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1. (WO2019002535) POLYMÈRES À EMPREINTE MOLÉCULAIRE CIBLANT LA PHÉNYLALANINE
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CLAIMS

1. A method for the preparation of molecular imprinted polymer (MIPs), which specifically bind L-phenylalanine (Phe) and L-Phe residues, said method comprising the steps of

a) polymerization of a mixture comprising

- 2-methylprop-2-enoic acid (MAA),

- l,4-bis(acryloyl)piperazine (DAP), and

- a template molecule consisting of L-Phe or a L-Phe derivative exposing a phenylalanine motif

in the presence of a catalyst and an oxidizing agent,

so as to obtain a cross-linked imprinted polymer,

b) if necessary subsequently fragmenting the cross-linked imprinted polymer to obtain a first fragmented polymer, and collecting the MIPs having particle sizes smaller than 63 μιη, c) optionally washing and drying the polymer fraction obtained from step b,

d) fragmenting the polymer fraction obtained from step b or c and collecting a second fragmented polymer having particle sizes in the range 150-250 nm,

e) subjecting the second fragmented polymer obtained from step d to affinity

chromatography where Phe constitutes the affinity tag in a chromatographic matrix, and f) recovering MIPs binding to Phe in step e.

2. The method according to claim 1, wherein the polymerization mixture in step a contains MAA and DAP in a molar ratio of 5-30.

3. The method according to claim 1 or 2, wherein the polymerization mixture in step a contains MAA and template molecule in a molar ratio of between 1.0-4.0, such as 1.1, about 1.2, about 1.3, about 1.4, about 1.5, about 1.6, about 1.7, about 1.8, about 1.9, about 2.0, about 2.1, about 2.2, about 2.3, about 2.4, about 2.5, about 2.6, about 2.7, about 2.8, about 2.9, about 3.0, about 3.1, about 3.2, about 3.3, about 3.4, about 3.5, about 3.6, about 3.7, about 3.8, about 3.9, and about 4.0.

4. The method according to any one of the preceding claims, wherein the template molecule is L-Phe derivative in the form of a peptide containing at least one L-Phe residue

5. The method according to claim 4, wherein the templated molecule is a dipeptide or a tri peptide.

6. The method according to claim 5, wherein the template molecule is selected from the group consisting of

Gly-L-Phe,

Ala-L-Phe,

L-Asp-L-Phe-OMe,

L-Asp-L-Phe,

Gly-Gly-L-Phe,

Ala-Gly-L-Phe,

Gly-Ala-L-Phe,

L-Phe-Gly,

L-Phe-Ala,

L-Phe-L-Phe,

L-Phe-Ala-L-Phe,

L-Phe-Gly-L-Phe.

7. The method according to claim 6, wherein the template molecule is Gly-L-Phe.

8. The method according to any one of the preceding claims, wherein the catalyst in step a) is selected from the group consisting of tetramethylethylenediamine, dimethylpiperazine, preferably tetramethylethylenediamine (TEMED).

9. The method according to any one of the preceding claims, wherein the oxidizing agent in step a) is selected from the group consisting of ammonium persulfate, potassium persulfate, and sodium thiosulfate, preferably ammonium persulfate (APS) .

10. The method according to any one of the preceding claims, wherein fragmentation in step b comprises grinding, milling, explosion, hammering, ball milling, cryo grinding, or collision homogenisation.

11. The method according to any one of the preceding claims, wherein the MIPs collected in step b have sizes in the 25-63 μιη range.

12. The method according to any one of the preceding claims, wherein step b entails collection of MIPs able to pass through a sieve with a 63 μιη cut-off.

13. The method according claim 12, wherein step b further entails collection of MIPs that are retained on a sieve with a 25 μιη cut-off.

14. The method according to any one of the preceding claims, wherein washing is performed at alternating pH with an organic solvent.

15. The method according to any one of the preceding claims, wherein the MIPs are packed in an HPLC column in step c) during washing under elevated pressure.

16. The method according to any one of the preceding claims, wherein fragmentation in step d is carried out in a ball mill or bead mill.

17. The method according to any one of the preceding claims, wherein collection of MIPs in step d is carried out by suspension of MIPs in an aqueous solvent such as water, incubation in an ultrasound bath, centrifugation, and isolation of the supernatant, which contains the 150-250 nm MIPs.

18. The method according to any one of the preceding claims, wherein step d entails washing to separate the MIPs from residual template molecule, such as a dialytic washing step or a washing step in an ultracentrifuge.

19. The method according to any one of the preceding claims wherein the affinity chromatography in step e) is carried out on a packed bed chromatographic column using a stationary chromatographic matrix, and where the MIPs are suspended in a buffered aqueous solvent.

20. The method according to any one of the preceding claims, wherein recovery in step f) comprises elution of the MIPs from the chromatographic matrix.

21. A molecular imprinted polymer (MIP), which specifically binds L-phenylalanine (Phe), wherein said MIP is comprised of polymerized methacrylic acid (MAA) cross-linked with 1,4-diacryloylpiperazine (DAP).

22. The MIP according to claim 21, wherein the molar ratio between MAA residues and DAP residues is between 5 and 30.

23. The MIP according to any one of claims 21-22, which is obtainable or obtained by the method according to any one of claims 1-20.

24. The molecular imprinted polymer according to any one of claims 21-23, which has a KD for binding to L-Phe of less than 10"7, such as less than 10"8, less than 9xl0"9, less than 8xl0"9, less than 7xl0"9, less than 6xl0"9, less than 5xl0"9, less than 4xl0"9, less than 3xl0~9,and less than 2xl0"9.

