# US 12,178,883 B2 · Alpha and gamma-D polyglutamated pemetrexed antifolates and uses thereof

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- Number: US 12,178,883 B2
- Document ID: US12178883B2
- Status: Granted
- Kind: B2
- Application number: 17/544,196
- Filed: 2021-12-07
- Published: 2022-03-24
- Granted: 2024-12-31
- Earliest claimed: 2016-08-12
- Assignees: L.E.A.F. Holdings Group LLC
- Applicants: L.E.A.F. Holdings Group LLC
- Inventors: Clet Niyikiza; Victor Mandla Moyo
- Web page: https://patentgrep.com/patent/US12178883B2

## CPC

- A61K 47/6913: {the liposome being modified on its surface by an antibody}
- A61K 9/1271: Non-conventional liposomes, e.g. PEGylated liposomes or liposomes coated or grafted with polymers (liposomes as conjugates {A61K47/6911})
- A61K 9/1277: Preparation processes; Proliposomes
- A61K 31/517: ortho- or peri-condensed with carbocyclic ring systems, e.g. quinazoline, perimidine
- A61K 31/519: ortho- or peri-condensed with heterocyclic rings
- A61K 45/06: Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
- A61K 47/10: Alcohols; Phenols; Salts thereof, e.g. glycerol; Polyethylene glycols [PEG]; Poloxamers; PEG/POE alkyl ethers
- A61K 47/545: {Heterocyclic compounds (A61K47/558 takes precedence)}
- 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 47/645: {Polycationic or polyanionic oligopeptides, polypeptides or polyamino acids, e.g. polylysine, polyarginine, polyglutamic acid or peptide TAT}
- A61K 47/6801: {Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent}
- A61K 47/6849: {the antibody targeting a receptor, a cell surface antigen or a cell surface determinant}
- A61K 47/6911: {the form being a liposome}
- A61P 19/02: for joint disorders, e.g. arthritis, arthrosis
- A61P 35/00: Antineoplastic agents
- C07K 16/28: against receptors, cell surface antigens or cell surface determinants
- A61K 2300/00: Mixtures or combinations of active ingredients, wherein at least one active ingredient is fully defined in groups A61K31/00 - A61K41/00
- B82Y 5/00: Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
- C07K 2317/73: Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
- C07K 2317/76: Antagonist effect on antigen, e.g. neutralization or inhibition of binding
- C07K 2317/92: Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
- Y02A 50/30: Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
- 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
- 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)
- B82Y: SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
- Y02A: TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE

## Abstract

The disclosure relates generally to polyglutamated antifolates, formulations containing liposomes filled with alpha or D-gamma polyglutamated antifolates, methods of making the polyglutamated antifolates and liposome containing formulations, and methods of using polyglutamated antifolates and liposome containing formulations to treat hyerproliferative disorders (e.g., cancer) and disorders of the immune system (e.g., an autoimmune disease such as rheumatoid arthritis).

## Claims

### Claim 1

A liposomal composition comprising polyglutamated pemetrexed (PMX) encapsulated by a pegylated liposome, wherein the polyglutamated PMX comprises at least 1 D-glutamate residue.

### Claim 2 (depends on 1)

The liposomal composition of claim 1, wherein the polyglutamated PMX comprises 3, 4, 5, 6, 7, 8, 9, or 2-10 D-glutamate residues.

### Claim 3 (depends on 1)

The liposomal composition of claim 1, wherein the polyglutamated PMX comprises 4, 5, 5-10, or more than 5, glutamate residues.

### Claim 4 (depends on 1)

The liposomal composition of claim 1, wherein the polyglutamated PMX comprises at least 3 glutamate residues linked by alpha carboxy group linkages.

### Claim 5 (depends on 1)

The liposomal composition of claim 1, wherein the polyglutamated PMX comprises at least 2 glutamate residues linked by gamma carboxy group linkages.

### Claim 6 (depends on 1)

The liposomal composition of claim 1, wherein the polyglutamated PMX comprises pentaglutamated PMX.

### Claim 7 (depends on 1)

The liposomal composition of claim 1, wherein the polyglutamated PMX comprises 4 D-gamma glutamate residues.

### Claim 8 (depends on 1)

The liposomal composition of claim 1, wherein the polyglutamated PMX comprises hexaglutamated PMX.

### Claim 9 (depends on 1)

The liposomal composition of claim 1, wherein the polyglutamated PMX comprises 5 D-gamma glutamate residues.

### Claim 10 (depends on 1)

The liposomal composition of claim 1, wherein the polyglutamated PMX comprises at least 1 D-glutamate residue and at least one L-glutamate residue.

### Claim 11 (depends on 1)

The liposomal composition of claim 1, wherein the liposome has a diameter in the range of 20 nm to 200 nm or 80 nm to 120 nm.

