Described is a multi drug multiligand conjugate for targeted drug delivery. The MDML conjugate contains a plurality of tripartite molecules linked to a central scaffold moiety, with each tripartite molecule comprising a targeting molecule, a therapeutic agent and a scaffold binding element. The MDML conjugate allows for more efficient delivery of therapeutic agents to the cells resulting in enhanced therapeutic efficiency. A model MDL conjugate is disclosed as well as method for the synthesis of the model conjugate.

Description of the Invention

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present disclosure relates generally to the field of drug delivery.

2. Description of the Related Art

Targeted drug delivery is a powerful strategy in the treatment and diagnosis of various human diseases, such as cancer, bacterial and viral infections and any disease expressing specific and targetable receptors, antigens or other markers. Targeted drug delivery has gained momentum during the past several years based on the specific delivery therapeutic molecules to malignant tissues comprising the disease state, such as the delivery of oncolytic agent to the neoplastic tissue. The ability to specifically target therapeutic molecules to specific cells of interest has advanced considerably during the past several years with the development of monoclonal antibodies and of synthetic small molecule peptides (SSMPs) capable of binding to tumor cell surface receptors. A number of drug, toxin, and radioisotope conjugates of tumor-recognizing molecules have been developed with some currently in clinical use.

Allergic reactions against large-molecule antibodies in humans due to the immunoreactivity of these proteins have hampered the desired development of antibody-drug conjugates for targeted delivery and much attention has been focused on the SSMPs which usually show no immunoreactivity due to smaller molecular sizes. With the exception of radiolabeled peptides, SSMPs have not been fully utilized as targeting drug delivery systems. Recently, the use of SSMPs in a targeted drug delivery systems with a paclitaxel-bombesin conjugate as a model compound was demonstrated (Safavy, U.S. Pat. No. 6,191,290, 2001; Safavy et al., J Med Chem, 1999, 42, 4919-4924). This and similar molecules will be referred to as single drug, single ligand (SDSL) conjugates. While effective at specifically delivering the therapeutic molecule to the target cell of interest, these SDSL conjugates have several limitations. First, the biological half-life of these SDSL conjugates is quite short, with the conjugates undergoing rapid clearance from the circulation (usually on the order of 1 to 5 minutes). As a result, only a fraction of the SDSL conjugates are taken up by the target cells, which results in a low therapeutic efficiency. Secondly, the ability to adapt SDSL conjugates to various disease states or to deliver different therapeutic molecules is limited. It would be beneficial if a conjugate were provided that is designed to deliver different therapeutic molecules to the target cells with a minimum of experimental manipulation.

Therefore, it would be advantageous to provide a pharmaceutical compound for use in a single-step drug delivery method that is capable of specifically delivering to target cells (via a targeting conjugate) multiple copies of the same or different therapeutic agent(s). This approach may result in higher therapeutic indices which may bring about a more significant response from the target cells. The present disclosure provides such a strategy and provides a method for the synthesis of a model conjugate as proof-of principle. This model conjugate was used in a tumor cell proliferation assay to demonstrate the drug-enhancing potential of this approach.

SUMMARY

The present disclosure shows the synthesis and design of two embodiments of a multidrug multiligand (MDML) conjugate which is capable of specifically delivering multiple doses of a therapeutic molecule to a target cell.

In one embodiment of the present disclosure, there is provided a pharmaceutical compound comprising a plurality of tripartite molecules in combination with a scaffold moiety. The tripartite molecules comprises a targeting molecule, a therapeutic agent, a scaffold binding element linked together by a central core molecule, the tripartite molecule being capable of binding to the scaffold moiety through complementary binding between the scaffold binding element and the scaffold moiety.

In another embodiment of the present disclosure, alternate methods of synthesizing the MDML conjugates are described. Specifically, the synthesis of one embodiment of the MDML conjugates, incorporating paclitaxel (a taxane derivative) as the therapeutic agent, a bombesin-derived peptide, BBN [7-13] as the targeting molecule, lysine or diamino benzoic acid as the core molecule and streptavidin/biotin as the scaffold moiety/scaffold binding element (the PTX-BBN[7-13]-BTN/SaV conjugate), is described in detail.

