U.S. patent application number 10/153145 was filed with the patent office on 2002-11-28 for treatment of hearing impairments.
Invention is credited to Gao, Wei-Qiang.
Application Number | 20020176859 10/153145 |
Document ID | / |
Family ID | 26721513 |
Filed Date | 2002-11-28 |
United States Patent
Application |
20020176859 |
Kind Code |
A1 |
Gao, Wei-Qiang |
November 28, 2002 |
Treatment of hearing impairments
Abstract
Compositions and methods are provided for prophylactic or
therapeutic treatment of a mammal for hearing impairments involving
neuronal damage, loss, or degeneration, preferably of spinal
ganglion neurons, by administration of a therapeutically effective
amount of a trkB or trkC agonist, particularly a neurotrophin, more
preferably NT-4/5. Also provided are improved compositions and
methods for treatments requiring administration of a pharmaceutical
having an ototoxic side-effect, wherein the improvement includes
administering a therapeutically effective amount of a trkB or trkC
agonist to treat the ototoxicity.
Inventors: |
Gao, Wei-Qiang; (Foster
City, CA) |
Correspondence
Address: |
KNOBBE MARTENS OLSON & BEAR LLP
620 NEWPORT CENTER DRIVE
SIXTEENTH FLOOR
NEWPORT BEACH
CA
92660
US
|
Family ID: |
26721513 |
Appl. No.: |
10/153145 |
Filed: |
May 21, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10153145 |
May 21, 2002 |
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09823717 |
Mar 30, 2001 |
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6429191 |
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09823717 |
Mar 30, 2001 |
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08778357 |
Jan 2, 1997 |
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6225282 |
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60044407 |
Jan 5, 1996 |
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Current U.S.
Class: |
424/146.1 ;
424/649; 514/162; 514/305; 514/36; 514/37; 514/41; 514/8.3;
514/8.4; 514/8.9 |
Current CPC
Class: |
C12N 2501/13 20130101;
C12N 2503/02 20130101; C12N 5/062 20130101; A61K 38/185
20130101 |
Class at
Publication: |
424/146.1 ;
514/12; 514/36; 514/37; 514/41; 514/162; 514/305; 424/649 |
International
Class: |
A61K 039/395; A61K
038/17; A61K 033/24 |
Claims
What is claimed is:
1. A method for treating damage to spiral ganglion neurons,
comprising exposing the neurons to a therapeutically effective
amount of a trkB or trkC agonist.
2. The method of claim 1, wherein treating damage to spiral
ganglion neurons reduces hearing loss.
3. The method of claim 1 further comprising administering a
therapeutically effective amount of a further trkB or trkC
agonist.
4. The method of claim 3, wherein the further trkB or trkC agonist
is identical to the trkB or trkC agonist of claim 1.
5. The method of claim 1, wherein the agonist is selected from the
group consisting of a neurotrophin, a chimeric neurotrophin, a
pantrophic neurotrophin, a small molecule agonist and an antibody
agonist or an antigen-binding fragment thereof.
6. The method of claim 5, wherein the neurotrophin is selected from
the group consisting of NT-4/5, BDNF and NT-3.
7. The method of claim 6, wherein the neurotrophin is NT-4/5.
8. The method of claim 5, wherein the pantrophic neurotrophin is
MNTS-1.
9. The method of claim 1, wherein the damage is caused by an
ototoxic agent.
10. The method of claim 9, wherein the ototoxic agent is a
pharmaceutical drug.
11. The method of claim 10, wherein the pharmaceutical drug is
selected from the group consisting of an aminoglycoside antibiotic,
a chemotherapeutic agent, a salicylate or salicylate-like compound,
a loop diuretic and a quinine or quinine-like compound.
12. The method of claim 11, wherein the aminoglycoside antibiotic
is selected from the group consisting of neomycin, paromomycin,
ribostamycin, lividomycin, kanamycin, amikacin, tobramycin,
viomycin, gentamicin, sisomicin, netilmicin, streptomycin,
dibekacin, fortimicin and dihydrostreptomycin.
13. The method of claim 10, wherein the ototoxic agent is an
anti-neoplastic drug.
14. The method of claim 13, wherein the ototoxic agent is cisplatin
or a cisplatin-like compound.
15. The method of claim 1, wherein the trkB or trkC agonist is
administered with an agent that promotes hair cell growth,
proliferation, survival or differentiation.
16. The method of claim 15, wherein the agent is retinoic acid or
retinoic acid in combination with TGF-.alpha..
17. A method for preventing damage to spiral ganglion neurons,
comprising exposing the neurons to a therapeutically effective
amount of a trkB or trkC agonist before exposing the spiral
ganglion neurons to a factor capable of damaging the neurons.
18. A method of treatment, comprising: administering a
chemotherapeutic drug; and admininistering a therapeutically
effective amount of a trkB or trkC agonist so that hearing
impairment induced by the chemotherapeutic agent is reduced,
wherein the chemotherapeutic drug is known to cause hearing
impairment.
19. The method of claim 18, wherein the chemotherapeutic agent is
cisplatin or a cisplatin analog.
20. The method of claim 19 comprising further administering a
therapeutically effective amount of a further trkB or trkC
agonist.
21. The method of claim 20, wherein the further trkB or trkC
agonist is identical to the trkB or trkC agonist of claim 18.
22. A method of treatment, comprising: administering an
aminoglycoside; and admininistering a therapeutically effective
amount of a trkB or trkC agonist so that hearing impairment induced
by the aminoglycoside is reduced, wherein the aminoglycoside is
known to cause hearing impairment.
23. The method of claim 22, wherein the aminoglycoside antibiotic
is selected from the group consisting of neomycin, paromomycin,
ribostamycin, lividomycin, kanamycin, amikacin, tobramycin,
viomycin, gentamicin, sisomicin, netilmicin, streptomycin,
dibekacin, fortimicin and dihydrostreptomycin.
24. The method of claim 23, further comprising administering a
therapeutically effective amount of a further trkB or trkC
agonist.
25. The method of claim 24, wherein the further trkB or trkC
agonist is identical to the trkB or trkC agonist of claim 22.
26. A method for reducing the ototoxic effect of an ototoxic agent
in a mammal, comprising exposing neurons in the mammal to a
thereupeutically effective amount of a trkB or trkC agonist,
wherein the ototoxic effect results from neuronal damage in the
mammal.
27. The method of claim 26, wherein the neuronal damage comprises
damage to spiral ganglion neurons.
28. The method of claim 26, wherein the neuronal damage further
comprises damage to vestibular ganglion neurons.
29. The method of claim 26, wherein the mammal is exposed to the
ototoxic agent before exposure to the trkB or trkC agonist.
30. The method of claim 29, wherein the mammal is exposed to the
ototoxic agent after exposure to the trkB or trkC agonist.
31. The method of claim 30, wherein the mammal is exposed to the
ototoxic agent and the trkB or trkC agonist at the same time.
32. The method of claim 26, further comprising administering a
therapeutically effective amount of a further trkB or trkC
agonist.
33. The method of claim 32, wherein the further trkB or trkC
agonist is identical to the trkB or trkC agonist of claim 26.
34. A kit for the treatment of hearing impairment in a mammal
comprising: a trkB or trkC agonist; and instructions for
administering to the mammal a therapeutically effective dose of the
trkB or trkC agonist if the mammal suffers from hearing impairment
caused by an ototoxic agent.
Description
REFERENCE TO RELATED APPLICATION
[0001] This application claims the benefit of priority under 35
U.S.C. .sctn.120 as a continuation of U.S. application Ser. No.
09/823,717, filed Mar. 30, 2001, which is a continuation of and
claims the benefit under 35 U.S.C. .sctn.120 of U.S. application
Ser. No. 08/778,357, now U.S. Pat. No. 6,225,282, filed Jan. 2,
1997, and under 35 U.S.C. .sctn.119(e) of U.S. Provisional
Application Serial No. 60/044,407, filed Jan. 5, 1996, the contents
of which are incorporated herein by reference.
BACKGROUND
[0002] 1. Technical Field
[0003] This application relates to methods for prophylactic and
therapeutic treatment of hearing impairments. More particularly,
the application relates to prevention or therapy of
ototoxin-induced hearing impairments by administration of
neurotrophins.
[0004] 2. Introduction
[0005] Hearing impairments are serious handicaps which affect
millions of people. Hearing impairments can be attributed to a wide
variety of causes, including infections, mechanical injury, loud
sounds, aging, and chemical-induced ototoxicity that damages
neurons and/or hair cells of the peripheral auditory system. The
peripheral auditory system consists of auditory receptors, hair
cells in the organ of Corti, and primary auditory neurons, the
spiral ganglion neurons in the cochlea. Spiral ganglion neurons
("SGN") are primary afferent auditory neurons that deliver signals
from the peripheral auditory receptors, the hair cells in the organ
of Corti, to the brain through the cochlear nerve. The eighth nerve
connects the primary auditory neurons in the spiral ganglia to the
brain stem. The eight nerve also connects vestibular ganglion
neurons ("VGN"), which are primary afferent sensory neurons
responsible for balance and which deliver signals from the utricle,
saccule and ampullae of the inner ear to the brain, to the
brainstem. Destruction of primary afferent neurons in the spiral
ganglia has been attributed as a major cause of hearing
impairments. Damage to the peripheral auditory system is
responsible for a majority of hearing deficits (Dublin, 1976;
Rybak, 1986; Lim, 1986; Pryor, 1994).
[0006] During embryogenesis, the vestibular ganglion, spiral
ganglion, and the otic vesicle are derived from the same neurogenic
ectoderm, the otic placode. The vestibular and auditory systems
thus share many characteristics including peripheral neuronal
innervations of hair cells and central projections to the brainstem
nuclei. Both of these systems are sensitive to ototoxins that
include therapeutic drugs, antineoplastic agents, contaminants in
foods or medicines, and environmental and industrial pollutants.
Ototoxic drugs include the widely used chemotherapeutic agent
cisplatin and its analogs (Fleischman et al., 1975; Stadnicki et
al., 1975; Nakai et al., 1982; Berggren et al., 1990; Dublin, 1976;
Hood and Berlin, 1986), commonly used aminoglycoside antibiotics,
e.g. gentamicin, for the treatment of infections caused by
Gram-negative bacteria, (Sera et al., 1987; Hinojosa and Lerner,
1987; Bareggi et al., 1990), quinine and its analogs, salicylate
and its analogs, and loop-diuretics.
[0007] The toxic effects of these drugs on auditory cells and
spiral ganglion neurons are often the limiting factor for their
therapeutic usefulness. For example, antibacterial aminoglycosides
such as gentamicins, streptomycins, kanamycins, tobramycins, and
the like are known to have serious toxicity, particularly
ototoxicity and nephrotoxicity, which reduce the usefulness of such
antimicrobial agents (see Goodman and Gilman's The Pharmacological
Basis of Therapeutics, 6th ed., A. Goodman Gilman et al., eds;
Macmillan Publishing Co., Inc., New York, pp. 1169-71 (1980) or
most recent edition). Aminoglycoside antibiotics are generally
utilized as broad spectrum antimicrobials effective against, for
example, gram-positive, gram-negative and acid-fast bacteria.
Susceptible microorganisms include Escherichia spp., Hemophilus
spp., Listeria spp., Pseudomonas spp., Nocardia spp., Yersinia
spp., Klebsiella spp., Enterobacter spp., Salmonella spp.,
Staphylococcus spp., Streptococcus spp., Mycobacteria spp.,
Shigella spp., and Serratia spp. Nonetheless, the aminoglycosides
are used primarily to treat infections caused by gram-negative
bacteria and, for instance, in combination with penicillins for the
synergistic effects. As implied by the generic name for the family,
all the aminoglycoside antibiotics contain aminosugars in
glycosidic linkage. Ototoxicity is a dose-limiting side-effect of
antibiotic administration. For example, nearly 75% of patients
given 2 grams of streptomycin daily for 60 to 120 days displayed
some vestibular impairment, whereas at 1 gram per day, the
incidence decreased to 25% (U.S. Pat. No. 5,059,591). Auditory
impairment was observed: from 4 to 15% of patients receiving 1 gram
per day for greater than 1 week develop measurable hearing loss,
which slowly becomes worse and can lead to complete permanent
deafness if treatment continues. Ototoxicity is also a serious
dose-limiting side-effect for cisplatin, a platinum coordination
complex, that has proven effective on a variety of human cancers
including testicular, ovarian, bladder, and head and neck cancer.
Cisplatin damages auditory and vestibular systems (Fleischman et
al., 1975; Stadnicki et al., 1975; Nakai et al., 1982; Carenza et
al., 1986; Sera et al., 1987; Bareggi et al., 1990). Salicylates,
such as aspirin, are the most commonly used therapeutic drugs for
their anti-inflammatory, analgesic, anti-pyretic and
anti-thrombotic effects. Unfortunately, they have ototoxic side
effects. They often lead to tinnitus ("ringing in the ears") and
temporary hearing loss (Myers and Bernstein, 1965). However, if the
drug is used at high doses for a prolonged time, the hearing
impairment can become persistent and irreversible, as reported
clinically (Jarvis, 1966).
[0008] Accordingly, there exists a need for means to prevent,
reduce or treat the incidence and/or severity of hearing
impairments involving auditory nerves, particularly that arising as
an unwanted side-effect of ototoxic therapeutic drugs including
cisplatin and its analogs, aminoglycoside antibiotics, salicylate
and its analogs, and loop diuretics. In addition, there exits a
need for methods which will allow higher and thus more effective
dosing with these ototoxicity-inducing pharmaceutical drugs, while
concomitantly preventing or reducing ototoxic effects caused by
these drugs. What is needed is a method that provides a safe,
effective, and prolonged means for prophylactic or curative
treatment of hearing impairments related to nerve damage, loss, or
degeneration, particularly ototoxin-induced. In addition there is
needed a rapid, reliable, and facile system for testing the effects
and mechanisms of ototoxic agents on hearing in animals, including
humans, and for testing the efficacy of therapeutics to prevent,
reduce or treat these impairments. The present invention provides a
method and system to achieve these goals and others as well.
SUMMARY
[0009] The present invention is based in part on the discovery
disclosed herein that administration of certain neurotrophins can
prevent or reduce gentamicin-, cisplatin-, or salicylate-induced
cell death of SGNs in dissociated cell culture and in cochlear
explant cultures in a dose-dependent manner. When neurotrophins or
other growth factors were added together with sodium salicylate,
gentamicin or cisplatin to the culture, SGNs were protected by
neurotrophin-4/5 (NT-4/5), brain-derived neurotrophic factor (BDNF)
and neurotrophin-3 (NT-3), but not by NGF or other growth factors,
including epidermal growth factor (EGF), basic fibroblast growth
factor (pFGF), FGF-5, FGF-7, insulin-like growth factor-1 (IGF-1),
platelet-derived growth factor (PDGF), transforming growth
factor-.alpha. (TGF-.alpha.), TGF-.beta.1, TGF-.beta.2,
TGF-.beta.3, TGF-.beta.5 and retinoic acid. Accordingly, it is one
object of the invention to provide a method for treating a mammal
to prevent, reduce, or treat the incidence of or severity of a
neuron-related hearing impairment, particularly an ototoxin-induced
or -inducible hearing impairment, by administering to a mammal in
need of such treatment a prophylactically or therapeutically
effective amount of a trkB or trkC agonist. The trkB or trkC
agonist is preferably a neurotrophin, more preferably NT-4/5, NT-3,
or BDNF, and most preferably NT-4/5, or a functional fragment or
derivative thereof, a chimeric neurotrophin, a pantropic
neurotrophin, or a small molecule or antibody agonist thereof.
[0010] According to the method of this invention a composition of
the invention can be administered at a suitable interval(s) either
prior to, subsequent to, or substantially concurrently with the
administration of or exposure to hearing-impairment inducing
neuronal damage, preferably ototoxin-induced or -inducible hearing
impairment. It is another object of the invention to provide a
method for treating a mammal to prevent, reduce, or treat
neuronal-damage-related hearing impairments, preferably an
ototoxin-induced hearing impairment, by administering to a mammal
in need of such treatment a composition containing a
prophylactically or therapeutically effective amount of the trkB or
trkC agonist in combination with a prophylactically or
therapeutically effective amount of a second trkB or trkC agonist
or an agent that acts synergistically or additively to enhance or
complement the prophylactic or therapeutic effect of the first trkB
or trkC agonist.
[0011] Also provided are improved compositions and methods for
treatments requiring administration of a pharmaceutical having an
ototoxic, hearing-impairing side-effect, wherein the improvement
includes administering (prophylactically or therapeutically) a
therapeutically effective amount of a trkB or trkC agonist to treat
the ototoxicity induced by the pharmaceutical. Accordingly, it is
an object of the invention to provide an improved composition
containing a trkB or trkC agonist, preferably a neurotrophin, more
preferably NT-4/5, NT-3, or BDNF, and most preferably NT-4/5, or a
functional fragment or derivative thereof, a chimeric neurotrophin,
a pantropic neurotrophin, or a small molecule or antibody agonist
thereof, in combination with an ototoxic, hearing-impairing
pharmaceutical drug for administration to a mammal. Such
combination compositions can further contain a pharmaceutically
acceptable carrier. The pharmaceutical composition will have lower
ototoxicity than the ototoxic pharmaceutical alone, and preferably,
will have a higher dosage of the ototoxic pharmaceutical than
typically used. Examples of such improved compositions include
cisplatin or other ototoxic neoplastic agent or an aminoglycoside
antibiotic(s) in combination with a trkB or trkC agonist.
[0012] Still further, the invention relates to the use in medicine
of compositions of the invention in cases of bacterial infection.
The present invention provides a solution to the art that has long
sought a therapy and a medicament which can treat the ototoxic
effects currently associated with certain antibiotics, and
particularly with the more popular and commonly used aminoglycoside
antibiotics without sacrificing the antimicrobial effectiveness of
the aminoglycosides.
[0013] Still further, the invention relates to the use in medicine
of compositions of the invention in cases of cancer. The present
invention provides a solution to the art that has long sought a
therapy and a medicament which can treat the ototoxic effects
currently associated with certain chemotherapeutics, and
particularly with the more popular and commonly used cisplatin
chemotherapeutics without sacrificing the antineoplastic
effectiveness of cisplatin or its analogs.
[0014] Still further, the invention relates to the use in medicine
of compositions of the invention in cases where anti-inflammation,
analgesic, or cardiovascular effects are desired. The present
invention provides a solution to the art that has long sought a
therapy and a medicament which can treat the ototoxic effects
currently associated with certain salicylate compounds, and
particularly with the more popular and commonly used aspirin,
without sacrificing the effectiveness of the salicylate
compounds.
[0015] Still further, the invention relates to the use in medicine
of compositions of the invention in cases where diuretics are
needed. The present invention provides a solution to the art that
has long sought a therapy and a medicament which can treat the
ototoxic effects currently associated with certain diuretics, and
particular with the more popular and commonly used loop-diuretics,
without sacrificing their diuretic effectiveness.
[0016] Still further, the invention relates to the use in medicine
of compositions of the invention in cases where quinine or
quinine-like compounds are needed. The present invention provides a
solution to the art that has long sought a therapy and a medicament
which can treat the ototoxic effects currently associated with
certain quinines without sacrificing their effectiveness.
[0017] Finally, it is an object of the invention to provide a
organotypic cochlear explant culture system that allows reliable,
rapid, and facile determination of the ototoxic effect of compounds
and the prophylactic or therapeutic effect of candidate
compositions and methods of the invention.
[0018] Additional objects and features of the invention will be
apparent to those skilled in the art from the following detailed
description and appended claims when taken in conjunction with the
figures.
[0019] Other aspects of the invention will become apparent from the
following detailed description and the claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0020] FIG. 1 is a histogram depicting the effects of NT-4/5 on SGN
survival. SGNs were prepared from P5 rats, plated and kept for 2
days in serum-free medium in the absence or presence of NT-4/5 at
different concentrations. Viable SGNs were identified by labeling
with a neurofilament monoclonal antibody (N52), viewed under a
Zeiss Axiophot microscope, and counted using a grid ocular reticule
covering an area of 1 mm.sup.2. For each culture, about 10 randomly
selected fields were counted. Data were collected from 3 to 5
cultures and normalized as a percentage of the number of viable
neurons in the control cultures in each of the experiments. The
error bars represent SEM. As compared to control cultures, NT-4/5
showed very significant survival-promoting effects on SGNs at all
doses (P<0.001, 2-tailed, unpaired t-test).
