The inventors have demonstrated that they can deliver therapeutic compositions to the inner ear of mammals via a variety of routes including the round window membrane, the semicircular canals, via cochleostomy and through the stapes footplate. Using pancaspase inhibitors, the inventors have shown that relatively large volumes of compositions can be injected with little to no hearing loss.

Description of the Invention

SUMMARY OF THE INVENTION

The present inventors have conducted studies where E1/E3 and E1/E3/E4 deleted adenoviral vectors (AD11D) carrying the green fluorescent protein (GFP) gene were injected into the round window, the basal turn of the cochlea (via a cochleostomy) or into the superior semicircular canal. Hearing was then tested 24 hours after viral gene transfer. Surprisingly, large vector titers in small volumes of fluid were well tolerated with the round window approach resulting in complete hearing preservation with transfer of GFP to hair cells and spiral ganglion cells. Injection of comparable doses of vector into a basal turn cochleostomy resulted in high-frequency hearing loss.

Most notably, when the technique was coupled with the addition of a pancaspase inhibitor, the combination protected hearing when larger volumes of fluid (e.g. greater than about 10% of the total inner ear volume) were administered to the inner ear. The inventors have published some of this work recently (Praetorius, M., et al., J. ORL 2003;65:211-214).

Research completed by the inventors and disclosed herein demonstrates that use of cell death inhibitors such as the caspase inhibitor zVAD-FMK (N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone) can prevent inner ear trauma caused by hydraulic/mechanical injury to the inner ear. Id.

Further research disclosed herein shows that the inventors were able to inject E1/E3/E4 deleted adenoviral vectors (AD11D) carrying the green fluorescent protein (GFP) gene were injected into a hole drilled into the stapes footplate with a laser, without any loss of hearing. Distribution of GFP activity was seen in the spiral ganglion, vestibular ganglion and isolated sections of the sensory epithelium, demonstrating that this approach is also effective in an animal model.

As such, it is an object of the present invention to provide a method for delivery of drugs or therapeutics into the inner ear of a mammal without significant loss of hearing.

It is a further object of this invention to treat the inner ear prophylactically to protect the inner ear from anoxia/sound trauma.

It is yet another object of the present invention to treat the inner ear of a mammal to protect the inner ear when the inner ear is opened and manipulated.

It is also another object of the present invention to prevent hearing loss in a mammal by pretreating the inner ear with alternate apoptosis inhibitors such as inhibitors of c-jun kinase, molecules altering the bcl-2/bax ratio or inhibitors of specific caspases or calpains.

It is yet another object of the present invention that the compositions and methods of use disclosed herein can be used to aid in cochlear implantation with hearing preservation by preventing damage to the inner ear during cochlear implantation.

It is a still further object of the present invention that the techniques disclosed can also be used in skull base surgery when the inner ear is opened to provide more extensive surgical approaches to the cranial vault.

It is another object of the present invention to use the technology disclosed herein to include infusion of apoptosis inhibitors during endoscopy of the inner ear so as to protect against potential hearing loss.

DETAILED DESCRIPTION AND PREFERRED EMBODIMENTS

Although preferred embodiments of the present invention are explained in detail, it is to be understood that other embodiments are possible. Accordingly, it is not intended that the invention is to be limited in its scope to the details of constructions and arrangement of components set forth in the following description or illustrated in the drawings. The invention is capable of other embodiments and of being practiced or carried out in various ways. Also, in describing the preferred embodiments, specific terminology will be resorted to for the sake of clarity. It is to be understood that each specific term includes all technical equivalents which operate in a similar manner to accomplish a similar purpose. Further, although the drawings are intended to illustrate the present invention, the drawings are not necessarily drawn to scale.

It has been shown that surgical trauma or manipulation of the inner ear, such as through cochlear implantation, can induce apoptosis in the hair cells of the inner ear and result in significant hearing loss.

