Mannose-receptor selective lysinylated cationic amphiphiles and a process for preparation thereof

Patent metadata

Field	Value
Publication number	US-9840530-B2
Application number	US-201314759144-A
Country	US
Kind code	B2
Filing date	Dec 27, 2013
Priority date	Jan 3, 2013
Publication date	Dec 12, 2017
Grant date	Dec 12, 2017

Abstract

The present invention relates to the mannose-receptor selective lysinylated cationic amphiphile and a process for preparation thereof. The compounds of the present invention can target DNA vaccines to antigen presenting cells (APCs) such as macrophages and dendritic cells (DCs), via mannose receptors expressed on the cell surface of APCs. The cationic amphiphiles disclosed herein show enhanced cellular and humoral immune response compared to their mannosyl counterparts in genetic immunization in mice. The present invention discloses that immunization with electrostatic complexes (lipoplexes) of DNA vaccines encoding melanoma antigens (gp100 and tyrosinase) and liposome of the presently described novel lysinylated cationic amphiphiles with mannose-mimicking shikimoyl head-groups provides long-lasting (100 days post melanoma tumor challenge) protective immunity in all immunized mice. Cationic amphiphiles with mannose-mimicking shikimoyl head-groups described in the present invention are likely to find future applications in the field of genetic immunization.

First claim

We claim:

1. A cationic amphiphile compound of formula I

wherein R is selected from the group consisting of shikimoyl, quinoyl, and mannosyl group,

R 1 and R 2 are each independently hydrogen or a lipophilic moiety and R 1 and R 2 are not hydrogen at the same time;
R 3 is independently hydrogen, C 1 -C 5 alkyl, C 1 -C 5 hydroxy alkyl and C 1 -C 5 amino alkyl;
X is optionally selected from chlorine or bromine;
wherein lipophilic moiety is selected from the group consisting of C 8-24 alkyl, mono-, di- and tri-unsaturated alkenyl.

2. The compound as claimed in claim 1 , wherein the compound is selected from the group consisting of:

3. The compound as claimed in claim 1 , wherein the compound is for use in in-vivo delivery of DNA vaccine.

4. A process for the preparation of compounds of formula I,

wherein R is selected from the group consisting of shikimoyl, quinoyl, and mannosyl group,

R 1 and R 2 are each independently hydrogen or a lipophilic moiety and R 1 and R 2 are not hydrogen at the same time;
R 3 is independently hydrogen, C 1 -C 5 alkyl, C 1 -C 5 hydroxy alkyl and C 1 -C 5 amino alkyl;
X is optionally selected from chlorine or bromine;
wherein lipophilic moiety is selected from the group consisting of C 8-24 alkyl, mono-, di- and tri-unsaturated alkenyl,
said process comprising the following steps:
(a) coupling of compound of formula II with L-lysine derivatives in a polar aprotic solvent to obtain compound of formula III, followed by acid deprotection to obtain compound of formula IV,

(b) coupling of compound of formula IV obtained from step (a), with shikimic acid, quinic acid or mannose to obtain compound of formula V,

(c) deprotecting compound of formula V obtained from step (b) to obtain compound of formula VI, followed by guanidinylation of formula VI in aprotic solvent to obtain compound of formula VII,

(d) quaternization of compound of formula VII obtained from step (c) with methyl iodide to obtain compound of formula VIII followed by base mediated deprotection in polar protic solvent to obtain compound of formula IX and finally extraction to obtain compound of formula I,

wherein R′ is selected from the group consisting of shikimoyl, quinoyl, and mannosyl group,

wherein R is selected from the group consisting of shikimoyl, quinoyl, and mannosyl group,

5. The process as claimed in claim 4 , wherein the compound of formula II is having 8-24 carbon atoms.

6. The process as claimed in claim 4 , wherein the polar aprotic solvent of step (a) and step (c) is selected from the group consisting of dichloromethane, dimethyl formamide, dimethylsulphoxide, pyridine, and triethyl amine.

7. The process as claimed in claim 4 , wherein base is selected from the group consisting of potassium carbonate, lithium hydroxide, sodium hydroxide, sodium carbonate, potassium hydroxide, sodium methoxide and potassium methoxide.

8. The process as claimed in claim 4 , wherein the polar protic solvent for base mediated deprotection in step (d) is selected from the group consisting of methanol, ethanol and mixture of water & methanol.

9. A formulation comprising the compound of formula I, a co-lipid, and a polyanionic compound along with physiologically acceptable additive.

10. The formulation as claimed in claim 9 , wherein, the formulation further comprises helper lipids which is selected from the group consisting of cholesterol, phosphatidylethanolaine and phosphatidylglycerol.

11. The formulation as claimed in claim 9 , wherein, the co lipid is selected from the group consisting of phosphatidylethanolamine, phosphatidylphosphocholine, neutral phosphatidyl ethanolamine, neutral phosphatidyl choline, phosphatidylglycerol, cholesterol, and 1,2-syn-dioleoyl-glycerolphosphatidylethanolamine (DOPE).

12. The formulation as claimed in claim 9 , wherein, the molar ratio of compound of formula I to co lipid ranges from 1:1 to 3:1.

13. The formulation as claimed in claim 9 , wherein, the polyanionic compound is selected from the group consisting of nucleic acid, a protein, an oligonucleotide, a peptide and a drug.

14. The formulation as claimed in claim 13 , wherein, the nucleic acid is selected from the group consisting of plasmid, a ribonucleic acid, a ribosomal RNA, antisense polynucleotide of RNA or DNA, polynucleotide of genomic DNA, cDNA and mRNA.

15. The formulation as claimed in claim 9 , wherein the formulation is administered via cutaneous, sub-cutaneous, intradermal, nasal, intravenous, intramuscular, intraperitonial and pulmonary route.

16. The formulation as claimed in claim 9 , wherein the formulation is administered intracellularly in the range from 25 to 100 microliters.

17. The formulation as claimed in claim 9 , wherein the formulation is administered to cells at a ratio 0.1 to 0.5 microgram of DNA to 50,000 cells.

18. The formulation as claimed in claim 9 , wherein the range of compound of formula I is from 9.0 to 0.3 microgram and lipid to DNA ratio ranges from 0.3:1 to 9:1.

19. A method for producing an immune response, the method comprising administering the formulation of claim 9 with a polynucleotide wherein said polynucleotide encodes an immunogen to at least one mouse thereby generating at least one immunized mouse.

Assignees

• Council Scient Ind Res

Inventors

• Garu Arup
• Moku Gopikrishna
• Agawane Sachin Barad
• Chaudhuri Arabinda

Classifications

• A61P37/04

  Immunostimulants · CPC title

• A61P37/02

  Immunomodulators · CPC title

• A61K9/1272

  comprising non-phosphatidyl surfactants as bilayer-forming substances, e.g. cationic lipids or non-phosphatidyl liposomes coated or grafted with polymers (lipids as modifying agents {A61K47/543}) · CPC title

• A61K9/1271

  Non-conventional liposomes, e.g. PEGylated liposomes or liposomes coated or grafted with polymers (liposomes as conjugates {A61K47/6911}) · CPC title

• C07C2601/14

  The ring being saturated · CPC title