[0021] FIG. 2 is a histogram depicting the effects of different
neurotrophins on SGN survival. SGNs were prepared from P5 rats and
maintained for 2 days in serum-free medium without or with
different neurotrophins at different concentrations. Quantitation
of viable SGNs was made in the same way as in FIG. 1 and the error
bars are SEM. When compared to control cultures, NT-4/5 and BDNF
showed very significant survival-promoting effects on SGNs at all
doses (P<0.001, 2-tailed, unpaired t-test). NT-3 also showed
significant effects (P<0.05 at 0.1 ng/ml; P<0.001 at doses
from 1 to 50 ng/ml). Difference between NT-3 and NT-4/5 or BDNF was
very significant at all doses (P<0.001). No statistical
difference was found between NT-4/5 and BDNF except the dose of 0.1
ng/ml (P<0.01).
[0022] FIG. 3 is a histogram depicting trkB-IgG protein inhibition
of the survival-promoting activity of NT-4/5 or BDNF. SGNs were
prepared from P5 rats and kept for 2 days in serum-free medium
containing 1 .mu.g/ml trkB-IgG and/or trkC-IgG alone or in
combination with 10 ng/ml neurotrophins. Quantitation of viable
SGNs was done in the same way as in FIG. 1. The error bars indicate
SEM.
[0023] FIG. 4 is a histogram depicting cisplatin dose-dependent
induction of neuronal cell death. SGNs were prepared from P5 rats,
plated in serum-free medium containing different doses of cisplatin
and assayed after 2 days in culture. Quantitation of viable SGNs
was made in the same way as in FIG. 1. The error bars are SEM.
[0024] FIG. 5 is a histogram depicting NT-4/5, BDNF and NT-3
protection of SGNs against cisplatin neurotoxicity. SGNs were
prepared from P5 rats and cultured for 2 days in serum-free medium
containing 1 .mu.g/ml, 2 .mu.g/ml or 4 .mu.g/ml of cisplatin alone
or with 10 ng/ml of different neurotrophins. Quantitation of viable
SGNs was done in the same way as in FIG. 1. The error bars stand
for SEM. The dashed horizontal line indicates the survival level of
normal cultures without cisplatin. Note that even in the cultures
containing 4 .mu.g/ml cisplatin. NT-4/5, BDNF and NT-3 showed very
significant protecting effects on SGNs as compared to the culture
containing cisplatin alone (P<0.001). While no difference was
found between cultures containing NT-4/5 and cultures containing
BDNF, the difference between cultures containing NT-3 and cultures
containing NT-4/5 or BDNF was statistically very significant
(P<0.001).
[0025] FIGS. 6A and 6B depict quantitation of toxic effects of
sodium salicylate on SGNs (FIG. 6A) and hair cells (FIG. 6B).
Cochlear explants were prepared from P3 rats, embedded and cultured
in 3-D collagen gels. Ototoxins were added at various
concentrations to 2-day cultures for 2 more days. After the
preparations were processed for double histochemistry with N52 and
phalloidin, they were examined under Zeiss Axiophot epifluorescent
microscope. For each culture, SGN peripheral axons and hair cells
were counted from 3-4 randomly selected fields in the middle turn
of each culture. Data were collected from 6 or more cultures for
each of the experimental groups. Two-tailed, unpaired t-test was
used for statistical analysis. Note that sodium salicylate
selectively induced degeneration of SGNs, but not hair cells loss.
The error bars are SEM and double asterisks (**) indicate
significant differences between experimental and control cultures
(p<0.001).
[0026] FIGS. 7A and 7B depict quantitation of toxic effects of
gentamicin on SGNs (FIG. 7A) and hair cells (FIG. 7B). The same
experimental procedures were performed as in FIGS. 6A and 6B. Note
that gentamicin primarily destroyed hair cells at low doses, but
started to induce damage to SGNs at doses higher than 1 mM. The
error bars stand for SEM and double asterisks (**) indicate that as
compared to the control cultures, p<0.001.
[0027] FIGS. 8A and 8B depict quantitation of toxic effects of
cisplatin on SGNs (FIG. 8A) and hair cells (FIG. 8B). The same
experimental procedures were performed as in FIGS. 6A and 6B. Note
that cisplatin damaged both hair cells and SGNs and showed a more
profound toxic effect on SGNs than on hair cells. The error bars
are SEM. * and ** indicate that as compared to the control
cultures, p<0.01 and p<0.001, respectively.
[0028] FIGS. 9A and 9B depict quantitation of protective effects of
NT-4/5 on SGNs (FIG. 9A) and hair cells (FIG. 9B). Cochlear
explants were prepared from P3 rats, embedded and cultured in a 3-D
collage gel. Sodium salicylate ("Sal"; 5 mM), gentamicin ("Gent"; 3
mM) or cisplatin ("Cis"; 4 .mu.g/ml) was added either alone or in
combination with NT-4/5 (20 ng/ml) to 2-day cultures and the
cultures were kept for 2 more days. The same experimental
procedures were performed as in FIGS. 6A and 6B. The error bars are
SEM and ** indicate that as compared to the cultures containing an
ototoxin alone, p<0.001. "Con" indicates control culture.
[0029] FIG. 10 depicts quantitation of neuroprotection of different
neurotrophins against sodium salicylate (Sal) and gentamicin
(Gent). Cochlear explants were prepared from P3 rats, embedded and
cultured in a 3-D collagen gel. Sodium salicylate (5 mM) or
gentamicin (3 mM) was added either alone or in combination with one
of the neurotrophins (20 ng/ml) to 2-day cultures and the cultures
were kept for 2 more days. The same experimental procedures were
performed as in FIGS. 6A and 6B. Note that NT-4/5, BDNF and NT-3,
but not NGF showed significant neuroprotection. The error bars are
SEM and ** indicate that as compared to the cultures containing an
ototoxin alone, p<0.001.
DETAILED DESCRIPTION
[0030] In general, the following words or phrases have the
indicated definition when used in the description, examples, and
claims:
[0031] "Treatment" refers to both therapeutic treatment and
prophylactic or preventative measures, wherein the object is to
prevent or slow down (lessen) neuron-damage-related hearing
impairment, preferably ototoxin-induced or inducible. Those in need
of treatment include those already experiencing a hearing
impairment, those prone to having the impairment, and those in
which the impairments are to be prevented. The hearing impairments
are due to neuronal damage, wherein the damage is caused by
infections, mechanical injury, loud sounds, aging, or, preferably,
chemical-induced ototoxicity, wherein ototoxins include therapeutic
drugs including antineoplastic agents, salicylates, quinines, and
aminoglycoside antibiotics, contaminants in foods or medicinals,
and environmental or industrial pollutants. Typically, treatment is
performed to prevent or reduce ototoxicity, especially resulting
from or expected to result from administration of therapeutic
drugs. Preferably a therapeutically effective composition is given
immediately after the exposure to prevent or reduce the ototoxic
effect. More preferably, treatment is provided prophylactically,
either by administration of the composition prior to or
concomitantly with the ototoxic pharmaceutical or the exposure to
the ototoxin.
[0032] By "ototoxic agent" in the context of the present invention
is meant a substance that through its chemical action injures,
impairs, or inhibits the activity of a component of the nervous
system related to hearing to in turn impair hearing. The list of
ototoxic agents that cause hearing impairments includes, but is not
limited to, neoplastic agents such as vincristine, vinblastine,
cisplatin, taxol, or dideoxy- compounds, e.g., dideoxyinosine;
alcohol; metals; industrial toxins involved in occupational or
environmental exposure; contaminants of food or medicinals; or
over-doses of vitamins or therapeutic drugs, e.g., antibiotics such
as penicillin or chloramphenicol, or megadoses of vitamins A, D, or
B6, salicylates, quinines and loop diuretics. Other toxic agents
that can cause ototoxicity-inducing hearing impairment can be
identified and characterized by methods as taught herein. By
"exposure to an ototoxic agent" is meant that the ototoxic agent is
made available to, or comes into contact with, a mammal. Exposure
to an ototoxic agent can occur by direct administration, e.g., by
ingestion or administration of a food, medicinal, or therapeutic
agent, e.g., a chemotherapeutic agent, by accidental contamination,
or by environmental exposure, e.g., aerial or aqueous exposure.
[0033] "Chronic" administration refers to administration of the
agent(s) in a continuous mode as opposed to an acute mode, so as to
maintain the initial anti-ototoxic effect for an extended period of
time.
[0034] "Mammal" for purposes of treatment refers to any animal
classified as a mammal, including humans, domestic and farm
animals, and zoo, sports, or pet animals, such as dogs, horses,
cats, cows, etc. Preferably, the mammal herein is human.
[0035] A "patient" for the purposes of the present invention
includes both humans and other mammals. Thus the methods are
applicable to both human therapy and veterinary applications.
[0036] "Non-immunogenic in a human" means that upon contacting the
polypeptide in a pharmaceutically acceptable carrier and in a
therapeutically effective amount with the appropriate tissue of a
human, no state of sensitivity or resistance to the polypeptide is
demonstrable upon the second administration of the polypeptide
after an appropriate latent period (e.g., 8 to 14 days).
[0037] By "hearing impairment" is meant a neurologic disorder,
oto-neurological in nature, typically sensorineural, but including
composite loss (both sensorineural and conductive loss), preferably
either a sensory or a neural (8th nerve related) hearing loss, and
most preferably a sensory loss (cochlear related), in which the
patient will display, complain of, or is diagnosed to have a
hearing loss. Conductive hearing loss is typically related to the
external or middle ear. These impairments of interest to the
present invention are those associated with damage, loss, or
degeneration of a neuron of the auditory system. Less preferably
such impairments can occur along with hair cell damage or
conductive hearing loss damage. Preferably affected are neurons of
the 8th nerve, cochlea and connecting neurons of the brainstem and
their temporal lobe connections. The loss can be unilateral. Hair
cells are epithelial cells possessing fine projections and located
in the maculae and the organ of Corti.
[0038] Hearing impairments relevant to the invention are preferably
sensory hearing loss due to end-organ lesions, e.g., acoustic
trauma, viral endolymphatic labyrinthitis, Meniere's disease. The
impairment can also be neural hearing loss due to events including
cerebellopontine angle tumors of the 8th nerve. Hearing impairments
include tinnitus, which is a perception of sound in the absence of
an acoustic stimulus, and may be intermittent or continuous,
wherein there is diagnosed a sensorineural loss. Hearing loss may
be due to bacterial or viral infection of the 8th nerve ganglia,
such as in herpes zoster oticus, purulent labyrinthitis arising
from acute otitis media, purulent meningitis, chronic otitis media,
sudden deafness including that of viral origin, e.g., viral
endolymphatic labyrinthitis caused by viruses including mumps,
measles, influenza, chickenpox, mononucleosis and adenoviruses. The
hearing loss can be congenital, such as that caused by rubella,
anoxia during birth, bleeding into the inner ear due to trauma
during delivery, ototoxic drugs administered to the mother,
erythroblastosis fetalis, and hereditary conditions including
Waardenburg's syndrome and Hurler's syndrome. The hearing loss can
be noise-induced, generally due to a noise greater than 85 decibels
(db) that damages the inner ear. Hearing loss includes presbycusis,
which is a sensorineural hearing loss occurring as a normal part of
aging, fractures of the temporal bone extending into the middle ear
and rupturing the tympanic membrane and possibly the ossicular
chain, fractures affecting the cochlea, and acoustic neurinoma,
which are tumors generally of Schwann cell origin that arise from
either the auditory or vestibular divisions of the 8th nerve.
Preferably, the hearing loss is caused by an ototoxic drug that
effects the auditory portion of the inner ear, particularly the
organ of Corti. Incorporated herein by reference are Chapters 196,
197, 198 and 199 of The Merck Index, 14th Edition, (1982), Merck
Sharp & Dome Research Laboratories, N.J. and related chapters
in the most recent edition) relating to description and diagnosis
of hearing impairments.
[0039] Tests are known and available for diagnosing hearing
impairments. Neuro-otological, neuro-ophthalmological, neurological
examinations, and electro-oculography can be used. (Wennmo et al.
Acta Otolaryngol (1982) 94:507-15). Sensitive and specific measures
are available to identify patients with auditory impairments. For
example, tuning fork tests can be used to differentiate a
conductive from a sensorineural hearing loss and determine whether
the loss is unilateral. An audiometer is used to quantitate hearing
loss, measured in decibels. With this device the hearing for each
ear is measured, typically from 125 to 8000 Hz, and plotted as an
audiogram. Speech audiometry can also be performed. The speech
recognition threshold, the intensity at which speech is recognized
as a meaningful symbol, can be determined at various speech
frequencies. Speech or phoneme discrimination can also be
determined and used an indicator of sensorineural hearing loss
since analysis of speech sounds relies upon the inner ear and 8th
nerve. Tympanometry can be used to diagnose conductive hearing loss
and aid in the diagnosis of those patients with sensorineural
hearing loss. Electrocochleography, measuring the cochlear
microphonic response and action potential of the 8th nerve, and
evoked response audiometry, measuring evoked response from the
brainstem and auditory cortex, to acoustic stimuli can be used in
patients, particularly infants and children or patients with
sensorineural hearing loss of obscure etiology. These tests serve a
diagnostic function as well as a clinical function in assessing
response to therapy.
[0040] Sensory and neural hearing losses can be distinguished based
on tests for recruitment (an abnormal increase in the perception of
loudness or the ability to hear loud sounds normally despite a
hearing loss ), sensitivity to small increments in intensity, and
pathologic adaptation, including stapedial reflex decay.
Recruitment is generally absent in neural hearing loss. In sensory
hearing loss the sensation of loudness in the affected ear
increases more with each increment in intensity than it does in the
normal ear. Sensitivity to small increments in intensity can be
demonstrated by presenting a continuous tone of 20 db above the
hearing threshold and increasing the intensity by 1 db briefly and
intermittently. The percentage of small increments detected yields
the "short increment sensitivity index" value. High values, 80 to
100%, is characteristic of sensory hearing loss, whereas a neural
lesion patient and those with normal hearing cannot detect such
small changes in intensity. Pathologic adaptation is demonstrated
when a patient cannot continue to perceive a constant tone above
the threshold of hearing; also known as tone decay. A Bekesy
automatic audiometer or equivalent can be used to determine these
clinical and diagnostic signs; audiogram patterns of the Type II
pattern, Type III pattern and Type IV pattern are indicative of
preferred hearing losses suitable for the treatment methods of the
invention. As hearing loss can often be accompanied by vestibular
impairment, vestibular function can be tested, particular when
presented with a sensorineural hearing loss of unknown etiology.
When possible diagnostics for hearing loss, such as audiometric
tests, should be performed prior to exposure in order to obtain a
patient normal hearing baseline. Upon exposure, particularly to an
ototoxic drug, audiometric tests should be performed twice a week
and continued testing should be done even after cessation of the
drug treatment since hearing loss may not occur until several days
after cessation.
[0041] In one embodiment the invention constitutes a method for
treating a mammal having or prone to a hearing impairment or
treating a mammal prophylactically to prevent or reduce the
occurrence or severity of a hearing impairment that would result
from exposure to a neuronal injury, loss, or degeneration,
preferably caused by an ototoxic agent, wherein a therapeutically
effective amount of a trkB or trkC agonist is administered to the
mammal. Preferably the agonist is a neurotrophin, more preferably
neurotrophin NT-4/5, NT-3, or BDNF, a functional fragment, fusion
or derivative thereof, such as a chimeric neurotrophin (having both
trkB and trkC agonism), a pantropic neurotrophin, or a small
molecule or antibody agonist thereof, as discussed in detail
herein. Most preferably the agonist is NT-4/5 or a chimeric or
pantropic variant thereof having at least both trkB and trkC
agonist activity. A preferred chimeric or pantropic neurotrophin
has a region conferring NT-3-receptor binding specificity and a
region conferring NT-4/5-receptor binding specificity. A preferred
pantropic neurotrophin is MNTS-1. In a preferred embodiment the
binding of a chimeric or pantropic neurotrophin to a neurotrophic
receptor is at least 80% of the binding of the natural neurotrophin
ligand to the receptor. When the patient is human, the
neurotrophins are preferably human neurotrophins or derived from
human neurotrophin sequences, in part to avoid or minimize
recognition of the agonist as foreign. Optionally, the trkB or trkC
agonist is administered alone or in combination. Additional
optional components include a hair cell growth factor or agonist,
which are compounds known to promote hair cell survival,
regeneration, growth, proliferation, or prevent or reduce
cytotoxicity of hair cells. The methods of the invention are
particularly effective when the hearing impairment is ototoxin
induced or inducible.
[0042] It is another object of the invention to provide a method
for treating a mammal to prevent, reduce, or treat a hearing
impairment, preferably an ototoxin-induced hearing impairment, by
administering to a mammal in need of such treatment a composition
containing a prophylactically or therapeutically effective amount
of the trkB or trkC agonist in combination with a prophylactically
or therapeutically effective amount of a second trkB or trkC
agonist or an agent that acts synergistically or additively to
enhance or complement the prophylactic or therapeutic effect of the
first trkB or trkC agonist. By "complement" is meant that the
second agonist agonizes the trk not recognized by the first agonist
such that the combination of first and second agonists achieves
agonism of both trkB and trkC. An example of one such embodiment of
this type is the use of both NT-4/5 and NT-3, which can be
administered as a single formulation or as separate
formulations.
[0043] In one embodiment is a method for treating a hearing
impairment wherein the ototoxicity results from administration of a
therapeutically effective amount of an ototoxic pharmaceutical
drug. Typical ototoxic drugs are chemotherapeutic agents, e.g.
antineoplastic agents, and antibiotics. Other possible candidates
include loop-diuretics, quinines or a quinine-like compound, and
salicylate or salicylate-like compounds.
[0044] The methods of the invention are particularly effective when
the ototoxic compound is an antibiotic, preferably an
aminoglycoside antibiotic. Ototoxic aminoglycoside antibiotics
include but are not limited to neomycin, paromomycin, ribostamycin,
lividomycin, kanamycin, amikacin, tobramycin, viomycin, gentamicin,
sisomicin, netilmicin, streptomycin, dibekacin, fortimicin, and
dihydrostreptomycin, or combinations thereof. Particular
antibiotics include neomycin B, kanamycin A, kanamycin B,
gentamicin C1, gentamicin C1a, and gentamicin C2.
[0045] Hearing impairments induced by aminoglycosides can be
prevented or reduced by the methods of the invention. Although the
aminoglycosides are particularly useful due to their rapid
bactericidal action in infections by susceptible organisms, their
use is limited to more severe, complicated infections because of
ototoxic and nephrotoxic side-effects. For this reason the
aminoglycosides are considered to have a low therapeutic/risk ratio
compared to other antibiotics used systemically. Damage to ganglion
neurons has been noted at high doses of aminoglycosides (Sera et
al., 1987) or after a delayed time (Lippe et al., 1995).
Aminoglycoside antibiotics were reported to cause direct neuronal
loss in the spiral ganglion in two young patients (Hinojosa and
Lerner, 1987).
[0046] Aminoglycosides are a class of compounds characterized by
the ability to interfere with protein synthesis in micro-organisms.
Aminoglycosides consist of two or more amino sugars joined in a
glycoside linkage to a hexose (or aminocyclitol) nucleus. The
hexose nuclei thus far known are either streptidine or
2-deoxystreptamine, though others may be anticipated.
Aminoglycoside families are distinguished by the amino sugar
attached to the aminocyclitol. For example, the neomycin family
comprises three amino sugars attached to the central
2-deoxystreptamine. The kanamycin and glutamicin families have only
two amino sugars attached to the aminocyclitol. Aminoglycosides
include: neomycins (e.g. neomycin B and analogs and derivatives
thereof), paromomycin, ribostamycin, lividomycin, kanamycins (e.g.
kanamycin A, kanamycin B, and analogs and derivatives thereof),
amikacin, tobramycin, viomycin, gentamicin (e.g., gentamicin C1,
gentamicin C1a, gentamicin C2, and analogs and derivatives
thereof), sisomicin, netilmicin, streptomycin, dibekacin,
fortimicin, and dihydrostreptomycin.
[0047] The aminoglycoside antibiotic which can be employed in
conjunction with the ototoxicity inhibiting compositions of the
invention is any aminoglycoside antibiotic. Examples of such
aminoglycoside antibiotics include kanamycin (Merck Index 9th ed.
#5132), gentamicin (Merck Index 9th ed. #4224), amikacin (Merck
Index 9th ed. #Al), dibekacin (Merck Index 9th ed. #2969),
tobramycin (Merck Index 9th ed. #9193), streptomycin (Merck Index
9th ed. #8611/8612), paromomycin (Merck Index 9th ed. #6844),
sisomicin (Merck Index 9th ed. #8292), isepamicin and netilmicin,
all known in the art. The useful antibiotics include the several
structural variants of the above compounds (e.g. kanamycin A, B and
C; gentamicin A, C1, C1a, C2 and D; neomycin B and C and the like).
The free bases, as well as pharmaceutically acceptable acid
addition salts of these aminoglycoside antibiotics, can be
employed.
