Bisphosphonate-Linked TrkB Agonist: Cochlea-Targeted Delivery of a Neurotrophic Agent as a Strategy for the Treatment of Hearing Loss

Article abstract of DOI:10.1021/acs.bioconjchem.8b00022

Hearing loss affects more than two-thirds of the elderly population, and more than 17% of all adults in the U.S. Sensorineural hearing loss related to noise exposure or aging is associated with loss of inner ear sensory hair cells (HCs), cochlear spiral ganglion neurons (SGNs), and ribbon synapses between HCs and SGNs, stimulating intense interest in therapies to regenerate synaptic function. 7,8-Dihydroxyflavone (DHF) is a selective and potent agonist of tropomyosin receptor kinase B (TrkB) and protects the neuron from apoptosis. Despite evidence that TrkB agonists can promote survival of SGNs, local delivery of drugs such as DHF to the inner ear remains a challenge. We previously demonstrated in an animal model that a fluorescently labeled bisphosphonate, 6-FAM-Zol, administered to the round window membrane penetrated the membrane and diffused throughout the cochlea. Given their affinity for bone mineral, including cochlear bone, bisphosphonates offer an intriguing modality for targeted delivery of neurotrophic agents to the SGNs to promote survival, neurite outgrowth, and, potentially, regeneration of synapses between HCs and SGNs. The design and synthesis of a bisphosphonate conjugate of DHF (Ris-DHF) is presented, with a preliminary evaluation of its neurotrophic activity. Ris-DHF increases neurite outgrowth in vitro, maintains this ability after binding to hydroxyapatite, and regenerates synapses in kainic acid-damaged cochlear organ of Corti explants dissected in vitro with attached SGNs. The results suggest that bisphosphonate-TrkB agonist conjugates have promise as a novel approach to targeted delivery of drugs to treat sensorineural hearing loss.

Full text of DOI:10.1021/acs.bioconjchem.8b00022

Article
pubs.acs.org/bc
Cite This: Bioconjugate Chem. XXXX, XXX, XXX−XXX
Bisphosphonate-Linked TrkB Agonist: Cochlea-Targeted Delivery of
a Neurotrophic Agent as a Strategy for the Treatment of Hearing
Loss
Judith S. Kempfle,†,‡,§ Kim Nguyen,∥ Christine Hamadani,†,‡ Nicholas Koen,†,‡ Albert S. Edge,†,‡
Boris A. Kashemirov,∥ David H. Jung,*,†,‡ and Charles E. McKenna*,∥
†Department of Otolaryngology and The Eaton-Peabody Laboratories, Massachusetts Eye and Ear Infirmary, Boston, Massachusetts
02114, United States
‡Department of Otology and Laryngology, Harvard Medical School, Boston, Massachusetts 02114, United States
§Department of Otolaryngology, University of Tubingen Medical Center, Tubingen 72076, Germany
̈
̈
∥Department of Chemistry, University of Southern California, Los Angeles, California 90089-0744, United States
S
* Supporting Information
ABSTRACT: Hearing loss affects more than two-thirds of the
elderly population, and more than 17% of all adults in the U.S.
Sensorineural hearing loss related to noise exposure or aging is
associated with loss of inner ear sensory hair cells (HCs),
cochlear spiral ganglion neurons (SGNs), and ribbon synapses
between HCs and SGNs, stimulating intense interest in
therapies to regenerate synaptic function. 7,8-Dihydroxyfla-
vone (DHF) is a selective and potent agonist of tropomyosin
receptor kinase B (TrkB) and protects the neuron from
apoptosis. Despite evidence that TrkB agonists can promote
survival of SGNs, local delivery of drugs such as DHF to the
inner ear remains a challenge. We previously demonstrated in an animal model that a fluorescently labeled bisphosphonate, 6-
FAM-Zol, administered to the round window membrane penetrated the membrane and diffused throughout the cochlea. Given
their affinity for bone mineral, including cochlear bone, bisphosphonates offer an intriguing modality for targeted delivery of
neurotrophic agents to the SGNs to promote survival, neurite outgrowth, and, potentially, regeneration of synapses between HCs
and SGNs. The design and synthesis of a bisphosphonate conjugate of DHF (Ris-DHF) is presented, with a preliminary
evaluation of its neurotrophic activity. Ris-DHF increases neurite outgrowth in vitro, maintains this ability after binding to
hydroxyapatite, and regenerates synapses in kainic acid-damaged cochlear organ of Corti explants dissected in vitro with attached
SGNs. The results suggest that bisphosphonate−TrkB agonist conjugates have promise as a novel approach to targeted delivery
of drugs to treat sensorineural hearing loss.
