# US 2019/0167807 A1 · Adipose Tissue-Targeting Polypeptide-Verbascoside-Nanogold Particle Derivative, Preparation Method and Application Thereof

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- Number: US 2019/0167807 A1
- Document ID: US20190167807A1
- Status: Published application
- Kind: A1
- Application number: 16/261,137
- Filed: 2019-01-29
- Published: 2019-06-06
- Earliest claimed: 2018-06-06
- Assignees: Affiliated Stomatological Hospital of Nanjing Medical University; Nanjing Medical University
- Applicants: Affiliated Stomatological Hospital of Nanjing Medical University; Nanjing Medical University
- Inventors: Yi YUAN; Yaqin ZHANG; Hongwei LI; Jindao WU; Heming WU; Liyong PU
- Web page: https://patentgrep.com/patent/US20190167807A1

## CPC

- A61K 47/6929: {the form being a nanoparticle, e.g. an immuno-nanoparticle}
- A61K 9/51: Nanocapsules; {Nanoparticles; (nanotubes A61K9/0092; polymeric micelles A61K9/1075; polymersomes A61K9/1273; pure drug nanoparticles A61K9/14; drug nanoparticles with adsorbed surface modifiers A61K9/141; conjugates, e.g. between drug and non-active nanoparticles, A61K47/50; preparations for in vivo diagnosis A61K49/00; with radioactive substances A61K51/00)}
- A61K 36/53: Lamiaceae or Labiatae (Mint family), e.g. thyme, rosemary or lavender
- A61K 47/60: the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
- A61K 47/64: Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent (peptidic linkers A61K47/65)
- A61K 47/6923: {the form being an inorganic particle, e.g. ceramic particles, silica particles, ferrite or synsorb}
- A61P 3/04: Anorexiants; Antiobesity agents
- A61K 9/0019: {Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner (non-active ingredients are additionally classified in A61K47/00)}
- B82Y 5/00: Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
- 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)
- A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- B82Y: SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES

## Abstract

Embodiments of the present disclosure provides a method of preparing an adipose tissue-targeting polypeptide-verbascoside-nanogold particle derivative, including: activating a carboxyl group of a cysteine residue at the C-terminus of an adipose tissue-targeting polypeptide to form an acyl chloride; reacting a verbascoside with a sodium hydroxide to obtain a sodium phenolate; esterifying the acyl chloride with the sodium phenolate to form an adipose tissue-targeting polypeptide-verbascoside derivative; and reacting the adipose tissue-targeting polypeptide-verbascoside derivative with PEG-modified nanogold particles. The present disclosure further discloses an adipose tissue-targeting polypeptide-verbascoside-nanogold particle derivative and the application of the adipose tissue-targeting polypeptide-verbascoside-nanogold particle derivative in preparing a drug for the treatment of obesity. The adipose tissue-targeting polypeptide-verbascoside-nanogold particle derivative has good biocompatibility, and capable of carrying drugs into the adipocytes, thereby greatly enhancing the availability of the drug and significantly improving the targeted anti-obesity activity of the drug.

## Claims

### Claim 1

A preparation method of an adipose tissue-targeting polypeptide-verbascoside-nanogold particle derivative, comprising:
activating a carboxyl group of a cysteine residue at a C-terminus of an adipose tissue-targeting polypeptide to form an acyl chloride, wherein the adipose tissue-targeting polypeptide comprises an amino acid sequence of CKGGRAKDC;
reacting a verbascoside with a sodium hydroxide to obtain a sodium phenolate;
esterifying the acyl chloride with the sodium phenolate to form an adipose tissue-targeting polypeptide-verbascoside derivative; and
modifying a nanogold particle with polyethylene glycol (PEG), mixing and reacting the adipose tissue-targeting polypeptide-verbascoside derivative with a PEG-modified nanogold particle, wherein the sulfydryl of the cysteine residue at the C-terminus of the adipose tissue-targeting polypeptide is connected to the nanogold particle with an Au—S bond.

### Claim 2 (depends on 1)

The preparation method of claim 1, wherein
a first amino protecting group is connected to a side chain of a second amino acid residue of the adipose tissue-targeting polypeptide, and a second amino protecting group is connected to a seventh amino acid residue of the adipose tissue-targeting polypeptide;
a guanidine protecting group is connected to the fifth arginine residue;
a carboxyl protecting group is connected to the eighth amino acid residue; and
a sulfhydryl protecting group is connected to the N-terminal amino acid residue.

### Claim 3 (depends on 2)

The preparation method of claim 2, wherein the amino protecting group is a 9-fluorenylmethoxycarbonyl; the guanidine protecting group is an amantadyl-oxygen carbonyl; the carboxyl protecting group is a methyl ester; and the sulfhydryl protecting group is a triphenylmethyl.

### Claim 4 (depends on 1)

The preparation method of claim 1, wherein a molar ratio of the adipose tissue-targeting polypeptide and the verbascoside is 1-5:1.

### Claim 5 (depends on 1)

The preparation method of claim 1, wherein the step of modifying the nanogold particle with PEG further comprising:
mixing the nanogold particle with SH-mPEG-COOH at 20-25° C.;
stirring and incubating for 12-24 hours; and
removing unreacted SH-mPEG-COOH after centrifugation and washing to obtain a precipitate, wherein the precipitate is the PEG-modified nanogold particle.

