The invention is directed to pharmaceutical compositions and methods for delivery of a therapeutic or diagnostic agent from one bodily compartment to one or more other bodily compartment by administering one of the following conjugates: a polymer having multiple functional groups at least one of which is covalently bound to a therapeutic or diagnostic agent, and at least one cell uptake promoter covalently bound to the therapeutic or diagnostic agent; or a polymer and at least one cell uptake promoter bound thereto; the polymer further comprising multiple functional groups at least one of which is covalently bound a therapeutic or diagnostic agent.

Description of the Invention

SUMMARY OF THE INVENTION

In its broadest aspect, the invention is directed to a method for delivery of a therapeutic agent or a diagnostic agent from an initial bodily compartment to at least one target bodily compartment, the method carried out by at least administering to the initial bodily compartment an effective transcompartmental delivery promoting amount of one of the following conjugates:

a) a polymer having multiple functional groups at least one of which is covalently bound to a therapeutic or diagnostic agent, and at least one cell uptake promoter is covalently bound to the therapeutic or diagnostic agent; or

b) a polymer and at least one cell uptake promoter bound thereto; the polymer further comprising multiple functional groups at least one of which is covalently bound to a therapeutic or diagnostic agent.

The conjugates described above include compounds having the general formulas: (X).sub.o--(Y).sub.m-(linker).sub.n a) where X is one or more transporter, receptor, binding or targeting ligands, including retro inverso peptides, which may be identical or non-identical; where Y is one or more of any therapeutic or diagnostic moieties, naturally occurring or artificial, including retro inverso peptides, which may be identical or nonidentical: where linker comprises polymer with functional groups and provides covalent bonds between linker and Y; and m, n, and o may be any independently varying integers, or more specifically may each independently vary from 1 to about 100: or (Y).sub.m-(linker).sub.n-(X).sub.o b) where X is one or more transporter, receptor, binding or targeting ligands, including retro inverso peptides, which may be identical or non-identical; where Y is one or more of any therapeutic or diagnostic moieties, naturally occurring or artificial, including retro inverso peptides, which may be identical or nonidentical; where linker comprises polymer with functional groups and provides covalent bonds between linker and X, and/or Y, or the combination thereof; and m, n, and o may be any independently varying integers, or more specifically may each independently vary from 1 to about 100.

The initial bodily compartment may be an extravascular or an intravascular site, which may be, by way of non-limiting examples, gastrointestinal tract, nasal, pulmonary, ocular, skin, organs, cells, tissues, bodily fluids, circulation, extracellular fluid, cerebrospinal fluid, ventricular fluid, lymphatic fluid, subdermal space, and intradermal space. The target bodily compartment may be, by way of non-limiting example, circulation, the central nervous system, the brain, the eye, or an intracellular environment. The target bodily compartment may be several compartments sequentially traversed by the conjugate of the invention, for example, an orally-delivered conjugate may pass from the initial intestinal luminal compartment across the intestinal epithelium into the circulation, from which it then may pass across the capillary endothelial cells into the central nervous system. In preferred embodiments, the intracellular environment is within an epithelial cell, an endothelial cell, a phagocytic cell, a lymphocyte, a neuron, or a cancer cell. An epithelial cell may be an intestinal cell; a phagocyte may be a macrophage.

The administering may be, for example, parenterally, ocularly, transmucosally or transdermally; transmucosally may be orally, nasally, pulmonarily, vaginally or rectally; parenterally may be intravenous, intra-arterial, intramuscular, intradermal, subcutaneous, intraperitoneal, intraventricular, or intracranial. Preferably, administering is orally.

The linker may be a linear or branched polymer, for example, poly(ethylene glycol), carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, an amino acid homopolymer, polypropylene oxide, a copolymer of ethylene glycol/propylene glycol, an ethylene/maleic anhydride copolymer, an amino acid copolymer, an amino acid copolymer of polyethylene glycol and an amino acid, a polypropylene oxide/ethylene oxide copolymer, and a polyethylene glycol/thiomalic acid copolymer. Poly(ethylene glycol) is preferred. Branched polyethylene glycol is most preferred. The linker may have a molecular weight ranging from about 200 to about 200,000 Daltons; preferably 2,000 to about 50,000 Daltons, and most preferably about 10,000 Daltons. The multiple thiol compounds are attached to said polymer at an interval, preferably the interval is every about 100 to about 10,000 Daltons; most preferably it is about 300 to about 5,000 Daltons.

The cell uptake promoter, transporter, receptor, binding or targeting ligand may be a vitamin such as, but not limited to, biotin, pantothenate, vitamin B6, or vitamin B12, or analogs thereof. It may also be a carbohydrate for which a transporter exists, such as for glucose and glucose derivatives. It may also be a chemotactic peptide such as a formyl-methionyl peptide. Examples of other peptide targeting agents with a range of size and amino acid order includes the peptide formyl-methionyl-leucyl-phenylalanine (fMLF) peptide and variants thereof which serves as a transport enhancing moiety and increases drug delivery into cells expressing the receptor for that peptide. fMLF is only one example of the class of formyl-methionyl peptides that binds to this receptor. Other examples include other formylmethionyl peptides and proteins capable of binding to the formyl peptide receptor on the surface of phagocytic cells, which also has been reported to bind to certain other, unrelated peptides lacking the formylmethionyl moiety, and these latter peptides unrelated to formylmethionyl peptides but capable of binding to the receptor are fully embraced herein. Other transport enhancing moieties may include Tat-biotin, retro-inverso (RI)-Tat, and RI-TAT-biotin. It may be a chemokine, such as RANTES or IL-2. It may also be a peptide such as Tat, penetratin or VEGF, or a membrane fusion peptide such as gp41. It may also be an enzyme such as neuraminidase. It may be an antibody or an antibody fragment with specific affinity for lymphocyte subpopulations, neurons or other cell types. Examples of such antibodies include antibodies to CD4, which may target helper T-cells, or CD44, which may target ovarian cancer cells. It may also be an antigen or epitope such as influenza virus hemagglutinin. It may also be a hormone such as estrogen, progesterone, LHRH, ACTH or growth hormone. It may also be an adhesion molecule such as ICAM, NCAM or a lectin. It may also be a lipid, such as myristic acid or stearic acid. It may be an oligonucleotide or an antisense oligonucleotide such as aptamers containing 5-(1-pentyl)-2'-deoxyuridine. These are merely non-limiting examples. Any of the cell uptake promoters embraced herein may be provided as a form which is capable of being covalently attached to a polymer or therapeutic agent as described above, such as through a functional or reactive group on the cell uptake promoter or by a chemical modification to provide one.