slowly degenerate.8 Notably, the cell bodies of SGNs persist
INTRODUCTION
■
long after hair cells and synapses have disappeared, for months
Age-related sensorineural hearing loss (SNHL) affects nearly
in mice5 and possibly decades in humans.9 Current treatment
two-thirds of adults 70 and older.1 SNHL is associated with
for SNHL relies solely on amplification of sound (hearing aids)
irreversible degeneration of the sensory cells of the auditory
or, in severe cases of SNHL, on electrical stimulation of
portion of the inner ear (cochlea), including hair cells (HCs)
remaining neurons (cochlear implants).10 However, such
modalities do not fully restore a biologic hearing experience.
The persistence of SGN cell bodies raises the possibility that
exogenous neurotrophins might support SGN survival, induce
neurite outgrowth, and even regenerate synapses with surviving
hair cells to improve hearing. Neurotrophins play a critical role
in SGN development and maintenance8,11 and have been
shown to promote SGN survival and enhance neurite
and spiral ganglion neurons (SGNs).2 Emerging evidence has
highlighted an additional, previously underappreciated etiology
of hearing loss−that of primary synaptopathy, or loss of
synapses between HCs and SGNs. Thus, noise exposure or
aging can lead to loss of ribbon synapses between SGNs and
HCs in murine models and humans.3−6 Primary synaptopathy
has been termed “hidden hearing loss” as it may underlie subtle
discriminatory functions of the ear, such as hearing in a noisy
environment, rather than grossly changing the thresholds at
which sound is detected.7 After synaptic degradation, or after
damage of the sensory epithelium of the cochlea with loss of
neurotrophic support, SGNs retract peripheral neurites and
Received: January 8, 2018
Revised: February 6, 2018
© XXXX American Chemical Society
A
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outgrowth and synaptogenesis.12−15 Strikingly, overexpression
of a neurotrophin within the cochlea via genetic manipulation16
and local application of neurotrophin protein to the cochlea17
have both been shown to regenerate cochlear synapses
following noise damage.
Current drug delivery methods to the inner ear include
diffusion across the round window membrane (RWM), direct
infusion via cochleostomy, or systemic delivery.18 With respect
to neurotrophin delivery, prior model systems have utilized
local methods to locally deliver viral vectors carrying a
neurotrophin gene or whole neurotrophin protein, such as
brain derived neurotrophic protein (BDNF) or neurotrophin-3
(NT-3), after damaging or destroying hair cells or synap-
ses.17,19−22 However, cochlear viral delivery in the setting of
hearing preservation has proven to be challenging, and delivery
of whole proteins across the intact human RWM, as opposed to
the rodent RWM, may similarly prove to be difficult.22,23 Small
molecule drugs, such as 7,8-dihydroxyflavone (DHF, 1), a
tropomyosin receptor kinase B (TrkB) agonist,24 provide a
potential alternative solution for regenerative treatment of
neurons. DHF effectively promotes SGN survival, supports
sprouting in vitro, and protects SGNs from in vivo degeneration
in the setting of connexin-26 mutation and hair cell loss.25,26
However, drug application of native DHF to the RWM may
have a debilitating limitation in that the delivered compound
may remain present for an insufficient amount of time before
being cleared by inner ear fluids.27,28
Figure 1. Molecular docking of 1 into the TrkB agonist site (PDB
code: 4AT3) using AutoDock Vina. A. Overview of 1 docked into the
binding site of TrkB. B. Zoomed view of the predicted preferred
binding pose of 1. C. Zoomed view comparing the predicted pose of 1
with the experimentally determined configuration of Cpd5n in the
TrkB active site.