25. The molecular imprinted polymer according to claim 24, where the KD is about 10"9.

26. A composition comprising molecular imprinted polymers according to any one of claims 21-24, said composition comprising a pharmaceutically acceptable carrier and/or diluent and/or excipient, wherein said composition is adapted for oral administration.

27. A method for the preparation of MIPs, which specifically bind L-Tyrosine (Tyr), said method comprising recovering MIPs that bind L-Tyrosine from an initial composition of MIPs that have been prepared using, as the template molecule during polymerization and cross-linking, a molecule comprising at least one phenylalanine motif and comprising no Tyr residues.

28. The method according to claim 27, wherein the template molecule is as defined in any one of claims 1 and 4-7.

29. The method according to claim 27 or 28, which comprises that the initial composition of MIPs is separated in at least 2 fractions, and recovering Tyr binding MIPs from a fraction, which is essentially free from MIPs that bind the Phe-containing capture probe.

30. The method according to any one of claims 27-29, which comprises the steps of a) polymerization of a mixture comprising

- 2-methylprop-2-enoic acid (MAA),

- l,4-bis(acryloyl)piperazine (DAP), and

- a template molecule defined in any one of claims 1 and 4-7

in the presence of a catalyst and an oxidizing agent,

so as to obtain a cross-linked imprinted polymer,

b) if necessary subsequently fragmenting the cross-linked imprinted polymer to obtain a first fragmented polymer, and collecting the fraction thereof having particle sizes smaller than 63 μιη,

c) optionally washing and drying the polymer fraction obtained from step b,

d) fragmenting the polymer fraction obtained from step c and collecting a second fragmented polymer having particle sizes in the range 150-250 nm, and

e) subjecting the second fragmented polymer obtained from step d to affinity

chromatography where

el) Tyr constitutes the affinity tag in a chromatographic matrix, or

e2) Phe constitutes the affinity tag in a chromatographic matrix, recovering MIPs not binding to Phe, and subjecting the MIPs not binding to Phe to further affinity chromatography where Tyr constitutes the affinity tag in a chromatographic matrix, and

f) recovering MIPs binding to Tyr in step e.

31. A molecular imprinted polymer (MIP), which specifically binds L-tyrosine (Tyr), wherein said MIP is comprised of polymerized methacrylic acid (MAA) cross-linked with 1,4-diacryloulpiperazine (DAP).

32. The MIP according to claim 31, wherein the molar ratio between MAA residues and DAP residues is between 5 and 30.

33. The MIP according to any one of claims 31-32, which is obtainable or obtained by the method according to any one of claims 27-30.

34. The molecular imprinted polymer according to any one of claims 31-33, which has a KD for binding to Tyr of less than 10"7, such as less than 10"8, less than 9xl0"9, less than 8xl0"9, less than 7xl0"9, less than 6xl0"9, less than 5xl0"9, less than 4xl0"9, less than 3xl0"9,and less than 2xl0"9.

35. The molecular imprinted polymer according to claim 34, where the KD is about 10"9.

36. A composition comprising molecular imprinted polymers according to any one of claims 31-34, said composition comprising a pharmaceutically acceptable carrier and/or diluent and/or excipient, wherein said composition is adapted for oral administration.

37. A method of treatment of phenylketonuria in a person in need thereof, said method comprising administering to a person in need thereof the MIPs according to any one of claims 21-24 or the composition according to claim 26 so as to deliver a daily effective dose of MIPs according to any one of claims 21-24.

38. A method for treatment of tyrosineamia and/or alkaptonuria, the method comprising administering to a person in need thereof

1) MIPs that bind phenylalanine, preferably the MIPs according to any one of claims 21-24 or the composition according to claim 26 so as to deliver a daily effective dose of MIPs according to any one of claims 21-24; and/or

2) MIPs that bind tyrosine according to any one of claims 31-34 or the composition according to claim 36, so as to deliver a daily effective dose of MIPs according to any one of claims 31- 34.

39. The method according to claim 38, wherein the treatment is carried out as an adjuvant therapy to nitisinone treatment of alkaptonuria or tyrosinemia-I.

40. The method according to any one of claims 37-39, wherein the daily effective dose is 1-35 g per 70 kg body weight.

41. The method according to claim 40, wherein the effective dose per meal is at most or about 34 g/70 kg, such as at most or about 33, at most or about 32, at most or about 31, at most or about 30, at most or about 29, at most or about 28, at most or about 27, at most or about 26, at most or about 25, at most or about 24 at most or about 23, at most or about 22, at most or about 21, at most or about 20, at most or about 19, at most or about 18, at most or about 17, at most or about 16, at most or about 15, at most or about 14, at most or about 13, at most or about 12, at most or about 11, at most or about 10, at most or about 9, at most or about 8, at most or about 7, at most or about 6, at most or about 5, at most or about 4, at most or about 3, at most or about 3, and at most or about 2 g/70 kg.

42. The method according to claim 40, wherein daily effective dose is 20-45 g / 70 kg, such as 25-40 g / 70 kg, and 30-35 g/ kg.

43. The method according to claim 40, wherein daily effective dose is about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31, about 32, about 33, about 34, about 35, about 36, about 37, about 38, about 39, about 40, about 41, about 42, about 43, about 44, about 45, about 46, about 47, about 48, about 49, about 50, about 51, about 52, about 53, about 54, about 55, about 56, about 57, about 58, about 59, about 60, about 61, about 62, about 63, about 64, about 65, about 66, about 67, about 68, about 69, about 70, about 71, about 72, about 73, about 74, about 75, about 76, about 77, about 78, about 79, about 80, about 81, about 82, about 83, about 84, about 85, about 86, about 87, about 88, about 89, about 90, about 91, about 92, about 93, about 94, about 95, about 96, about 97, about 98, about 99, about 100, about 101, about 102, about 103, about 104, and about 105 g per 70 kg body weight.