Protein occlusion for delivery of small molecules

Details Protein occlusion for delivery of small molecules Protein occlusion for delivery of small molecules

1998-07-16

2002-06-18

Azpuru, Carlos (Department: 2165)

Drug, bio-affecting and body treating compositions

Preparations characterized by special physical form

Implant or insert

C424S045000, C424S430000, C424S433000, C424S434000, C424S436000, C424S443000, C424S451000, C424S464000

Reexamination Certificate

active

06406710

ABSTRACT:

TECHNICAL FIELD OF THE INVENTION
The present invention relates to complexes between (1) a target-binding moiety; (2) a cavity-forming moiety; and (3) a pharmacological compound to be delivered to a target, wherein the pharmacological compound is occluded inside of the cavity-forming moiety, but not covalently bound to either the target-binding moiety or the cavity-forming moiety. The complexes of this invention may be used as to deliver a pharmacological compound to cells, tissues, organs, viruses, microorganisms or other surfaces that are characterized by an entity that binds the target-binding moiety portion of the complex. The present invention also relates to pharmaceutical compositions comprising the non-covalent complexes of this invention. The invention also relates to methods of delivering a pharmacological compound to a target in a patient. The present invention also relates to the use of the complexes of this invention for the separation of chemical entities from their chiral forms or contaminants.
BACKGROUND OF THE INVENTION
The formulation and specific delivery of agonists and antagonists to their targets is of fundamental importance in modern pharmacology. Frequently, the pharmacological activity at the target (e.g., cytotoxicity towards malignant tissue in cancer therapy) is complicated by undesired activity at other sites (e.g., cytotoxicity towards healthy tissue).
To improve selective delivery at the molecular level, the specificity of monoclonal antibodies has been exploited for the delivery of antibody/protein fusions to target cells. The use of antibodies as carrier vehicles confers high specificity for the target, but has the disadvantage that release of the protein from the antibody when the fusion reaches the target site is difficult and inefficient. Moreover, antibody/protein fusions often raise neutralizing antibodies which prevent the fusions from reaching its target, thus rendering treatment ineffective. Finally, the type of compound that can be effectively fused to antibodies is mostly limited to larger compounds, such as cytotoxic peptides or proteins. It is extremely difficult to covalently link inorganic or organic molecules to proteins at precise ratios and without loss of protein function.
An alternative method to improve selective delivery at the molecular level is to take advantage of the known specificity of receptor/peptide ligand interactions and make ligand/toxin fusions that selectively destroy cells displaying the cognate receptor. This method also has the disadvantages and limitations of the antibody/toxin fusions. Accordingly, there is a great need for a target-specific delivery system that can overcome these problems, especially one that is useful for the selective delivery of small molecules to desired targets.
Many enzymes contain cavities or channels at their catalytic centers. Such cavities are filled with water molecules that can be exchanged with other solvents (Y. Kita et al., “Contribution of the surface free energy perturbation to protein-solvent interactions”
Biochemistry
33, pp. 15178-89 (1994)]. Certain enzymes, such as Na,K-ATPase, whose active sites accommodate charged substrates can also occlude small inorganic cations [E. Or et al., “Effects of competitive sodium-like antagonists on Na,K-ATPase suggest that cation occlusion from the cytoplasmic surface occurs in two steps”
J. Biol. Chem.,
268, pp. 16929-37, (1993)]. Moreover, water filled pockets that appear to be completely buried in the interior of a protein have also been seen in crystal structures of enzymes. The reasons for the existence of empty or water-filled cavities within the tightly packed hydrophobic interior of a protein remain unknown. In the case of a subtilisin, the water was proposed to become trapped as the newly synthesized protein is folded [J. T. Pedersen et al., “Cavity mutants of Savinase. Crystal structures and differential scanning calorimetry experiments give hints of the function of the buried water molecules in subtilisins”
J. Mol. Biol.,
242, pp. 193-202 (1994)].
The most extensively characterized hydrophobic cavity in the core of an enzyme is that of T4 lysozyme [B. W. Matthews et al., “Studies on protein stability with T4 lysozyme”
Adv. Protein Chem.,
46, pp. 249-278 (1995)]. The presence of this cavity provided a useful tool for the study of the importance of hydrophobic forces in protein packing. Using directed mutagenesis, Matthews et al. altered the volume of hydrophobic amino acid side-chains protruding into the cavity and, by diffusion, altered the occupancy of the cavity by solvents of increasing hydrophobicity. The consequences of these modifications on the size and shape of the cavity as well as the structure, function and stability of T4 lysozyme were determined by X-ray crystallography, calorimetry and binding measurements (A. E. Eriksson et al., “A cavity-containing mutant of T4 lysozyme is stabilized by buried benzene”
Nature,
355, pp. 371-3 (1992); A. E. Eriksson et al., “Similar hydrophobic replacements of Leu99 and Phe153 within the core of T4 lysozyme have different structural and thermodynamic consequences”
J. Mol. Biol.
229, pp. 747-769 (1993); A. Morton et al., “Energetic origins of specificity of ligand binding in an interior nonpolar cavity of T4 lysozyme”
Biochemistry
34, pp. 8564-75, (1995); A. Morton et al., “Specificity of ligand binding in a buried nonpolar cavity of T4 lysozyme: linkage of dynamics and structural plasticity”
Biochemistry,
34, pp. 8576-88 (1995)].
Through this work, it became apparent that, within limits, the cavity in the hydrophobic core of T4 lysozyme can be modified or occupied by hydrophobic solvents with little effect on the overall structure of the protein. Such modified cavities could accommodate compounds as large as 161 Å
3
, with dissociation constants in the 14 to 500 micromolar range—range comparable to many enzyme-substrate binding constants [A. Morton et al. (1995), supra].
The presence of cavities in the hydrophobic cores of non-enzymatic proteins has largely been ignored, since no apparent significance could be ascribed to their presence. For example, the presence of a cavity in the subunit interface of nerve growth factor (NGF) was not reported in a document describing that compound's crystal structure (N. Q. McDonald et al. “New protein fold revealed by a 2.3-Å resolution crystal structure of nerve growth factor”
Nature
354, pp. 411-14 (1991)]. More recently, however, in a comparison of the structure of a Brain Derived Neurotrophic Factor (BDNF)/Neurotropain-3 (NT-3) heterodimer with that of the NGF homodimer, the preservation of a cavity in both structures was observed. It was postulated that the purpose of that cavity may be to facilitate structural change upon receptor binding [C. R. Robinson et al., “Structure of the Brain Derived Neurotrophic Factor/Neurotrophin 3 Heterodimer”
Biochemistry,
34, pp. 4139-36 (1995)]. Cavities have also been reported in the structures of other cytokines, such as IL-1 and fibroblast growth factor-&bgr;, FGF&bgr;. With the availability of cavity-locating computer programs [G. J. Kleywegt et al.,
Acta Crystallogr.,
D50, pp. 178-185 (1994)] and the increasing number of crystal structures being resolved, it is expected that the existence of cavities will be documented in many more proteins. As in the case of the BDNF/NT-3 heterodimer interface, cavities may also be present at the interfaces of other multi-protein complexes, such as the TGF-&bgr; covalent dimer and the CNTF/receptor complex or at complexes of proteins with other macromolecules, such as a fibroblast growth factor (FGF)/heparin complex.
SUMMARY OF THE INVENTION
The present invention solves the problems set forth above by providing complexes between (1) a target-binding moiety; (2) a cavity-forming moiety; and (3) a pharmacological compound to be delivered to a target, wherein the pharmacological compound is occluded inside of the cavity-forming moiety, but is not cova

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