We have therefore sought to identify a novel method to
anchor neurotrophic activity in the cochlea. Bisphosphonates
(BPs), such as risedronate (Ris) or zoledronate (Zol), whose
chemical structures mimic pyrophosphate, are characterized by
strong affinity to bone mineral and are widely used clinically to
treat resorptive bone disorders such as osteoporosis.29 Prior
work has demonstrated that BPs can conjugate to fluorescent
dyes to image bone30,31 and to antibiotics for bone-targeted
drug delivery.32 We previously showed that 6-FAM-Zol, a
fluorescein−BP conjugate,33,35 readily enters the mammalian
cochlea through the RWM and labels the modiolus and osseus
spiral lamina, the location of SGNs.34,36
Figure 2. Structures of COOH-DHF, 2, linkers 3 and 4, and RIS-
DHF, 5.
In this study, we report the design and synthesis of a novel
BP conjugate with DHF (Ris-DHF, 5) and evaluate its
potential for bone-targeted neurotrophic stimulation of
damaged SGNs and for reinnervation of cochlear hair cells.
considered 1,6-diaminohexane (3) as the linker molecule and
carried out preliminary synthetic studies using this approach
(details, Supporting Information) but ultimately turned to a
more polar and water-soluble PEG linker, 15-amino-4,7,10,13-
tetraoxopentadecanoic acid (4). Based on our previous studies
showing that fluorescently labeled Ris and Zol bind strongly to
cochlear bone,39 we chose Ris as the BP, adapted for linking by
attachment of a 1-amino-2-hydroxypropane-3-diyl group at the
pyridyl nitrogen, which led to the target compound, 5 (Ris-
DHF, Figure 2).
Synthesis. The synthesis of 1 has been previously reported
by Sun et al.40 A similar route was followed to prepare 2,
beginning from tribenzylation of 2,3,4-trihydroxybenzophenone
(6) followed by aldol condensation of the product 7 with 4-
carboxybenzaldehyde (replacing benzaldehyde in the literature
synthesis of 1) in 20% aq. KOH/EtOH at 50 °C, giving the
adduct 8 which was then reacted with TFA to remove the 2-
benzyl group, forming 9 (Scheme 1). This sequence of steps, in
reversing the recommended order in the 1 synthesis,40
improved the overall yield of 9 from <20% to ∼50%.
Cyclization promoted by heating with a catalytic amount (0.1
equiv) of I2 in DMSO then gave the dibenzyl-protected p-
carboxyflavone 10, which was converted to 2 by treatment with
1 atm H2 over Pd/C:Pd(OH)2/C in 1:1 THF:EtOH.
RESULTS AND DISCUSSION
■
Chemistry. Design of a Bisphosphonate-Modified TrkB
Agonist. As the TrkB agonist, we selected 1 (Figure 1) due to
its well established specificity and potency.24 To determine a
suitable site for linker attachment (i.e., compatible with
retention of agonist activity), 1 was modeled into the crystal
structure of TrkB (PDB code 4AT3)37 after deletion of the
cocrystallized Cpd5n (a TrkB inhibitor), using AutoDock
Vina.38 This study revealed several potential binding
interactions of 1 in the agonist binding site, including 7-OH
hydrogen bonding with GLU604 and π-stacking of the phenyl
ring with PHE565 (Figure 1A and 1B) (cf. Cpd5n bound
within the site, Figure 1C). In this preferred pose, the phenyl
ring of 1 (circled in red) is facing outward from the binding
pocket, suggesting permissible modification at the 3- or 4-
carbon.
Subsequent docking results (data not shown) with the 4-
phenyl carboxyl 7,8-DHF derivative 2 (COOH-DHF, Figure 2)
encouraged us to select it as the linkable form of 1. We initially
B
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Scheme 1. Synthesis of COOH-DHF, 2
Scheme 2. Synthesis of RIS-DHF, 5
Conversion of 10 to its activated N-succinyl ester 11 allowed
facile amidation with the PEG linker 4 in anhydrous DMF (to
12), and N-succinyl esterification of the distal free linker
carboxyl acid group in this intermediate (12 to 13) followed by
removal of the two remaining benzyl groups, again by catalytic
hydrogenation, provided 14 (Scheme 2). This compound was
reacted with the linkable Ris derivative 1530 in H2O/THF (pH
adjusted to 8.3 with Na2CO3) to give the final target compound
5 (13%).
