In vitro method and apparatus for analysing the behaviour of substances in simulated physiological environment

• US 10,458,891 B2
• Filed: 08/19/2015
• Issued: 10/29/2019
• Est. Priority Date: 08/20/2014
• Status: Active Grant

• Current Assignee: Hoffmann-La Roche Incorporated (Roche Holding AG)
• Original Assignee: Hoffmann-La Roche Incorporated (Roche Holding AG)
• Inventors: Mahler, Hanns-Christian, Jere, Dhananjay Jagdish, Patel, Sulabh
• Primary Examiner(s): West, Paul M.
• Application Number:
  US15/504,865
• Publication Number:
  US 20170268976A1
• Time in Patent Office: 1,532 Days
• CPC Class Codes: G01N 13/00 Investigating surface or boundary effects, e.g. wetting power; Investigating diffusion effects; Analysing materials by determining surface, boundary, or diffusion effects scanning-probe techniques or apparatus G01Q
• CPC Class Codes: G01N 2013/003 Diffusion; diffusivity between liquids
• CPC Class Codes: G01N 33/15 Medicinal preparations ; Physical properties thereof, e.g. dissolubility
• CPC Class Codes: G01N 33/559 through a gel, e.g. Ouchterlony technique

Abstract

The invention refers to an in vitro method and apparatus for analyzing the behavior of substances in simulated physiological environment. The method comprises the steps of providing a first fluid, a gel matrix and a second fluid, separating the first fluid and the gel matrix by at least one first semi-permeable membrane and separating the gel matrix and the second fluid by at least one second semi-permeable membrane. The method further comprises the steps of injecting a substance into the first fluid, letting the substance migrate from the first fluid through the at least one first semi-permeable membrane, through the gel matrix, through the at least one second semi-permeable membrane and into the second fluid, and determining clearance of the substance from the first fluid.

Claims

1. An in vitro method for analyzing the behaviour of substances in simulated physiological environment, the method comprising the steps of:

providing a first fluid, a gel matrix and a second fluid;

separating the first fluid and the gel matrix by at least one first semi-permeable membrane;

separating the gel matrix and the second fluid by at least one second semi-permeable membrane;

injecting a substance into the first fluid;

letting the substance migrate from the first fluid through the at least one first semi-permeable membrane, through the gel matrix, through the at least one second semi-permeable membrane and into the second fluid; and

determining clearance of the substance from the first fluid, wherein the first fluid is vitreous humor and the second fluid is a buffer solution.

2. A method according to claim 1, wherein the step of determining clearance of a substance from the first fluid is performed by measuring a substance concentration in the first fluid, in the second fluid or in the first and in the second fluid.

3. A method according to claim 1, further comprising the step of varying the Molecular Weight Cut Off (MWCO) of the at least one first or of the at least one second semi-permeable membrane, or varying the composition of the gel matrix.

4. A method according to claim 1, wherein the injected substance comprises molecules having a size in a range between about 100 Da and about 400 kDa.

5. A method according to claim 1, wherein the at least one first membrane has a Molecular Weight Cut Off (MWCO) substantially corresponding to the Retinal Exclusion Limit (REL).

6. A method according to claim 1, wherein the step of injecting a substance into the first fluid comprises injecting a substance via a substance delivery system into the first fluid, thereby releasing the substance into the first fluid in a delayed manner.

7. A method according to claim 1, wherein the substance is at least one of a macromolecule, a drug formulation, an excipient, a protein or a combination thereof.

8. A method according to claim 1, wherein the buffer solution is a physiologically relevant buffer solution.

9. A method according to claim 1, further comprising the step of varying a viscosity of the gel matrix or varying a concentration of a component of the gel matrix.

10. A method according to claim 1, wherein the injected substance comprises molecules having a size in a range between about 1 kDa and about 250 kDa.

11. A method according to claim 1, wherein the injected substance comprises molecules having a size in a range between 4 kDa and 150 kDa.

12. A method according to claim 1, comprising in vitro testing a substance and determining data on stability or bioavailability of the substance.

13. A method according to claim 12, wherein the substance is a drug formulation.

14. An apparatus for analyzing the behaviour of molecules in simulated physiological environment, the apparatus comprising

a first compartment for receiving a first fluid,

a second compartment for receiving a gel matrix, and

a third compartment for receiving a second fluid;

the apparatus further comprising

a first support for supporting at least one first semi-permeable membrane, and

a second support for supporting at least one second semi-permeable membrane, the second support being arranged at a distance from the first support, wherein

the first support is arranged between the first compartment and the second compartment, and

wherein the second support is arranged between the second compartment and the third compartment, wherein the first support forms a porous wall of the first compartment and of the second compartment, wherein the second support forms a porous wall of the second compartment and of the third compartment, and wherein a shape and size of the first support is adapted to the form and size of a retina.

15. An apparatus according to claim 14, wherein at least one first semi-permeable membrane is arranged on the first support and at least one second semi-permeable membrane is arranged on the second support, the at least one first semi-permeable membrane having a Molecular Weight Cut Off (MWCO) being smaller than or equal to the Molecular Weight Cut Off (MWCO) of the at least one second semi-permeable membrane.

16. An apparatus according to claim 14, wherein the Molecular Weight Cut Off (MWCO) of the at least one first semi-permeable membrane substantially corresponds to the Retinal Exclusion Limit (REL).

17. An apparatus according to claim 14, wherein at least the first support has a concave shape.

18. An apparatus according to claim 14, comprising or being made of glass.

19. An apparatus according to claim 14, further comprising a cover for closing an opening of the first compartment.