[0048] For the purpose of this disclosure, the terms
"pharmaceutically acceptable acid addition salt" shall mean a mono
or poly salt formed by the interaction of one molecule of the
aminoglycoside antibiotic with one or more moles of a
pharmaceutically acceptable acid. Included among those acids are
acetic, hydrochloric, sulfuric, maleic, phosphoric, nitric,
hydrobromic, ascorbic, malic and citric acid, and those other acids
commonly used to make salts of amine-containing
pharmaceuticals.
[0049] Accordingly, the methods and compositions of the invention
find use for the prevention and treatment of opportunistic
infections in animals and man which are immunosuppressed as a
result of either congenital or acquired immunodeficiency or as a
side-effect of chemotherapeutic treatment. According to an
alternate embodiment of the present invention, a trkB or trkC
agonists is used advantageously in combination with a known
antimicrobial agent to provide improved methods and compositions to
prevent and/or treat diseases induced by gram positive bacteria
including, but not limited to: Staphylococcus aureus, Streptococcus
pneumonia, Hemophilus influenza; gram negative bacteria including,
but not limited to: Escherichia coli; Bacterium enteritis,
Francisella tularensis; acid-fast bacteria including, but not
limited to Mycobacterium tuberculosis, and Mycobacterium leprae.
Use of a combination of an antimicrobial agent together with a trkB
or trkC agonist is advantageous with antibacterial aminoglycosides
such as gentamicin, streptomycin, and the like which are known to
have serious ototoxicity, which reduce the usefilness of such
antimicrobial agents. Use of trkB or trkC agonist in combination
with such agents permits a lower dosage of the toxic antimicrobial
agents while still achieving therapeutic (antibacterial)
effectiveness.
[0050] In some embodiments the trkB or trkC agonist is
co-administered with an ototoxin. For example, an improved method
is provided for treatment of infection of a mammal by
administration of an aminoglycoside antibiotic, the improvement
comprising administering a therapeutically effective amount of a
trkB or trkC agonist to the patient in need of such treatment to
reduce or prevent ototoxin-induced hearing impairment associated
with the antibiotic. In yet another embodiment is provided an
improved method for treatment of cancer in a mammal by
administration of a chemotherapeutic compound, the improvement
comprises administering a therapeutically effective amount of a
trkB or trkC agonist to the patient in need of such treatment to
reduce or prevent ototoxin-induced hearing impairment associated
with the chemotherapeutic drug.
[0051] Also provided herein are methods for promoting spiral
ganglion neuron survival upon, prior to, or after exposure to an
agent or effect that is capable of inducing a sensorineural hearing
impairment. Such agents and effects are those described herein. The
method includes the step of administering to the neuron an
effective amount of trkB or trkC agonist or other compositions
containing same as discussed herein. Preferably, the method is used
upon, prior to, or after exposure to a hearing-impairing
ototoxin.
[0052] In one embodiment the methods of treatment are applied to
hearing impairments resulting from the administration of a
chemotherapeutic agent to treat its ototoxic side-effect. Ototoxic
chemotherapeutic agents amenable to the methods of the invention
include, but are not limited to an antineoplastic agent, including
cisplatin or cisplatin-like compounds, taxol or taxol-like
compounds, and other chemotherapeutic agents believed to cause
ototoxin-induced hearing impairments, e.g., vincristine, an
antineoplastic drug used to treat hematological malignancies and
sarcomas.
[0053] In one embodiment the methods of the invention are applied
to hearing impairments resulting from the administration of
salicylate, e.g., aspirin, or a salicylate-like compound, to treat
its ototoxic side-effect. Salicylates, such as aspirin, are the
most commonly used therapeutic drugs for their anti-inflammatory,
analgesic, anti-pyretic and anti-thrombotic effects. Unfortunately,
they have ototoxic side effects. They often lead to tinnitus
("ringing in the ears") and temporary hearing loss (Myers and
Bernstein, 1965). Clinical and animal studies have suggested that
the site of action of this drug is in the cochlea (Myers and
Bernstein, 1965; McCabe and Dey, 1965). Electrophysiological
recordings in animals indicate salicylates result in an increase in
hearing suprathreshold, which suggests damage to hair cells
(Boettcher et al., 1989; Boettcher and Salvi, 1991). However,
postmortem examination of a patient who received large doses of
aspirin showed normal hair cell numbers, but a significant loss of
SGNs (De Moura and Hayden, 1968). The present experiments clearly
demonstrate for the first time, see Examples below, the selective
toxic effects of salicylates on auditory neurons, but not on hair
cells. Interestingly, the dosage (3 mM) of salicylate which starts
to induce significant damage to SGNs in the present study is
comparable to the peak serum and perilymph levels that result in
hearing losses (Myers and Bernstein, 1965; Woodford et al., 1978;
Boettcher et al., 1990). The finding that sodium salicylate at both
low and high concentrations exclusively induces degeneration of
SGNs and their axons, but not hair cells suggests a possibility
that the initial hearing disorder caused by salicylates may result
from neuronal dysfunction, perhaps axonal degeneration in the
eighth nerve or abnormality in SGNs (Wittmaack, 1903). Once the
drug is stopped, the damaged axons may regenerate successfully and
restore the normal auditory function. Such initial axonal
degeneration is therefore reversible. However, if the drug is used
at high doses for a prolonged time, the neuronal impairment will
become more severe and SGN death occurs. As a consequence, the
hearing impairment may become persistent and irreversible, as
reported clinically (Jarvis, 1966).
[0054] In one embodiment the methods of the invention are applied
to hearing impairments resulting from the administration of quinine
and its synthetic substitutes, typically used in the treatment of
malaria, to treat its ototoxic side-effect
[0055] In another embodiment the methods of the invention are
applied to hearing impairments resulting from administration of a
diuretic to treat its ototoxic side-effect. Diuretics, particularly
"loop" diuretics, i.e. those that act primarily in the Loop of
Henle, are candidate ototoxins. Illustrative examples, not limiting
to the invention method, include furosemide, ethacrynic acid, and
mercurials. Diuretics are typically used to prevent or eliminate
edema. Diuretics are also used in nonedematous states such as
hypertension, hypercalcemia, idiopathic hypercalciuria, and
nephrogenic diabetes insipidus.
[0056] In one embodiment the trkB or trkC agonist is administered
prior to administration or exposure to a hearing-impairing event
such as exposure to an ototoxin.
[0057] In another embodiment the trkB or trkC agonist is
administered with an agent that promotes hair cell growth,
proliferation, regeneration, or survival. As shown herein, low
concentrations of gentamicin preferentially kills hair cells while
the damage to the ganglion neurons is not significant. High
concentrations of gentamicin induces degeneration of ganglion
neurons as well as hair cells. Accordingly, this dual toxicity of
aminoglycosides can be treated by the methods of the invention,
preferably with compositions of the invention.
[0058] Preparation and Identification of Agonists
[0059] Agonists to trkB or trkC can be prepared by using the known
family of ligands for trkB or trkC. Survival of developing sensory
neurons is dependent upon trophic factors derived from their target
tissues (Davies et al., 1986). Generally, a neurotrophin is a
protein involved in the development, regulation and maintenance of
the nervous system, and in particular of neurons. Currently, there
are at least five known important neurotrophic factors: nerve
growth factor (NGF), neurotrophin-3 (NT-3), neurotrophin-4 (NT-4/5,
also sometimes called neurotrophin-5 (NT-5) or NT-4/5),
brain-derived neurotrophic factor (BDNF), and ciliary neurotrophic
factor (CNTF). The best characterized mammalian neurotrophic
factors are members of the nerve growth factor (NGF) family of
proteins, and are called neurotrophins. These include NGF
(Levi-Montalcini, 1987), brain-derived neurotrophic factor (BDNF)
(Barde et al., 1982; Leibrock et al., Nature (1989) 341:149)
neurotrophin-3 (NT-3) (Hohn et al., Nature, 344: 339 (1990);
Maisonpierre et al., Science, 247: 1446 (1990); Rosenthal et al.,
Neuron, 4: 767 (1990); copending U.S. Ser. No. 07/494,024 filed
Mar. 15, 1990; U.S. Ser. No. application Ser. No. 07/490,004, filed
Mar. 7, 1990; Ernfors et al., 1990; Jones and Reichardt, 1990) and
neurotrophin-4/5 (NT-4/5) (Berkemeier et al., 1991; Ip et al.,
1992) and neurotrophin-6 (NT-6). While NT-6 is newly cloned from
Xenopus (Gotz et al., 1994) and is less well understood, it is now
well accepted that the other four mammalian neurotrophins exert
their biological functions through activation of high-affinity
binding receptors, the trks (Barbacid, 1993; Snider, 1994). Each of
the neurotrophins binds to specific high-affinity receptors, the
trks (Klein et al., 1990; Kaplan et al., 1991; Klein et al., 1991a;
Klein et al., 1991b; Soppet et al., 1991; Squinto et al., 1991;
Lamballe et al., 1991; Tsoulfas et al., 1993; Ip et al., 1993). For
example, NGF selectively binds to trkA, BDNF and NT-4/5 to trkB,
and NT-3 to trkC. Although neurotrophins exert their main effects
through binding to the trks, they also bind to the NGF low affinity
receptor, P75. Recent studies indicate that the binding of NGF to
P75 may enhance the trkA-mediated signal transduction pathway
(Davies et al., 1993a; Verdi et al, 1994; Barker and Shooter, 1994;
Clary and Reichardt, 1994).
[0060] Neurotrophins transduce intracellular signalling at least in
part through the ligand-dependent activation of a class of tyrosine
kinase-containing receptors of M.sub.r=140-145,000 known as the
trks (Martin-Zanca, et al. (1989); Kaplan, et al. (1991) Nature;
Klein, et al. (1991a); Kaplan, et al. (1991) Science); Klein, et
al. (1991b) Cell; Soppet, et al. (1991); Squinto, et al. (1991);
Lamballe, et al. (1991); Tsoulfas, et al. (1993)). Thus, the signal
transduction pathway of neurotrophins is initiated by this
high-affinity binding to and activation of specific tyrosine kinase
receptors and subsequent receptor autophosphorylation
(Cordon-Cardo, et al. (1991)). Although there is some degree of
cross-receptor interaction between the neurotrophins and the
different trks, the predominant specificity appears to be NGF/trkA,
BDNF/trkB, and NT-3/trkC while NT-4/5 appears to interact primarily
with trkB as efficiently as BDNF (see above and Klein, et al
(1992); Klein, et al. (1989)).
[0061] Expression of trkB, trkC and p75 mRNAs in embryonic
cochleovestibular ganglia (Ylikoski et al., 1993; Schecterson and
Bothwell, 1994) and BDNF and NT-3 mRNAs in the inner ear structures
(Pirvola et al., 1992; Wheeler et al., 1994; Schecterson and
Bothwell, 1994) has been reported. However, the expression of
neurotrophin receptors at the protein level has not been well
determined, particularly in postnatal tissue.
[0062] DNA sequences encoding NGF, BDNF and NT-3 have all been
isolated (Ullrich et al., Nature 303:821-825; Hyman et al., WO
91/03568; Hohn et al., WO 91/03569; and Kaisho et al., FEBS Letters
266:187-191). Researchers have transformed animal and non-animal
hosts with these sequences in order to express the
neurotrophins.
[0063] Researchers have expressed human NGF, BDNF and NT-3 in
mammalian expression systems. Bruce and Heinrich (1989,
Neurobiology of Aging 10:89-94) expressed a DNA sequence encoding
the complete precursor for hNGF in COS cells and detected hNGF
dimer in the conditioned medium. However, they could not determine
the efficiency at which pre-pro-hNGF was converted to mature hNGF.
Kakinuma et al. (EP 0 414 151, 1991) expressed active hNGF in CHO
cells. Hyman et al. (WO 91/03568, 1991) expressed HBDNF in CHO
cells. Nakahama et a/ (EP 0 386 752, 1990) and Hohn et al. (WO
91/03569, 1991) expressed hNT-3 in COS cells.
[0064] U.S. Pat. Nos. 5,235,043 and 5,229,500 disclose human BDNF
sequence and methods for its production and formulation.
Applicant's U.S. patent application Ser. No. 08/581,662 entitled
"Treatment of Balance Impairments" is also incorporated herein by
reference.
[0065] NT-4/5, and its chimeric or pantropic neurotrophins, are
most preferred agonists for use in the methods and compositions of
the present invention. Its human gene and amino acid sequence are
known (U.S. Pat. No. 5,364,769, which is incorporated herein by
reference). NT-4/5 is defined to be a polypeptide encoded by the
known mature human NT-4/5 nucleotide sequence set forth in U.S.
Pat. No. 5,364,769, fragments thereof having greater than about 5
residues comprising an immune epitope or other biologically active
site of NT-4/5, amino acid sequence variants of said sequence,
wherein an amino acid residue has been inserted N- or C-terminal
to, or within, said sequence or its fragment as defined above,
and/or amino acid sequence variants of said sequence or its
fragment as defined above wherein an amino acid residue of said
sequence or fragment thereof has been substituted by another
residue, including other animal species of NT-4/5 such as rat
preproNT-4/5, and derivatives of NT-4/5 or its fragments as defined
above wherein the NT-4/5 or its fragments have been covalently
modified by substitution with a moiety other than a naturally
occurring amino acid; provided, however, that such fragment or
variant is novel and unobvious over the prior art, and is not NGF,
BDNF, or NT-3 of any animal species or any known fragment of such
NGF, BDNF, or NT-3. Mature NT-4/5 amino acid sequence variants
generally will be about 75% (and usually >85%) homologous on an
identical residue basis after aligning (introducing any necessary
spaces) to provide maximum homology.
[0066] NT-4/5 nucleic acid is defined as RNA or DNA which encodes a
NT-4/5 polypeptide or which hybridizes to such DNA and remains
stably bound to it under stringent conditions and is greater than
about 10 bases in length; provided, however, that such hybridizing
nucleic acid is novel and unobvious over any prior art nucleic acid
including that which encodes or is complementary to nucleic acid
encoding BDNF, NT-3, or NGF. Stringent conditions are those which
(1) employ low ionic strength and high temperature for washing, for
example, 0.15 M NaCl/0.015 M sodium citrate/0.1% NaDodSO.sub.4 at
50.degree. C., or (2) use during washing a denaturing agent such as
formamide, for example, 50% (vol/vol) formamide with 0.1% bovine
serum albumin/0. 1% Ficoll/0. 1% polyvinylpyrrolidone/50 mM sodium
phosphate buffer at pH 6.5 with 750 mM NaCl, 75 mM sodium citrate
at 42.degree. C.
[0067] DNA encoding NT-4/5 is obtained from brain tissue cDNA
libraries, or genomic DNA, or by in vitro synthesis. Hybridizing
nucleic acid generally is obtained by in vitro synthesis.
Identification of NT-4/5 DNA most conveniently is accomplished by
probing human cDNA or genomic libraries by labeled oligonucleotide
sequences selected from the known sequence in accord with known
criteria, among which is that the sequence should be of sufficient
length and sufficiently unambiguous that false positives are
minimized. Typically, a .sup.32P-labeled oligonucleotide having
about 30 to 50 bases is sufficient, particularly if the
oligonucleotide contains one or more codons for methionine or
tryptophan. Isolated nucleic acid will be DNA that is identified
and separated from contaminant nucleic acid encoding other
polypeptides from the source of nucleic acid. The nucleic acid may
be labeled for diagnostic purposes.
[0068] Amino acid sequence variants of NT-4/5 are prepared by
introducing appropriate nucleotide changes into the NT-4/5 DNA, or
by in vitro synthesis of the desired NT-4/5. Such variants include,
for example, deletions from, or insertions or substitutions of,
residues within the amino acid sequence for human NT-4/5. Any
combination of deletion, insertion, and substitution is made to
arrive at the final construct, provided that the final construct
possesses the desired characteristics. The amino acid changes also
may result in further modifications of NT-4/5 upon expression in
recombinant hosts, e.g. introducing or moving sites of
glycosylation, or introducing membrane anchor sequences (in
accordance with U.S. Ser. No. 07/083,757, filed Aug. 6, 1987, which
is equivalent to PCT WO 89/01041 published Feb. 9, 1989).
[0069] There are two principal variables in the construction of
amino acid sequence variants: the location of the mutation site and
the nature of the mutation. These are variants may represent
naturally occurring alleles (which will not require manipulation of
the NT-4/5 DNA) or predetermined mutant forms which are made by
mutating the DNA, either to arrive at an allele or a variant that
is not found in nature. In general, the location and nature of the
mutation chosen will depend upon the NT-4/5 characteristic to be
modified. For example, candidate NT-4/5 antagonists or super
agonists will be initially selected by locating sites that are
identical or highly conserved among NGF, BDNF, NT-3, and NT-4/5.
These sites then will be modified in series, e.g., by (1)
substituting first with conservative choices and then with more
radical selections depending upon the results achieved, (2)
deleting the target residue, or (3) inserting residues of the same
or different class adjacent to the located site, or combinations of
options 1-3.
[0070] One helpful technique is called "ala scanning". Here, a
residue or group of target residues are identified and substituted
by alanine or polyalanine. Those domains demonstrating functional
sensitivity to the alanine substitutions then are refined by
introducing further or other variants at or for the sites of
alanine substitution. Obviously, such variations which, for
example, convert NT-4/5 into NGF, BDNF, or NT-3 are not included
within the scope of this invention, nor are any other NT-4/5
variants or polypeptide sequences that are not novel and unobvious
over the prior art. Thus, while the site for introducing an amino
acid sequence variation is predetermined, the nature of the
mutation per se need not be predetermined. For example, to optimize
the performance of a mutation at a given site, ala scanning or
random mutagenesis is conducted at the target codon or region and
the expressed NT-4/5 variants are screened for the optimal
combination of desired activity.
[0071] Amino acid sequence deletions generally range from about 1
to 30 residues, more preferably about 1 to 10 residues, and
typically are contiguous. Deletions may be introduced into regions
of low homology among BDNF, NGF, NT-3, and NT-4/5 to modify the
activity of NT-4/5. Deletions from NT-4/5 in areas of substantial
homology with BDNF, NT-3, and NGF will be more likely to modify the
biological activity of NT-4/5 more significantly. The number of
consecutive deletions will be selected so as to preserve the
tertiary structure of NT-4/5 in the affected domain, e.g.,
beta-pleated sheet or alpha helix.
[0072] Amino acid sequence insertions include amino- and/or
carboxyl-terminal fusions ranging in length from one residue to
polypeptides containing a thousand or more residues, as well as
intrasequence insertions of single or multiple amino acid residues.
Intrasequence insertions (i.e., insertions within the mature NT-4/5
sequence) may range generally from about 1 to 10 residues, more
preferably 1 to 5, most preferably 1 to 3. An example of a terminal
insertion includes fusion of a heterologous N-terminal signal
sequence to the N-terminus of the NT-4/5 molecule to facilitate the
secretion of mature NT-4/5 from recombinant hosts. Such signals
generally will be homologous to the intended host cell and include
STII or lpp for E. coli, alpha factor for yeast, and viral signals
such as herpes gD for mammalian cells. Other insertions include the
fusion of an immunogenic polypeptide such as a bacterial or yeast
protein to the N- or C-termini of NT-4/5.
[0073] The third group of variants are those in which at least one
amino acid residue in the NT-4/5 molecule, and preferably only one,
has been removed and a different residue inserted in its place. In
some embodiments substitutions of one to five amino acids are made.
In yet another embodiment one to three amino acids are substituted.
In some preferred embodiments two amino acid substitutions are
made. The substitutions can be chosen from the table herein. An
example is the replacement of arginine and lysine by other amino
acids to render the NT-4/5 resistant to proteolysis by serine
proteases, thereby creating a more stable NT-4/5 analogue. The
sites of greatest interest for substitutional mutagenesis include
sites where the amino acids found in BDNF, NGF, NT-3, and NT-4/5.
are substantially different in terms of side chain bulk, charge or
hydrophobicity, but where there also is a high degree of homology
at the selected site within various animal analogues of NGF, NT-3,
and BDNF (e.g., among all the animal NGFs, all the animal NT-3s,
and all the BDNFs). This analysis will highlight residues that may
be involved in the differentiation of activity of the trophic
factors, and therefore, variants at these sites may affect such
activities. Examples of such NT-4/5 sites, numbered from the mature
N-terminal end, and exemplary substitutions include NT-4/5
(G.sub.78.fwdarw.K, H, Q or R) and NT-4/5 (R.sub.85.fwdarw.E, F, P,
Y or W). Other sites of interest are those in which the residues
are identical among all animal species' BDNF, NGF, NT-3, and
NT-4/5, this degree of conformation suggesting importance in
achieving biological activity common to all four factors. These
sites, especially those falling within a sequence of at least 3
other identically conserved sites, are substituted in a relatively
conservative manner. Such conservative substitutions are shown in
Table 1 under the heading of preferred substitutions. If such
substitutions result in a change in biological activity, then more
substantial changes, denominated exemplary substitutions in Table
1, or as further described below in reference to amino acid
classes, are introduced and the products screened.