The functional group on the polymer may be any of a number of moieties which may serve for reacting the various components of the conjugates together, that is, for binding the therapeutic agent and, in the cased in which the cell uptake promoter is bound to the polymer, for binding the cell uptake promoter. Non-limiting examples include a ketone, an ester, a carboxylic acid, an aldehyde, an alcohol, a thiol, or an amine, but these are merely illustrative of the invention. Conjugation of the therapeutic agent and cell uptake promoter may be achieved using bifunctional cross-linking agents, oxidation in the case of two sulfhydral groups forming the cross-link, or any other means for covalently linking the aforementioned components. The functional groups may be activated groups, such as N-ethylmaleimide, which will react directly with a moiety to form a covalent bond. Labile bonds, such as disulfide bonds which are reducible in vivo, are preferred, or enzyme-attackable bonds such as ester bonds. Thioether bonds are also embodied herein. Non-cleavable bonds are also embraced herein, wherein the therapeutic agent is active when covalently bound to the composition of the invention. The bond between the polymer and therapeutic or diagnostic agent, and the bond between the polymer and the cell uptake promoter (in that particular embodiment) are independently labile or non-labile. Where the cell uptake promoter is bound to the therapeutic agent, it may be bound through a labile or non-labile bond. One of skill in the art will readily determine the activity of the therapeutic agent when bound to the polymer and/or cell uptake promoter and construct the composition of the invention to maximize both transcompartmental transport and activity of the therapeutic agent at the desired compartment or compartments. As will be seen below, the lability of the labile bond may be adjusted to maximize delivery to the desired compartment(s) and maximize pharmacological activity of the therapeutic agent at that compartment(s). Moreover, it would be desirable that the polymer contain orthogonal functional groups, such that the number of substituent groups on the polymer can be specified and well controlled during manufacturing. Also, by controlling the addition of appended groups to one or more specific functional groups on the polymer backbone, a monodisperse product, defined as a population of molecules having the same molecular mass, may be readily achieved. By definition, orthogonal refers to chemical groups that can be involved in specific chemical reactions independently of one another. By way of non-limiting examples, when working with peptides, the two most commonly used orthogonal groups are the amino group (--NH.sub.2) and the thiol group (--SH). Reagents are available that will react with only amino groups or thiol groups, but not with both. In manufacturing a particular conjugate, one may begin with a scaffold that contains amino and thiol groups, each present in integer numbers. The scaffold may be a peptide, such as Lys-Cys-Cys-Cys. The amino acid Cys has a thiol group, so this peptide can react with 3_molar equivalents of a thiol specific reagent, such as maleimide-PEG to give the product: Lys-Cys(PEG)-Cys(PEG)-Cys(PEG) where by convention the thiol and maleimide groups are understood to be present but not specifically written.

The amino acid Lys has one amino group, but there is one amino group present due to the peptide backbone structure. Therefore, this peptide can react with two equivalents of an amino group specific reagent, such as the N-hydroxysuccinimide activated ester of biotin to give: (biotin)Lys(biotin)-Cys(PEG)-Cys(PEG)-Cys(PEG)(SEQ ID NO: 12)

where by convention, the biotin that reacts due to the peptide backbone structure is written at the extreme left and the biotin associated with the Lys is written in parentheses.

Thus, a peptide acting as a scaffold of the formula: (Lys).sub.n-(Cys).sub.m can be derivatized using two orthogonal reactions to give a product with exactly n+1 copies of the amine-reactive chemical and m copies of the thiol-reactive chemical. By being orthogonal, these 2 reactions can be carried out with either the thiol or the amino reaction first and without regard to any significant improper cross-reaction occuring.

An additional methodology is to use orthogonal protecting groups, such as in the peptide: Cys(t-butyl)-Cys(trityl)-Cys(trityl). All 3 thiol groups in this peptide are blocked from reacting with thiol-specific reagents. However, treatment with reducing agent (e.g. dithiothreitol at pH 8) will remove the t-butyl group to give: Cys-Cys(trityl)-Cys(trityl) which may be reacted with maleimide-PEG to give: Cys(PEG)-Cys(trityl)-Cys(trityl). Then treatment with acid will remove the trityl group to give: Cys(PEG)-Cys-Cys which may be reacted with maleimide-biotin to give: Cys(PEG)-Cys(biotin)-Cys(biotin).

The acid treatment and dithiothreitol treatment may be performed in the reverse order. This peptide still has an amino group available, such as for reacting with amine-reactive fluorescein isothiocyanate to give: (fluorescein)-Cys(PEG)-Cys(biotin)-Cys(biotin). Similarly, the Fmoc and tBoc protecting groups for amines are orthogonal in that the first is base-labile and the second is acid-labile, such as in the peptide: (Fmoc)Lys(tBoc)-Cys(t-butyl)-Cys(trityl) which can accomodate 4 separate reactions.

In a preferred embodiment, multiple cell uptake promoter molecules and multiple therapeutic agent molecules are bound to a branched polymer. In a non-limiting example, a conjugate of the invention with multiple branches and cell uptake promoters on each branch, provide effective delivery. Moreover, combined drug therapy using a composition of the invention may provide a fixed delivery ratio between two or more compounds desirably delivered at a particular ratio, and avoids the problem of varied pharmacokinetics using alternate routes of administration

The therapeutic agent may be any pharmaceutically useful compound that may be bound via a functional group thereon to the composition of the invention. Such agents may be therapeutic agents of many types, such as bioactive proteins, peptides, including, but not limited to, retro inverso (RI) peptides, small-molecule compounds, antisense oligonucleotides, and the like. The invention is not limited in any way as to the nature of the therapeutic agent component of the compositions. If no functional group is present on the compound, it may be derivatized to bear one. As noted above, the compositions and methods of the invention provide transcompartmental delivery of such compounds.

The diagnostic agent may be any diagnostically useful compound that may be bound via a functional group thereon to the composition of the invention. Diagnostic moieties having reporter molecules that can be detected by imaging equipment may include radioactive, paramagnetic, fluorescent or radioopaque chemical entities. Specific examples include iodinated sugars that are used as radioopaque agents, and can be appended to linker backbones using ester or other linkages as described above. Additional diagnostic examples include the use of radioactive metal complexes such as Technetium-99m in coordination compounds such as types of, e.g. .sup.99mTc-Tetrofosmin or .sup.99mTc-Sestamibi, which are used in various types of scintigraphic imaging. Peptidic or other chelating groups can be used to prepare and append chelators that are able to coordinate isotopes of this type.

