Title:  Dendrimer constructs and metal complexes thereof having superoxide dismutase activity

United States Patent:  6,410,680

Assignee:  DendriMolecular, Inc. (San Carlos, CA)

Filed:  June 21, 2000

A metal-dendrimer complex in which the metal active site is deep within the three-dimensional dendrimer construct, has superoxide dismutase-like activity. In particular embodiments the complex is a copper(II)-dendrimer complex and the dendrimer construct is a dendritic polypeptide, and the copper(II) is complexed with imidazole groups provided by histidine side groups within the dendritic construct. The dendrimer construct has a generally globular shape, and the branched chains nearer the surface are sufficiently densely packed to restrict the movement of larger biomolecules into the dendritic construct toward the metal active sites. Smaller molecules such as the superoxide anion (O₂^.multidot.-) move freely from the milieu into the dendritic complex and to the metal active sites, where the dismutation of superoxide to hydrogen peroxide is effected; and smaller molecules such as hydrogen peroxide move freely out from the dendritic complex to the milieu. Also, a method for treating or preventing a disease condition associated with oxidative stress includes administering such a metal-dendrimer complex to a subject in need of treatment, in a form and by a route of administration suitable for bringing the complex to the site of the condition.

SUMMARY OF THE INVENTION

In one general aspect the invention features a dendrimer construct having a core and two or more branched arms projecting outwardly from the core. The arms include internal branched units and terminal moieties; the terminal moieties constitute an outermost surface of the dendrimer construct. The arms include at least one metal ion binding site enclosed within the outermost surface. The outermost surface of the dendrimer construct is sufficiently densely packed to restrict the movement of larger molecules from the milieu into the dendritic construct, and the surface is sufficiently porous to permit free movement of smaller molecules from the milieu into the dendrimer construct and to the metal ion binding site and out from the dendrimer construct to the milieu.

In another general aspect the invention features a metal-dendrimer complex in which a metal active site is enclosed within the surface of the dendrimer construct. The dendrimer construct includes a core and two or more branched arms projecting outwardly from the core. The arms include internal branched units and terminal moieties; the terminal moieties constitute the outer surface of the dendrimer construct. The arms include at least one metal ion binding site associated with one or more internal branched units and enclosed within the outermost surface, and a metal ion is complexed at the metal ion binding site to form the metal active site. The outermost surface of the dendrimer construct is sufficiently densely packed to restrict the movement of larger molecules from the milieu into the dendritic construct Smaller molecules such as the superoxide anion (O₂^.multidot.-) move freely from the milieu into the dendrimer construct and to the metal active site, where the dismutation of superoxide to hydrogen peroxide is effected; and smaller molecules such as hydrogen peroxide move freely out from the dendrimer construct to the milieu.

In some embodiments the metal ion in the complex is an ion of a transition metal such as copper, manganese, or iron; in particular embodiments the metal ion is an ion of copper, particularly copper(II); or is an ion of iron, particularly iron(III).

In some embodiments the dendrimer construct results from sequential monomer addition in a divergent synthesis, beginning from a core and constructing the branched arms by proceeding outwardly through successive generations (the core being the zeros^th generation). One mode of such divergent synthesis proceeds by sequential addition of monomers using a protection-deprotection scheme. The core may have two (divalent), three (trivalent), four (tetravalent) or more reactive moieties, providing points of attachment for, respectively, two, three, four or more branched arms; usually the core is divalent or trivalent.

A The monomers or branching units making up the arms may be 1.fwdarw.2 branching or 1.fwdarw.3 branching. The dendrimer construct may have all structurally similar arms, and each arm may contain similar repeat internal branch units (except for one or more branch units with which the ion binding site is associated); or each arm may contain dissimilar internal branch units. Or, the dendrimer construct may have structurally different arms, each having either similar or dissimilar internal branch units or terminal moieties.

Preferred internal branch units, at least in the vicinity of metal active sites, include structures that are less likely to be degraded by the presence of the superoxide anion (O₂^.multidot.-) or of its derivatives such as the highly reactive OH^.multidot. radical which may exist transiently near the metal reactive site, or hydrogen peroxide.

In some embodiments the dendrimer construct is a dendritic polypeptide or a dendritic polyamidoamine. Suitable monomers include any of various L-, D-, or DL-.alpha.-amino acids carrying functional groups in the side chains R, where R has the general formula (CH₂)_n NH₂, where n=1-7; or the general formula (CH₂)_n COOH, where n=1-7.