1TABLE 1 Preferred Original Residue Exemplary Substitutions
Substitutions Ala (A) val; leu; ile val Arg (R) lys; gln; asn lys
Asn (N) gln; his; lys; arg gln Asp (D) glu glu Cys (C) ser ser Gln
(Q) asn asn Glu (E) asp asp Gly (G) pro pro His (H) asn; gln; lys;
arg; arg Ile (I) leu; val; met; ala; phe; norleucine leu Leu (L)
norleucine; ile; val; met; ala; phe Ile Lys (K) arg; gln; asn Arg
Met (M) leu; phe; ile leu Phe (F) leu; val; ile; ala leu Pro (P)
gly gly Ser (S) thr thr Thr (T) ser ser Trp (W) tyr tyr Tyr (Y)
trp; phe; thr; ser phe Val (V) ile; leu; met; phe; ala; norleucine
leu
[0074] Sites particularly suited for conservative substitutions
include, numbered from the N-terminus of the mature NT-4/5, R11,
G12, E13, V16, D18, W23, V24, D26, V40, L41, Q54, Y55, F56, E58,
T59, G77, R79, G80, H85, W86, A99, L100, T101, W110, R111, W112,
1113, R114, I115, D116, and T118. Cysteine residues not involved in
maintaining the proper conformation of NT-4/5 also may be
substituted, generally with serine, in order to improve the
oxidative stability of the molecule and prevent aberrant
crosslinking. Sites other than those set forth in this paragraph
are suitable for deletional or insertional studies generally
described above.
[0075] Substantial modifications in function or immunological
identity are accomplished by selecting substitutions that differ
significantly in their effect on maintaining (a) the structure of
the polypeptide backbone in the area of the substitution, for
example, as a sheet or helical conformation, (b) the charge or
hydrophobicity of the molecule at the target site, or (c) the bulk
of the side chain. Naturally occurring residues are divided into
groups based on common side chain properties:
[0076] (1) hydrophobic: norleucine, met, ala, val, leu, ile;
[0077] (2) neutral hydrophilic: cys, ser, thr;
[0078] (3) acidic: asp, glu;
[0079] (4) basic: asn, gln, his, lys, arg;
[0080] (5) residues that influence chain orientation: gly, pro;
and
[0081] (6) aromatic: trp, tyr, phe.
[0082] Non-conservative substitutions will entail exchanging a
member of one of these classes for another. Such substituted
residues also may be introduced into the conservative substitution
sites set forth above or, more preferably, into the remaining
(non-conserved) sites.
[0083] Examples of NT-4/5 variants include
NT-4/5(65NAE67.fwdarw.NAS or NAT) (this adds an N-linked
glycosylation site); NT-4/5(R83-Q94); NT-4/5(G1-C61) (variants so
depicted are fragments containing the residues indicated);
NT-4/5(G1-C17); NT-4/5(C17-C61); NT-4/5(C17-C78); NT-4/5(C17-C90);
NT-4/5(C17-C119); NT-4/5(C17-C121); NT-4/5(R11-R27);
NT-4/5(R11-R34); NT-4/5(R34-R53); NT-4/5(C61-C78); NT-4/5(R53-C61);
NT-4/5(C61-C119); NT-4/5(C61-C78); NT-4/5(C78-C119);
NT-4/5(C61-C90); NT-4/5(R60-C78); NT-4/5(K62-C119);
NT-4/5(K62-K91); NT-4/5(R79-R98); NT-4/5(R83-K93);
NT-4/5(T101-R111); NT-4/5(G1-C121) VLTVKRVRR; NT-4/5(V40-C121)
VLTVKRVRR; NT-4/5(V40-C121) SLTIKRIRA; NT-4/5(V40-C121) TLSRKAGRRA;
DDDSPIARRGEISVCDSVSDWVSAPDKDTAVDIKGDDVMVLKKVGINHSV;
NT-4/5(V40-C121); hNGF(S1-V48) NT-4/5(V40-C121) hNGF(V109-A120);
BDNF(R7-Q48) NT-4/5(V40-C121) BDNF(V110-R119); NT-4/5(.DELTA.C78);
NT-4/5(.DELTA.C61); NT-4/5(.DELTA.Q54-.DELTA.T59) (variants
depicted in this fashion comprise deletions of the indicated span
of residues, inclusive); NT-4/5(.DELTA.R60-.DELTA.D82);
NT-4/5(.DELTA.H85-.DELTA.S88); NT-4/5(.DELTA.W86-.DELTA.T101);
NT-4/5(R53.fwdarw.H); NT-4/5(K91.fwdarw.H); NT-4/5(V108.fwdarw.F);
NT-4/5(R84.fwdarw.Q, H, N, T, Y or W); and NT-4/5(D116.fwdarw.E, N,
Q, Y, S or T).
[0084] Also included is NT-4/5 wherein position 70 is substituted
with an amino acid residue other than G, E, D or P; position 71
with other than A, P or M; and/or position 83 with other than R, D,
S or K; as well as cyclized NT-4/5 fragments, including cyclic
polypeptides comprising the sequences IKTG, EIKTG, EIKTGN, SPV,
SPVK, HQV, KSS, KSSA, YAEHKS, RYAEHKS, RYAEHKSH, YAEHKSH, ANRTS,
NRT, ANRT, NRTS, KEA, KEAR, KEARP, IDDK, SENN, TSENN, TSENNK or
KLVG.
[0085] Also within the scope hereof are BDNF, NT-3, and NGF amino
acid variants having analogous structures to the NT-4/5 variants
set forth herein. For example, the analogous positions of NGF,
NT-3, and BDNF are substituted with a residue other than D, E, or
P, respectively, in analogy to the same mutation at position 70 of
NT-4/5.
[0086] DNA encoding NT-4/5 variants preferably is prepared by
site-specific mutagenesis of DNA that encodes an earlier prepared
variant or a nonvariant version of NT-4/5. Site-specific
mutagenesis allows the production of NT-4/5 variants through the
use of specific oligonucleotide sequences that encode the DNA
sequence of the desired mutation, as well as a sufficient number of
adjacent nucleotides, to provide a primer sequence of sufficient
size and sequence complexity to form a stable duplex on both sides
of the deletion junction being traversed. Typically, a primer of
about 20 to 25 nucleotides in length is preferred, with about 5 to
10 residues on both sides of the junction of the sequence being
altered. In general, the technique of site-specific mutagenesis is
well known in the art, as exemplified by publications such as
Adelman et al., DNA, 2: 183 (1983).
[0087] As will be appreciated, the site-specific mutagenesis
technique typically employs a phage vector that exists in both a
single-stranded and double-stranded form. Typical vectors useful in
site-directed mutagenesis include vectors such as the M13 phage,
for example, as disclosed by Messing et al., Third Cleveland
Symposium on Macromolecules and Recombinant DNA, Editor A. Walton,
Elsevier, Amsterdam (1981), the disclosure of which is incorporated
herein by reference. These phage are readily commercially available
and their use is generally well known to those skilled in the art.
Also, plasmid vectors that contain a single-stranded phage origin
of replication (Veira et al., Meth. Enzymol., 153: 3 [1987]) may be
employed to obtain single-stranded DNA. Alternatively, nucleotide
substitutions are introduced by synthesizing the appropriate DNA
fragment in vitro and amplifying it by polymerase chain reaction
(PCR) procedures known per se in the art.
[0088] In general, site-directed mutagenesis in accordance herewith
is performed by first obtaining a single-stranded vector that
includes within its sequence a DNA sequence that encodes the
relevant protein. An oligonucleotide primer bearing the desired
mutated sequence is prepared, generally synthetically, for example,
by the method of Crea et al., Proc. Natl. Acad. Sci. (USA), 75:
5765 (1978). This primer is then annealed with the single-stranded
protein-sequence-containing vector, and subjected to
DNA-polymerizing enzymes such as E. coli polymerase I Klenow
fragment, to complete the synthesis of the mutation-bearing strand.
Thus, a heteroduplex is formed wherein one strand encodes the
original non-mutated sequence and the second strand bears the
desired mutation. This heteroduplex vector is then used to
transform appropriate cells such as JM101 cells and clones are
selected that include recombinant vectors bearing the mutated
sequence arrangement.
[0089] After such a clone is selected, the mutated region may be
removed and placed in an appropriate vector for protein production,
generally an expression vector of the type that is typically
employed for transformation of an appropriate host.
[0090] Most deletions and insertions, and substitutions in
particular, are not expected to produce radical changes in the
characteristics of the NT-4/5 molecule, and single substitutions
will preserve at least one immune epitope in the NT-4/5
polypeptide.
[0091] Since it is often difficult to predict in advance the
characteristics of a variant NT-4/5, it will be appreciated that
some screening will be needed to select the optimal variant. One
can screen for enhanced trophic activity, differential neuron cell
type specificity, stability in recombinant cell culture or in
plasma (e.g. against proteolytic cleavage), possession of
antagonist activity, oxidative stability, ability to be secreted in
elevated yields, and the like. For example, a change in the
immunological character of the NT-4/5 molecule, such as affinity
for a given antibody, is measured by a competitive-type
immunoassay. Changes in the enhancement or suppression of
neurotrophic activities by the candidate mutants are measured by
dendrite outgrowth or explant cell survival assays. Modifications
of such protein properties as redox or thermal stability,
hydrophobicity, susceptibility to proteolytic degradation, or the
tendency to aggregate with carriers or into multimers are assayed
by methods well known in the art.
[0092] Trypsin or other protease cleavage sites are identified by
inspection of the encoded amino acid sequence for paired basic
residues, e.g. combinations of adjacent arginyl and lysinyl
residues. These are rendered inactive to protease by substituting
one of the residues with another residue, preferably a basic
residue such as glutamine or a hydrophobic residue such as serine;
by deleting one or both of the basic residues; by inserting a
prolyl residue immediately after the last basic residue; or by
inserting another residue between the two basic residues. A variant
NT-4/5 typically is made by site-specific mutagenesis of the native
NT-4/5-encoding nucleic acid, expression of the variant nucleic
acid in recombinant cell culture, and, optionally, purification
from the cell culture, for example, by bioassay of the variant's
activity or by immunoaffinity adsorption on a rabbit polyclonal
anti-NT-4/5 column (to absorb the variant by binding it to at least
one remaining immune epitope). Small fragments, on the order of 40
residues or less, are conveniently made by in vitro methods.
[0093] The NT-4/5-encoding nucleic acid, whether variant or cDNA,
then is ligated into a replicable vector for further cloning or for
expression. Vectors are useful for performing two functions in
collaboration with compatible host cells (a host-vector system).
One function is to facilitate the cloning of the nucleic acid that
encodes the NT-4/5, i.e., to produce usable quantities of the
nucleic acid. The other function is to direct the expression of
NT-4/5. One or both of these functions are performed by the
vector-host system. The vectors will contain different components
depending upon the function they are to perform as well as the host
cell that is selected for cloning or expression.
[0094] Each vector will contain nucleic acid that encodes NT-4/5 as
described above. Typically, this will be DNA that encodes the
NT-4/5 in its mature form linked at its amino terminus to a
secretion signal. This secretion signal preferably is the NT-4/5
presequence that normally directs the secretion of NT-4/5 from
human cells in vivo. However, suitable secretion signals also
include signals from other animal NT-4/5, signals from NGF, NT-2,
or NT-3, viral signals, or signals from secreted polypeptides of
the same or related species.
[0095] If the signal sequence is from another NT molecule, it may
be the precursor sequence spanning from the initiating methionine
(M) residue of NT-2, NT-3, or NGF up to the arginine (R) residue
just before the first amino acid of the mature protein, or a
consensus or combination sequence from any two or more of those
precursors taking into account homologous regions of the
precursors. The DNA for such precursor region is ligated in reading
frame to DNA encoding the mature NT-4/5.
[0096] Expression and cloning vectors contain a nucleic acid
sequence that enables the vector to replicate in one or more
selected host cells. Generally, in cloning vectors this sequence is
one that enables the vector to replicate independently of the host
chromosomes, and includes origins of replication or autonomously
replicating sequences. Such sequences are well-known for a variety
of bacteria, yeast and viruses. The origin of replication from the
well-known plasmid pBR322 is suitable for most gram negative
bacteria, the 2.mu. plasmid origin for yeast and various viral
origins (SV40, polyoma, adenovirus, VSV or BPV) are useful for
cloning vectors in mammalian cells. Origins are not needed for
mammalian expression vectors (the SV40 origin may typically be used
only because it contains the early promoter). Most expression
vectors are "shuttle" vectors, i.e. they are capable of replication
in at least one class of organisms but can be transfected into
another organism for expression. For example, a vector is cloned in
E. coli and then the same vector is transfected into yeast or
mammalian cells for expression even though it is not capable of
replicating independently of the host cell chromosome.
[0097] DNA also is cloned by insertion into the host genome. This
is readily accomplished with bacillus species, for example, by
including in the vector a DNA sequence that is complementary to a
sequence found in bacillus genomic DNA. Transfection of bacillus
with this vector results in homologous recombination with the
genome and insertion of NT-4/5 DNA. However, the recovery of
genomic DNA encoding NT-4/5 is more complex than that of an
exogenously replicated vector because restriction enzyme digestion
is required to excise the NT-4/5 DNA.
[0098] Expression and cloning vectors should contain a selection
gene, also termed a selectable marker. This is a gene that encodes
a protein necessary for the survival or growth of a host cell
transformed with the vector. The presence of this gene ensures that
any host cell which deletes the vector will not obtain an advantage
in growth or reproduction over transformed hosts. Typical selection
genes encode proteins that (a) confer resistance to antibiotics or
other toxins, e.g. ampicillin, neomycin, methotrexate or
tetracycline, (b) complement auxotrophic deficiencies, or (c)
supply critical nutrients not available from complex media, e.g.
the gene encoding D-alanine racemase for bacilli.
[0099] A suitable selection gene for use in yeast is the trp1 gene
present in the yeast plasmid YRp7 (Stinchcomb et al., 1979, Nature
282:39; Kingsman et al., 1979, Gene 7:141; or Tschemper et al,
1980, Gene 10:157). The trp1 gene provides a selection marker for a
mutant strain of yeast lacking the ability to grow in tryptophan,
for example, ATCC No. 44076 or PEP4-1 (Jones, 1977, Genetics
85:12). The presence of the trp1 lesion in the yeast host cell
genome then provides an effective environment for detecting
transformation by growth in the absence of tryptophan. Similarly,
Leu2 deficient yeast strains (ATCC 20,622 or 38,626) are
complemented by known plasmids bearing the Leu2 gene.
[0100] Examples of suitable selectable markers for mammalian cells
are dihydrofolate reductase (DHFR) or thymidine kinase. Such
markers enable the identification of cells which were competent to
take up the NT-4/5 nucleic acid. The mammalian cell transformants
are placed under selection pressure which only the transformants
are uniquely adapted to survive by virtue of having taken up the
marker. Selection pressure is imposed by culturing the
transformants under conditions in which the concentration of
selection agent in the medium is successively changed, thereby
leading to amplification of both the selection gene and the DNA
that encodes NT-4/5. Amplification is the process by which genes in
greater demand for the production of a protein critical for growth
are reiterated in tandem within the chromosomes of successive
generations of recombinant cells. Increased quantities of NT-4/5
are synthesized from the amplified DNA.
[0101] For example, cells transformed with the DHFR selection gene
are first identified by culturing all of the transformants in a
culture medium which lacks hypoxanthine, glycine, and thymidine. An
appropriate host cell in this case is the Chinese hamster ovary
(CHO) cell line deficient in DHFR activity, prepared and propagated
as described by Urlaub and Chasin, 1980, Proc. Nat'l. Acad. Sci.
USA 77:4216. A particularly useful DHFR is a mutant DHFR that is
highly resistant to MTX (EP 117,060A). This selection agent can be
used with any otherwise suitable host, e.g. ATCC No. CCL61 CHO-K1,
notwithstanding the presence of endogenous DHFR. The DHFR and
NT-4/5-encoding DNA then is amplified by exposure to an agent
(methotrexate, or MTX) that inactivates the DHFR. One ensures that
the cell requires more DHFR (and consequently amplifies all
exogenous DNA) by selecting only for cells that can grow in
successive rounds of ever-greater MTX concentration. Alternatively,
hosts co-transformed with genes encoding NT-4/5, wild-type DHFR,
and another selectable marker such as the neo gene can be
identified using a selection agent for the selectable marker such
as G418 and then selected and amplified using methotrexate in a
wild-type host that contains endogenous DHFR.
[0102] Other methods, vectors and host cells suitable for
adaptation to the synthesis of NT-4/5 in recombinant vertebrate
cell culture are described in M. J. Gething et al., Nature
293:620-625 (1981); N. Mantei et al., Nature 281:40-46 (1979); and
A. Levinson et al., EP 1 17,060A and 1 17,058A. A particularly
useful plasmid for mammalian cell culture expression of NT-4/5 is
pRK5 (EP pub. no. 307,247) or pSVI6B (U.S. Ser. No. 07/441,574
filed Nov. 22, 1989).
[0103] Expression vectors, unlike cloning vectors, should contain a
promoter which is recognized by the host organism and is operably
linked to the NT-4/5 nucleic acid. Promoters are untranslated
sequences located upstream from the start codon of a structural
gene (generally within about 100 to 1000 bp) that control the
transcription and translation of nucleic acid under their control.
They typically fall into two classes, inducible and constitutive.
Inducible promoters are promoters that initiate increased levels of
transcription from DNA under their control in response to some
change in culture conditions, e.g. the presence or absence of a
nutrient or a change in temperature. At this time a large number of
promoters recognized by a variety of potential host cells are well
known. These promoters are operably linked to NT-4/5-encoding DNA
by removing them from their gene of origin by restriction enzyme
digestion, followed by insertion 5' to the start codon for NT-4/5.
This is not to say that the genomic NT-4/5 promoter is not usable.
However, heterologous promoters generally will result in greater
transcription and higher yields of expressed NT-4/5.
[0104] Nucleic acid is operably linked when it is placed into a
functional relationship with another nucleic acid sequence. For
example, DNA for a presequence or secretory leader is operably
linked to DNA for a polypeptide if it is expressed as a preprotein
which participates in the secretion of the polypeptide; a promoter
or enhancer is operably linked to a coding sequence if it affects
the transcription of the sequence; or a ribosome binding site is
operably linked to a coding sequence if it is positioned so as to
facilitate translation. Generally, operably linked means that the
DNA sequences being linked are contiguous and, in the case of a
secretory leader, contiguous and in reading phase. Linking is
accomplished by ligation at convenient restriction sites. If such
sites do not exist then synthetic oligonucleotide adaptors or
linkers are used in accord with conventional practice.
[0105] Promoters suitable for use with prokaryotic hosts include
the .beta.-lactamase and lactose promoter systems (Chang et al.,
1978, Nature 275:615; and Goeddel et al., 1979, Nature 281:544),
alkaline phosphatase, a tryptophan (trp) promoter system (Goeddel,
1980, Nucleic Acids Res. 8:4057 and EPO Appln. Publ. No. 36,776)
and hybrid promoters such as the tac promoter (H. de Boer et al.,
1983, Proc. Nat'l. Acad. Sci. USA 80:21-25). However, other known
bacterial promoters are suitable. Their nucleotide sequences have
been published, thereby enabling a skilled worker operably to
ligate them to DNA encoding NT-4/5 (Siebenlist et al. 1980, Cell
20:269) using linkers or adaptors to supply any required
restriction sites. Promoters for use in bacterial systems also will
contain a Shine-Delgarno (S.D.) sequence operably linked to the DNA
encoding NT-4/5.
[0106] Suitable promoting sequences for use with yeast hosts
include the promoters for 3-phosphoglycerate kinase (Hitzeman et
al., 1980, J. Biol. Chem. 255:2073) or other glycolytic enzymes
(Hess et al., 1968, J. Adv. Enzyme Reg. 7:149; and Holland, 1978,
Biochemistry 17:4900), such as enolase, glyceraldehyde-3-phosphate
dehydrogenase, hexokinase, pyruvate decarboxylase,
phosphofructokinase, glucose-6-phosphate isomerase,
3-phosphoglycerate mutase, pyruvate kinase, triosephosphate
isomerase, phosphoglucose isomerase, and glucokinase.
[0107] Other yeast promoters, which are inducible promoters having
the additional advantage of transcription controlled by growth
conditions, are the promoter regions for alcohol dehydrogenase 2,
isocytochrome C, acid phosphatase, degradative enzymes associated
with nitrogen metabolism, metallothionein,
glyceraldehyde-3-phosphate dehydrogenase, and enzymes responsible
for maltose and galactose utilization. Suitable vectors and
promoters for use in yeast expression are further described in R.
Hitzeman et al., EP 73,657A. Yeast enhancers also are
advantageously used with yeast promoters.
