hydrogel) or lipophilic (ie, oleogel), whose composition is adapted to the physicochemical characteristics of the permeant, in particular by the addition of adjuvants. The hydrogels advantageously comprise:

as a solvent: water, a mixture of water / polar cosolvents or even water / surfactants,

as a gelling agent, alone or in combination:

o natural gelling agents: eg, collagen, hyaluronan, chondroitin sulfate, agarose, gelatin artificial gelling agents: eg, chitosan, cellulose ethers (eg, methylcellulose), cellulose derivatives (eg, carboxymethylcellulose), synthetic gelling agents: eg, carbopol; polyethylene glycols; polyvinyl alcohol

Oleogels advantageously comprise:

as a solvent: mineral oils (e.g., paraffin oil) or vegetable oils

(e.g., sweet almond oil),

as a gelling agent, alone or in combination:

o natural gelling agents: e.g., cholesterol ester,

synthetic gelling agents: e.g., sorbitan ester.

In a particular embodiment, the recipient compartment is in the form of a multi-well plate, each of the wells being filled with a pre-molded semi-solid medium ready for use. Each well is intended to receive a sealing ring over its entire periphery, advantageously by sliding, whose upper end cooperates with the lower end of the donor compartment by means of a thread or by sliding. The membrane is held between the ring and the donor compartment by the thread or overlay after the sliding. As before, the ring has at least one orifice arranged at its thread or the covering area to allow the evacuation of air at the time of assembly.

The subject of the invention is also the use of a liquid, semi-solid, thermoreversible or non-thermoreversible receiving medium in a multi-compartment diffusion cell as described above.

The invention and the advantages resulting therefrom will emerge from the following exemplary embodiments, in support of the appended figures.

Figure 1 is a schematic representation of a Franz cell

Figure 2 is a schematic representation of a monocompartmental diffusion cell of the invention according to a first embodiment wherein the sealing of the device is provided by a threading system. Figure 3 is a schematic representation of a monocompartmental diffusion cell of the invention according to a second embodiment wherein the sealing of the device is provided by a threading system.

Figure 3bis is a schematic representation of a monocompartmental diffusion cell of the invention according to the same embodiment as that of Figure 3 where the sealing of the device is provided by a sliding system.

FIG. 4 is a schematic representation of a multicompartmental diffusion cell of the invention according to a preferred embodiment.

FIG. 5 is a schematic representation of the diffusion measurement steps of a molecule in a diffusion cell according to the invention.

Figure 6 is a lidocaine titration curve done by three different cells Franz cell respectively, Quixsep ^® system and cell of the invention.

Figure 7 is a curve methylprednisolone metering performed by three different cells Franz cell respectively, Quixsep ^® system and cell of the invention. Figure 8 is a titration curve of mitomicyne C done by three different cells Franz cell respectively, Quixsep ^® system and cell of the invention.

Example 1: Franz Cell Figure 1 schematically shows a static vertical cell called Franz cell. This cell consists of a recipient compartment (1) containing a recipient fluid maintained at 37 ° C stirred by means of a magnetic stirrer (2) and a donor compartment (3), surmounted where appropriate, for the determination of the substances volatile, by an activated carbon filter (4). The two compartments are separated by a biological, artificial or synthetic membrane (5) resting on a grid (6). The receiving compartment also has a neck (7) intended to allow sampling over time, either manually or automatically. The procedure used for these assays is described in particular in the two following references: OECD / OECD, OECD Guideline for the testing of chemicals. Skin Absorption: in vitro method, in 428, O.f.E.C.-o.a. Development, Editor. 2004: Paris.

OECD / OECD, OECD Guidance Notes on Dermal Absorption, in Draft 22 October 2010, OfEC-oa Development, Editor. 2010. Example 2 Monompartmental Diffusion Cell of the Invention According to a First Embodiment

As shown in FIG. 2, the cell comprises a donor compartment (8) in the form of a hollow cylinder intended to receive the chemical compound or compounds to be tested. The cylinder (8) has at its lower end a male thread (9) intended to cooperate with the female internal thread (not shown in the figure) of the receiving compartment (10). The membrane (11) is maintained between the donor compartment (8) and the recipient compartment (10). The recipient compartment (10) contains a semi solid medium (12) intended to come into contact with the lower face of the membrane (11).

Example 3 and 3bis: Monompartmental diffusion cell of the invention according to a second embodiment

As shown in FIG. 3, the cell comprises a sealing ring (13) provided with a female thread and one or more orifices (14) at this thread to allow the evacuation of air during the assembly of the CDD. The donor compartment is a cylinder (8) provided with a male thread (9) for sealing. The recipient compartment is a hollow base (15) intended to contain the semi-solid receiving medium. In an alternative embodiment (Figure 3bis), the sealing ring is threadless and securing with the compartments is by sliding (16).

Example 4 Multicompartmental Diffusion Cell of the Invention

In this embodiment shown in FIG. 4, the recipient compartment is in the form of a multiwell plate (17) (18). Each well contains a precolded receiving medium (22). The donor compartments (19) are assembled on the sealing rings (20) by inserting the membrane (21), then placed individually in the wells (18). The donor compartment (19) is filled with a preparation liquid, semi-solid or solid of one or more chemical compounds. Figure 5 illustrates a method of measuring the diffusion of a molecule in a cell of the invention.

The use of CDD of the invention in multiwell mode comprises five steps. Step 1: An unrepresented lid protecting the multi-well plate and the precolded receiving media is removed.

Step 2: Sealing rings (20) and donor compartments (19) are placed in front of each of the wells.

Step 3: A membrane sample (21) is inserted on the upper part of the ring (20).

Step 4: The compartment (19) is screwed into the ring (20) surmounted by the membrane sample (21).