In still yet another embodiment of the present disclosure there is provided a method of treating an individual having a disease by administering the MDML conjugates of the present disclosure to an individual in an amount sufficient to treat the disease. Because of the higher therapeutic index achieved by the MDML conjugates, they may be administered at a lower concentration range than that of the un-conjugated therapeutic agents by means of injection or intravenous infusion. By disease it is meant any abnormal condition of an organ or other part of an organism.

In another embodiment of the present disclosure, there is provided a method of administering the MDML conjugates of the present disclosure to an individual as a diagnostic reagent. Because of the higher therapeutic index achieved by the MDML conjugates, they may be administered at a lower concentration range than that of the un-conjugated therapeutic agents by means of injection or intravenous infusion.

In still another embodiment of the present disclosure, a kit is provided to aid in the treatment of individuals having a particular disease

Other and further aspects, features, and advantages of the present disclosure will be apparent from the following description of the presently preferred embodiments of the invention.

DETAILED DESCRIPTION

The present disclosure illustrates that the effect of a given therapeutic agent may be enhanced by incorporation of the therapeutic agent into a MDML conjugate capable of delivering a plurality of therapeutic agents to specific target cells of interest by virtue of a plurality of targeting molecules incorporated into the same conjugate. The present disclosure describes methods for the design, synthesis and delivery of MDML conjugates for the treatment of various human disease states. Cancer is the exemplary human disease discussed below and cited in the examples, but this disclosure should not be interpreted to limit only to the treatment of cancer as one of ordinary skill in the art would be able to adapt the teachings of the disclosure to treatment of alternate disease states. It will be understood by those of skill in the art that the MDML conjugate described can be used to treat a variety of disease states by incorporating the desired targeting molecule(s) and therapeutic agent(s).

The MDML conjugate described in the present disclosure comprise a plurality of tripartite molecules in combination with a scaffold moiety. The tripartite molecules each comprise a targeting molecule, a therapeutic agent, a scaffold binding element linked together by a central core molecule. The core molecule provides the framework for construction of the tripartite molecule. The core molecule can be any molecule that provides sufficient functional groups to link together the targeting molecule, the therapeutic agent and the scaffold binding element in a functional manner. The functional groups should be positioned in a manner to minimize steric hindrance between the targeting molecule, the therapeutic agent and the scaffold binding element. In one embodiment, the core molecule is the amino acid lysine. In an alternate embodiment, the core molecule is diamino benzoic acid. Other core molecules may be used as described.

The tripartite molecule is capable of binding to the scaffold moiety through complementary binding between the scaffold binding element and the scaffold moiety. The scaffold moiety provides the framework to join the tripartite molecules together and allows for the delivery of multiple doses of the therapeutic agent to the target cell of interest.

The targeting molecule is any molecule that can direct the MDML conjugate to a defined population of cells of interest (target cells). The targeting molecule may be, but is not limited to, a peptide, a receptor ligand peptide (meaning a peptide that is designed to specifically bind to one or more designated receptor molecules), an antibody or antibody fragment. Exemplary targeting molecules include, but are not limited to, bombesin/gastrin-releasing peptide (BBN/GRP) receptor-recognizing peptide, a somatostatin receptor recognizing peptide, an epidermal growth factor receptor recognizing peptide, a monoclonal antibody or a portion thereof, or polyclonal antibody or a portion thereof, or a receptor recognizing carbohydrate, or any combination of the above. Examples of receptor recognizing polypeptides that bind to the BBN/GRP receptor, the somatostatin receptor and/or other receptors are peptides comprising the sequence of the BBN[7-13] peptide shown in SEQ ID NO. 1 (Gln Trp Ala Val Gly His Leu), the BNN[7-14] peptide shown in SEQ ID NO. 2 (Gln Trp Ala Val Gly His Leu Met) and a semi-synthetic derivative of BBN peptide shown in SEQ ID NO. 3 (Gly D-Tyr Gln Trp Ala Val bAla His Phe Nle). The targeting molecule incorporated into each tripartite molecule may be the same or may be different. The targeting molecule need not direct the MDML conjugate to only one cell type. The selection of a particular targeting molecule will depend on the cell population to be targeted and is within the ordinary skill in the art.