### Claim 12 (depends on 1)

The liposomal composition of claim 1, wherein the liposome is formed from liposomal components and wherein: the liposomal components comprise:
(a) at least one of an anionic lipid and a neutral lipid;
(b) at least one member selected from the group consisting of: DSPE; DSPE-PEG; DSPE-PEG-maleimide; HSPC; HSPC-PEG; cholesterol; cholesterol-PEG; and cholesterol-maleimide; or
(c) at least one selected from the group consisting of: DSPE; DSPE-PEG-FITC; DSPE-PEG-maleimide; cholesterol; and HSPC.

### Claim 13 (depends on 12)

The liposomal composition of claim 12, wherein one or more liposomal components further comprises at least one steric stabilizer selected from the group consisting of polyethylene glycol (PEG); poly-L-lysine (PLL); monosialoganglioside (GM1); poly(vinyl pyrrolidone) (PVP); poly(acrylamide) (PAA); poly(2-methyl-2-oxazoline); poly(2-ethyl-2-oxazoline); phosphatidyl polyglycerol; poly[N-(2-hydroxypropyl) methacrylamide]; amphiphilic poly-N-vinylpyrrolidones; L-amino-acid-based polymer; or polyvinyl alcohol.

### Claim 14 (depends on 1)

The liposomal composition of claim 1, wherein the PEG of the liposome has a number average molecular weight (Mn) of 200 to 5000 daltons.

### Claim 15 (depends on 1)

The liposomal composition of claim 1, wherein the liposome:
(a) is anionic or neutral,
(b) has a zeta potential that is less than or equal to zero,
(c) has a zeta potential that is between 0 to −150 mV, or
(d) has a zeta potential that is between −30 to −50 mV.

### Claim 16 (depends on 1)

The liposomal composition of claim 1, wherein the liposome is cationic.

### Claim 17 (depends on 1)

The liposomal composition of claim 1, wherein the liposome has an interior space and the interior space comprises the polyglutamated PMX and an aqueous pharmaceutically acceptable carrier, optionally wherein the aqueous pharmaceutically acceptable carrier (a) is trehalose, (b) comprises citrate buffer at a concentration of between 5 to 200 mM and a pH of between 2.8 to 6, or (c) comprises a total concentration of sodium acetate and calcium acetate of between 50 mM to 500 mM.

### Claim 18 (depends on 1)

The liposomal composition of claim 1, wherein the liposome comprises less than 200,000 molecules or between 10,000 to 100,000 molecules of polyglutamated PMX.

### Claim 19 (depends on 1)

The liposomal composition of claim 1, wherein the polyglutamated PMX comprises at least 2 D glutamate residues and the liposome has a diameter in the range of 20 nm to 200 and a zeta potential that is less than or equal to zero.

### Claim 20 (depends on 19)

The liposomal composition of claim 19, wherein the polyglutamated PMX comprises 3, 4, 5, 6, 7, 8, 9, or 2-10 D-glutamate residues.

### Claim 21 (depends on 1)

The liposomal composition of claim 1, which further comprises a targeting moiety attached to one or both of a PEG and the exterior of the liposome, optionally by a covalent bond, and wherein the targeting moiety has a specific affinity for a surface antigen on a target cell of interest.

### Claim 22 (depends on 21)

The liposomal composition of claim 21, wherein the targeting moiety is a polypeptide, or one or more selected from the group consisting of: an antibody, a humanized antibody, an antigen binding fragment of an antibody, a single chain antibody, a single-domain antibody, a bi-specific antibody, a synthetic antibody, a pegylated antibody, and a multimeric antibody.

### Claim 23 (depends on 21)

The liposomal composition of claim 21, wherein the targeting moiety binds the antigen with an equilibrium dissociation constant (Kd) in a range of 0.5×10−10 to 10×10−6 as determined using surface plasmon resonance analysis.

### Claim 24 (depends on 21)

The liposomal composition of claim 21, wherein the targeting moiety specifically binds one or more folate receptors selected from the group consisting of: folate receptor alpha (FR-α), folate receptor beta (FR-β), and folate receptor delta (FR-δ).

### Claim 25 (depends on 1)

The liposomal composition of claim 1, wherein each liposome comprises from 30 to 200 targeting moieties.

### Claim 26 (depends on 1)

The liposomal composition of claim 1, further comprising one or more of an immunostimulating agent, a detectable marker and a maleimide, wherein the immunostimulatory agent, the detectable marker or the maleimide is attached to said PEG or the exterior of the liposome.

### Claim 27 (depends on 26)

The liposomal composition of claim 26, wherein the immunostimulating agent is at least one selected from the group consisting of: a fluorescein; a fluorescein isothiocyanate (FITC); a dinitrophenol (DNP); a beta glucan; a beta-1,3-glucan; and a beta-1,6-glucan.

### Claim 28 (depends on 1)

The liposomal composition of claim 1, further comprising a hapten or at least one cryoprotectant selected from the group consisting of mannitol; trehalose; sorbitol; and sucrose.

### Claim 29 (depends on 1)

The liposomal composition of claim 1, which is in unit dosage form.

### Claim 30 (depends on 1)

A pharmaceutical composition comprising the liposomal composition of claim 1.