# US 11,028,203 B2 · Molecular imprinted polymers targeting phenylalanine

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- Number: US 11,028,203 B2
- Document ID: US11028203B2
- Status: Granted
- Kind: B2
- Application number: 16/624,578
- Filed: 2018-06-29
- Published: 2020-05-07
- Granted: 2021-06-08
- Earliest claimed: 2017-06-29
- Assignees: Mipsalus ApS
- Applicants: Mipsalus ApS
- Inventors: Klaus Gregorius; Nicolas Otto Krogh
- Web page: https://patentgrep.com/patent/US11028203B2

## CPC

- C08F 220/18: with acrylic or methacrylic acids
- A61K 9/0053: {Mouth and digestive tract, i.e. intraoral and peroral administration}
- A61K 31/787: containing heterocyclic rings having nitrogen as a ring hetero atom
- B01J 20/268: {Polymers created by use of a template, e.g. molecularly imprinted polymers}
- C08F 2/22: Emulsion polymerisation
- C08F 2/32: Polymerisation in water-in-oil emulsions
- C08F 226/06: by a heterocyclic ring containing nitrogen
- C07K 1/22: Affinity chromatography or related techniques based upon selective absorption processes
- C07K 5/06: Dipeptides
- C07K 5/08: Tripeptides
- C08F: MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
- A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D)
- B01J: CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00)

## Abstract

Disclosed is agents and methods that target metabolism malfunctions, inborne as well as acquired, as well as methods for preparation of the agents. In particular, the invention relates to methods for preparing molecular imprinted polymers with high binding capacity for phenylalanine or tyrosine, MIPs that bind phenylalanine or tyrosine, and methods for treating phenylketonuria, alkaptonuria, and hypertyrosinemia.

## Claims

### Claim 1

A method for the preparation of molecular imprinted polymers (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),
1,4-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 μm,
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).

### Claim 2 (depends on 1)

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

### Claim 3 (depends on 1)

The method according to claim 1, wherein the polymerization mixture in step a) contains MAA and template molecule in a molar ratio of between 1.0-4.0.

### Claim 4 (depends on 1)

The method according to claim 1, wherein the template molecule is L-Phe derivative in the form of a peptide containing at least one L-Phe residue.

### Claim 5 (depends on 4)

The method according to claim 4, wherein the template molecule is a dipeptide or a tripeptide.

### Claim 6 (depends on 5)

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, and
L-Phe-Gly-L-Phe.

### Claim 7 (depends on 6)

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

### Claim 8 (depends on 1)

The method according to claim 1, wherein the catalyst in step a) is selected from the group consisting of tetramethylethylenediamine, dimethylpiperazine, preferably tetramethylethylenediamine (TEMED).

### Claim 9 (depends on 1)

The method according to claim 1, 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).

### Claim 10 (depends on 1)

The method according to claim 1, wherein fragmentation in step b) comprises grinding, milling, explosion, hammering, ball milling, cryo grinding, or collision homogenisation.

### Claim 11 (depends on 1)

The method according to claim 1, wherein the MIPs collected in step b) have sizes in the 25-63 μm range.

### Claim 12 (depends on 1)

The method according to claim 1, wherein step b) entails collection of MIPs able to pass through a sieve with a 63 μm cut-off.

### Claim 13 (depends on 12)

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

### Claim 14 (depends on 1)

The method according to claim 1, wherein washing is performed at alternating pH with an organic solvent.

### Claim 15 (depends on 1)

The method according to claim 1, wherein the MIPs are packed in an HPLC column in step c) during washing under elevated pressure.

### Claim 16 (depends on 1)

The method according to claim 1, wherein fragmentation in step d) is carried out in a ball mill or bead mill.

### Claim 17 (depends on 1)

The method according to claim 1, wherein collection of MIPs in step d) is carried out by suspension of MIPs in an aqueous solvent, incubation in an ultrasound bath, centrifugation, and isolation of the supernatant, which contains the 150-250 nm MIPs.

### Claim 18 (depends on 1)

The method according to claim 1, wherein step d) entails washing to separate the MIPs from residual template molecule.

### Claim 19 (depends on 1)

The method according to claim 1, 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.

### Claim 20 (depends on 1)

The method according to claim 1, wherein recovery in step f) comprises elution of the MIPs from the chromatographic matrix.