The therapeutic agent can be any diagnostic agent or any pharmaceutical agent, or a combination thereof. Therapeutic agents include, but not limited to, drugs, anti-tumor agents, toxins, radionuclides, metallic nuclei or a combination of the above. Exemplary therapeutic agents include taxane molecule. By taxane molecule it is meant to include any taxane derivatives such as paclitaxel (taxol) and toxotere (docetaxel) and their analogues. In one embodiment describe, the therapeutic agent is paclitaxel. The therapeutic agent incorporated into each tripartite molecule may be the same or may be different. The selection of a particular therapeutic agent will depend on the particular disease to be treated or condition to be diagnosed and is within the ordinary skill in the art.

The linker molecules may be any molecule capable of linking the targeting molecule, the therapeutic agent and the scaffold binding moiety to a central molecule. The linker molecules may reduce the effects of steric hindrance and increase the overall yield of the synthetic reaction. However, the use of a linker molecule is optional. In the embodiment described polyethylene glycol (PEG) was used as the linker. The chemical and biological properties of PEG molecules have been extensively studied and some useful characteristics of this polymer have been noted. These include aqueous as well as organic solubilities, lack of immunogenicity, and favorable blood clearance patterns and in vivo behavior. Furthermore, PEG molecules are available in a wide range of chemistries, facilitating the formation of the tripartite molecules. In the embodiment disclosed, a heterobifunctional derivative of the PEG was used in this synthesis, allowing the PEG to connect the lysine and targeting molecule, therapeutic agent and scaffold binding moiety as described below in Example 1 and FIG. 1A (see Original Patent).

The scaffold binding element and the scaffold moiety may be any set of molecules that are capable of specific, complementary, covalent or non-covalent binding to one another. Exemplary binding pairs include, but are not limited to, poly(amino acids), peptides, polypeptides, proteins, carbohydrates, biotin/avidin, biotin/streptavidin, hapten/antibody, ligand/receptor, enzyme/inhibitor and lectin/carbohydrate. The biotin/streptavidin system has been used in this disclosure as the model scaffold binding element/scaffold moieties. Avidin (isolated from egg whites) and streptavidin (isolated from Streptomyces avidinii), each have a very high binding affinity for biotin. The streptavidin-biotin system represents the strongest non-covalent biological interaction known between a protein and a ligand. Streptavidin has a lower non-specific tissue binding and absorption. Both avidin and streptavidin are capable of binding 4 molecules of biotin. Modified forms of avidin and streptavidin may also be used in methods of this disclosure. Biotin includes, but is not limited to biotin which has been modified by the addition of alkyl groups, and biotin derivatives such as active esters, amines, hydrazides and thiol groups with the complimentary reactive groups on polymers being amines, acyl and alkyl leaving groups, carbonyl groups and alkyl halides or Michael-type acceptors.
 

Claim 1 of 13 Claims

1. A compound comprising a plurality of tripartite molecules linked to a scaffold moiety, with each tripartite molecule comprising a targeting molecule for directing the compound to a defined population of cells, a therapeutic agent and a scaffold binding element capable of complementary interaction with the scaffold moiety, each of the targeting molecule, the therapeutic agent and the scaffold binding element being joined by a linker molecule to a central core molecule, wherein said scaffold moiety is selected from the group consisting of avidin and streptavidin, said scaffold binding element is biotin, said linker molecule is polyethylene glycol, said central core molecule is selected from the group consisting of lysine and diamino benzoic acid, said targeting molecule is selected from the group consisting of SEQ ID NO. 1, SEQ ID NO. 2 and SEQ ID NO. 3, said targeting molecule being the same or being different for each tripartite molecule, and said therapeutic agent is selected from the group consisting of paclitaxel and toxotere, said therapeutic agent being the same or being different for each tripartite molecule.

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