average neurite outgrowth of 0.248 mm ( SEM 0.0483) for
control samples. Average outgrowth for treated samples was
0.294 mm ( 0.0308) for Ris, 0.445 mm ( SEM 0.0506) for
DHF, and 0.421 mm ( 0.0490) for Ris-DHF. The relative ratio
of treated samples to control demonstrated a significant
increase of fiber length after treatment with DHF and Ris-
DHF compared to Ris alone or control (Figure 3A-B). DHF
promoted a slightly longer average neurite outgrowth than Ris-
DHF, but this difference was not statistically significant (p =
0.32). Neurite outgrowth with Ris alone did not significantly
differ from control. To gain insight into the mechanism of
action of Ris-DHF, we investigated whether Ris-DHF acts
through TrkB, the receptor for BDNF and DHF. TrkB is
phosphorylated upon binding to BDNF41,42 or DHF,43 and we
identified phosphorylated TrkB along elongating neurites of
SGNs treated with Ris-DHF (Figure 3C). Treatment with
ANA12, a noncompetitive inhibitor of TrkB,44 appeared to
abrogate SGN neurite outgrowth in the presence of DHF or
Ris-DHF (Figure 3D). Taken together, these results suggest
that the ability of DHF to promote spiral ganglion neurite
outgrowth via TrkB was preserved following conjugation to Ris
in the hybrid Ris-DHF molecule.
Compound 5 was purified by gradient RP-HPLC (0.1 M
triethylammonium bicarbonate buffer (pH 7.0) containing 10%
and then 75% of acetonitrile). A pKa calculation (Marvin
Sketch 15.12.14.0) indicated that the 7-OH group (red, Scheme
1
2) is deprotonated at pH 7 (pKa calculated, 6.6), thus the H
NMR sample was coevaporated with 70% MeOH, 0.1 M
ammonium acetate buffer (pH 5.0) and then dissolved in D2O,
which provided good peak resolution. 5 was further
characterized by MS and 31P NMR spectroscopy.
Biology. Ris-DHF Promotes Spiral Ganglion Neurite
Outgrowth in Vitro. To study the effect of Ris-DHF on spiral
ganglion neurite outgrowth, postnatal (P4) SGNs were plated
in culture and subjected to treatment with 400 nM of Ris, DHF,
Ris-DHF, or control with DMSO for 48 h (Figure 3).
Immunohistochemical and quantitative analysis of neuron-
specific class III Tubulin (TuJ1) positive neurites revealed an
Ris-DHF Promotes Neurite Outgrowth after Binding to
Hydroxyapatite. To evaluate the functional ability of Ris-DHF
to promote neurite outgrowth when bound to hydroxyapatite
C
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Figure 3. Spiral ganglion neurite outgrowth in vitro. A. Immunohistochemical analysis of cochlear SGNs after 48 h in culture, treated with 400 nM
DHF, 400 nM Ris, 400 nM Ris-DHF, or DMSO as a control. Neurons were stained with neural marker TuJ1 (red); nuclei are labeled with DAPI
(blue). Scale bar represents 100 μm. Neurite outgrowth length was measured with 3D Neurite Tracer, and total traces were processed and rendered
into a tagged 3D skeleton. B. Relative ratio of neurite lengths across experiments (n = 7) was calculated compared to control. Results are expressed
as mean SEM (* represents p < 0.05). C. Phosphorylated TrkB (pTrkB) puncta visualized along elongated neurites. D. 10 nM ANA12, an
inhibitor of TrkB, decreases DHF- and Ris-DHF-mediated neurite outgrowth. Results are expressed as relative outgrowth ratios to Control with
ANA12 (n = 4). Results are expressed as mean SEM (* represents p < 0.05). E. Structure of ANA12.
(HA, the primary component of bone matrix), we preincubated
HA nanoparticles for 1 h with DHF, Ris, Ris-DHF, or DMSO.
For easier visualization of particles, we added a small amount of
Fam-Ris, a fluorescent derivative of risedronate,30 to all
samples. Washed particles were then plated with SGNs for 48
h in regular growth medium to evaluate neurite outgrowth
driven by HA-bound drug. Analysis and quantification revealed
that HA-bound Ris-DHF promoted significantly more out-
growth as compared to HA-bound Ris or control. Ris promoted
some neurite outgrowth compared to untreated control,
suggesting a minor positive effect of Ris on neurites. As
expected, DHF preincubation with HA did not produce a
significant increase in neurite outgrowth (Figure 4A and B).