[0108] NT-4/5 transcription from vectors in mammalian host cells is
controlled by promoters obtained from the genomes of viruses such
as polyoma, cytomegalovirus, adenovirus, retroviruses, hepatitis-B
virus and most preferably Simian Virus 40 (SV40), or from
heterologous mammalian promoters, e.g. the actin promoter. The
early and late promoters of the SV40 virus are conveniently
obtained as an SV40 restriction fragment which also contains the
SV40 viral origin of replication (Fiers et al., 1978, Nature
273:113). Of course, promoters from the host cell or related
species also are useful herein.
[0109] Transcription of NT-4/5-encoding DNA by higher eukaryotes is
increased by inserting an enhancer sequence into the vector. An
enhancer is a nucleotide sequence, usually about from 10-300 bp,
that acts on a promoter to increase its transcription and does so
in a manner that is relatively orientation and position
independent. Many enhancer sequences are now known from mammalian
genes (globin, elastase, albumin, .alpha.-fetoprotein and insulin).
Typically, however, one will use an enhancer from a eukaryotic cell
virus. Examples include the SV40 enhancer on the late side of the
replication origin (bp 100-270), the cytomegalovirus early promoter
enhancer, the polyoma enhancer on the late side of the replication
origin, and adenoviral enhancers. The enhancer may be spliced into
the vector at a position 5' or 3' to the NT-4/5-encoding sequence,
but is preferably located at a site 5' from the promoter.
[0110] Expression vectors used in eukaryotic host cells (yeast,
fungi, insect, plant, animal, human or nucleated cells from other
multicellular organisms) will also contain sequences necessary for
the termination of transcription and for stabilizing the mRNA. Such
sequences are commonly available from the 5' and, occasionally 3'
untranslated regions of eukaryotic or viral DNAs or cDNAs. These
regions contain regions that are transcribed as polyadenylated
segments in the untranslated portion of the mRNA encoding NT-4/5.
The 3' untranslated regions also include transcription termination
sites.
[0111] Suitable host cells for cloning or expressing the vectors
herein are the prokaryote, yeast or higher eukaryote cells
described above. Suitable prokaryotes include gram negative or gram
positive organisms, for example E. coli or bacilli. A preferred
cloning host is E. coli 294 (ATCC 31,446) although other gram
negative or gram positive prokaryotes such as E. coli B, E. coli
X1776 (ATCC 31,537), E. coli W3110 (ATCC 27,325), pseudomonas
species, or Serratia Marcesans are suitable.
[0112] In addition to prokaryotes, eukaryotic microbes such as
filamentous fungi or yeast are suitable hosts for NT-4/5-encoding
vectors. Saccharomyces cerevisiae, or common baker's yeast, is the
most commonly used among lower eukaryotic host microorganisms.
However, a number of other genera, species and strains arc commonly
available and useful herein.
[0113] Suitable host cells for the expression of NT-4/5 are derived
from multicellular organisms. Such host cells are capable of
complex processing and glycosylation activities. In principle, any
higher eukaryotic cell culture is workable, whether from vertebrate
or invertebrate culture, although cells from mammals such as humans
are preferred. Propagation of such cells in culture is per se well
known. See Tissue Culture, Academic Press, Kruse and Patterson,
editors (1973). Examples of useful mammalian host cell lines are
VERO and HeLa cells, Chinese hamster ovary cell lines, the W138,
BHK, COS-7, MDCK cell lines and human embryonic kidney cell line
293.
[0114] Host cells are transformed with the above-described
expression or cloning vectors and cultured in conventional nutrient
media modified as is appropriate for inducing promoters or
selecting transformants containing amplified genes. The culture
conditions, such as temperature, pH and the like, suitably are
those previously used with the host cell selected for cloning or
expression, as the case may be, and will be apparent to the
ordinary artisan.
[0115] Covalent modifications of NT-4/5 molecules are included
within the scope of this invention. Variant NT-4/5 fragments having
up to about 40 residues may be conveniently prepared by in vitro
synthesis. In addition, covalent modifications are introduced into
the molecule by reacting targeted amino acid residues of the NT-4/5
polypeptide with an organic derivatizing agent that is capable of
reacting with selected side chains or the N- or C-terminal
residues.
[0116] Cysteinyl residues most commonly are reacted with
.alpha.-haloacetates (and corresponding amines), such as
chloroacetic acid or chloroacetamide, to give carboxymethyl or
carboxyamidomethyl derivatives. Cysteinyl residues also are
derivatized by reaction with bromotrifluoroacetone,
.alpha.-bromo-.beta.-(5-imidozoyl)propionic acid, chloroacetyl
phosphate, N-alkylmaleimides, 3-nitro-2-pyridyl disulfide, methyl
2-pyridyl disulfide, p-chloromercuribenzoate,
2-chloromercuri-4-nitrophenol, or
chloro-7-nitrobenzo-2-oxa-1,3-diazole.
[0117] Histidyl residues are derivatized by reaction with
diethylpyrocarbonate at pH 5.5-7.0 because this agent is relatively
specific for the histidyl side chain. Para-bromophenacyl bromide
also is useful; the reaction is preferably performed in 0.1M sodium
cacodylate at pH 6.0.
[0118] Lysinyl and amino terminal residues are reacted with
succinic or other carboxylic acid anhydrides. Derivatization with
these agents has the effect of reversing the charge of the lysinyl
residues. Other suitable reagents for derivatizing
.alpha.-amino-containing residues include imidoesters such as
methyl picolinimidate; pyridoxal phosphate; pyridoxal;
chloroborohydride; trinitrobenzenesulfonic acid; O-methylisourea;
2,4-pentanedione; and transaminase-catalyzed reaction with
glyoxylate.
[0119] Arginyl residues are modified by reaction with one or
several conventional reagents, among them phenylglyoxal,
2,3-butanedione, 1,2-cyclohexanedione, and ninhydrin.
Derivatization of arginine residues requires that the reaction be
performed in alkaline conditions because of the high pK.sub.a of
the guanidine functional group. Furthermore, these reagents may
react with the groups of lysine as well as the arginine
epsilon-amino group.
[0120] The specific modification of tyrosyl residues may be made,
with particular interest in introducing spectral labels into
tyrosyl residues by reaction with aromatic diazonium compounds or
tetranitromethane. Most commonly, N-acetylimidizole and
tetranitromethane are used to form O-acetyl tyrosyl species and
3-nitro derivatives, respectively. Tyrosyl residues are iodinated
using .sup.125I or .sup.131I to prepare labeled proteins for use in
radioimmunoassay, the chloramine T method described above being
suitable.
[0121] Carboxyl side groups (aspartyl or glutamyl) are selectively
modified by reaction with carbodiimides (R'--N.dbd.C.dbd.N--R')
such as 1-cyclohexyl-3-(2-morpholinyl-4-ethyl) carbodiimide or
1-ethyl-3-(4-azonia-4,4-dimethylpentyl) carbodiimide. Furthermore,
aspartyl and glutamyl residues are converted to asparaginyl and
glutaminyl residues by reaction with ammonium ions.
[0122] Derivatization with bifunctional agents is useful for
crosslinking NT-4/5 to a water-insoluble support matrix or surface
for use in the method for purifying anti-NT-4/5 antibodies, and
vice versa. Commonly used crosslinking agents include, e.g.,
1,1-bis(diazo-acetyl)-2-phenyleth- ane, glutaraldehyde,
N-hydroxysuccinimide esters, for example, esters with
4-azidosalicylic acid, homobifunctional imidoesters, including
disuccinimidyl esters such as
3,3'-dithiobis(succinimidylpropionate), and bifunctional maleimides
such as bis-N-maleimido-1,8-octane. Derivatizing agents such as
methyl-3-[(p-azidophenyl)dithio]propioimidate yield
photoactivatable intermediates that are capable of forming
crosslinks in the presence of light. Alternatively, reactive
water-insoluble matrices such as cyanogen bromide-activated
carbohydrates and the reactive substrates described in U.S. Pat.
Nos. 3,969,287; 3,691,016; 4,195,128; 4,247,642; 4,229,537; and
4,330,440 are employed for protein immobilization.
[0123] Glutaminyl and asparaginyl residues are frequently
deamidated to the corresponding glutamyl and aspartyl residues.
Alternatively, these residues are deamidated under mildly acidic
conditions. Either form of these residues falls within the scope of
this invention.
[0124] Other modifications include hydroxylation of proline and
lysine, phosphorylation of hydroxyl groups of seryl or threonyl
residues, methylation of the .alpha.-amino groups of lysine,
arginine, and histidine side chains (T. E. Creighton, Proteins:
Structure and Molecular Properties, W.H. Freeman & Co., San
Francisco, pp. 79-86 [1983]), acetylation of the N-terminal amine,
and amidation of any C-terminal carboxyl group. NT-4/5 also is
covalently linked to nonproteinaceous polymers, e.g. polyethylene
glycol, polypropylene glycol or polyoxyalkylenes, in the manner set
forth in U.S. Ser. No. 07/275,296 or U.S. Pat. Nos. 4,640,835;
4,496,689; 4,301,144; 4,670,417; 4,791,192 or 4,179,337.
[0125] NT-4/5 preferably is recovered from the culture medium as a
secreted protein, although it also may be recovered from host cell
lysates when directly expressed without a secretory signal. When
NT-4/5 is expressed in a recombinant cell other than one of human
origin, the NT-4/5 is thus completely free of proteins of human
origin. However, it is necessary to purify NT-4/5 from recombinant
cell proteins in order to obtain preparations that are
substantially homogeneous as to protein. As a first step, the
culture medium or lysate is centrifuged to remove particulate cell
debris. NT-4/5 thereafter is purified from contaminant soluble
proteins, for example, by fractionation on immunoaffinity or ion
exchange columns; ethanol precipitation; reverse phase HPLC;
chromatography on silica or on a cation exchange resin such as
DEAE; chromatofocusing; SDS-PAGE; ammonium sulfate precipitation;
or gel electrophoresis using, for example, Sephadex G-75. NT-4/5
variants in which residues have been deleted, inserted or
substituted are recovered in the same fashion as native NT-4/5,
taking account of any substantial changes in properties occasioned
by the variation. For example, preparation of an NT-4/5 fusion with
another protein, e.g. a bacterial or viral antigen, facilitates
purification because an immunoaffinity column containing antibody
to the antigen can be used to adsorb the fusion. A protease
inhibitor such as phenyl methyl sulfonyl fluoride (PMSF) also may
be useful to inhibit proteolytic degradation during purification,
and antibiotics may be included to prevent the growth of
adventitious contaminants. One skilled in the art will appreciate
that purification methods suitable for native NT-4/5 may require
modification to account for changes in the character of NT-4/5 or
its variants upon expression in recombinant cell culture.
[0126] The trkB and trkC receptor DNA sequences are known. The
receptors can be expressed to obtain a soluble form of the receptor
by identifying the extracellular domain and excising the
transmembrane domain therefrom). The soluble form of the receptor
can then be used to screen for trkB or trkC binding molecules,
preferably small organic molecules, that are candidate agonists for
receptor activity. Screening of agonists uses, for example,
transformed cells expressing trkB or trkC receptor. Further or
alternative screening uses the assays taught herein.
[0127] As discussed above variants of native neurotrophins are made
that act as agonists. The receptor binding site(s) of a
neurotrophin are determined by binding studies. These regions can
be subcloned and tested for agonist activity. Such regions can be
also be constructed into larger molecules using known protein
engineering techniques, such as template-assembly synthesis.
Standard mutagenesis techniques (deletion or radical substitution
of appropriate nucleic acids) are used to identify such regions and
to create mutants for testing for agonism. Agonist activity can be
determined by several means, including the assays described
herein.
[0128] Chimeric or pantropic neurotrophins that bind either trkB or
trkC or preferably both are suitable for use in the methods and
compositions of the invention. By the term "pantropic
neurotrophins" or "pantropic neurotrophic factors", or grammatical
equivalents, herein is meant a neurotrophin which, unlike naturally
occurring neurotrophins, has multiple neurotrophin specificities.
That is, it contains domains which confer different neurotrophin
specificities. WO 95/33829 and corresponding U.S. Ser. No.
08/253,937, are hereby incorporated by reference for describing,
making and using pantropic neurotrophic factors suitable for
practicing the present invention. The discussions herein pertaining
to NT-4/5 or pantropic neurotrophin synthesis, design, expression
and use apply to chimeric and other neurotrophins as well. In one
embodiment, this means that the pantropic neurotrophins of the
present invention will bind to a variety of neurotrophic receptors.
Thus, for example, naturally occurring NGF, which is the natural or
native ligand for the trkA receptor, does not bind appreciably to
either the trkB or trkC receptor with high affinity; for example,
NGF binds to these receptors with a 500-1000 fold lower K.sub.D
than BDNF or NT-3, respectively. However, a pantropic NGF, i.e. a
pantropic neurotrophin whose amino acid backbone is based on NGF,
may bind to at least the trkA, trkB and p75 receptor.
Alternatively, a pantropic NGF will bind to the trkA, trkC and p75
receptor. One preferred embodiment allows the binding of the trkA,
trkB, trkC and p75 receptor. Similarly, naturally occurring BDNF
and NT-4/5, which are the natural ligands for the trkB receptor, do
not bind appreciably to either the trkA or trkC receptor as above.
Thus pantropic BDNF or NT-4/5 will bind to trkB and any combination
of trkA, trkC and p75, as shown above for pantropic NGF.
[0129] In alternative embodiments, the naturally occurring
neurotrophin will bind with poor affinity to several neurotrophin
receptors. In this embodiment, the pantropic neurotrophin binds to
these receptors with affinities higher than normally found, similar
to the affinities seen for the natural ligand. For example, NT-3
binds strongly to trkC, and weakly to trkA and trkB. Thus, a
pantropic NT-3 binds to trkC with its normal binding affinity, and
will bind to either trkA with an affinity similar to the trkA
natural ligand, NGF, or to trkB with an affinity similar to the
trkB natural ligands BDNF or NT-4/5, or both.
[0130] In a preferred embodiment, methods of treatment use a
chimeric or pantropic neurotrophin or variant with a binding
affinity for neurotrophin receptors at least about 50-60%,
preferably about 75-80%, and even more preferably about 90%, and
most preferably 100% of the binding affinity of the natural ligand.
Thus, a pantropic NGF will bind to the trkB or trkC receptor with
at least 50% of the binding of BDNF or NT-4/5, or NT-3,
respectively. This affinity is measured by a variety of ways, as
will appreciated by those skilled in the art. The preferred method
is the use of competition assays, as shown in (Hulme, et al.) and
in Example 2. Generally, binding affinities are reported as
IC.sub.50, that is, the concentration of unlabeled competitor which
inhibits 50% of the binding of labeled ligand to the receptor.
[0131] In alternative embodiments, the pantropicity of the
neurotrophin is measured not by binding affinity to neurotrophin
receptors, but rather by the neuronal survival or neurite outgrowth
assays. Thus, all neurotrophins support the survival of embryonic
neural crest-derived sensory neurons. Survival of embryonic
sympathetic neurons is only supported by NGF, while survival of
placode-derived sensory neurons is supported by NT-3 and BDNF
(Grotz et al., 1992). Survival of sensory neurons of the dorsal
root ganglion is supported by both NGF and BDNF. NT-3 elicits
neurite outgrowth of sensory neurons from dorsal root ganglion,
sympathetic chain ganglia, and nodose ganglion, as well as supports
survival of nodose ganglia neurons and dorsal root ganglion
neurons. Thus, neuronal survival assays or neurite outgrowth assays
can be run to determine the pantropicity of the pantropic
neurotrophins.
[0132] Thus, neurotrophin specificity is determined by the
neurotrophin receptor binding, and the neuronal survival assays
and/or neurite outgrowth assays. Thus, a pantropic neurotrophin
with NGF specificity means a neurotrophin which exhibits at least
the binding characteristics, neuronal survival assay specificity,
or the neurite outgrowth assay specificity of NGF. Similarly, a
pantropic neurotrophin with BDNF, NT-3 or NT-4/5 specificity
exhibits at least the binding characteristics, neuron survival
assay specificity, or neurite outgrowth assay specificity of BDNF,
NT-3 or NT-4/5, respectively.
[0133] In an additional embodiment, pantropic neurotrophins are
made by constructing covalent heterodimers. Normally, neurotrophins
are homodimers, comprising two identical monomers which are
non-covalently associated. In this embodiment, as outlined below,
pantropicity is conferred by each monomer containing domains which
confer different neurotrophic specificity. Alternatively,
pantropicity may be created by covalently attaching two different
neurotrophins with different specificities to create a covalent
heterodimer. Thus, for example, a NGF monomer may be covalently
attached to a NT-3 monomer, resulting in a pantropic neurotrophin
with both NGF and NT-3 specificity. Similarly, covalent
heterodimers may be made with any combination of NGF, NT-3, NT-4/5,
BDNF or CNTF to create pantropic neurotrophins with at least two
specificities. In addition, this procedure may be done with
monomers which are themselves pantropic, resulting in covalent
dimers of any combination of pantropic and single specificity
monomers. Thus, a pantropic covalent dimer may be a homodimer of
two pantropic monomers. However, to be included within the
definition of the present invention, the pantropic covalent dimer
must have at least two, and preferably three, neurotrophin
specificities.
[0134] The covalent attachment is preferably done as a direct
fusion of the nucleic acid, such that when the protein is
expressed, the C-terminus of the first monomer is attached directly
to the N-terminus of the second monomer, creating a single nucleic
acid encoding the dimer. In alternative embodiments, a linker may
be used, such as short repeats of glycine, or glycine and serine;
for example, a linker such as gly-gly or gly-gly-ser-gly-gly may be
used. This is done using techniques well known in the art. Other
techniques for the covalent attachment of proteins are well known
in the art.
[0135] Pantropic neurotrophins accomplish pantropic binding, or, as
discussed above, pantropic neuronal survival, by containing domains
which confer neurotrophin receptor specificity or binding. A domain
may be defined in one of two ways. In the first embodiment, a
domain is a portion of the neurotrophin which confers some
neurotrophic specificity. In this embodiment, a single monomer of
the pantropic neurotrophin contains one or several domains which
confer different specificities. The domains can range in size from
a single amino acid to about 10-15 amino acids. The domain may be
comprised of a combination of amino acids from a different
neurotrophin than the host neurotrophin, i.e. a domain from one
neurotrophin may be substituted into a second neurotrophin,
conferring pantropicity to the second neurotrophin. Alternatively,
the domain may result from amino acid substitutions which are not
based on homology to existing neurotrophins, as outlined below. In
the preferred embodiment, the domain comprises a continuous
sequence of amino acids; that is, a single stretch of amino acids
is replaced. In other embodiments, the domain may be comprised of
discontinuous amino acids; for example, several regions within the
neurotrophin may confer specificity, and thus replacements at
several positions within the neurotrophin are necessary for
pantropicity.
[0136] In some embodiments, there is more than one domain within a
neurotrophin which can confer neurotrophic specificity, which will
depend on the particular neurotrophin. BDNF, for example, has a
number of domains which appear to confer BDNF specificity. The
present invention shows that a single amino acid change in NT-3,
from aspartic acid at position 15 to an alanine, confers BDNF
specificity to NT-3. This domain can also be imported into the NGF
and NT-4/5 sequences at the positions that correspond to position
15 in NT-3; i.e. position 16 in NGF or position 18 in NT-4/5. It
should be understood that the corresponding amino acids are
determined by an examination of the alignment of the sequences as
depicted in U.S. Pat. No. 5,364,769. In addition to this domain,
there are other domains within BDNF which confer BDNF specificity.
For example, the substitution of the BDNF sequence from positions
78 to 88 (QCRTTQSYVR), or from positions 93-99 (SKKRIG) may confer
BDNF specificity (55).
[0137] Similarly, NT-3 has a number of domains which may confer
NT-3 specificity when substituted into a different neurotrophin. A
number of residues of NT-3 have been shown to be important in NT-3
trkC receptor binding as well as bioactivity assays. Specifically,
mutations at positions R103, D105, K80, Q83, E54, R56, T22, Y51,
V97, Y11, E7, R8, E10 and R68 all contribute to NT-3 specificity,
since mutations at these positions in NT-3 cause decreases in NT-3
activity. Of these, K80, Q83, T22, and V97 are within variable
regions, and the rest are found within constant regions. In
addition, residues in the vicinity of the residues may also give
NT-3 specificity. In some embodiments, changes in the constant
regions may also give NT-3 specificity. Alternatively, mutations at
positions R31 and E92 caused increases in NT-3 binding;
specifically, R31A and E92A NT-3 showed increased trkC binding.
These mutations can be directly imported into neurotrophins besides
NT-3, using the procedures described below. The amino acids at any
of these positions may be changed, as outlined below.