In a preferred embodiment, the functional groups on the polymer are thiol groups, and in a further preferred embodiment, the therapeutic agent also has a thiol group or is derivatized to have a thiol group. Thus, a preferred conjugate of the invention may be a) a polymer to which multiple thiol compounds each comprising a thiol group are bound, and at least one therapeutic or diagnostic agent comprising a thiol group bound to said polymer through a disulfide bond, and wherein said therapeutic or diagnostic agent comprising a thiol group, further comprises a cell uptake promoter bound thereto; or b) a polymer and a cell uptake promoter conjugated thereto, the polymer having multiple thiol compounds bound thereto, and at least one therapeutic or diagnostic agent comprising a thiol group bound to said polymer through a disulfide bond.

In a preferred embodiment, the multiple thiol groups are attached to said polymer at an interval. Preferably, the interval is about 100 to about 10,000 Daltons. The thiol compound may be, by way of non-limiting example, cysteamine, 1-amino-2-methyl-2-propanethiol, or 1-amino-2-propanethiol.

The cell uptake promoter may be biotin, pantothenate, vitamin B6, vitamin B12, a carbohydrate, a chemokine, a membrane fusion peptide, a lipid, an oligonucleotide, an antisense oligonucleotide, an enzyme, a hormone, an adhesion molecule, a peptide or protein, a formyl-methionyl peptide, a retro inverso peptide or protein, or an antibody molecule or antibody fragment, but it is not so limiting.

The therapeutic or diagnostic agent comprising a thiol group may be a synthetic or naturally-occurring protein or peptide. It may also be a therapeutic agent or a diagnostic agent with or modified to have a thiol group, or be conjugatable to a thiol group, such a modified antisense oligonucleotide or a thioamide-moiety-containing therapeutic agent. It may be a small-molecule compound with a pharmacological activity. It may be a retro-inverso form of a biologically-active peptide, retro-inverso form possessing the same or similar biological activity but possessing other desirable characteristics such as decreased susceptibility to enzymatic attack or metabolic enzymes. In one non-limiting example, the peptide comprises a Tat-inhibitory polypeptide, comprising an amino acid sequence of formula I: R-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg-X-(biotin)-Cys-NHz (SEQ ID NO:1), and biologically and pharmaceutically acceptable salts thereof, stereo, optical and geometrical isomers thereof where such isomers exist, as well as the pharmaceutically acceptable salts and solvates thereof, wherein R comprises the residue of a carboxylic acid or an acetyl group; and X is a Cys or Lys residue. Examples of the foregoing include N-acetyl-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg-Cys-(biotin)-Cys-NH.sub.2 (SEQ ID NO:2) N-acetyl-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg-Lys-(biotin)-Cys-NH.sub.2 (SEQ ID NO:3) N-acetyl-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg-D-Cys-(biotin)-Cys-NHz (SEQ ID NO:4) N-acetyl-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg-D-Lys-(biotin)-Cys-- NH.sub.2 (SEQ ID NO:5) N-acetyl-Gln-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg-D-Lys-(biotin)-Cys-NH.sub.2 (SEQ ID NO:6); N-acetyl-Arg-Lys-Lys-Arg-Arg-Pro-Arg-Arg-Arg-Cys-(biotin-Cys-NH.sub.2 (SEQ ID NO:7); or N-acetyl-DCys-DLys-(biotin)-DArg-DArg-DArg-DGln-DArg-DArg-DLys-DLys-DArg-- NH.sub.2 (SEQ ID NO: 8) or biologically and pharmaceutically acceptable salts thereof.

The number of copies of the therapeutic or diagnostic agent and the cell uptake promoter on the composition of the invention may be independently varied depending on the initial and target compartments, the desired pharmacokinetics of the product, and other factors. In some cases, the cell uptake promoter is a retro inverso peptide that acts as a transport enhancing moiety which increases therapeutic drug delivery into cells expressing receptors for the retro inverso peptide. In some case, the transport enhancing moiety may also serve as a therapeutic agent itself.

DETAILED DESCRIPTION OF THE INVENTION

As used herein, a bodily compartment refers to any place or location in or on the body, including extravascular and intravascular sites. Intravascular sites as related to bodily compartments refers to a site within a blood vessel. Extravascular sites or bodily compartments refer to all other areas within or outside of the body not associated directly with a blood vessel, such as but not limited to the gastrointestinal tract, nasal, pulmonary, ocular, skin surface, intradermal, subcutaneous, an organ, cell, tissue, non-blood bodily fluid such as cerebrospinal fluid, lymphatic fluid, etc.

In Ser. No. 09/044,411, now U.S. Pat. No. 6,258,774, incorporated herein by reference, certain of the inventors herein described the utility of an intracellular delivery system for a therapeutic agent comprising a thiol group, using a conjugate of a polymer comprising thiol groups disulfide linked to the therapeutic agent, and a cell uptake promoter such as biotin covalently bound to the therapeutic agent or to the polymer. The cell uptake promoter enhances the intracellular access of the conjugate, and the susceptibility of the disulfide bond between the therapeutic agent and the polymer under reducing conditions within the cell results in release of the therapeutic agent within the cell, where it may exert its desirable effect. The present application is directed to further properties, aspects, compositions and uses of the conjugates therein described, for example, in delivery of therapeutic agents across the intestinal mucosa, across the blood-brain barrier, and enhancement of transport by altering the number of therapeutic agent molecules and/or cell uptake promoter molecules on the polymer, as well as altering the size and branching of the polymer. Further types of bonds, both labile and non-labile, between the polymer, the therapeutic agent and the cell uptake promoter are described. By following the teachings herein with regard to the nature of the polymer, therapeutic agent and cell uptake promoter, one can readily design a conjugate composition capable of delivery from a preselected initial bodily compartment, such as the gastrointestinal tract, to a target bodily compartment, such as the central nervous system. The initial bodily compartment may be accessed through oral or parenteral delivery, such as but not limited to oral, nasal, pulmonary, rectal, vaginal and transdermal; parenteral includes, but is not limited to, intravenous, intraarterial, intramuscular, intradermal, intraocular, subcutaneous, intraperitoneal, intraventricular, intraorbital and intracranial administration.

Applicants have found that the conjugates of the invention are capable of transcytosis across various cell types, in order to deliver the therapeutic agent across one or more compartments. In some cases, the conjugate remains intact after passage and is available for further targeting to a second target cell or organ. In some cases, the therapeutic agent may upon passage across a target cell become partially or fully released from the conjugate, in which it may desirably act locally or within the target compartment, or other locations contiguous with the target compartment. Moreover, several target compartments may be traversed by the compositions of the invention, such as an orally-absorbable compound which passes from the intestinal lumen into the circulation, and then from the circulation across capillary endothelial cells into the central nervous system. Thus, more than one barrier may be traversed by the compositions herein.