In some embodiments the metal ion is complexed with imidazole groups provided by L-histidine within the dendrimer construct Particular such embodiments include dendrimer constructs having 1,4-diaminobutane ["DAB"] as a core and having L-lysine ["K"] as a monomer for each generation except that generation in which L-histidine ["H"] is used to provide imidazole coordination sites for copper ions. In some such embodiments 16 histidine residues are provided and, accordingly, histidine is employed as the monomer at the fourth generation; these include the D₆ construct (DAB)K₂ K₄ K₈ H₁₆ K₁₆ K₃₂, the D₇ construct (DAB)K₂ K₄ K₈ H₁₆ K₁₆ K₃₂ K₆4, and the D₈ construct (DAB)K₂ K₄ K₈ H₁₆ K₁₆ K₃₂ K₆4 K₁₂₈. Where, as in these embodiments, 16 imidazole groups are provided, the number of copper(II) ions that may be complexed in each molecule may be in the range from as few as 1 to as many as 4, more usually from 1 to 3 or from 1 to 2. In some embodiments the N(1) of the imidazole group is alkylated with an iodoalkane having the general formula I(CH₂)_n H(n=1-8). (See, A. Noordam et al. 1978, Recl. Trav. Chim. Pays-Bas, Vol 97, pp. 293-95.)

In another general aspect the invention features a method for treating or preventing a disease condition associated with oxidative stress, by administering to a subject in need of treatment a metal-dendrimer complex according to the invention, in a form and by a route of administration suitable for bringing the complex to the site of the condition. A disease condition associated with oxidative stress, as that expression is used herein, is one in which oxidative stress is a primary cause or in which oxidative stress is secondary to the condition but contributes significantly to disease pathology.

In some embodiments the disease condition to be treated is an oxidative stress associated disease of the gastrointestinal tract and the metal-dendrimer complex is administered orally for topical treatment within the gastrointestinal lumem. As will be appreciated, movement of molecules across the mucosa can be influenced by molecular size as well as various other properties, and absorption can be effected differently in different regions of the digestive tract. See, e.g., McMartin et al. 1987, Jour. Pharm. Sci, Vol. 76, pages 535 ff.; Peters et al. 1987, Jour. Pharm. Si., Vol. 76, pages 857 ff. A preferred metal-dendrimer complex for topical treatment in the digestive tract has a higher molecular weight, usually in the range about 50 kd to about 100 kd or greater, so that absorption of the complex across the digestive tract mucosa, and clearance of the complex through the kidneys, will be limited. In some embodiments a metal-dendrimer complex for treatment in the digestive tract has a molecular weight in the range about 50 daltons to about 100 daltons.

The water-soluble metal-dendrimer complexes according to the invention can be useful in treatment of any of a variety of disease conditions associated with oxidative stress. For example, a Copper(II)-dendritic peptide complex having molecular size in the range greater than about 50 kd can also be administered rectally, or by intravenous, intraperitoneal, intramuscular, or subcutaneous injection, for treatment of damage associated with oxidative stress in the digestive tract.

The dendrimer construct will not be a substrate for enzymatic degradation Moieties in the outermost tiers, on the surface of the dendritic construct, can be modified to facilitate affinity of the complex to cell surfaces or tissues, and particular modifications can be used to provide affinity for selected types of cells or tissues. The surface of the dendritic construct can be modified, for example by use of agents such as polyethylene glycol (see, e.g., C. O. Beauchamp et al. 1978, Anal. Biochem., Vol. 131, pp. 25-33) to reduce inununogenicity of the complex; or, for example, by use of alkyl groups to facilitate affinity of the construct to cell surfaces or tissues or to facilitate permeability of the construct across membrane bilayers; or, for example, by conjugation to the dendrimer construct of selected cationic peptide fragments, which have a high aiffity for heparin-like proteoglycans in targeted cells or tissues (see, e.g., M. Inoue et al 1991, Jour. Biol. Chem., Vol. 266, pp. 16409-14), to promote accumulation of the complexes at one or more targeted disease sites. The molecular size of the dendrimer complex can be readily controlled

Interaction of proteins or protein fragments with the surface of the dendrimer construct is expected not to substantially alter or destroy the tertiary structure of metal active site. Even where a surface-modified metal-dendrimer complex according to the invention is employed, small molecules such as the O₂^.multidot.- radical can diffuse relatively unimpeded to the active site, where they participate in the redox reaction. The metal active site is situated deeply within the dendrimer construct. The extremely reactive and harmful hydroxyl radical (OH^.multidot.), which is an expected product of Fenton chemistry at the active site, is rapidly converted to hydrogen peroxide before it can leave the dendrimer complex to the surrounding milieu because the hydroxyl radical has a short life time (.about.70 ns; I. Saito et al. 1990, Chemistry of Active Oxygens, chapter 1, p. 4, M. Misono, ed., Chemical Review No.7, Chemical Society of Japan, Tokyo (in Japanese)) and the rate constant k for the dismutation reaction OH^.multidot. +OH^.multidot..fwdarw.H₂ O₂ is extremely high (k=5.times.10^-9 M^-1 S^-1 ; B. Halliwell et al., supra, p. 57).

Claim 1 of 23 Claims

What is claimed is:

1. A metal-dendrimer complex having superoxide dismutase-like activity, comprising a dendrimer construct having a globular shape defining a surface, and having at least one metal ion complexed at a metal ion binding site situated within the volume defined by the surface of the construct, sufficiently deeply to impede diffusion from the metal ion binding site to the milieu surrounding the dendrimer construct surface of any hydroxyl radical generated by reaction of superoxide radical at said metal ion binding site.

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