It is believed that the hearing loss that results after trauma to the inner ear is due to apoptosis induced through caspases activation. Therefore, if one could inhibit the subsequent activation of caspases after surgical intervention, one could reduce or eliminate the loss of hearing that often accompanies such trauma.

Apoptosis is particularly prominent during the development of an organism, where cells that perform transitory functions are programmed to die after their function no longer is required. In addition, apoptosis can occur in cells that have undergone major genetic alterations, thus providing the organism with a means to rid itself of defective and potentially cancer forming cells. Apoptosis also can be induced due to exposure of an organism to various external stimuli, including, for example, bacterial toxins, ethanol and ultraviolet radiation. Chemotherapeutic agents for treating cancer also are potent inducers of apoptosis.

At present and herein defined, the "caspase family" is known to comprise 12 members, caspases 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 and 12 (as reviewed by Cryns and Yuan, 1998, supra); one of these, caspase 9, is described as comprising the prototype caspase-recruitment domain (CARD), and thus the term "caspase" refers to any of caspases 1 through 12. See also U.S. Pat. Nos. 6,177,259 and 6,228,603 and herein incorporated into the specification as if set forth in their entireties. Mammalian caspases are homologues of the product of the C. elegans cell-death gene ced-3 and have been shown to play important roles in regulating apoptosis (Cryns and Yuan, 1998, supra). A cowpox virus cytokine response modifier gene (crmA) encodes a serpin that is a specific inhibitor of two mammalian caspases, caspase-1 and caspase-8 (Zhou et al., 1997, J. Biol. Chem., 272: 7797-7800).

The caspases previously were referred to as the "Ice" proteases, based on their homology to the first identified member of the family, the interleukin-1.beta. (IL-1.beta.) converting enzyme (Ice), which converts the inactive 33 kiloDalton (kDa) form of IL-1.beta. to the active 17.5 kDa form. The Ice protease was found to be homologous to the Caenorhabditis elegans ced-3 gene, which is involved in apoptosis during C. elegans development, and transfection experiments showed that expression of Ice in fibroblasts induced apoptosis in the cells.

A role for the caspases in apoptosis has been demonstrated by showing that overexpression of each of the identified caspases in various cell types results in apoptosis of the cell. In addition, expression in cells of CrmA, which is expressed by cowpox virus, was shown to protect the cells from undergoing cell death in response to various inducers of apoptosis by inhibiting caspase-1 activity. CrmA also was shown to bind caspase-3 and to inhibit proteolysis of the poly (ADP-ribose) polymerase (PARP) due to caspase-3, whereas a CrmA point mutant lacking the ability to bind caspase-3 did not inhibit proteolysis. PARP, as well as other cellular proteins including lamin B, topoisomerase I and .beta.-actin, are degraded during apoptosis of a cell.

Caspase Inhibitors

Caspases have been shown to be inhibited by certain peptide fragments which presumably bind to the active site of the enzyme. The synthetic tetrapeptide VAD (Val-Ala-Asp) coupled to fluoromethylketone (VAD-fmk) or the N-benzyloxycarbonyl-derivative (zVAD-fmk) are synthetic permeable inhibitors of caspases (caspase-1) that have the same spectrum of activity as derivatives of YVAD (Tyr-Val-Ala-Asp). This tetrapeptide coupled to fluoromethylketone (YVAD-fmk) is a synthetic inhibitor of caspases. The aldehyde derivative (YVAD-CHO) is another inhibitor. The N-Acetyl-derivatives of these compounds (Ac-YVAD-AFC, Ac-YVAD-CHO) and the N-benzyloxycarbonyl-derivatives (indicated by the prefix z: ZYVAD-AFC, ZYVAD-AMC, zYYAD-fmk) as well as chloromethyl ketone derivatives (cmk; YVAD-CMK) are also used.

Another derivative (Ac-VAD-CMK; N-Acetyl-VAD-chloromethyl ketone derivative) is another inhibitor with a broad spectrum. zVAD-fmk has been shown also to inhibit efficiently cathepsin B activity in vitro and in tissue culture cells at concentrations used to demonstrate the involvement of caspases and thus appears to have non-specific effects.