Target cells, tissues and organs include, for example, the central nervous system, which requires the conjugate to cross the endothelial cell tight junctions of the brain capillaries. Another target is circulation, for which access is gained from the gastrointestinal tract by delivery of the conjugate across the intestinal epithelium. A preferred embodiment of the invention is the oral delivery into the circulation of protein or peptide therapeutic agents bound to the aforementioned conjugate, administered orally. A second preferred embodiment is the delivery of therapeutic agents across the blood-brain barrier in the form of a conjugate as described above. Other targets include macrophages, a reservoir for HIV infection, and tumor cells. Targeting of particular agents, such as chemotherapeutic agents, to tumor cells is a further embodiment of the present invention.

Moreover, the methods and compositions of the present invention also take advantage of the known selective accumulation in tumors of macromolecular conjugates of chemotherapeutic agents, increasing their cytotoxicity for the tumor and decreasing their systemic toxicity for the host [see Seymour L W, Miyamoto H, Maeda H, Brereton M, Strohalm J, Ulbrich K, Duncan R. Influence of molecular weight on passive tumour accumulation of a soluble macromolecular drug carrier, Eur. J. Cancer 31A(5): 766-770 (1995), and Soyez H, Seymour L W, Schacht E. Macromolecular derivatives of N,N-di-(2-chloroethyl)4-phenylene diamine mustard. 2. In vitro cytotoxicity and in vivo anticancer efficacy, J. Control. Release 57(2): 187-196 (1999)].

Cell uptake promoters include but are not limited to various vitamins and other molecules which are recognized by and are transported by various transporters. These include, but are not limited to, various forms of biotin, various forms of vitamin B.sub.6, various forms of vitamin B.sub.12. Other examples of cell uptake promoters include sugars which are recognized and taken up by specific cell surface transporters. Others include chemotactic peptides such as formyl-methionyl peptides, and in a preferred embodiment, formyl-methionine-leucine-phenylalanine, which specifically targets phagocytic cells. The cell uptake promoter embraces analogs and derivatives of the foregoing and related molecules which are covalently attachable to the polymer, therapeutic or diagnostic agents of the invention. For example, a variety of biotin derivatives with functional or reactive groups are available from Sigma Chemical Co., and a skilled artisan can readily prepare a cell uptake promoter with a functional or reactive group for conjugation, wherein the cell uptake promoter on the conjugate retains its cell uptake promoting activity. As noted herein, multiple copies of the cell uptake promoter may be used on a polymer herein bearing one or more copies of the therapeutic or diagnostic agent, or multiple copies of a therapeutic or diagnostic agent conjugated to a cell uptake promoter may be present on the polymer.

With regard to the cell uptake promoter being chemotactic N-formyl-methionyl peptides that can specifically bind to surface receptors on phagocytic cells, as will be seen below, a single copy of N-formyl-methionine-leucine-phenylalanine (fMLF) covalently linked to a poly(ethylene glycol)-based polymer displayed reduced binding avidity (Kd=190 nM) for differentiated HL-60 cells relative to free fMLF (Kd=28-nM). Increasing the number of fMLF residues attached to a single polymer up to eight results in enhanced avidity for these cells (Kd=0.18 nM), that appears to be independent of whether the polymer backbone is linear or branched. However, no polymer showed enhanced ability to activate phagocytic cells, relative to the free peptide (EC.sub.50=5 nM), as measured by transient stimulation of release of calcium ions from intracellular stores into the cytoplasm. A polymer bearing four fMLF and four digoxigenin residues showed specific enhancement in binding to differentiated HL-60 cells and mouse peritoneal macrophages in situ relative to a polymer lacking fMLF; no such enhancement was seen in binding to receptor negative lymphocytic Jurkat cells. These results suggest that multiple fMLF residues linked to a drug-delivery polymer can be used to target appended drugs to phagocytic cells with relatively little toxicity due to cellular activation.

Therapeutic agents as defined herein include any prophylactically- or therapeutically-useful compound which is possesses or can be derivatized to possess a functional group through which the therapeutic agent may be conjugated to a polymer of the invention. These include various biologically-active proteins, polypeptides and peptides, which may be naturally-occurring, artificial, and comprise other molecular substituents other than amino acids, including oligonucleotides such as antisense oligonucleotides. While such therapeutic agents are known in the art, several non-limiting examples include growth factors such as insulin, anti-HIV peptides such as Tat inhibitor (see below), erythropoietin, growth hormone, interferon, immunoglobulin, parathyroid hormone, calcitonin, enkephalin, and endorphin. Other therapeutic agents include those which are modified to be able to form a functional or reactive group. Examples of such groups include but are not limited to a thiol (sulfhydryl) group. Such therapeutic agents also include those which possess a thiol group or effectively have a thiol group which may be conjugated to a polymer of the invention, such as a thioamide moiety. Examples of thioamide-moiety-containing therapeutic agents are described in co-pending application Ser. No. 09/621,109, incorporated herein by reference in its entirety. Such compounds include but are not limited to UC781; R82150; HBY097; troviridine; S2720; UC38 and 2',3'-dideoxy-3'-fluoro-4-thiothymidine.

In one embodiment, the conjugates of the invention have been found to be particularly useful for the transport and subsequent delivery of active therapeutic or diagnostic agents across the intestine, the blood-brain barrier, or across both barriers. As will be seen in the examples below, the conjugates of the invention are capable of apical to basal transport in intestinal epithelial cells, and the same directional transport in endothelial cells. These studies demonstrate the utility of the conjugates of the invention for permitting oral delivery of therapeutic or diagnostic agents into the circulation of an animal, as well as the transport of the conjugate from the circulation across the endothelial cell barrier of the brain, and into the central nervous system. These examples demonstrate further the release of the active therapeutic or diagnostic agent from the conjugate through in-vivo reduction of the disulfide bond, and thus the desired pharmacological activity, although the invention is not limited to reducible conjugates and embraces those in which the therapeutic or diagnostic agent is active in a conjugate with or without reduction, enzymatic cleavage or hydrolysis. Of course, access to the endothelium or to other cell types within the body may be provided by oral delivery of the conjugates of the invention, or by other means such as parenteral administration.

Moreover, by adjusting the copy number of both the therapeutic or diagnostic agent and the cell uptake promoter on the selected polymer, and the environment of the reducible disulfide bond between the therapeutic or diagnostic agent and the polymer, the pharmacokinetics, transport and delivery properties of the conjugate may be selected for a particular target cell type, persistence of the conjugate in transit or terminal bodily compartments, particular reducing environment which frees the active therapeutic or diagnostic agent, among other parameters, can be selected to maximize the therapeutic or diagnostic value of the conjugate for its particular utility. The skilled artisan, by the guidance provided herein, will be able to prepare and administer a composition of the invention for the desired end use.

