Title: Drug delivery to the
inner ear and methods of using same
United States Patent: 7,387,614
Issued: June 17, 2008
Hinrich (Reisterstown, MD)
Assignee: University of
Maryland, Baltimore (Baltimore, MD)
Appl. No.: 10/895,418
Filed: July 21, 2004
Web Seminars -- Pharm/Biotech/etc.
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
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
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
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.
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
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.
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
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.
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.
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.
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.
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.
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