Treatment of cells with caspase inhibitors can inhibit characteristic biochemical and morphological events associated with cell death by apoptosis. There are numerous references to these compounds in the literature. See Fernandes-Alnemri T., Cancer Research 55(24): 6045-6052 (1995); Fernandes-Alnemri T., PNAS (USA) 93(15): 7464-7469 (1996); Muzio M. et al., Cell 85(6): 817-827 (1996); Nicholson D W et al., Nature (London) 376(6535): 37-43 (1995); Rotonda J et al., Nat, Struct. Biol. 3(7): 619-625 (1996); Schotte P et al., FEBS Lett. 442(1): 117-121 (1999); Talanian R V et al., JBC 272(15): 9677-9682 (1997).

It has also been found that caspases may be inhibited by another family of proteins called Inhibitors of Apoptosis proteins (IAP). Liston et al., Nature 379:349-353 (1996). Ambrosini et al., Nat. Med. 3:917-921 (1997); Bertin et al., J. Virology 70:6251-6259 (1996); Birnbaum et al., J. Virology 68:2521-2528 (1994); Roy et al., EMBO J. 16:6914-6925 (1997). X-linked inhibitory protein (XIAP), as well as the inhibitory proteins cIAP-1 and cIAP-2 block two distinct pathways of caspase activation by inhibiting different caspases, and are described in detail in U.S. Pat. No. 6,228,603 and herein incorporated by reference herein as if fully set forth in its entirety.

The baculovirus inhibitor of apoptosis protein repeat (BIR) is a domain of tandem repeats separated by a variable length linker that seems to confer cell death-preventing activity. Eight other genes (BIRC1, BIRC1.1, BIRC2, BIRC5, BIRC6.3, BIRC6.4, BIRC7, BIRC8) in the database also contain this motif. The BIR domain is found in proteins belonging to the IAP (inhibitor of apoptosis proteins) family.

Definitions

Biologically active agent" or "biologically active substance" refers to a chemical substance, such as a small molecule, macromolecule, or metal ion, that causes an observable change in the structure, function, or composition of a cell upon uptake by the cell. Observable changes include increased or decreased expression of one or more mRNAs, increased or decreased expression of one or more proteins, phosphorylation of a protein or other cell component, inhibition or activation of an enzyme, inhibition or activation of binding between members of a binding pair, an increased or decreased rate of synthesis of a metabolite, increased or decreased cell proliferation, and the like.

As used herein, the terms, inject, administer, deliver are synonymous and mean the transfer of the composition being referred to from one reservoir or repository to a tissue, cell, or part of an organ, tissue, fluid or space.

The terms "therapeutic agent", "therapeutic composition", and "therapeutic substance" as well as "protein of interest" refer, without limitation, to any composition that can be used to the benefit of a mammalian species. Such agents may take the form of ions, small organic molecules, peptides, proteins or polypeptides, oligonucleotides, and oligosaccharides, for example. As defined herein, a therapeutic protein of interest can be any protein, protein fragment, peptide or peptide fragment that can be used to the benefit of a mammalian species.

The term "peptide" as used herein refers to a compound made up of a single chain of D- or L-amino acids or a mixture of D- and L-amino acids joined by peptide bonds. Generally, peptides contain at least two amino acid residues and are less than about 50 amino acids in length. D-amino acids are represented herein by a lower-case one-letter amino acid symbol (e.g., r for D-arginine), whereas L-amino acids are represented by an upper case one-letter amino acid symbol (e.g., R for L-arginine).

The term "protein" as used herein refers to a compound that is composed of linearly arranged amino acids linked by peptide bonds, but in contrast to peptides, has a well-defined conformation. Proteins, as opposed to peptides, generally consist of chains of 50 or more amino acids.

"Polypeptide" as used herein refers to a polymer of at least two amino acid residues and which contains one or more peptide bonds. "Polypeptide" encompasses peptides and proteins, regardless of whether the polypeptide has a well-defined conformation.