While the preferred embodiments of the invention consist of a therapeutic or diagnostic agent, polymer and cell uptake promoter (the latter bound either to the therapeutic or diagnostic agent or the polymer), and the therapeutic or diagnostic agent reversibly bound to the polymer by way of a labile or non-labile bond, such as but not limited to a disulfide bond, the conjugate may consist of only the therapeutic or diagnostic agent and the polymer, or the therapeutic or diagnostic agent and the cell uptake promoter. Applicants have found enhanced transport of therapeutic agents bound to a polymer of the invention, preferably multiple copies of the therapeutic agent bound to a polymer but it is not necessarily so limited.

Furthermore, the delivery of therapeutic or diagnostic agents into various other desirable target cell types is demonstrated herein, particularly in cancer cells, and in certain cells types for which particular targeting or therapeutic or diagnostic agents is desirable, such as macrophages for the treatment of HIV infection. The targeted delivery and release of the compound within particular cell types provides a therapeutically or diagnostically effective drug at the desired site, rather than elsewhere in the body or at levels too low to be therapeutically or diagnostically effective to achieve the desired therapeutic, diagnostic or prophylactic effect. It has been found that N-formyl-methionyl peptides such as fMLP provide the selective targeting to phagocytic cells such as but not limited to macrophages, to achieve the targeted delivery of therapeutic or diagnostic agents for the aforementioned purposes.

It is a further aspect of the invention to increase the amount of therapeutic or diagnostic agent that can be delivered to a target site by adjusting the number of copies of the therapeutic or diagnostic agent and the number of copies of the cell uptake promoter on the polymer, as well as adjust the molecular weight of the polymer to suit the particular application.

Furthermore, in order to elucidate the structural features that potentially govern the interaction between biotinylated-PEGs and SMVT, or other cellular components, the interactions of linear biotin-PEG-3400 and branched biotin-PEGs differing in size and shape with the human biotin transporter, hSMVT, were determined in CHO cells overexpressing SMVT (CHO/SMVT). Specifically, the branched biotin-PEGs used were 3 arm/10 kDa, 4 arm/10 kDa, 8 arm/10 kDa and 8 arm/20 kDa. The molecular weight of each branched biotin-PEG molecule is evenly distributed among all the arms. Thus, biotin-PEG-8 arm/10 kDa consists of .about.1250 monomers in each arm, while biotin-PEG-8 arm/20 kDa consists of .about.2500 monomers per arm. Therefore, these molecules vary considerably in their size based on their differential arm-length. In contrast, biotin-PEG-3 arm/10 kDa, and 8 arm/10 kDa would vary in their shape. The uptake of these compounds was evaluated in CHO/hSMVT cells in order to avoid potentially confounding transporters that may be present in Caco-2 cells. Computational techniques were used to explore the influence of conformational flexibility on the physical properties of the substrates. Various structural, geometric and topological descriptors of the compounds were estimated and their correlation with kinetic parameters of transport determined.

The present results suggest that the linear and branched biotin-PEGs of varying shapes and sizes exhibit unique interactions with SMVT with different relative affinities towards SMVT. The maximal affinity of the biotin-PEGs towards SMVT is exhibited within a specific window of topological and structural properties, outside of loss of affinity in paralleled by an increase in the net SMVT-mediated cellular uptake. However, Applicants are not bound by said theory.

In another study using endothelial cells, the following table (see Original Patent) illustrates the effect of ratio of the number of biotins in a poly(ethylene glycol) molecule on permeability to the blood-brain barrier. Various branched (3 arm/10 kDa, 4 arm/10 kDa, 8 arm/10 kDa and 8 arm/20 kDa) biotin-PEGs were evaluated at a fixed concentration (10 .mu.M) using bovine brain microvessel endothelial cells (BBMECs), an in vitro BBB model. Non-biotinylated PEGs were used in the control studies. Permeabilities of biotin conjugated 3 arm/10 kDa, 4 arm/10 kDa, 8 ar/10 kDa and 8 arm/20 kDa PEGs were 4.8-, 11-, 15- and 19-fold greater, respectively, than the permeabilites of their controls. Transport of the non-biotinylated PEGs were significantly lower (p<0.005) in all cases. There is a positive relationship between the number of branches and the transport of the biotin-PEGs, as shown in Table 1 (see Original Patent).

The present invention embraces various ratios between the number of cell uptake promoter molecules and the polymer molecule, including, for example, in branched polymers from one cell uptake promoter per branch to one cell uptake promoter per branched polymer molecule, to linear or branched polymers with repeating cell uptake promoter molecules at intervals. The same parameters apply to the therapeutic or diagnostic agent. As noted herein, the ratios may be readily selected to maximize both delivery at the desired target and amount of therapeutic or diagnostic agent delivered at the target.