Qualitatively, we observed neurite outgrowth paths in response
to HA-bound Ris-DHF to be more convoluted than in response
to Ris-DHF in solution (compare Figure 3A and 4A). Higher
power magnification revealed that neurites both terminated
upon HA-Ris-DHF pellets and traveled past the pellets (Figure
4C). Taken together, these data demonstrate that neurite
outgrowth driven by Ris-DHF is maintained after binding to
HA.
Ris-DHF Promotes Regeneration of Cochlear Ribbon
Synapses in Vitro. To assess the ability of Ris-DHF to
stimulate synaptic regeneration, we adapted a well-established
in vitro model for cochlear synaptopathy.45 Organ of Corti
(OC) explants with attached neurons were dissected and plated
and then treated with kainic acid (KA) to induce excitotoxic
damage to ribbon synapses.45 After 2 h of treatment, synaptic
damage and neurite retraction were confirmed (data not
shown), and explants were treated with soluble DHF, Ris, Ris-
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Figure 4. Prebinding to hydroxyapatite bone matrix does not inhibit 400 nM Ris-DHF-promoted neurite outgrowth in vitro. A. ∼500 μg HA
nanoparticles prebound to 400 nM of Ris, DHF, Ris-DHF, or DMSO control were plated with SGNs for 48 h. Total traces were processed and
rendered into a tagged 3D skeleton (white). B. Neurite outgrowth length was measured via 3D Neurite Tracer software, and each neurite was
individually traced through its z-stack. Relative ratio of neurite lengths across total experiments (n = 8) was calculated. Results are expressed as mean
SEM (* represents p < 0.05; ** represents p < 0.01). C. Neurites were observed to both grow past (single blue arrowhead) and terminate upon
(double blue arrowheads) HA-Ris-DHF nanoparticles.
DHF, or DMSO as control for 24 h. After fixation, regenerated
synapses were visualized using immunohistochemistry and
confocal microscopy (Figure 5). The juxtaposition of pre- and
postsynaptic portions of the ribbon synapse was considered to
represent a fully regenerated synapse. The number of synapses
was then quantified per inner hair cell (IHC) (Figure 5C). Each
experiment was performed with an untreated control explant
(no kainic acid, KA−), to evaluate synapse survival in culture at
baseline. KA− explants maintained 12.5 synapses/IHC
( 0.5671) on average. Spontaneous synaptic regeneration
was assessed with OC explants treated with KA alone (KA+).
KA+ explants demonstrated very little spontaneous synaptic
outgrowth and the regeneration of ribbon synapses within the
OC in vitro. Furthermore, prebinding of Ris-DHF to HA, which
simulates bone binding, preserves the ability of Ris-DHF to
direct neurite outgrowth. These results highlight the promise of
BPs as drug delivery molecules for the inner ear and outline a
novel small molecule approach to the regeneration of cochlear
synapses between HCs and SGNs.
The delivery of drugs to the inner ear presents unique
opportunities and challenges.46 Systemic administration may be
relatively straightforward but has the potential for greater
systemic side effects and decreased concentration within the
inner ear following delivery. Local delivery to the cochlea across
the RWM via intratympanic injection is common in outpatient
clinical use in humans for the treatment of sudden hearing loss
and Meniere’s disease, although it remains unclear if the drugs
administered in such treatments are uniquely able to cross the
oval and round windows into the inner ear. Local delivery via
cochleostomy has the potential for the highest delivery levels.
However, opening the cochlea presents inherent risks to
hearing that may be unacceptably high for patients with useful
hearing, such as patients with synaptopathy. Our previous work
demonstrated that BPs can enter the mammalian cochlea
following delivery to the RWM and that such delivery could be
achieved in a nonototoxic manner.35 As the cochlea is encased
in bone, bisphosphonates may represent a novel way to achieve
regeneration (1.57
treatment with Ris-DHF significantly increased the number of
regenerated synapses compared to KA+ (8.20 0.5828
0.4989 synapses/IHC). Additional
synapses/IHC). Unconjugated DHF regenerated at a com-
parable level (DHF: 9.20 0.7865 synapses/IHC; p value =
0.12 relative to Ris-DHF). Ris alone had a modest regenerative
effect on synapses, albeit significantly lower (5.34
0.7629
synapses/IHC) when compared to DHF or Ris-DHF. Taken
together, these results suggest that the hybrid molecule Ris-
DHF promotes synaptic regeneration after damage in vitro.
