COVALENT CONJUGATES BETWEEN ENDOPEROXIDES AND TRANSFERRIN AND LACTOFERRIN RECEPTOR-BINDING AGENTS

Abstract

The invention relates to covalent conjugates between endoperoxides and small peptides and organic compounds that bind to molecular cavities on the transferrin or lactoferrin receptor, and the use of compositions comprising these conjugates to treat cancer, hyperproliferative disorders, inflammatory diseases, and infections.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing aspects and many of the attendant advantages of this invention will become more readily appreciated as the same become better understood by reference to the following detailed description, when taken in conjunction with the accompanying drawings, wherein:

FIG. 1 shows a schematic diagram for transferrin receptor-targeted delivery of artemisinin.

FIG. 2 shows a schematic diagram of the synthesis of artemisinin-peptide conjugates, which are generally prepared by reacting a carbonyl-containing peptide with artemisinin derivatives that carry a hydrazine (R,R′—N—NH₂) or aminoxy (R—O—NH₂) group.

FIG. 3 shows the effect of artemisinin-tagged peptide HAIYPRH on cultured human Molt-4 lymphobastoid leukemia cells and normal human lymphocytes. Cell viability was determined at 24, 48, and 72 hours in the presence of different concentrations (3.1, 6.2, 12.4 μM) of artemisinin-tagged peptide. The artemisinin-tagged peptide killed Molt-4 cells very effectively at low micromolar concentrations without affecting normal lymphocytes (FIGS. 3A and 3B). The IC₅₀ values of ART-peptide and ART2-peptide on Molt-4 cells were 4.23±0.09 μM and 0.86±0.10 μM at 72 hrs, respectively (FIGS. 3C and 3D). ART-peptide and ART2-peptide were peptides covalently attached with one and two artemisinin moieties, respectively. The artemisinin-tagged peptides were virtually non-toxic to normal leukocytes (IC₅₀>10,000 uM, data not shown). Under the same assay condition, DHA showed IC₅₀ values of 5.3±0.26 μM and 43±22 μM for Molt-4 cells and normal leukocytes, respectively. Therefore, tagging to the TfR-binding peptide significantly improved the anti-cancer potency and selectivity of artemisinin. The in vitro cancer cytotoxicity of the artemisinin-tagged peptides is comparable to or higher than that of the other commonly used anti-cancer drugs, except that the artemisinin-tagged peptides show much higher cancer/normal cell selectivity. FIGS. 3E and 3F show the effect of ART-peptide and ART2-peptide on medulloblastoma cells (DAOY cells). Both compounds are effective against this cell line, and it is clear that ART2-peptide is more effective than ART-peptide.

FIG. 4 shows the effect of artemisinin-tagged peptide HAIYPRH on breast tumors in an in vivo cancer model. Rats with breast tumors that were previously induced by injections with MTLn3 breast cancer cells and between 5 mm to 10 mm in diameter were treated daily with the artemisinin-tagged peptide for 5 days. The artemisinin-tagged peptide was dissolved in a phosphate buffer at pH 7.4. 0.1 mL of the buffer containing 0.5 mg of the tagged-peptide was injected daily via a tail vein. Controls were injected with 0.1 mL of the buffer daily. The length, width, and height of ellipsoidal tumor were measured with a caliper. The tumor volume was calculated using the formula: length×width×height×7/6. The results presented in the figure are expressed as percent change in tumor volume from the first day of tagged-peptide treatment for each rat. Intravenous injection of the peptide significantly retarded the growth of breast tumors in the rat.

Claims

1. A compound comprising at least one endoperoxide containing moiety covalently linked to a receptor binding agent that binds to a transferrin receptor or a lactoferrin receptor on a cell without interfering with transferrin or lactoferrin binding to the same receptor.

2. The compound of claim 1, wherein the endoperoxide containing moiety comprises an endoperoxide bridge.

3. The compound of claim 2, wherein the endoperoxide bridge is a 1,2,4-trioxane bridge or a 1,2,4,5-tetraoxane bridge.

4. The compound of claim 3, wherein the endoperoxide containing moiety is an artemisinin-related endoperoxide.

5. The compound of claim 1, wherein the receptor binding agent is a peptide.

6. The compound of claim 5, wherein the peptide is selected from the group consisting of SEQ ID NO: 1 and SEQ ID NO: 2.

7. A composition comprising a compound comprising at least one endoperoxide containing moiety covalently linked to a receptor binding agent that binds to a transferrin receptor or a lactoferrin receptor on a cell without interfering with transferrin or lactoferrin binding to the receptor, and a pharmaceutically acceptable carrier.

8. A method of treating a human or animal subject for a disease or condition selected from the group consisting of cancer; restenosis; proliferative eye, kidney, and skin diseases; precancerous hyperplastic conditions, benign prostatic hyperplasia (BPH), benign breast disease (BBD); autoimmune disease; arthritis; graft rejection; inflammatory bowl disease; proliferation induced after medical procedures; an infection by a pathogenic organism that has a receptor for transferrin or lactoferrin; topical bacterial infections; gingivitis; skin infections; eye infections; and malaria; comprising administering to said subject a therapeutically effective amount of a composition comprising a compound comprising at least one endoperoxide containing moiety covalently linked to a receptor binding agent that binds to a transferrin receptor or a lactoferrin receptor on a cell without interfering with transferrin or lactoferrin binding to the same receptor.

9. The method of claim 8, wherein the endoperoxide containing moiety comprises an endoperoxide bridge.

10. The method of claim 9, wherein the endoperoxide bridge is a 1,2,4-trioxane bridge or a 1,2,4,5-tetraoxane bridge.

11. The method of claim 10, wherein the endoperoxide containing moiety is an artemisinin-related endoperoxide.

12. The method of claim 8, wherein receptor binding agent is a peptide.

13. The method of claim 8, wherein the subject is human.

14. The method of claim 8, wherein the disease or condition is selected from the group consisting of cancer, precancerous hyperplastic conditions, BPH, and BPD.

15. The method of claim 8, comprising administering an effective amount of the covalent conjugate in combination with an agent that increases iron transport into cells.

16. The method of claim 15, wherein the agent that increases iron transport into cells is selected from the group consisting of insulin, insulin-like growth factor I, epidermal growth factor, erythropoietin, interleukin-4, interleukin-10, interleukin-13, and dexrazoxane.