# US 8,772,235 B2 · Compounds having peptides conjugated to bone targeting moieties and methods of making and using thereof

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- Number: US 8,772,235 B2
- Document ID: US8772235B2
- Status: Granted
- Kind: B2
- Application number: 13/501,652
- Filed: 2010-10-12
- Published: 2012-10-25
- Granted: 2014-07-08
- Earliest claimed: 2009-10-14
- Assignees: The Governors of the University of Alberta
- Applicants: Michael R. Doschak; Krishna Hari Bhandari
- Inventors: Michael R. Doschak; Krishna Hari Bhandari
- Web page: https://patentgrep.com/patent/US8772235B2

## CPC

- A61K 47/50: the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K 38/23: Calcitonins
- A61K 47/548: {Phosphates or phosphonates, e.g. bone-seeking (phospholipids A61K47/544)}
- A61P 19/08: for bone diseases, e.g. rachitism, Paget's disease
- A61P 19/10: for osteoporosis
- A61P 29/00: Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
- A61P 35/00: Antineoplastic agents
- 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

## Abstract

Described herein are compositions having a peptide sequence that includes at least one bone targeting moiety, wherein the bone targeting moiety is bonded to the peptide sequence by a linker, wherein the peptide sequence is calcitonin, and wherein the composition is neutral or a pharmaceutically acceptable salt or ester thereof. In one aspect, calcitonin inhibits or slows osteoclast mediated resorptive bone loss. The compounds described herein can be used in a number of therapeutic applications including treating or preventing conditions associated with bone loss, which include, but are not limited to, osteoporosis, Paget's disease, osteolytic tumors, Rheumatoid Arthritis, Psoriatic Arthritis, Ankylosing Spondylitis, Osteoarthritis, osteopenia, and hypercalcemia. Also described herein are the methods of making these compositions that prevent or treat conditions associated with bone loss and methods of preventing bone fractures.

## Claims

### Claim 1

A composition comprising a peptide sequence and at least one bone targeting moiety, wherein the bone targeting moiety is bonded to the peptide sequence by a linker, wherein the peptide sequence comprises calcitonin, wherein the linker is a sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sulfo-SMCC) and the bone targeting moiety is a residue of {2-[(3-mercaptopropyl)thio]ethane-1,1-diyl}bis(phosphonic acid), and wherein the composition is neutral or a pharmaceutically acceptable salt or ester thereof.

### Claim 2 (depends on 1)

The composition of claim 1, wherein the peptide sequence comprises vertebrate calcitonin.

### Claim 3 (depends on 2)

The composition of claim 2, wherein vertebrate calcitonin comprises salmon calcitonin, human calcitonin, pig calcitonin, eel calcitonin, ray fish salmon calcitonin, bovine calcitonin, chicken calcitonin, rat calcitonin, mouse calcitonin, bastard halibut or olive flounder calcitonin, dog calcitonin, sardine calcitonin, humpback salmon calcitonin, or any combination thereof.

### Claim 4 (depends on 1)

The composition of claim 1, wherein the peptide sequence comprises a peptide sequence at least 90% identical to SEQ ID NO 1, SEQ ID NO 2, SEQ ID NO 3, SEQ ID NO 4, SEQ ID NO 5, SEQ ID NO 6, SEQ ID NO 7, SEQ ID NO 8, SEQ ID NO 9, SEQ ID NO 10, SEQ ID NO 11, SEQ ID NO 12, SEQ ID NO 13, SEQ ID NO 14, and SEQ ID NO 15.

### Claim 5 (depends on 1)

The composition of claim 1, wherein the linker is covalently bonded to a reactive amino group present in the peptide sequence.

### Claim 6 (depends on 1)

The composition of claim 1, wherein the linker is covalently bonded to the N-terminus of the peptide sequence.

### Claim 7 (depends on 1)

The composition of claim 1, wherein the peptide sequence comprises at least one lysine residue, and the linker is covalently bonded to the lysine residue by an amino group in lysine.

### Claim 8 (depends on 1)

The composition of claim 1, wherein the peptide sequence comprises at least two lysine residues, and the linker is covalently bonded to the lysine residue by an amino group in lysine.