Viral Vectors

A number of different gene therapy vectors have been tried in the inner ear. Adenoviral vectors are the best characterized because they are easy to produce and can carry a large amount of DNA. Several genes driven by different promoters can be transferred through this system. Although adenovirus is easy to use, but it has a limited expression time and has been associated with adverse immune reactions. Adeno-associated virus (AAV) is a smaller vector with more limited capacity that may allow long-term expression of transferred genes. It is more difficult to produce than adenovirus but has the advantage of not being associated with any known human disease. Herpes virus vectors have been used in a variety of applications and are capable of carrying large gene payloads. Theses vectors have the potential of maintaining long-term expression of the transferred gene and are particularly suited for targeting neurons. Liposomal or nonviral gene delivery uses charged lipids or polymers to condense the DNA to be transferred. Unfortunately at present liposomal delivery is less efficient than viral methods of gene transfer.

Adeno-Associated Viral Vectors

AAV is a parvovirus that carries an approximately 4500-bp genome flanked by terminal repeat sequences. These sequences are needed for the initiation of DNA replication and viral packaging. The native virus can exist in a lytic life cycle or can integrate into the host cell's DNA as a provirus. A helper virus (adenovirus) is needed for the virus to complete the lytic cycle or for production of an AAV vector. The existence of a latent state allows this vector potentially to be used for treatments that require long expression periods. The size of the genes that can be transferred using this system is limited by the overall size of the vector particle. About 95% of the AAV native genome can be replaced. It is not clear, however, whether the recombinant vectors that are produced truly integrate into the host genome. One of the great advantages of this packaging system is that it is not associated with any known human disease, making it a safe vector to use (Lalwani A. K., et al., Adv. Otorhinolaryngol., 2002;61:28-33). Recent developments in AAV vectorology include production of more concentrated vector stocks, which is particularly important for applications in the inner ear where a there are limitations on the volume of vector that can be delivered.

Herpes Simplex Vectors

Herpes simplex-derived vectors provide an ideal method for gene transfer to neurons. The most commonly used vector in this category is derived from herpes simplex type I (HSV 1). The native virus is able to infect both dividing and post-mitotic cells and has a broad tissue tropism. This virus also can assume a latent state in neuronal cells and exists as an episome in the nucleus of neurons. Problems relating to integration of the viral genome into the host's DNA are therefore not an issue. Generally cells infected with these viruses also escape immune surveillance. HSV 1 is a 152-kb double-stranded DNA virus coated in an envelope consisting of 12 glycoproteins. The genome contains more than 70 open reading frames. Because of the large size of the genome, vectors derived from HSV 1 can carry multiple large genes. During its natural life cycle, HSV 1 infects epithelial cells and fibroblasts in the skin and enters a lytic phase. The resulting released viruses fuse with local nerve fibers and through retrograde transport arrive at the neuronal cell body. A lytic cycle can then recur, or the virus can enter a latent stage in the nucleus of the neuron (Glorioso J. C. et al., Annu. Rev. Microbiol., 1995;49:675-710). During this stage only latency-associated RNAs are produced. Some vectors have been derived that take advantage of this virus's biology to link gene expression to latency promoters, thereby producing long-term stable gene expression in post-mitotic neurons. Replication-deficient vectors derived from HSV 1 have been produced by deleting ICP4, and growing vectors on a permissive cell line. Multiple generations of more advanced vectors have been produced by deleting additional early genes and growing the vector on engineered cell lines.

Retroviral Vectors

Retroviruses are RNA viruses and were the first viruses to be used for gene therapy. Their basic genetic structure allowed construction of a helper-free packaging system that carried the viral genes gag, pol, and env in trans. These vectors yield long-term expression of transferred genes but raise some potential concerns regarding insertional mutagenesis. Thus insertion of the pro-viral genome into the hosts DNA may cause mutations. These vectors are well characterized but have the disadvantage of being able to enter only dividing cells. Currently a new type of retroviral vector, lentiviral vectors, has been developed. These vectors are derived from HIV and simian immunodeficiency virus and are able to infect non-dividing cells. Lentiviral vectors are seen as having significant potential to provide long-term stable expression of transferred genes (Van De Water T. R., et al., Ann. NY Acad. Sci., 1999;884:345-60).