[0138] NGF has a number of domains which may confer NGF specificity
when substituted into a different neurotrophin. The N-terminal
amino acids of NGF confer NGF specificity when substituted for the
N-terminal residues of NT-3. Specifically, the 7 N-terminal amino
acids (SSSHPIF) of NGF may be substituted for the 6 N-terminal
amino acids of NT-3 (YAEHKS), resulting in a pantropic NT-3 with
NGF specificity. The exact number of NGF N-terminal residues is not
crucial; as shown in the Examples, and particularly in Example 3,
the histidine at amino acid position 4 appears to be quite
important for NGF specificity; thus from about 4 to about 10
N-terminal residues may be exchanged although in some embodiments,
a single amino acid change will be sufficient. Similarly, a number
of other residues of NGF have been shown to be important in NGF
trkA receptor binding as well as bioactivity assays. For example,
there are a number of residues which, when mutated, lose NGF
activity. This shows the importance of the residue for NGF
specificity. These residues include, but are not limited to, H4,
P5, V18, V20, G23, D30, Y52, R59, R69, H75, Y79, T81, and R103. Of
these, D30, R59, Y79, and T81 are in "variable regions", i.e.
regions which vary between the different neurotrophins, with the
remainder in constant regions. In some embodiments, the variable
region residues are more likely to cause NGF specificity, since
constant region residues may be important for general structure and
characteristics, and may not confer specificity. However, as shown
above for the D15A mutation, mutations in the constant regions can
confer specificity as well. Furthermore, there are a number of
amino acid substitutions in NGF which increase NGF binding and/or
bioactivity. Accordingly, these substitutions may be imported into
other neurotrophin backbones to confer NGF specificity. These
residues include, but are not limited to, E11, F12, D24, E41, N46,
S47, K57, D72, N77, H84, D105, and K115.
[0139] Once identified, the residues important in neurotrophin
specificity can be replaced by any of the other amino acid residues
using techniques described in the examples and well-known
techniques for site-directed mutagenesis. Generally, the amino
acids to be substituted are chosen on the basis of characteristics
understood by those skilled in the art. For example, when small
alterations in the characteristics are desired, substitutions are
generally made as discussed above.
[0140] In the context of a covalent heterodimer, a domain may also
refer to the entire neurotrophin monomer. Thus, a pantropic
covalent heterodimer can be comprised of a domain which confers
NT-3 specificity, i.e. the NT-3 monomer, covalently attached to a
domain that confers BDNF specificity, i.e. the BDNF monomer.
Similarly, an NT-3 monomer may be paired with an NGF monomer, or an
NGF monomer may be paired with a BDNF monomer. In addition,
covalent heterodimers may be made with NT-4/5 and CNTF monomers as
well. In these embodiments, the domain is large, and generally
comprises most or all of the wild-type neurotrophin amino acid
sequence.
[0141] In one embodiment, the agonist is a pantropic or chimeric
NT-3. In this context, a pantropic NT-3 is a pantropic neurotrophin
which has an amino acid sequence homologous to the amino acid
sequence of NT-3, with domains which confer other neurotrophin
specificities. In the preferred embodiment, the domains are
substituted for NT-3 residues; that is, some number of amino acids
are deleted from the NT-3 sequence, and an identical or similar
number of amino acids are substituted, conferring an additional
specificity. For example, the MNTS-1 (multiple neurotrophic
specificities-1) pantropic NT-3 comprises the first 7 amino acids
of NGF replacing the 6 N-terminal residues of NT-3, plus the D15A
substitution. The MNTS-1 pantropic NT-3 has NT-3, NGF, and BDNF
specificities, and also binds to the p75 receptor. Other pantropic
NT-3s are made using minimal changes within the N-terminus. For
example, since H4 and P5 are conserved among NGFs, and 2
hydrophobic residues in positions 6 and 7 are conserved, the
following variants are made: 1) YASHPIF-hNT-3; 2) YAHPIF-hNT-3; 3)
YASHPIS-hNT-3; 4) YAEHPIF-hNT-3; 5) YAQHPIF-hNT-3. When the D15A
substitution is added, the resulting neurotrophins exhibit NGF,
NT-3 and BDNF specificity. Alternatively, replacing the variable
region 2 or 3 or 4, or combinations, of NT-3 with the corresponding
region from NGF gives a pantropic neurotrophin with both NT-3 and
NGF specificity.
[0142] A pantropic NGF can be made with a D16A substitution, which
confers BDNF specificity, plus substitutions in the pre-variable
region 1 (VI 8E+V20L+G23T) and in variable region 4
(Y79Q+T81K+H84Q+F86Y+K88R). Alternatively, the substitutions in the
pre-variable region 1 can be made with only single amino acid
substitutions in variable region 4; for example, V18E+V20L+G23T and
one of Y79Q, T81K, H84Q, F86Y, or K88R may be made.
[0143] In a preferred embodiment, the agonist is a chimeric or
pantropic NT-4/5, preferably made with a trkC binding region. NGF
specificity may be conferred on NT-4/5 by replacing the N-terminal
9 amino acids of NT-4/5 with the N-terminal 7 amino acids of
NGF.
[0144] In one embodiment, binding to the p75 receptor by the
pantropic neurotrophin has been substantially diminished or
eliminated. For example, there are a variety of amino acid residues
which contribute to p75 binding, in which mutations result in
diminished p75 binding. In NT-3, mutations at positions R68, Y11,
K73, R114, K115, Y51, K73, R31 and H33 and in NGF, mutations at
positions F12, 131, K32, K34, K50, Y52, R69, K74, K88, L112, S113,
R114, and K115 all result in diminished p75 binding. Since F12,
131, K50, Y52, R69, and K74 are all within constant regions of the
neurotrophins; these changes are expected to alter p75 binding in
the other neurotrophins as well. The other residues may be altered
as well.
[0145] In addition to the amino acid changes outlined above, those
skilled in the art understand that some variability of the amino
acid sequence is tolerated without altering the specificity and
characteristics of the neurotrophin. Thus, pantropic neurotrophins
can have amino acid substitutions, insertions or deletions compared
to the wild-type sequences which do not affect pantropicity but are
merely variations of the sequence. In some embodiments, these
mutations will be found within the same positions identified as
important to specificity; i.e. in some cases, neutral mutations may
be made without changing neurotrophin specificity.
[0146] The pantropic neurotrophins of the present invention can be
made in a variety of ways, using recombinant technology as
discussed above. In a preferred embodiment, the pantropic
neurotrophins of the invention are expressed in mammalian cells.
Mammalian expression systems are also known in the art. In one
embodiment, pantropic neurotrophins are produced in yeast cells.
Yeast expression systems are well known in the art, and include
expression vectors for Saccharomyces cerevisiae, Candida albicans
and C. maltosa, Hansenula polymorpha, Kluyveromyces fragilis and K.
lactis, Pichia guillerimondii and P. pastoris, Schizosaccharomyces
pombe, and Yarrowia lipolytica. The methods of introducing
exogenous nucleic acid into yeast hosts, as well as other hosts, is
well known in the art, and will vary with the host cell used. In a
preferred embodiment, pantropic neurotrophins are expressed in
bacterial systems. Expression vectors for bacteria are well known
in the art, and include vectors for Bacillus subtilis, E. coli,
Streptococcus cremoris, and Streptococcus lividans, among others.
The bacterial expression vectors are transformed into bacterial
host cells using techniques well known in the art, such as calcium
chloride treatment, electroporation, and others. In one embodiment,
pantropic neurotrophins are produced in insect cells. Expression
vectors for the transformation of insect cells, and in particular,
baculovirus-based expression vectors, are well known in the art.
Materials and methods for baculovirus/insect cell expression
systems are commercially available in kit form; for example the
"MaxBac" kit from Invitrogen in San Diego. Recombinant baculovirus
expression vectors have been developed for infection into several
insect cells. For example, recombinant baculoviruses have been
developed for Aedes aegypti, Autographa californica, Bombyx mori,
Drosophila melangaster, Spodoptera frugiperda, and Trichoplusia
ni.
[0147] Once expressed, chimeric or pantropic neurotrophins are used
as neurotrophic factors. These chimeric or pantropic neurotrophins
may be utilized in various compositions, assays, and therapeutic
applications of the invention.
[0148] For use in the assays of the invention the agonist can be
labeled. By "labeled" herein is meant an agonist that has at least
one element, isotope or chemical compound attached to enable the
detection of the neurotrophin bound to a neurotrophin receptor. In
general, labels fall into three classes: a) isotopic labels, which
may be radioactive or heavy isotopes; b) immune labels, which may
be antibodies or antigens; and c) colored or fluorescent dyes. The
labels may be incorporated into the neurotrophin at any position.
Once labelled, the neurotrophins are used to detect neurotrophin
receptors, either in vitro or in vivo. For example, the presence of
neurotrophin receptors can be an indication of the presence of
certain cell types, useful establishing and in scoring the assays.
That is, a subpopulation of certain cell types may be shown by the
binding of the labeled neurotrophin to the cells via the
receptors.
[0149] Additionally, the neurotrophins are useful as standards in
assays of the invention. For example, the activity of a variant
neurotrophin in any particular assay may be determined using known
neurotrophin standards, and then the variant neurotrophin may be
used in the diagnosis and quantification of neurotrophins and other
agonists.
[0150] As will be understood by those skilled in the art, the
pantropic neurotrophins of the present invention can replace other
neurotrophic factors which are used as media components in the
cultures as taught herein and in the methods of treatment taught
herein. The amount of the pantropic neurotrophins to be added can
be easily determined using standard assays.
[0151] Purification of Agonists
[0152] Techniques used for separating the agonist from impurities
depend on which particular agonist is being employed. These
procedures may include, for example, one or more steps selected
from immunoaffinity chromatography, ion-exchange column
fractionation (e.g., on DEAE or matrices containing carboxymethyl
or sulfopropyl groups), chromatography on Blue-Sepharose, CM
Blue-Sepharose, MONO-Q, MONO-S, lentil lectin-Sepharose,
WGA-Sepharose, Con A-Sepharose, Ether Toyopearl, Butyl Toyopearl,
Phenyl Toyopearl, or protein A Sepharose, SDS-PAGE chromatography,
silica chromatography, chromatofocusing, reverse phase HPLC (e.g.,
silica gel with appended aliphatic groups), gel filtration using,
e.g., Sephadex molecular sieve or size-exclusion chromatography,
chromatography on columns that selectively bind the trkB or trkC
agonist, such as trkB or trkC receptors or antibody-affinity, and
ethanol or ammonium sulfate precipitation. A protease inhibitor may
be included in any of the foregoing steps to inhibit proteolysis.
Examples of suitable protease inhibitors include
phenylmethylsulfonyl fluoride (PMSF), leupeptin, pepstatin,
aprotinin, 4-(2-aminoethyl)-benzenesulfonyl fluoride
hydrochloride-bestatin, chymostatin, and benzamidine.
[0153] Therapeutic Compositions and Administration of Agonists
[0154] Agonists to trkB or trkC alone, in combination with each
other, or optionally in combination with ototoxic pharmaceuticals,
are believed to find use as drugs for in vivo treatment of mammals,
ex vivo treatments involving transplant or assays involving organs
such as during perfusion, and in vitro assays and screening
methods. For example, the trkB or trkC agonist alone or in
combination with each other will be useful in treating hearing
impairments in cases where pharmaceutical drugs are limited in
their dosage or display side-effect of a oto-neurological hearing
impairment.
[0155] In the preferred embodiment, the neurotrophin(s) is
administered to a patient to treat neural-related (associated with
neuron degeneration, damage or loss) hearing impairment,
prophylactically or therapeutically. Preferably hair cell loss or
damage is not present or not at a significant level that would
hinder hearing recovery. Specific examples include, but are not
limited to neuropathies, and other conditions characterized by
necrosis, damage, or loss of neurons affecting hearing, whether
caused by trauma, injury, aging, noise, environmental toxins, or
ototoxic pharmaceutical drugs. For example, neuropathies associated
with certain conditions such as diabetes, AIDS, or chemotherapy may
be treated using the compositions and methods of the present
invention.
[0156] Therapeutic formulations of agonist(s) (and optionally
ototoxic pharmaceutical drug) for treating hearing impairments are
prepared for storage by mixing the agonist(s) or drug having the
desired degree of purity with optional physiologically acceptable
carriers, excipients, or stabilizers (Remington's Pharmaceutical
Sciences, 16th edition, Oslo, A., Ed., [1980]), in the form of
lyophilized cake or aqueous solutions. Acceptable carriers,
excipients, or stabilizers are non-toxic to recipients at the
dosages and concentrations employed, and include buffers such as
phosphate, citrate, and other organic acids; antioxidants including
ascorbic acid; low molecular weight (less than about 10 residues)
polypeptides; proteins, such as serum albumin, gelatin, or
immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone;
amino acids such as glycine, glutamine, asparagine, arginine or
lysine; monosaccharides, disaccharides, and other carbohydrates
including glucose, mannose, or dextrins; chelating agents such as
EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming
counterions such as sodium; and/or nonionic surfactants such as
Tween, Pluronics, or polyethylene glycol (PEG).
[0157] The agonist(s) are also suitably linked to one of a variety
of nonproteinaceous polymers, e.g., polyethylene glycol,
polypropylene glycol, or polyoxyalkylenes, in the manner set forth
in U.S. Pat. Nos. 4,640,835; 4,496,689; 4,301,144; 4,670,417;
4,791,192 or 4,179,337.
[0158] The agonist(s) to be used for in vivo administration must be
sterile. This is readily accomplished by filtration through sterile
filtration membranes, prior to or following lyophilization and
reconstitution. The agonist(s) ordinarily will be stored in
lyophilized form or in solution. Preferably, it is free or
substantially free (at least 80%, preferably at least 90%, more
preferably at least 95%, and even more preferably at least 99%
pure) of contaminating polypeptides from the purification
source.
[0159] Therapeutic agonist compositions generally are placed into a
container having a sterile access port, for example, an intravenous
solution bag or vial having a stopper pierceable by a hypodermic
injection needle.
[0160] The agonist(s) is administered in an acute or chronic
fashion, as may be required, for prophylactic and therapeutic
applications, by a number of routes including: injection or
infusion by intravenous, intraperitoneal, intracerebral,
intramuscular, intradermally, intraocular, intraarterial,
subcutaneously, or intralesional routes, topical administration,
orally if an orally active small molecule is employed, using
sustained-release systems as noted below, or by an indwelling
catheter using a continuous administration means such as a pump, by
patch, or implant systems, e.g., intracerebral implantation of a
sustained-release vehicle. Agonist(s) is administered continuously
by infusion or by periodic bolus injection if the clearance rate is
sufficiently slow, or by administration into the blood stream,
lymph, CNS or spinal fluid. A preferred administration mode is
directly to the affected portion of the ear or vestibule,
topically, and, preferably to the affected neurons, so as to direct
the molecule to the source and minimize side effects of the
agonists.
[0161] Neurotrophin, preferably NT-4/5, can be injected through
chronically implanted cannulas or chronically infused with the help
of osmotic minipumps. Subcutaneous pumps are available that deliver
proteins through a small tubing to the appropriate area. Highly
sophisticated pumps can be refilled through the skin and their
delivery rate can be set without surgical intervention. Examples of
suitable administration protocols and delivery systems involving a
subcutaneous pump device or continuous infusion through a totally
implanted drug delivery system are those used for the
administration of dopamine, dopamine agonists, and cholinergic
agonists to Alzheimer patients and animal models for Parkinson's
disease described by Harbaugh, J. Neural Transm. Suppl., 24:
271-277 (1987) and DeYebenes et al., Mov. Disord., 2: 143-158
(1987), the disclosures of which are incorporated herein by
reference. It is envisioned that it may be possible to introduce
cells actively producing agonist into areas in need of increased
concentrations of agonist.
[0162] Suitable examples of sustained-release preparations include
semipermeable matrices of solid hydrophobic polymers containing the
protein, which matrices are in the form of shaped articles, e.g.,
films, or microcapsules. Examples of sustained-release matrices
include polyesters, hydrogels (e.g.,
poly(2-hydroxyethylmethacrylate) as described by Langer et al., J.
Biomed. Mater. Res., 15: 167-277 [1981] and Langer, Chem. Tech.,
12: 98-105 [1982] or poly(vinylalcohol)), polylactides (U.S. Pat.
No. 3,773,919, EP 58,481), copolymers of L-glutamic acid and gamma
ethyl-L-glutamate (Sidman et al., Biopolymers, 22: 547-556 [1983]),
non-degradable ethylene-vinyl acetate (Langer et al., supra),
degradable lactic acid-glycolic acid copolymers such as the Lupron
Depot.TM. (injectable microspheres composed of lactic acid-glycolic
acid copolymer and leuprolide acetate), and
poly-D-(-)-3-hydroxybutyric acid (EP 133,988). The agonist(s) also
may be entrapped in microcapsules prepared, for example, by
coacervation techniques or by interfacial polymerization (for
example, hydroxymethylcellulose or gelatin-microcapsules and
poly-[methylmethacylate] microcapsules, respectively), in colloidal
drug delivery systems (for example, liposomes, albumin
microspheres, microemulsions, nano-particles and nanocapsules), or
in macroemulsions. Such techniques are disclosed in Remington's
Pharmaceutical Sciences, 16th edition, Osol, A., Ed., (1980).
[0163] While polymers such as ethylene-vinyl acetate and lactic
acid-glycolic acid enable release of molecules for over 100 days,
certain hydrogels release proteins for shorter time periods. When
encapsulated proteins remain in the body for a long time, they may
denature or aggregate as a result of exposure to moisture at
37.degree. C., resulting in a loss of biological activity and
possible changes in immunogenicity. Rational strategies can be
devised for protein stabilization depending on the mechanism
involved. For example, if the aggregation mechanism is discovered
to be intermolecular S-S bond formation through thiodisulfide
interchange, stabilization may be achieved by modifying sulfhydryl
residues, lyophilizing from acidic solutions, controlling moisture
content, using appropriate additives, and developing specific
polymer matrix compositions.
[0164] Sustained-release agonist(s) compositions also include
liposomally entrapped agonist(s). Liposomes containing agonist(s)
are prepared by methods known per se: DE 3,218,121; Epstein et al.,
Proc. Natl. Acad. Sci. USA, 82: 3688-3692 (1985); Hwang et al.,
Proc. Natl. Acad. Sci. USA, 77: 4030-4034 (1980); EP 52,322; EP
36,676; EP 88,046; EP 143,949; EP 142,641; Japanese patent
application 83-118008; U.S. Pat. Nos. 4,485,045 and 4,544,545; and
EP 102,324. Ordinarily the liposomes are of the small (about
200-800 Angstroms) unilamellar type in which the lipid content is
greater than about 30 mol. % cholesterol, the selected proportion
being adjusted for the optimal agonist therapy. A specific example
of a suitable sustained-release formulation is in EP 647,449.
[0165] An effective amount of agonist(s) to be employed
therapeutically will depend, for example, upon the therapeutic
objectives, the route of administration, the species of the
patient, and the condition of the patient. Accordingly, it will be
necessary for the therapist to titer the dosage and modify the
route of administration as required to obtain the optimal
therapeutic effect. As is known in the art, adjustments for age as
well as the body weight, general health, sex, diet, time of
administration, drug, interaction and the severity of the disease
may be necessary, and will be ascertainable with routine
experimentation by those skilled in the art. A typical daily dosage
of trkB or trkC agonist used alone might range from about 1
.mu.g/kg to up to 100 mg/kg of patient body weight or more per day,
depending on the factors mentioned above, preferably about 10
.mu.g/kg/day to 10 mg/kg/day. Typically, the clinician will
administer agonist until a dosage is reached that repairs,
maintains, and, optimally, reestablishes neuron function to relieve
the hearing impairment. Generally, the agonist is formulated and
delivered to the target site at a dosage capable of establishing at
the site an agonist level greater than about 0.1 ng/ml, more
typically from about 0.1 ng/ml to 5 mg/ml, preferably from about 1
to 2000 ng/ml. In a specific embodiment of the invention, a
effective pharmaceutical composition, for example for promoting the
survival of SGNs, may provide a local neurotrophin protein
concentration of between about 1 and 100 ng/ml, preferably 5 to 25
ng/ml, and more preferably, between 10 and 20 ng/ml. The progress
of this therapy is easily monitored by conventional assays and
neurological diagnostic methods.
[0166] If two agonists are administered together, they need not be
administered by the same route, nor in the same formulation.
However, they can be combined into one formulation as desired. In a
preferred embodiment NT-4/5 optionally is combined with or
administered in concert with or formed as a pantropic neurotrophin
with a neurotrophic agonist to trkC. Both agonists can be
administered to the patient, each in effective amounts, or each in
amounts that are sub-optimal but when combined are effective.
Preferably such amounts are about 10 .mu.g/kg/day to 10 mg/kg/day
of each. In another preferred embodiment, the administration of
both agonists is by injection using, e.g., intravenous or
subcutaneous means, depending on the type of agonist employed.