In the studies herein, the intestinal transport properties of another large peptide, RI-K(biotin)-Tat9, are characterized. HIV-1 tat protein enters cells and transactivates the HIV-1 long terminal repeat (LTR) when added exogenously to cell culture media (A. D. Frankel and C. O. Pabo. Cellular uptake of the tat protein from human immunodeficiency virus, Cell. 55:1189-1193 (1988); D. A. Mann and A. D. Frankel. Endocytosis and targeting of exogenous HIV-1 Tat protein, EMBO J. 10:1733-1739 (1991)). It has been suggested that adsorptive endocytosis (D. A. Mann and A. D. Frankel. Endocytosis and targeting of exogenous HIV-1 Tat protein, EMBO J. 10:1733-1739 (1991)) or specific cell-surface proteins are involved in the uptake of HIV-1 tat (B. E. Vogel, S. J. Lee, A. Hildebrand, W. Craig, M. D. Pierschbacher, F. Wong-Staal, E. Ruoslahti. A novel integrin specificity exemplified by binding of the alpha v beta 5 integrin to the basic domain of the HIV Tat protein and vitronectin, J. Cell Biol. 121:461-468 (1993); D. A. Brake, C. Debouck, and G. Biesecker. Identification of an Arg-Gly-Asp (RGD) cell adhesion site in human immunodeficiency virus type 1 transactivation protein, tat, J. Cell Biol. 111: 1275-1281 (1990); B. S. Weeks, K. Desai, P. M. Loewenstein, M. E. Klotman, P. E. Klotman, M. Green, and H. K. Kleinman. Identification of a novel cell attachment domain in the HIV-1 Tat protein and its 90-kDa cell surface binding protein, J. Biol. Chem. 268:5279-5284 (1993)). The mechanisms behind the uptake of HIV-1 tat remain controversial, however, the ability of tat to enter cells and to serve as a carrier for heterologous proteins is well established (D. A. Mann and A. D. Frankel. Endocytosis and targeting of exogenous HIV-1 Tat protein, EMBO J. 10:1733-1739 (1991); S. Fawell, J. Seery, Y. Daikh, C. Moore, L. L. Chen, B. Pepinsky, and J. Barsoum. Tat-mediated delivery of heterologous proteins into cells, Proc. Natl. Acad. Sci. USA 91:664-668 (1994); D. C. Anderson, E. Nichols, R. Manger, D. Woodle, M. Barry, A. R. Fritzberg. Tumor cell retention of antibody Fab fragments is enhanced by an attached HIV TAT protein-derived peptide, Biochem. Biophys. Res. Commun. 194:876-884 (1993); R. B. Pepinsky, E. J. Androphy, K. Corina, R. Brown J. Barsoum. Specific inhibition of a human papillomavirus E2 trans-activator by intracellular delivery of its repressor, DNA Cell Biol. 13:1011-1019 (1994)). Chen et al. (L. L. Chen, A. D. Frankel, J. L. Harder, S. Fawell, J. Barsoum, B. Pepinsky. Increased cellular uptake of the human immunodeficiency virus-1 Tat protein after modification with biotin, Anal Biochem. 227:168-175 (1995)) reported a six-fold increase in uptake of tat by the addition of hydrophobic biotin groups. The amount of tat protein that reaches the intracellular compartment, however, is quite low (D. A. Brake, C. Debouck, and G. Biesecker. Identification of an Arg-Gly-Asp (RGD) cell adhesion site in human immunodeficiency virus type 1 transactivation protein, tat, J. Cell Biol. 111: 1275-1281 (1990)). Choudhury et al. (I. Choudhury, J. Wang, A. B. Rabson, S. Stein, S. Pooyan, S. Stein, and M. Leibowitz. Inhibition of HIV-1 replication by a Tat RNA-binding domain peptide analog, J. AIDS Human Retrovir. 17:104-111 (1998)) synthesized a 10-amino acid tat inhibitor, N-acetyl-L-Arg-L-Lys-L-Lys-L-Arg-L-Arg-L-Gln-L-Arg-L-Arg-L-Arg-L-Cys-NH.s- ub.2, denoted Tat9-C, consisting of the 9 amino acid RNA binding domain of Tat linked to a C-terminal cysteine, with its amino terminus acetylated and its carboxy terminus amidated. They showed that Tat9-C, upon S-biotinylation on the cysteine residue (Tat9-C(biotin)) was taken up 30-fold more efficiently by Jurkat cells than was Tat9-C (3% versus 0.1%, respectively). Subsequently, Tat9-K(biotin), which resembles Tat9-C(biotin) except that the cysteine-S-biotin moiety is replaced by lysine-e-N-biotin, was synthesized (I. Choudhury, J. Wang, S. Stein, A. Rabson, and M. J. Leibowitz. Translational effects of peptide antagonists of Tat protein of human immunodeficiency virus type 1, J. Gen. Virol. 80: 777-782 (1999)). Tat9-K(biotin) and Tat9-C(biotin) showed similar ability to compete with Tat protein binding to the TAR domain of viral RNA preventing Tat-dependent gene expression in cultured cells (I. Choudhury, J. Wang, S. Stein, A. Rabson, and M. J. Leibowitz. Translational effects of peptide antagonists of Tat protein of human immunodeficiency virus type 1, J. Gen. Virol. 80: 777-782 (1999)). The replacement of L-amino acids by their D-stereoisomers generally resulted in a reduced ability of Tat9-K(biotin) to bind to the TAR RNA (J. Wang. Development of an HIV-1 Tat antagonist based on TAR RNA as the strategic target. Ph.D. Thesis, Rutgers, The State University of New Jersey, New Brunswick (1997)). The inventors herein found that a retro-inverso (RI) derivative of Tat9-K(biotin), denoted RI-K(biotin)-Tat9, had comparable TAR RNA binding activity to Tat9-K(biotin) (J. Wang, S. Pooyan, M. J. Leibowitz, S. Stein, unpublished results). RI-K(biotin)-Tat9, with its lack of L-amino acids and blocked termini was found to be highly resistant to proteolysis in 10% fetal calf serum. With its reversed order of mirror-image amino acids, it should have steric similarity to Tat9-K(biotin), differing only in the polarity of the underlying peptide backbone. Therefore, RI-K(biotin)-Tat9 was used for some of the studies described herein. In the present study, a mechanistic evaluation of the transport of RI-K(biotin)-Tat9 was performed to determine its suitability for oral administration. The absorptive transport kinetics of RI-K(biotin)-Tat9 was characterized using Caco-2 cell monolayers, a widely used and well established model for investigating drug transport. The specific interactions between RI-K(biotin)-Tat9 and the biotin transporter, SMVT, were determined using CHO cells overexpressing hSMVT (CHO/hSMVT). The current results suggest that a novel strategy for enhancing the intestinal absorption of large peptides may be to add a targeting moiety such as biotin in order to significantly alter its absorption pathway and significantly enhance intestinal permeability.