Adenoviral vectors are most commonly based on adenovirus serotype 5, a double-stranded DNA vector of 35 kilobase (kb) of which 30 kb can be replaced in current constructs. Early-generation vectors carry deletions of early genes E1 and E3, the function of which is supplied by engineered cell lines in trans. Adenovirus is a non-enveloped virus that attaches to cells by two main mechanisms. The fiber coat protein of the vector binds the coxsackie adenovirus receptor, and penton proteins bind cell-surface integrins. The vector then enters the cell through endocytosis and completes its life cycle as a non-integrating nuclear episome.

Newer-generation adenovectors include deletions of additional early genes, in particular, deleted of the E4 region. They have several advantages for use in the inner ear. E1/E3/E4-deleted vectors have been shown to be less cell disrupting in other delivery systems and hence less toxic following delivery to the inner ear. The E4 region of adenovirus encodes several proteins that modulate the host cell's function. In studies of primary endothelial cells, E1/E3/E4-deleted vectors have been shown to be less perturbing than adenovirus vectors containing E4 (Qian H. Set al., Circ. Res. 2001;88(9):911-7; Rafii S. et al., Circ. Res. 2001;88(9):903-10; Ramalingam R., et al., J. Virol. 1999;73(12):10183-90. Studies in human embryonic lung fibroblasts have also suggested that E1/E3/E4 vectors reduce the risk of cell perturbation (Hobbs WE, et al., J. Virol. 2001;75(7):3391-403).

First generation replication-deficient adenoviral vectors of the present invention were constructed with the E1A, E1B and a portion of E3 were deleted from the viral genome, and the E. coli .beta.-galactosidase gene (.beta.-gal) was inserted under control of the Cytomegalovirus (CMV) promoter (U.S. Pat. Nos. 5,168,062 and 5,385,839) and designated AD.lacZ (Davidson et al., Nat. Genet. 1993; 3:219-223; Li et al., Ophthalmol. Vis. Sci. 1994; 35:2543-2549). Other promoters can be used, such as platelet-derived growth factor (PDGF), neuron-specific enolase (NSE), and elongation factor 1alpha (EF-1alpha), as well as mouse and human Cytomegalovirus and chicken .beta.-actin (Luebke A. E., et al., Hum. Gene Ther., 2001; 12:773-781).

Newer-generation adenoviral vectors of the present invention were constructed similarly to the vectors above except that the E4 region was also deleted, along with other modifications that significantly lower the expression of viral genes in transduced cells, and which do not change cellular gene expression (Brough et al. J. Virol. 1996; 71:6496-6501; Ramalingam et al., Blood 1999; 93:2396-2944; Kanzaki et al., Hear. Res. 2002; 169:112-120). All references described in the specification are hereby incorporated by reference herein as if fully set forth in their entireties.
 

Claim 1 of 4 Claims

1. A method for delivering a pharmaceutical composition to the inner ear of a mammal without damaging hearing comprising the steps of: a) visualizing the ear canal; b) making an incision parallel to the annulus and elevating the tympanic membrane; c) preserving and identifying the chorda tympani nerve; d) visualizing the stapes footplate in the ear; e) drilling a hole in the center of the stapes footplate with a diameter sufficiently large to allow the flow of perilymphatic fluid; f) injecting about 0.25 .mu.L-10.0 .mu.L of a pharmaceutical composition for treatment of the inner ear of a mammal; and g) filling the hole in the stapes footplate.

____________________________________________
If you want to learn more about this patent, please go directly to the U.S. Patent and Trademark Office Web site to access the full patent.