Typically, the clinician will administer the agonist(s) until a
dosage is reached that achieves the desired effect for treatment of
the hearing impairment. The progress of this therapy is easily
monitored by conventional assays.
[0167] The two types of agonists, if used together, may be
formulated together in an appropriate carrier vehicle to form a
pharmaceutical composition that preferably does not contain cells.
In one embodiment, the buffer used for formulation will depend on
whether the composition will be employed immediately upon mixing or
stored for later use, since long-term storage may bring into issue
stability such as solubility and aggregation that can be addressed
by altering the pH. The final preparation may be a stable liquid or
lyophilized solid.
[0168] The agonist(s) optionally is combined with or administered
in concert with ototoxic pharmaceutical drugs. Initially the drugs
are administered in conventional therapies known for the ototoxic
pharmaceutical. Adjustments to the therapies are at the discretion
of the skilled therapist to titrate dosages and conditions that
decrease ototoxicity-related hearing while maintaining, and
preferably improving, treatment outcomes with the ototoxic
pharmaceutical drug.
[0169] Accordingly, methods for preventing or reducing ototoxicity
of an aminoglycoside antibiotic or other ototoxic pharmaceutical
are disclosed herein, which comprise the administration of an
effective dose of a trkB or trkC agonist. In addition, provided
herein are compositions having reduced ototoxicity as a result of
incorporation of the ototoxicity-inhibiting trkB or trkC agonists
of the present invention. These pharmaceutical compositions
comprise an effective ototoxicity-inhibiting amounts of agonists as
described herein, therapeutically effective amounts of the ototoxic
pharmaceutical drug, e.g. aminoglycosides antibiotic,
anti-neoplastic agent such as cisplatin, and optionally a
pharmaceutically acceptable carrier and/or vehicle which would be
familiar to one skilled in the pharmaceutical arts. The actual
amounts of ototoxic pharmaceutical drug employed will range from
those given in standard references for prescription drugs, e.g.
"Physicians Desk Reference" (1995), "Drug Evaluations" AMA, 6th
Edition (1986); to amounts somewhat larger since the ototoxicity
potential is reduced in these compositions.
[0170] The effective amounts of such agents, if employed, will be
at the physician's or veterinarian's discretion. Dosage
administration and adjustment is done to achieve the best
management of hearing (and when used in conjunction with an
ototoxic pharmaceutical drug, the indication for the ototoxic
drug). The dose will additionally depend on such factors as the
type of drug used and the specific patient being treated. Typically
the amount employed will be the same dose as that used if the drug
were to be administered without agonist; however, lower doses may
be employed depending on such factors as the presence of
side-effects, the condition being treated, the type of patient, and
the type of agonist and drug, provided the total amount of agents
provides an effective dose for the condition being treated. For
example, a test dose may be 5 mg, which is then ramped up to 10-20
mg per day, once a day, to 25 mg twice per day (BID) or three times
per day (TID), and may be titrated to 50 mg BID or TID as the
patient tolerates it. Tolerance level is estimated by determining
whether decrease in hearing impairment is accompanied by signs of
observed side-effects. A discussion of the dosage, administration,
indications and contraindications associated with ototoxic
pharmaceuticals optionally used with the neurotrophins in the
methods of the invention can be found in the Physicians Desk
Reference, Medical Economics Data Production Co., Montvale, N.J.
(1995).
[0171] In preferred embodiments therapeutic formulations contain
NT-4/5, a fragment, variant, or pantropic, and can be prepared for
storage by mixing NT-4/5 having the desired degree of purity with
optional physiologically acceptable carriers, excipients or
stabilizers (Remington's Pharmaceutical Sciences, supra,) in the
form of lyophilized cake or aqueous solutions.
[0172] The compositions herein also can suitably contain other
growth factors, most preferably hair cell growth factors, or
combination of factors, for example retinoic acid or retinoic acid
in combination with TGF-.alpha.. Such growth factors, including
peptide growth factors, are suitably present in an amount that is
effective for the purpose intended, i.e., to promote survival,
growth, proliferation, regeneration, restoration or recovery of
hair cells when desired, and optionally, to enhance growth or
recovery of auditory neurons, e.g., spiral ganglia. Although the
present results indicate that particular neurotrophins have strong
protective effects on SGNs, they did not protect hair cells from
the ototoxic drugs. Accordingly, if hair cell loss due to
ototoxicity is significant, hearing impairment recovery can be
improved by new hair cell growth, survival, proliferation, or
regeneration. While not to be constrained by the mechanism
forwarded, it is believed that two mechanisms may be available
damage of hair cells by aminoglycosides (Lim, 1986; Rybak, 1986).
One is the reversible blockage of transduction channels on the hair
cells. The other mechanism is impairment of the cell maintenance
machinery required for cell repair and survival. The latter is
believed to be irreversible and probably occurs intracellularly
through a mechanism which inhibits certain enzymes such as
phosphatidylinositol phospholipase C, affecting degradation of
phospholipids and membrane permeability (Schacht, 1986; Au et al.,
1987). Agents that antagonize the reversible blockage of
transduction channels on the hair cells, by aminoglycosides for
example, are suitable for use in the compositions of the invention
and methods of their use. Agents that antagonize ototoxin-induced
membrane permeability changes of hair cells, by aminoglycosides for
example, are suitable for use in the compositions of the invention
and methods of their use. Agents that promote the growth or
proliferation of new hair cells, agents that support survival of
hair cells, and agents that support regeneration of hair cells are
suitable for use in the compositions of the invention and in
methods of their use. Recent studies have suggested possible
candidates (Forge et al., 1993; Cotanche and Lee, 1994; Tsue et
al., 1994a; Cotanche and Lee, 1994; Kelley et al., 1995). For
example, diffusible factors such as TGF-.alpha. and EGF (Lambert,
1994; Yamashita and Oesterle, 1995) or components derived from
antibiotic treated inner ear tissue (Tsue et al., 1994b) stimulate
proliferation of supporting cells. Retinoic acid alone or in
combination with TGF-.alpha. facilitates hair cell regeneration in
vitro (Lefebvre et al., 1993, 1995).
[0173] The effectiveness of treating hearing impairments with the
methods of the invention can be evaluated by the following signs of
recovery, including recovery of normal hearing function, which can
be assessed by known diagnostic techniques including those
discussed herein, and normalization of nerve conduction velocity,
which is assessed electrophysiologically.
[0174] In another embodiment, agonist compositions of the invention
are used during clinical cochlear implants to keep or improve
viability of spiral ganglion neurons. Preferably a combination of a
trkB and a trkC neurotrophin and a hair cell growth factor(s) will
be used, either alone or in combination with a cochlear
implant.
[0175] Cochlear Explants
[0176] In one embodiment of the invention is provided a method of
assaying for a trkB or trkC agonist that provides spiral ganglion
neuron protection or survival from an ototoxin. The assay steps
include culturing a cochlear explant, administering a trkB or trkC
agonist to the culture, administering an ototoxin to the culture,
and determining the amount of protection or survival compared to a
control culture to which the trkB or trkC agonist was not
administered.
[0177] In a preferred embodiment of the invention is provided an
organotype cochlear explant culture that utilizes a 3-D collagen
matrix cultures and maintains its normal, in vivo architecture to
provide an auditory assay system. The spiral ganglion remain
attached. The explant is cultured in three-dimensional ("3-D")
collagen gel in serum free medium.
[0178] Embedding the cochlear explants in the 3-D collagen was
better for maintaining their normal architecture than floating the
explants or placing the explants on a monolayer substrate, since
the explant tissue could be kept unfolded and cell migration out of
the tissue could be limited. By using neurofilament (N52) and
phalloidin-FITC conjugate double labeling, the integrity of SGNs
and the hair cells in the cochlea was demonstrated. Cochlear
explants prepared according to the invention maintained normal
architecture in the 3-D collagen gel cultures as observed by
Nomarski micrographs. The SGNs remained in their normal locations.
No gross cell death of SGNs or hair cells occurred under this
culture condition. The peripheral axons of the SGNs which innervate
the hair cells in the organ of Corti were seen to maintain
consistently a radial projection pattern, as seen in vivo.
[0179] Organotypic cultures of postnatal cochlear explants provided
herein, in which the afferent innervation of hair cells by primary
auditory neurons are intact, are useful to examine ototoxicity of
different classes of ototoxins, including ototoxic pharmaceutical
drugs, for example, salicylate, gentamicin, and cisplatin, and to
search for or test candidate agents that protect against the
ototoxic effect. To determine if an ototoxin is able to induce
degeneration of SGNs and/or hair cells in the cochlear explant
cultures, the ototoxin, preferably an ototoxic pharmaceutical drug,
is added at different concentrations to the culture after allowing
the culture to recover from the in vitro explant. Histochemical
double-labeling of the cochlear explant cultures with a
neurofilament antibody (Texas red-mediated) and phalloidin
(FITC-conjugated) can be used to compare control cultures
(untreated) with cultures treated with ototoxin. While the
neurofilament antibody (Texas red-mediated) labels the somata and
peripheral axons of SGNs, the phalloidin-FITC conjugate stains the
stereocillia bundles of hair cells. Cell count of the remaining
hair cells and SGNs can be done to determine and quantify ototoxic
effect. Since the density of the axons of SGNs is a reliable index
of the number of surviving SGNs, in one embodiment the number of
the SGN axons from a given length (e.g., 100 .mu.m) in the middle
of the cochlea is counted. Phalloidin-labeled hair cells can also
be counted in the same way.
[0180] Organotypic culture of cochlear explants offers advantages
to explore the mechanism of actions of ototoxins (Anniko and Sobin,
1986), to discover protective agents (Richardson and Russell, 1991)
and to search for hair cell growth factors (Lefebvre et al., 1993),
as it keeps the afferent neuronal innervation of hair cells intact
and appears to follow closely the normal development pathway
(Sobkowicz et al., 1975; Kelley et al., 1993; Rastel et al., 1993;
Kelley et al., 1995). According to the present invention, provided
herein is a reliable, rapid, and facile method of testing the
effects of ototoxic agents and the drugs that prevent, reduce or
treat these ototoxic effects. As exemplified herein an organotypic
culturing of postnatal cochlear explants in a 3-D collagen matrix
in well defined, serum-free medium provides these advantages
without the need for a cumbersome Maximov slide assembly (Sobkowicz
et al., 1975; Rastel et al., 1993) or undefined medium. Embedding
the cochlear explants in the 3-D collagen was better for
maintaining the normal architecture than floating the explants or
placing the explants on a monolayer substrate, since the explant
tissue could be kept unfolded and cell migration out of the tissue
could be limited.
[0181] The protective effect of a trkB or trkC agonist, preferably
a neurotrophin, against ototoxicity can be readily determined in
the organotypic cultures of postnatal cochlear explants described
herein. The explants and methods of the invention can be used to
find agents that can protect SGNs and hair cells from an ototoxin.
The protective agonist can be added prior to, concomitant with, or
subsequent to addition of the ototoxin to the culture.
[0182] The following examples are offered by way of illustration
and not by way of limitation. The disclosures of all citations in
the specification are expressly incorporated herein by
reference.
EXAMPLES
Example I
NT-4/5 is a Potent and Specific Survival Factor for SGNs in
Culture
[0183] The effects of trkB or trkC agonists to enhance neuronal
survival was determined using SGNs in cell culture. Texas red
microscopy was used to show neuro filament immunohistochemistry on
who lemount of P5 cochlear tissue. When cochlear tissue containing
both the spiral ganglion and organ of Corti was immunolabeled with
the monoclonal antibody N52 against neurofilament protein (200 kd),
the somata and processes of the SGNs were intensively stained.
Neuronal innervations of hair cells in the organ of Corti from the
cell somata in the spiral ganglion were easily seen. Since both the
somata and processes of SGNs were heavily labeled and their
innervation of hair cells was evident, this antibody was used to
identify SGNs in the dissociated cell cultures.
[0184] Bright field microscopy was used to demonstrate
neurofilament protein immunocytochemistry in the SGN cultures in
serum-free medium alone or in medium containing 10 ng/ml of NT-4/5,
respectively. Identical results (not shown) were also obtained with
a different neuronal marker, a monoclonal antibody 3A10 (Furley et
al., 1990). SGNs were dissociated from postnatal day 5 (P5) rat
cochleae and plated in defined serum-free medium. After spiral
ligament and stria vascularis tissues were removed, the remaining
spiral ganglia were incubated in a mixture of 0.125% trypsin and
0.125% collagenase for 25 min at 37.degree. C. The enzyme was
inactivated with a mixture of 0.005% soybean trypsin inhibitor
(Sigma) and 0.005% DNase (Worthington) before trituration with
0.05% DNase in Eagle's Basal Medium ("BME"). The dissociated cells
were preplated on a 35 mm untreated tissue culture dish for 25 min
to enrich the neuronal population. Under these experimental
procedures, about 10% of the cell population were spiral ganglion
neurons ("SGNs") as determined by immunocytochemistry with a
monoclonal antibody (N52; Boehringer) against neurofilaments (200
kd; Boehringer). The cell suspension was finally plated on
polylysine (500 .mu.g/ml)/laminin (10 .mu.g/ml) coated 16-well
LabTek slides in 200 .mu.l of serum-free medium (BME plus
insulin-transferrin-sodium selenite media supplement (Sigma
I-1884), 1% BSA, 2 mM glutamine, and 5 mg/ml glucose) without
antibiotics as modified from Baired et al. (1992). Cells were
plated at a density of 80,000/well. In control cultures about 75%
of SGNs died after 2 days, presumably due to a lack of growth
factors. Addition of NT-4/5 to the culture greatly enhanced the
number of surviving SGNs in a dose-dependent manner (FIG. 1). A
wide range of doses (from 0.1 ng/ml to 50 ng/ml) were tested and a
maximal effect of approximately 3-fold increase was seen at a
concentration of 10 ng/ml. Neurofilament immunocytochemical
staining revealed that cultured SGNs showed bipolar or Y-shaped
branching patterns, as seen in vivo.
[0185] Effects of other neurotrophins including NGF, BDNF, and NT-3
on SGN survival in vitro were examined. Among the four
neurotrophins, BDNF was equal in potency to NT-4/5 at
concentrations from 1-50 ng/ml (P<0.001 as compared to the
control; FIG. 2). At 0.1 ng/ml, NT-4/5 was more potent than BDNF
(P<0.01). NT-3 also displayed a significant survival-promoting
effect (P<0.05 at 0.1 ng/ml; P<0.001 at higher doses),
although this effect was less potent than NT-4/5 or BDNF at all
doses (0.1-50 ng/ml) (P<0.001). In contrast, NGF showed no
detectable effects in SGN cultures at all doses examined (FIG.
2).
[0186] To test whether NT-4/5, BDNF and NT-3 have synergistic
effects, NT-4/5 was added together with BDNF or NT-3 into the
culture; no additive effects were observed at saturated
concentrations (FIG. 2).
[0187] To determine if SGNs respond to other growth factors,
epidermal growth factor (EGF), basic fibroblast growth factor
(PFGF), transforming growth factor-.beta.1 (TGF-.beta.1),
TGF-.beta.2, TGF-.beta.3, or TGF-.beta.5 was added to the cultures.
No survival-promoting effects were seen, indicating selective
responses of SGNs to NT-4/5, BDNF and NT-3.
Example II
SGNs Make TrkB Protein, the High-Affinity Binding Receptor for BDNF
and NT-4/5
[0188] To determine whether SGNs make trkB protein, the
high-affinity binding receptor for BDNF and NT-4/5, trkB
immunohistochemistry was performed on cross sections of P5 spiral
ganglion with a polyclonal antiserum against trkB extracellular
domain. Dual-immunohistochemistry was performed on cross sections
of the spiral ganglion with trkB, trkA, or P75 and neurofilament
protein antibodies. Texas red microscopy was used to show the
staining pattern of antibodies against trkB, trkA and P75,
respectively. Fluorescent microscopy was used to show the
immunostainings of neurofilament antibody (N52) in the same
sections as for Texas red microscopy. The SGN tissue was immersed
in 4% paraformaldehyde (0.1M phosphate buffer, pH 7.4) for 1 hour.
After the preparations were cryoprotected with a 30% sucrose
solution, cross sections were cut on a cryostat. The sections were
first blocked with a 10% normal goat serum in 1% Triton X-100 in
phosphate buffered saline ("PBS") for 20 minutes and then incubated
with a mixture of a monoclonal antibody (N52) against neurofilament
200 kD (Boehringer, 5 .mu.g/ml) and a rabbit antibody against
extracellular trkB (anti-trkB.sub.23- 36; 2 .mu.g/ml; Yan et al.,
1994; Gao et al., 1995), a trkA antiserum, or a P75 antiserum in
PBS containing 3% normal goat serum and 1% Triton X-100 overnight
at 4.degree. C. FITC-conjugated goat anti-mouse and Texas
red-conjugated goat anti-rabbit secondary antibodies (1:70-100;
Cappel) were then used to reveal the double labeling pattern in the
sections. For neurofilament immunohistochemistry on cochlear
wholemounts, the preparations were incubated with primary antibody
for 2 days at 4.degree. C. and then Texas red-conjugated goat
anti-mouse antiserum (1:100; Cappel) was used to reveal the
staining pattern. For neurofilament immunocytochemistry, SGN
cultures were fixed in 4% paraformaldehyde (in 0.1M phosphate
buffer, pH 7.4) for 30 minutes, washed in PBS (pH 7.4), and the
immunostainings were performed with a biotinylated sheep anti-mouse
secondary antibody and a streptavidin-horse radish peroxidase
conjugate (1:200: Amersham Life Science) as described in Gao et al.
(1995b).
[0189] N52 intensely labeled both somata and processes of SGNs in
the spiral ganglions. SGN somata and processes were heavily labeled
by trkB and P75 antibodies. In contrast, trkA antiserum (1:10,000;
Clary et al., 1994) failed to detect the presence of trkA protein
in these neurons. These results indicate that the effects of NT-4/5
and BDNF on these neurons are direct.
[0190] When the sections of spiral ganglion were double-labeled
with a monoclonal antibody (N52) against neurofilament protein and
trkB or trkA antiserum, the majority of the neurofilament-positive
SGNs were also labeled by the trkB antiserum, but not the trkA
antiserum, suggesting that most SGNs produce trkB, but not trkA
proteins. It is of interest that SGNs were also immunoreactive to
an antiserum against P75 (1:10,000; Weskamp and Reichardt, 1991),
the low-affinity receptor for all neurotrophins. It appeared that
all SGNs made the P75 protein. In addition, both trkB and P75
immunostainings extended into the cochlear epithelium; however, the
immunoreactivities were confined to the afferent nerve terminals of
SGNs, being undetectable in the hair cells.
Example III
TrkB-IgG Fusion Protein Blocks the Survival-Promoting Activity of
NT-4/5
[0191] To provide further evidence that NT-4/5 acts specifically on
the SGNs, trkB-IgG fusion protein, a specific antagonist for NT-4/5
and BDNF (Shelton et al., 1995), was added to the SGN cultures
containing NT-4/5 or BDNF. As shown in FIG. 3, addition of the
trkB-IgG fusion protein completely inhibited the survival-promoting
effects of NT-4/5 and/or BDNF. As expected, the trkB-IgG fusion
protein did not block the activity of NT-3. On the other hand,
trkC-IgG fusion protein, a specific antagonist for NT-3 (Shelton et
al., 1995), abolished the effects of NT-3, but failed to block the
effects of NT-4/5 and/or BDNF (FIG. 3). The specific blocking
effects of trkB-IgG or trkC-IgG on NT-4/5 and BDNF or NT-3,
respectively, were also seen when more than one neurotrophin was
present in the culture. In addition, trkB-IgG and/or trkC-IgG
themselves did not show. detectable effects on the survival of SGNs
in normal cultures (FIG. 3). These experiments considered together
confirmed the specificity of trkB-IgG and trkC-IgG and indicate
that the survival-promoting effects of NT-4/5 on SGNs are specific
and result from agonism of trkB or trkC.
Example IV
NT-4/5, BDNF and NT-3, but not NGF, Protect SGNs Against Cisplatin
Neurotoxicity
[0192] The ability of trkB or trkC agonists to protect neurons from
ototoxicity was determined using SGNs in cell culture. When
cisplatin was added to the SGN cultures, it induced a
dose-dependent inhibition of SGN survival (FIG. 4). At a dose of 4
.mu.g/ml or higher, virtually all SGNs died in the culture within 2
days. In contrast, cisplatin at a concentration of 4 .mu.g/ml had
no inhibitory effects on survival of postnatal neurons harvested
from central nervous system such as cerebellar granule neurons or
hippocampal neurons in vitro; the majority of cerebellar granule
neurons and hippocampal neurons survived and elaborated neurites in
the culture containing 4-6 .mu.g/ml of cisplatin, suggesting that
SGNs are selectively vulnerable to cisplatin.