### Claim 9 (depends on 1)

The composition of claim 1, wherein the peptide sequence comprises at least one lysine residue, and the linker is covalently bonded to the lysine residue by an amino group in lysine and the linker is bonded to the N-terminus of the peptide sequence.

### Claim 10 (depends on 1)

The composition of claim 1, wherein the linker comprises a water soluble crosslinker.

### Claim 11 (depends on 1)

The composition of claim 1, wherein the linker comprises a heterofunctional crosslinker, a homofunctional crosslinker, or a combination thereof.

### Claim 12 (depends on 1)

The composition of claim 1, wherein the linker has at least one group capable of reacting with a nucleophile.

### Claim 13 (depends on 1)

The composition of claim 1, wherein the peptide sequence is a salmon calcitonin peptide.

### Claim 14 (depends on 1)

A pharmaceutical composition comprising the composition of claim 1 and a pharmaceutically acceptable carrier.

### Claim 15

A method of making a composition comprising:
(a) reacting at least one amine group present in a peptide sequence with at least one linker, wherein the linker is a sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sulfo-SMCC), to form a peptide linker intermediate, and
(b) reacting the peptide linker intermediate with a bisphosphonate containing compound, wherein the bisphosphonate containing compound is {2-[(3-mercaptopropyl)thio]ethane-1,1-diyl}bis(phosphonic acid).

### Claim 16 (depends on 15)

The method of claim 15, wherein the peptide sequence comprises salmon calcitonin, human calcitonin, pig calcitonin, eel calcitonin, ray fish salmon calcitonin, bovine calcitonin, chicken calcitonin, rat calcitonin, mouse calcitonin, bastard halibut or olive flounder calcitonin, dog calcitonin, sardine calcitonin, humpback salmon calcitonin, or any combination thereof.

### Claim 17 (depends on 15)

The method of claim 15, wherein the peptide sequence comprises a sequence at least 90% identical to SEQ ID NO 1, SEQ ID NO 2, SEQ ID NO 3, SEQ ID NO 4, SEQ ID NO 5, SEQ ID NO 6, SEQ ID NO 7, SEQ ID NO 8, SEQ ID NO 9, SEQ ID NO 10, SEQ ID NO 11, SEQ ID NO 12, SEQ ID NO 13, SEQ ID NO 14, and SEQ ID NO 15.

### Claim 18 (depends on 15)

The method of claim 15, wherein the ratio of the peptide sequence to linker comprises a 1:3, a 1:5, a 1:7, or a 1:10 mol/mol ratio.

### Claim 19 (depends on 15)

The method of claim 15, wherein the reaction of step (a) comprises from 10 to 60 minutes at room temperature.

### Claim 20 (depends on 15)

The method of claim 15, wherein the bisphosphonate containing compound comprises a thiol bisphosphonate compound.

### Claim 21 (depends on 15)

The method of claim 15, wherein the ratio of the peptide linker intermediate to the bisphosphonate containing compound comprises a 1:3, a 1:5, a 1:7, a 1:10, or a 1:20 mol/mol ratio.

### Claim 22 (depends on 15)

The method of claim 15, wherein step (b) comprises reacting the peptide linker intermediate with the bisphosphonate containing compound from 2 hours to 24 hours at room temperature.

### Claim 23 (depends on 1)

A method of treating loss of bone mass in a subject comprising administering the composition of claim 1 to the subject having a condition that causes loss of bone mass.

### Claim 24 (depends on 1)

A method for preventing a loss of bone in a subject comprising administering to the subject the composition of claim 1.

### Claim 25 (depends on 23)

The method of claim 23, wherein the subject comprises a mammal.

### Claim 26 (depends on 23)

The method of claim 23, wherein the subject comprises a human.

### Claim 27 (depends on 23)

The method of claims 23, wherein the condition comprises osteoporosis, Paget's disease, osteolytic tumors, Rheumatoid Arthritis, Psoriatic Arthritis, Ankylosing Spondylitis, Osteoarthritis, hypercalcemia, osteopenia, or any combination thereof.

### Claim 28 (depends on 1)

A method for reducing bone loss comprising contacting the bone with the composition of claim 1.

### Claim 29 (depends on 1)

A method for preventing bone fractures comprising contacting the bone with the composition of claim 1.