SMVT, the sodium dependent multivitamin transporter, which transports the water soluble vitamins biotin, pantothenate and lipoic acid, is a protein of 635 amino acids with 12 transmembrane domains. It is expressed in the placenta, intestine, brain, liver, lung, kidney and heart in rats, rabbits and humans. Further, within the intestinal tract, different variants of SMVT (variant I) are also expressed in the duodenum (II), jejunum (II, IV), ileum (II, III), and colon (III). In mammalian cells, the cDNA-induced uptake of physiological substrates biotin, pantothenate and lipoate is electrogenic and sodium dependent with a Na.sup.+:vitamin stoichiometry of 2:1 (P. D. Prasad, H. Wang, W. Huang, Y-J. Fei, F. H. Leibach, L. D. Devoe, and V. Ganaphthy. Molecular and functional characterization of the intestinal Na.sup.+-dependent multivitamin transporter, Arch. Biochem. Biophys. 366:95-106 (1999)). The physiological aspects involved in the cellular entry of intact HIV-1 Tat protein (86 amino acid residues) and fragments such as residues 1-36 or 36-72 have been extensively documented. However, the specific transport pathways for cellular uptake were not identified. In the present study, a novel Tat inhibitor, RI-K(biotin)-Tat9, with substantial anti-HIV-1 activity was synthesized. As will be seen in the examples below, the absorptive transport of RI-K(biotin)-Tat9 through Caco-2 cell monolayers was found to be concentration dependent and saturable suggesting the involvement of a carrier mediated transport pathway. Inhibition studies implicate the involvement of SMVT. The expression of SMVT in Caco-2 cell monolayers was validated by using a functional assay and known competitive substrates and verified using RT-PCR. The K.sub.m value (11.28 .mu.M) calculated for biotin transport in the current study is consistent with those obtained from human intestinal brush-border membrane vesicles (5.26 .mu.M), colonic epithelial NCM460 cells (19.7 .mu.M), rat small intestine (8.77 .mu.M) and other reports suggesting the involvement of SMVT (T. Y. Ma, D. L. Dyer, and H. M. Said. Human intestinal cell line Caco-2: A useful model for studying the cellular and molecular regulation of biotin uptake, Biochim. Biophys. Acta. 1189: 81-88 (1994); P. D. Prasad, S. Ramamoorthy, F. H. Leibach, and V. Ganaphthy. Characterization of a sodium-dependent vitamin transporter mediating the uptake of pantothenate, biotin and lipoate in human placental choriocarcinoma cells, Placenta 18: 527-533 (1997); H. M. Said, R. Redha, and W. Nylander. A carrier-mediated, Na+ gradient-dependent transport for biotin in human intestinal brush-border membrane vesicles, J. Amer. Physiol. 253: G631-G636 (1987); H. M. Said, R. Redha, W. Nylander. Biotin transport in basolateral membrane vesicles of human intestine, Gasteroenterology 94: 1157-1163 (1988); H. M. Said, A. Ortiz, E. McCloud, D. Dyer, M. P. Moyer, and S. Rubin. Biotin uptake by human colonic epithelial NCM460 cells: a carrier-mediated process with pantothenic acid, Amer. J. Physiol. 275: C1365-C1371 (1998); P. D. Prasad, H. Wang, W. Huang, Y-J. Fei, F. H. Leibach, L. D. Devoe, and V. Ganaphthy. Molecular and functional characterization of the intestinal Na+-dependent multivitamin transporter, Arch. Biochem. Biophys. 366:95-106 (1999)). The absorptive transport of RI-K-Tat9, which lacks biotin, across Caco-2 cell monolayers was modest (0.8.times.10.sup.-7-1.times.10.sup.-6 cm/s) and not mediated by a carrier system, as indicated by its lack of concentration dependence. The chemical modification of RI-K-Tat9 to RI-K(biotin)-Tat9 resulted in a significant increase (3.2-fold; p<0.001) in the absorptive permeability (at 1 .mu.M). More importantly, RI-K(biotin)-Tat9 transport was concentration dependent and saturable, suggestive of a carrier mediated transport pathway. The absorptive transport was also temperature dependent with an estimated E.sub.a of 9.11 kcal/mole. It is generally believed that E.sub.a values for active carrier-mediated transport range from 7 to 25 kcal/mole while the E.sub.a value for passive diffusion is less than 4 kcal/mole (I. J. Hidalgo and R. T. Borchardt. Transport of a large neutral amino acid (phenylalanine) in a human intestinal epithelial cell line: Caco-2, Biochim. Biophys. Acta 1028:25-30 (1990)). The E.sub.a value in the present study was more than 2 fold greater than that required for passive diffusion, indicating the active, carrier-mediated transport of RI-K(biotin)-Tat9. The presence of a carrier system was further evident from the significantly larger P.sub.c (3.22.times.10.sup.-6 cm/s) component compared to the P.sub.m (0.57.times.10.sup.-6 cm/s). The specific interactions between RI-K(biotin)-Tat9 and SMVT were confirmed using hSMVT transfected CHO cells. The presence and expression of SMVT in the CHO/SMVT cells was confirmed by molecular and functional assays. The uptake of RI-K(biotin)-Tat9 and biotin was significantly higher (p<0.01) in CHO/hSMVT cells than in the vector transfected CHO/pSPORT cells with similar K.sub.m values (1.00 and 1.39 .mu.M, respectively). It appears that, upon biotinylation, the transport properties of the passively absorbed RI-K-Tat9 were modified, rendering RI-K(biotin)-Tat9 a substrate of SMVT. While Caco-2 cell transport entails passage of compounds across the apical and basolateral membranes, the non-polarized CHO cells represent a model of apical membrane uptake process. The similarity in the K.sub.m values for RI-K(biotin)-Tat9 transport from the Caco-2 (3.27 .mu.M) and CHO/hSMVT (1.00 .mu.M) cell studies suggests that the SMVT protein may be located in the apical domain of Caco-2 cells or there are transporters on both cellular domains. Further studies are required to determine the localization of SMVT.

As mentioned earlier, the Caco-2 cell monolayer transport of RI-K-Tat9 was low (P.sub.e.about.0.9.times.10.sup.-6 cm/s) and the likely result of a passive diffusion process. Upon biotinylation, despite the greater overall permeability of RI-K(biotin)-Tat9, its passive permeability component (P.sub.m.about.0.57.times.10.sup.-6 cm/s) was not statistically different (p>0.05) from that of RI-K-Tat9. The apparent lack of increase in the P.sub.m of RI-K(biotin)-Tat9 could be the result of confounding factors, such the presence or absence of other potential transport pathways in the Caco-2 cell model (e.g., active secretory transport) or the realization that transport occurs by means of paths of least resistance in intact systems. The consequent inability of Caco-2 cell monolayers to discriminate between the relative intrinsic contributions of active and passive transport pathways could result in inaccurate estimates of P.sub.m. On the other hand, CHO cells, being non-polarized with minimal transporter expression, do not suffer from these drawbacks. Accordingly, in CHO/hSMVT cells, the J.sub.max of RI-K(biotin)-Tat9 uptake in CHO/hSMVT cells substantially decreased (from 675.95 to 227.26 pmol/mg protein/10 min) when the P.sub.m component was included in the non-linear regression model. RI-K(biotin)-Tat9 uptake in CHO/pSPORT cells was also significantly higher (p<0.01) than RI-K-Tat9 uptake in CHO/hSMVT and CHO/pSPORT (<1 pmol/mg protein/10 min) cells, indicating the cellular uptake enhancing properties of biotin even in the absence of SMVT. This result is consistent with earlier reports on the increased uptake of biotinylated Tat peptides in Jurkat and HL3T1 cells, presumably due to the enhanced hydrophobicity gained by adding biotin to the peptide.