### Claim 6 (depends on 1)

The preparation method of claim 1, wherein a molar ratio of the adipose tissue-targeting polypeptide-verbascoside derivative and the PEG modified nanogold particle is 106:9−5.

### Claim 7

An adipose tissue-targeting polypeptide-verbascoside-nanogold particle derivative, wherein the derivative is prepared by a preparation method comprising:
activating a carboxyl group of a cysteine residue at a C-terminus of an adipose tissue-targeting polypeptide to form an acyl chloride; wherein the adipose tissue-targeting polypeptide has an amino acid sequence of CKGGRAKDC;
reacting a verbascoside with a sodium hydroxide to obtain a sodium phenolate;
esterifying the acyl chloride with the sodium phenolate to form an adipose tissue-targeting polypeptide-verbascoside derivative;
modifying nanogold particles with polyethylene glycol (PEG), mixing and reacting the adipose tissue-targeting polypeptide-verbascoside derivative with PEG-modified nanogold particles; wherein the sulfydryl of the cysteine residue at the C-terminus of the adipose tissue-targeting polypeptide is connected to the nanogold particles with an Au—S bond,
wherein a first amino protecting group is connected to a side chain of a second amino acid residue of the adipose tissue-targeting polypeptide, and a second amino protecting group is connected to a seventh amino acid residue of the adipose tissue-targeting polypeptide; a guanidine protecting group is connected to the fifth arginine residue; a carboxyl protecting group is connected to the eighth amino acid residue;
and a sulfhydryl protecting group is connected to the N-terminal amino acid residue;
wherein the amino protecting group is a 9-fluorenylmethoxycarbonyl, the guanidine protecting group is an amantadyl-oxygen carbonyl, the carboxyl protecting group is a methyl ester, and the sulfhydryl protecting group is a triphenylmethyl;
wherein a molar ratio of the adipose tissue-targeting polypeptide and the verbascoside is 1-5:1;
wherein the step of modifying the nanogold particles with PEG further comprises:
mixing the nanogold particles with SH-mPEG-COOH at 20-25° C.;
stirring and incubating for 12-24 hours; and
removing unreacted SH-mPEG-COOH after centrifugation and washing to obtain a precipitate, wherein the precipitate is the PEG-modified nanogold particles; and
wherein a molar ratio of the adipose tissue-targeting polypeptide-verbascoside derivative and the PEG modified nanogold particles is 106:9−5.

### Claim 8 (depends on 7)

The adipose tissue-targeting polypeptide-verbascoside-nanogold particle derivative of claim 7, wherein the derivative is used for preparing a drug of treating obesity.

### Claim 9 (depends on 8)

The adipose tissue-targeting polypeptide-verbascoside-nanogold particle derivative of claim 8, wherein the drug is administered through an injection.

### Claim 10

A preparation method of an adipose tissue-targeting polypeptide-verbascoside-nanogold particle derivative, comprising:
step (1), activating a carboxyl group of a cysteine residue at a C-terminus of an adipose tissue-targeting polypeptide to form an acyl chloride; wherein the adipose tissue-targeting polypeptide has an amino acid sequence of CKGGRAKDC;
step (2), reacting a verbascoside with a sodium hydroxide to obtain a sodium phenolate;
step (3), esterifying the acyl chloride with the sodium phenolate to form an adipose tissue-targeting polypeptide-verbascoside derivative;
step (4), modifying nanogold particles with polyethylene glycol (PEG), mixing and reacting the adipose tissue-targeting polypeptide-verbascoside derivative with PEG-modified nanogold particles; wherein the sulfydryl of the cysteine residue at the C-terminus of the adipose tissue-targeting polypeptide is connected to the nanogold particles with an Au—S bond;
the step (1) further including: dissolving the polypeptide in Dimethyl Sulfoxide (DMSO) to form a polypeptide solution; adding the polypeptide solution into a bottle, dripping N, N-dimethylformamide (DMF) and thionyl chloride into the bottle and stirring evenly; and reacting at 70-75° C. for 1-2 hours to obtain the acyl chloride.
the step (2) further including: dissolving the verbascoside and the sodium hydroxide in methanol respectively to form two solutions; mixing the two solutions; reacting at room temperature to generate the sodium phenolate; wherein a chemical structure formula of the verbascoside is
wherein a molar ratio of the verbascoside to the sodium hydroxide is 1:4;
the step (3) further including: dissolving the sodium phenolate obtained in the step (2) in first deionized water to form a sodium phenolate solution; dripping the acyl chloride obtained in the step (1) into the sodium phenolate solution; stirring and reacting at 10-15° C. for 1.5-2 hours; washing, suction filtering and drying to obtain adipose tissue-targeting polypeptide; wherein a molar ratio of the adipose tissue-targeting polypeptide obtained in the step (1) to the verbascoside obtained in step (2) is 1-5:1; and
the step of modifying the nanogold particles with PEG further including: mixing the nanogold particles with a SH-mPEG-COOH solution at 20-25° C., stirring and incubating for 12-24 hours; removing unreacted SH-mPEG-COOH by centrifugation and washing to obtain a precipitate; dissolving the precipitate in second deionized water; wherein a molar ratio of the adipose tissue-targeting polypeptide-verbascoside derivative to the PEG modified nanogold particles is 106:9−5.