[0193] To determine if NT-4/5 could protect the SGNs from cisplatin
neurotoxicity, 10 ng/ml NT-4/5 was added along with 3 different
doses of cisplatin. At concentrations of 1 .mu.g/ml or 2 .mu.g/ml
cisplatin, the percentage of surviving SGNs was higher than that in
the control cultures lacking cisplatin (FIG. 5). This suggests that
NT-4/5 promotes SGN survival and protects SGNs from cisplatin
neurotoxicity. At 4 1 .mu.g/ml of cisplatin, NT-4/5 still
significantly protected SGNs from cell death (P<0.001), although
the survival level was only 70% of control cultures in the absence
of cisplatin (FIG. 5),
[0194] Equivalent protective effects against cisplatin
neurotoxicity were observed for 10 ng/ml BDNF. NT-3 at 10 ng/ml
also displayed significant protective effects (P<0.001), but was
less potent compared to NT-4/5 or BDNF (P<0.001). Combinations
of NT-4/5 and BDNF or of NT-4/5 and NT-3 did not show additive
effects as compared to that of NT-4/5 alone. Finally, NGF at 10
ng/ml did not show any protective effects (FIG. 5).
Example V
Cochlear Explant Cultures
[0195] An organotype cochlear explant culture that utilizes a 3-D
collagen matrix cultures and maintains its normal, in vivo
architecture. Cochleae were dissected from postnatal day 3 (P3)
Wistar rats. After spiral ligament and stria vascularis tissues
were removed, the remaining cochlear tissue containing the spiral
ganglion and organ of Corti was cut into 3 pieces according to the
basal, middle and apical turns. The explants were then embedded in
a three-dimensional ("3-D") collagen matrix of a droplet (20 .mu.l)
of freshly made collagen gel which was placed on the bottom of a 35
mm Nunc tissue culture dish, modified from what described
previously (Gao et al., 1991) as follows. Rat tail collagen (type
I, Collaborative Research) was mixed with 10.times. BME medium and
2% sodium carbonate in a ratio of 10:1:1 and placed on ice just
before use. The collagen matrix containing the cochlear explants
were incubated at 37.degree. C. for 5-10 min until it gelled. The
matrix was then cultured in defined serum-free medium using
sufficient medium to cover the explants (2 ml of serum-free medium
(BME plus serum-free supplement (Sigma I-1884), 1% BSA, 2 mM
glutamine, and 5 mg/ml glucose; containing no antibiotics). The
culture medium was changed every other day thereafter.
[0196] Embedding the cochlear explants in the 3-D collagen was
better for maintaining the normal architecture than floating the
explants or placing the explants on a monolayer substrate, since
the explant tissue could be kept unfolded and cell migration out of
the tissue could be limited. Cochlear explants prepared according
to the invention maintained normal architecture in the 3-D collagen
gel cultures as observed by Nomarski micrographs of cochlear tissue
dissected from P3 rats and grown for 2 days in vitro at low and
high magnifications. Fluorescence microscopy showed the phalloidin
staining of stereocillia bundles of hair cells in the cochlear
explant cultured for 4 days. The cultured cochlear explants
maintained normal laminar structures including the somata of the
spiral ganglion, 1 row of inner hair cells, and 3 rows of outer
hair cells in the organ of Corti. The SGNs and hair cells in the
explants grew well and maintained their normal connectivity, such
as the afferent innervation of hair cells by SGNs and stereocillia
bundles of hair cells, as revealed by double histochemical labeling
with a monoclonal antibody against neurofilament protein and a
phalloidin-FITC conjugate. The SGNs and hair cells remained in
their normal locations. No supernumerary rows of hair cells or
gross cell death of SGNs and hair cells occurred under this culture
condition. With the neurofilament antibody staining, the peripheral
axons of the SGNs which innervate the hair cells in the organ of
Corti were seen to maintain consistently a radial projection
pattern, as seen in vivo.
Example VI
Ototoxicity in Cochlear Explant Cultures
[0197] Organotypic cultures of postnatal cochlear explants provided
herein, in which the afferent innervation of hair cells by primary
auditory neurons are intact, were used to examine ototoxicity of
three different classes of clinical drugs represented by
salicylate, gentamicin, and cisplatin. To determine if ototoxins
were able to induce degeneration of SGNs and hair cells in the
cochlear explant cultures, ototoxic therapeutic drugs were added to
the culture at different concentrations. Histochemical
double-labeling of the cochlear explant cultures with a
neurofilament antibody (Texas red-mediated) and phalloidin
(FITC-conjugated) was used to compare control cultures (untreated)
with cultures treated with sodium salicylate, gentamicin, or
cisplatin. While the neurofilament antibody (Texas red-mediated)
labeled the somata and peripheral axons of SGNs, the
phalloidin-FITC conjugate stained the stereocillia bundles of hair
cells. Three cultures per experimental paradigm were studied in
each individual experiment. Three or more separate repetitions of
the experiment were conducted to validate the ototoxic effect.
[0198] Sodium salicylate was found to specifically induce neuronal
degeneration, but not hair cell loss, in the cochlear explant
cultures. Inclusion of sodium salicylate, an analog of aspirin, at
a concentration of 5 mM in the culture induced massive death of
SGNs and degeneration of their peripheral axons. In contrast, all
hair cells remained intact. The sodium salicylate induced SGN
degeneration was dose dependent (FIG. 6A). As the density of the
radial peripheral axons of SGNs appeared to be a reliable index of
the number of surviving SGNs, the number of the peripheral axons of
SGNs from a given length (100 .mu.m) in the middle turn of the
cochlea was counted for different experimental groups and plotted
in FIG. 6A. Phalloidin-labeled hair cells were also counted in the
same way (FIG. 6B). In the control culture, there were
approximately 24 radial peripheral axons and about 53 hair cells
within a 100 .mu.m length of the middle turn of the cochlea. In the
presence of sodium salicylate, the number of SGN peripheral axons
decreased sharply (FIG. 6A), but the number of hair cells remained
the same (FIG. 6B). Even at a high concentration (10 mM), no
significant hair cell loss was observed (FIG. 6B).
[0199] When the aminoglycoside gentamicin was added to the cochlear
explant cultures, it preferentially destroyed hair cells at low
doses. At concentrations ranging from 0.01 to 0.5 mM, a substantial
number of hair cells in the cochlea were killed (FIG. 7B), but the
SGNs were not significantly damaged (P >0.05; FIG. 7A). However,
at higher concentrations (1-5 mM), gentamicin not only eliminated
virtually all hair cells (FIG. 7B), it started to induce SGN
degeneration as well (FIG. 7A). Debris of dead hair cells was
easily seen in the region of organ of Corti. Nevertheless, even at
a concentration of 5 mM, still approximately 40% of the SGNs
remained intact (FIG. 7A). Similar results were observed when
neomycin, another aminoglycoside, was added to the culture (data
not shown), suggesting a common toxic mechanism amongst
aminoglycosides.
[0200] It has been reported that cisplatin induces hair cell loss
in the organ of Corti (Fleischman et al., 1975; McAlpine and
Johnstone, 1990). In the dissociated cell cultures reported herein,
cisplatin induced SGN cell death, indicating a direct toxic effect
on SGNs. Cisplatin ototoxicity was further studied by adding
various concentrations of cisplatin to the cochlear explant
cultures of the invention in which innervation of hair cells by
SGNs were intact. Cisplatin damaged both SGNs and hair cells at a
wide range of concentrations (1-10 .mu.g/ml) (FIGS. 8A, 8B).
However, it showed a more profound toxic effect on SGNs than on
hair cells. At a concentration of 0.5 mg/ml, cisplatin induced a
significant degeneration of SGNs and their peripheral axons
(p<0.01, FIG. 5A). In contrast, hair cell loss was not
statistically significant (p>0.05, FIG. 8B). When the cisplatin
dose was raised to 1-2 .mu.g/ml, between 20-30% of hair cells was
destroyed while about 50% of SGNs degenerated; the damages to both
SGNs and hair cells became significant (p<0.001; FIGS. 8A, 8B).
At a concentration of 4 .mu.g/ml, cisplatin destroyed most of the
SGNs and about 55% hair cells still remained intact. At even higher
concentrations (8-10 .mu.g/ml), this drug destroyed a majority of
hair cells as well (FIG. 8B).
Example VII
Protective Effects Of Neurotrophins In Cochlear Explant
Cultures
[0201] The protective effect of neurotrophins against ototoxicity
was determined in the organotypic cultures of postnatal cochlear
explants described in the Examples above. Sodium salicylate
(Sigma), gentamicin sulfate (Sigma) or cisplatin (Bristol-Myers
Squibb) was added at various concentrations to 2-day cultures of
cochlear explants. A human recombinant neurotrophin (Genentech,
Inc.) or other growth factors, alone or in combination, was added
to the culture at the same time the ototoxin was added. Three
cultures per experimental paradigm were studied in each individual
experiment. Three or more separate repetitions of the experiment
were conducted to demonstrate the neuroprotective effect.
[0202] Two days after treatment with ototoxins or co-treatment with
ototoxins and neurotrophins (or other growth factors), the cochlear
explant cultures were fixed in 4% paraformaldehyde in 0.1 M
phosphate buffer (pH 7.4) for 30 min, washed in PBS, and subjected
to double histochemical staining of the cochlear explant cultures.
After incubation with the monoclonal antibody N52 against
neurofilament protein (200 kd) in 1% Triton X100 containing 3%
normal goat serum ("NGS") for 2 days at 4.degree. C., the explants
were incubated with a Texas red-conjugated goat anti-mouse
secondary antibody (1:70; Cappel) at room temperature for 40 min.
The preparations were then stained with FITC-conjugated phalloidin
(0.5 .mu.g/ml; Sigma) for 45 min, washed in PBS and mounted in
Fluoromount-G (Southern Biotechnology Associates, Birmingham, Ala.)
which contains an anti-fading agent.
[0203] Quantitative analysis of the numbers of peripheral axons of
SGNs and hair cells in the cochlear explants were performed. The
N52 and phalloidin double-labeled cochlear explants were examined
under a Zeiss Axiophot epifluorescent microscope. As the density of
radial peripheral axons of SGNs reflects the number of surviving
SGNs, the number of these axons was counted using a grid ocular
reticule covering a distance of 100 .mu.m in the middle turns only.
The surviving hair cells were counted in a similar manner. The
density of the radial axons in the apical turn was a little higher
than that in the basal turn and hair cells in the apical turn were
somewhat more resistant to ototoxins. For each culture, SGN
peripheral axons and hair cells were counted from 3-4 randomly
selected fields in the middle turn of each culture. Data were
collected from 6 or more cultures for each of the experimental
groups. Two-tailed, unpaired t-test was used for statistical
analysis.
[0204] The three types of drugs induced differential damage to
auditory neurons and hair cells in the cochlea as shown in the
above Examples. Sodium salicylate (3-10 mM) specifically induced
degeneration of auditory neurons, but not hair cell loss.
Gentamicin (0.01-0.5 mM) preferentially caused hair cell death,
though loss of auditory neurons was also seen at higher
concentrations (1-5 mM). In contrast, addition of cisplatin to the
culture resulted in degeneration of both auditory neurons and hair
cells at a wide range of doses tested (1-10 .mu.g/ml), with more
profound damage to auditory neurons than to hair cells. When
neurotrophins and other growth factors were added to the culture
together with the ototoxins, neuronal degeneration was prevented by
neurotrophin-4/5 ("NT-4/5"), brain-derived neurotrophic factor
("BDNF") and neurotrophin-3 ("NT-3"), but not by NGF or other
growth factors. In contrast, the hair cell loss caused by cisplatin
or gentamicin was not prevented by the presence of neurotrophins.
While other growth factors, including epidermal growth factor (EGF)
(Collaborative Research), basic fibroblast growth factor
(.beta.FGF) (Gibco), FGF-5 (R & D Systems), FGF-7 (R&D
Systems), insulin-like growth factor-1 (IGF-1) (Genentech),
platelet-derived growth factor (PDGF) (Gibco), transforming growth
factor-.alpha. (TGF-.alpha.) (R & D Systems), TGF-.beta.1
(Genentech), TGF-.beta.2, TGF-.beta.3, TGF-.beta.5 (R & D
Systems) or retinoic acid (Sigma), did not show detectable
protective effects on either SGNs or hair cells (data not shown),
specific neurotrophins--those that bind trkB or trkC--displayed
significant protection for SGNs against the three ototoxins. When
NT-4/5 was added at a concentration of 20 ng/ml together with an
ototoxin, degeneration of SGNs and their peripheral axons was
prevented as determined by histochemical double labeling (a
neurofilament antibody (Texas red-mediated) and phalloidin
(FITC-conjugated)) of cochlear explant cultures of a culture
co-treated with sodium salicylate (5 mM) and NT-4/5 (20 ng/ml), a
culture co-treated with gentamicin (3 mM) and NT-4/5 (20 ng/ml),
and a culture co-treated with cisplatin (4 .mu.g/ml) and NT-4/5 (20
ng/ml). As compared to the ototoxicity observed in the Examples
above, NT-4/5 protected SGNs, but not hair cells, from all of the
three ototoxins. However, hair cell loss caused by gentamicin or
cisplatin was not stopped by the presence of NT-4/5. Quantitation
of protective effects of NT-4/5 on SGNs and hair cells against the
three ototoxins is shown in FIGS. 9A and 9B. The presence of NT-4/5
in control cultures did not enhance the number of SGNs or hair
cells (p>0.05), though it significantly protected SGNs from the
three ototoxins (p<0.001; FIGS. 9A and 9B). The protective
effects on SGNs but not on hair cells were also observed with BDNF
and NT-3 (FIG. 10). In contrast, NGF did not show any detectable
protection (FIG. 10). The finding that the three ototoxins tested
induced differential damage to auditory neurons and hair cells in
the cochlea, suggests (a hypothesis forwarded solely for discussion
and not necessarily as the underlying fact of the present
invention) that salicylates, aminoglycosides and chemotherapeutic
agents act through different mechanisms. Nonetheless, as show
herein, all are treatable with compositions and methods of the
invention.
[0205] Neurotrophins are members of the NGF family of proteins.
They have been widely shown to regulate the differentiation and
survival of developing neurons (Korsching, 1993, Gao et al., 1995a)
as well as to aid in the repairing or recovery of adult CNS neurons
from injury and toxins (Hefti, 1986; Knusel et al., 1992; Yan et
al, 1992; Gao et al., 1995b). They exert their biological functions
through activation of high-affinity binding receptors, the trks
with high characteristic specificity (Barbacid, 1993; Snider,
1994). Previous studies indicate that the SGNs express specific trk
genes (Ylikoski et al., 1993; Schecterson and Bothwell, 1994). As
reported herein, SGNs express specific trk proteins (see also
Vazquez et al., 1994). Hair cells express certain neurotrophin
genes (Pirvola et al., 1992; Schecterson and Bothwell, 1994;
Wheeler et al., 1994). In dissociated cell culture systems, as
shown herein, specific neurotrophins promote survival of SGNs
(Vazquez et al., 1994; Lefebvre et al., 1994). As demonstrated for
the first time herein, neurotrophins protect SGNs from cisplatin
ototoxicity. Similarly, as demonstrated for the first time herein,
these neurotrophins also protect vestibular ganglion neurons from
gentamicin in vitro. The results presented with organotypic
cochlear explants are consistent with the dissociated neuronal
culture findings. As the organotypic culture keeps the afferent
innervation of hair cells by SGNs intact, it better represents the
in vivo system and, consequently, allows exploration of the
mechanism of actions of ototoxins and, most importantly, provides a
system to discover and test candidate protective agents. While
NT-4/5, BDNF and NT-3 protect SGNs against all three ototoxins,
they are particularly preferred to prevent salicylate-induced
hearing disorders.
[0206] Salicylates, such as aspirin, are the most commonly used
therapeutic drugs for their anti-inflammatory, analgesic,
anti-pyretic and anti-thrombotic effects. Unfortunately, they have
ototoxic side effects. They often lead to tinnitus ("ringing in the
ears") and temporary hearing loss (Myers and Bernstein, 1965).
Although clinical and animal studies suggest that the site of
action of this drug is in the cochlea (Myers and Bernstein, 1965;
McCabe and Dey, 1965), the mechanism for the ototoxicity of
salicylates is still unclear. Electrophysiological recordings in
animals indicate salicylates result in an increase in hearing
suprathreshold, which suggests damage to hair cells (Boettcher et
al., 1989; Boettcher and Salvi, 1991). However, postmortem
examination of a patient who received large doses of aspirin showed
normal hair cell numbers, but a significant loss of SGNs (De Moura
and Hayden, 1968). The results presented in this specification
clearly demonstrate for the first time the selective toxic effects
of salicylates on auditory neurons, but not on hair cells.
Interestingly, the dosage (3 mM) of salicylate which starts to
induce significant damage to SGNs in the present study is
comparable to the peak serum and perilymph levels that result in
hearing losses (Myers and Bernstein, 1965; Woodford et al., 1978;
Boettcher et al., 1990). The finding that sodium salicylate at both
low and high concentrations exclusively induces degeneration of
SGNs and their axons, but not hair cells suggests a possibility
that the initial hearing disorder caused by salicylates may result
from neuronal dysfunction, perhaps axonal degeneration in the
eighth nerve or abnormality in SGNs (Wittmaack, 1903). Once the
drug is stopped, the damaged axons may regenerate successfully and
restore the normal auditory function. Such initial axonal
degeneration is therefore reversible. However, if the drug is used
at high doses for a prolonged time, the neuronal impairment will
become more severe and SGN death occurs. As a consequence, the
hearing impairment may become persistent and irreversible, as
reported clinically (Jarvis, 1966).
[0207] It is interesting to note that while many previous studies
suggested that the primary action site of cisplatin is the hair
cell (Fleischman et al., 1975; McAlpine and Johnstone, 1990; Pryor,
1994), the experiments presented in this specification indicates
for the first time that cisplatin damages both hair cells and SGNs,
and that at low concentrations it shows a more profound toxic
effects on SGNs than hair cells. Neurotoxicity of cisplatin was
also noted on proprioceptive sensory neurons in vivo (Thompson et
al., 1984) and in vitro (Windebank et al., 1994; Konings et al.,
1994), resulting in peripheral sensory neuropathy (Siegal and Haim,
1990; Gao et al., 1995b). The action of cisplatin on cancer cells
is to interfere with DNA synthesis as it induces inter- and intra-
strand cross linking of DNA molecules. In a rat model of
cisplatin-induced peripheral neuropathy, cisplatin also induces
cytoplasmic patching of neurofilament protein (Gao et al., 1995b),
one of the components of the cytoskeletal complex. Although not
meant to be limiting to the invention, it is suggested that the
susceptibility of SGNs and hair cells to cisplatin may be
attributable to the fact that they are highly enriched in
cytoskeletal components, such as neurofilament and actin. In
support of this notion, cisplatin has been reported to affect the
intermediate and microfilament cytoskeletal network in cultured
squamous carcinoma cells (Kopf-Maier and Muhlhausen, 1992). The
results of the cochlear explants in the above Examples support the
notion that cisplatin directly damages ganglion neurons, as
observed in the dissociated cell cultures in the Examples
herein.
[0208] Although the present results indicate that particular
neurotrophins have strong protective effects on SGNs, they did not
protect hair cells from the ototoxic drugs. In addition, none of
the presently known growth factors including EGF, .beta.FGF, FGF-5,
FGF-7, IGF-1, PDGF, TGF-.alpha., TGF-.beta.1, TGF-.beta.2,
TGF-.beta.3, TGF-.beta.5 or retinoic acid shows protective effects
on SGNs or hair cells. These results suggest that most presently
known growth factors are not critical for protection or maintenance
of hair cells. If hair cell loss due to ototoxicity is significant,
hearing recovery could be improved by new hair cell growth or
regeneration. Recent studies have suggested possible candidates
(Forge et al., 1993; Cotanche and Lee, 1994; Tsue et al., 1994a;
Cotanche and Lee, 1994; Kelley et al., 1995). For example,
diffusible factors such as TGF-.alpha. and EGF (Lambert, 1994;
Yamashita and Oesterle, 1995) or components derived from antibiotic
treated inner ear tissue (Tsue et al., 1994b) stimulate
proliferation of supporting cells. Retinoic acid alone or in
combination with TGF-.alpha. facilitates hair cell regeneration in
vitro (Lefebvre et al., 1993, 1995). As taught herein,
neurotrophins will provide for prevention of neuronal cell death
after injury or insult by ototoxins. Furthermore, although cochlear
implants have been performed clinically with patients benefiting
from the cochlear implants, a gradual SGN loss still occurs (Leake
et al., 1992). Neurotrophins will prove to be important in keeping
ganglion neurons alive in cochlear implants. A combination of
specific neurotrophins and hair cell growth factors or cochlear
implants will prove preferably competent for recovery or repairing
of hearing loss caused by ototoxins or injury.
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