The substrate specificity of SMVT and its specific interaction with RI-K(biotin)-Tat9 were demonstrated by studying the inhibition of RI-K(biotin)-Tat9 transport across Caco-2 cells and uptake in CHO/hSMVT cells in the presence of biotin, biocytin and desthiobiotin. While all three compounds competitively inhibited the Caco-2 cell transport of RI-K(biotin)-Tat9, they also significantly lowered (p<0.001) RI-K(biotin)-Tat9 uptake in CHO/hSMVT cells, thereby confirming their specific interaction with SMVT. Previous reports have indicated the requirement of a free carboxyl group on substrates for efficient interactions with SMVT. In these studies, biotin uptake was significantly inhibited by unlabeled biotin, pantothenate, thioctic acid and desthiobiotin compared to biocytin, biotin methyl ester, and thioctic acid amide (the latter three being compounds with a blocked or no carboxyl group). Other reports suggest that the keto group at the second position of the imidazole ring is essential for substrate-SMVT interactions, based on the inability of iminobiotin and diaminobiotin to inhibit biotin uptake. It is believed that SMVT interacts primarily, but not exclusively, with the carboxylic side-chain, which is observed in the known substrates biotin, lipoic acid (containing a valeric carboxyl group) and pantothenic acid (containing a propionic carboxyl group). In the current studies, we observed that RI-K(biotin)-Tat9, a compound in which the valeric carboxyl group is blocked, was indeed a substrate for SMVT. Additionally, we observed that biocytin (biotin conjugated to lysine through an amide bond) substantially inhibited RI-K(biotin)-Tat9 transport across Caco-2 cells. Although RI-K(biotin)-Tat9 does not meet the hypothesized requirements of SMVT substrates as outlined in earlier reports, our results point towards the involvement of SMVT in its absorptive transport. The reason behind this apparent incongruity could be the inconclusive characterization of SMVT substrates in the previous reports. For instance, the requirement of a free carboxyl group on the valeric acid moiety has been emphasized. Yet, pantothenic acid and short chain fatty acids, compounds with non-valeric carboxyl groups, have been reported to significantly inhibit biotin transport. The inhibitory properties of biocytin observed in the present study may well be due to the free carboxyl group on the lysine moiety.

Although the foregoing results show that appending biotin as a targeting moiety may be applied as a novel and useful strategy to enhance the intestinal absorption of large peptides, due to the high affinity, low capacity nature of SMVT (Km values of substrates are typically in the low micromolar range), the resultant saturation of the transporter may limit the dose of drug that can be delivered via this transporter. In order to overcome this drawback, we evaluated the ability of a poly(ethylene glycol) (PEG)-based biopolymeric delivery vehicle to maximize the therapeutic or diagnostic payload of the peptide. As described above, the PEGylated delivery vehicle (or conjugate) was designed to (i) carry multiple copies of a drug, (ii) have an extended half-life in blood or extracellular fluid, (iii) enhance cellular uptake of the drug and subsequently (iv) release the appended drug molecules inside the cell. In a preliminary study, the conjugate, containing multiple copies of an 11-amino acid Tat-peptide with an appended biotin molecule, N-acetyl-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg-Lys(biotin)-Cys-NH.sub.2 (SEQ ID NO: 3), displayed 5-fold greater potency (compared to the single copy Tat-peptide) in preventing Tat-dependent gene expression in a cultured cell system (which was not tested for expression of SMVT).

RI refers to the retro-inverso form of a peptide or protein, in which the protein is chemically synthesized from all D-amino acids in a sequence that is of the opposite polarity to that of the natural protein. It is slowly becoming accepted that the retro-inverso form of a peptide most often has the full biological activity inherent to the natural peptide. With improvements in solid phase peptide synthesis and fragment condensation technology, it is becoming possible to extend the size range from peptides to proteins. Since the structure/activity relationships are unique for every protein, some research may be required to modify the retro-inverso protein into a form with suitable biological activity. For example, the charge status of the N- or C-terminus might have to be changed. Use of the retro-inverso form is important to this invention in order to protect against digestion. For proteins or peptides that are naturally resistant to digestion by conditions in the intestinal environment, the naturally-occurring form of the peptide or protein may be used. The invention is not so limited as to the type of therapeutic or diagnostic agent that is the portion of the compound of the invention, only that its desired biological activity is achieved by oral delivery to the body. Other peptide analogs, such as but not limited to peptoids, may also be used in the practice of the present invention.

The transporter provides the enhanced oral uptake and delivery of the compounds of the invention from the lumen of the intestine into the circulation of the animal. As will be seen in the examples herein, which are merely illustrative of the invention and notsoever limiting as to scope, one or more (up to 8) copies of the transporter, the vitamin biotin, were chemically linked to poly(ethylene glycol) (PEG) polymers ranging in molecular weight from 10 to 20 kDa. Carrier-mediated transport, involving the intestinal multivitamin transport protein, was demonstrated. The flux was found to increase with increasing copy number of biotins on PEG. The flux is in the range indicative of good oral bioavailability. In a separate study, a biotin moiety was linked directly to an RI-peptide which in turn was linked (8 copies) to a PEG carrier. Again, the flux was in the range useful for oral bioavailability for both the biotin-linked peptide and its multicopy version on PEG. This biotin-mediated transport can be extended to proteins, as well as to other therapeutic or diagnostic agents, including but not limited to polynucleotides, oligonucleotides, lipids, small-molecule compounds, and other macromolecules and small molecules.

The linker may be used to connect the transporter to the therapeutic or diagnostic agent. By way of example, merely illustrative of the invention but not limiting the scope thereof, PEG polymers containing multiple attachment sites can be appended with one or more biotin groups to yield conjugates capable of being transported across the intestinal epithelia. The linkage of a protein therapeutic or diagnostic agent to this PEG-biotin conjugate still allows the functioning of the biotin transporter, thereby providing delivery of the protein across the intestinal barrier. The protein may be attached to the PEG vehicle by a disulfide bond for eventual release of the protein from its carrier vehicle or by a more biostable bond if the appended PEG-biotin has no deleterious effect on the functioning of that protein drug. However, having a PEG polymer on a therapeutic protein can be useful to extend half-life in vivo by inhibiting renal or hepatic clearance (especially for a small peptide or a small antisense DNA).
 

Claim 1 of 11 Claims

1. A transcompartmental delivery promoting composition comprising: a) a branched PEG having multiple arms, each having a functional group, at least one peptide selected from the group consisting of SEQ ID NOS: 1-8 independently covalently bound to one of said arms, and at least one cell uptake promoter consisting of formyl-methionyl-leucyl phenylalanine (fMLF) covalently bound to said peptide selected from the group consisting of SEQ ID NOS: 1-8; or b) a branched PEG having multiple arms, each having a functional group, at least one cell uptake promoter consisting of formyl-methionyl-leucyl phenylalanine (fMLF) independently covalently bound to one of said arms; and at least one peptide selected from the group consisting of SEQ ID NOS: 1-8 independently covalently bound to one of said arms.
 

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