Step 5: The assembly (19-20-21) is assembled in the well (18). The insertion is facilitated by the presence of the orifice on the ring (20) for expelling the air during the introduction of the assembly (19-20-21) in the well (18). The compartment (19) is filled with a liquid, semi-solid or solid preparation of one or more chemical compounds (23).

In another embodiment, the multi-well plate does not contain the precured semi-solid medium and it is sold separately. Under these conditions, each of the recipient wells is first filled before assembling the donor compartments and the membrane by means of the ring.

Example 5 Permeability study with the cell according to the first embodiment

The purpose of this study was to determine and compare the permeability of three molecules across bladder wall maintained in three different devices (Table 1):

1. Franz CCD

2. CCD of the invention

3. Dialysis Device (Quixsep ^® )

Table 1: Characteristics of devices for studying permeability.

CDD of CDD of

QuixSe devices

Franz the invention

Vdonneur (CDl) 1.00 5.00 0.40

Receiver (CDl) 9.00 2.77 60

Area (cm ² ) 0.78 6.16 1.54

Area / Receiver (Ass ^"1 ) 0.09 2.22 0.03 The bladder wall samples used were from pork bladder. Aqueous solutions of lidocaine (1.67 mg.mL ^"1 ), methylprednisolone (0.33 mg.mL ^{" 1} ) and mitomycin C (1.33 mg.mL ^"1 ) were prepared in physiological saline (NaCl 0). , 9%) and then introduced into donor compartments (Figure 2, Table 2).

The CSD recipient Franz medium and the dialysis liquid Quixsep ^® device is a sodium chloride solution to 0.9%>. The semi-solid medium of the CDD of the invention was an agar gel (composition: peptone 5.0 g, yeast extract 2.5 g, glucose 1.0 g, agar: 15.0 g, qsp 1 liter of purified water). The permeation patterns of lidocaine, methylprednisolone, and mitomycin C through porcine bladder samples held in three different devices are shown in Figures 5, 6, and 7, respectively. The permeation profiles have numerous analogies confirming the technical feasibility of a CDD permeability study with a semi-solid receiving medium. The permeability coefficients of lidocaine, methylprednisolone and mitomycin C are shown in Table 2.

Table 2: Coefficients of permeability of lidocaine, methylprednisolone and mitomycin C across the pork bladder calculated from permeation profiles obtained using three diffusion devices. Each value is the mean ± standard deviation of 3 to 5 experimental determinations.

Devices 10 ^b x Coefficient of permeability (cm.s ^_1 )

Lidocaine Methylprednisolone Mitomycin C

CDD of Franz 5.33 ± 0.9 2.99 ± 0.82 8.23 ± 1.20

CDD of the invention 6.62 ± 0.57 7.34 ± 3.16 5.35 ± 0.35

QuixSep ^® 9.30 ± 1.15 4.01 ± 1.48 13.95 ± 5.66

In other words, the invention makes it possible to propose a simple diffusion cell to manufacture and comprising, as a receiving medium, a semi-solid substance, which is a gel, which makes it easier to implement.

Claims

1 / Diffusion cell comprising, intended to be separated by a biological, artificial or synthetic membrane, a donor compartment intended to contain a composition of chemical compound (s) to be assayed and a recipient compartment intended to contain a collecting collecting medium the quantity of chemical compound that has passed through the biological, artificial or synthetic membrane, characterized in that:

the recipient compartment (17) is in the form of a multi-well plate

(18)

the donor compartment is in the form of a plurality of individual compartments (19) whose number is equal to the number of wells,

the membrane is divided into as many fragments (21) as wells (18),

and in that it furthermore has means for positioning and securing (20) membrane fragments (21) between each donor compartment (19) and each well (18).

21 Diffusion cell according to claim 1, characterized in that the donor compartments (19) are in tubular form and in that the positioning and securing means of the membrane are in the form of a ring (20) .

3 / Cell diffusion according to claim 2, characterized in that the outer diameters of the donor compartments (19) and inner rings (20) are such that the donor compartment cooperates by sliding with the ring.

4 / diffusion cell according to claim 3, characterized in that the ring (20) has an outer diameter less than the inner diameter of the well (18) and in that its depth is less than that of the well. 5 / Cell broadcast according to claim 4, characterized in that the ring is provided in its upper part with a support flange on the edge of the well.

6 / diffusion cell according to one of claims 1 to 5, characterized in that each donor compartment is provided with an individual lid. 7 / A method for measuring the diffusion of a molecule through a biological, artificial or synthetic membrane implementing the cell according to one of claims 1 to 6, according to which:

all or some of the wells are filled with a receiving medium,

the content of the recipient medium is analyzed into the molecule to be tested.

8 / Method according to claim 7, characterized in that the receiving medium is in semi-solid form at a temperature between 30 and 40 ° C and in liquid form at room temperature (20 to 25 ° C). Method according to claim 8, characterized in that the semi-solid medium, at a temperature of between 30 and 40 ° C, has a viscosity similar to that of the dermis.

10 / Method according to one of claims 8 or 9, characterized in that the semi-solid medium is in the form of a gel containing between 1 and 30% by weight of at least one gelling polymer.

1 1 / Method according to claim 10, characterized in that the gelling polymer is a poloxamer. 12 / Method according to claim 1 1, characterized in that the poloxamer is poloxamer 407.

13 / Method according to one of claims 7 to 12, characterized in that the biological membrane is in the form of an isolated tissue (epithelium, endothelium, connective tissue) or an organ sample (skin, stomach intestine, bladder, lungs) humans, animals or plants.

14 / Use of the recipient medium defined in one of claims 7 to 12 in the diffusion cell object of one of claims 1 to 6.