We have shown that a rationally designed bisphosphonate-
DHF molecule retains neurotrophic properties of native DHF,
both with respect to the promotion of spiral ganglion neurite
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Figure 5. Cochlear synapse regeneration after Ris-DHF treatment in an in vitro synaptopathy model. A. Organ of Corti (OC) explants treated with
0.5 mM KA for 2 h followed by 24 h incubation with Ris 400 nM, DHF 400 nM, or Ris-DHF 400 nM. Untreated explants (KA-) or KA only treated
explants (KA+) served as controls. Presynaptic synapse was stained with CtBP2 (red), which also labeled inner hair cell (IHC) cell bodies.
Postsynaptic synapse (neural synapse portion) was visualized with PSD95 (green). Hair cell bodies were labeled with myosin VIIa (Myo7a, white).
B. Regrowth of neurites following 400 nM Ris-DHF treatment at 12 h and 24 h. Neurites are visualized in white with antineurofilament antibody
(NF). C. Computed postprocessing within 3D cubical space allowed for quantification of juxtaposed pre- and postsynaptic labels. D. Quantified
synapses per IHC across 5 experiments compared to controls (n = 5) at 24 h. Results are expressed as mean SEM (n.s., not significant; **
represents p < 0.01).
long-term cochlear drug delivery using well established clinical
techniques for minimally invasive local delivery.
molecule approach we describe may therefore hold several
critical advantages with respect to clinical application. First,
small molecules may be more likely than proteins to cross the
RWM, particularly in the setting of the more complex RWM in
humans relative to rodents.23 Second, we have chosen to
leverage the bony anatomy of the cochlea through use of a
tailored BP as an anchor for long-term stimulation of SGNs. In
this regard, our previous work examining BP delivery to the
cochlea has demonstrated that BPs have a high affinity for the
osseous spiral lamina of the cochlea, which lies in close
proximity to SGNs.35 The prolonged binding of BP conjugates
to bone31 suggests that this approach may provide a way to
overcome some of the described barriers to inner ear drug
delivery, including distribution of drug among the cochlear
fluids and elimination of drug from the cochlea.28
With respect to the inner ear, BPs appear to promote SGN
survival in the setting of osteoprotegerin deficiency,47
suggesting that they may have positive effects in the context
of SGN survival and synaptogenesis beyond simply holding
molecules of interest in place. In this regard, our results show
that Ris modestly promotes neurite outgrowth following
prebinding to HA. Further, Ris mediates regeneration of
cochlear synapses following KA treatment, although at a lower
level than either DHF or Ris-DHF. In the context of our
ANA12 inhibitor data, Ris-mediated neurite outgrowth appears
to act independently of TrkB, suggesting that Ris may perform
this function via a parallel pathway for SGN survival or
outgrowth.
Because of the critical roles of neurotrophins in development
and maintenance of cochlear wiring,8,11 there has been intense
interest in the therapeutic use of neurotrophins to promote
SGN survival and synaptic regeneration. Genetically modified
mice that overexpressed a neurotrophin within the cochlea
established the theoretical viability of such an approach, as
these animals demonstrated an ability to regenerate cochlear
synapses after noise trauma.16 In this regard, the therapeutic
approaches explored thus far have involved intracochlear
delivery of viral particles expressing neurotrophin genes,19
local topical delivery of neurotrophin proteins to the RWM,17,20
and local intracochlear delivery of neurotrophin proteins in the
context of a cochlear implant.21 Local delivery of DHF has also
been shown to promote SGN survival after massive hair cell
loss.25 Of these approaches, however, only topical delivery of
neurotrophin proteins to the RWM has been accomplished in
animals in the context of hearing preservation. The small
EXPERIMENTAL PROCEDURES
■
General Information. All reagents, including 4-carbox-
ybenzaldehyde and mono-tBoc-protected 1,6-diaminohexane
(18, Supporting Information), were purchased from Sigma-
Aldrich or Alfa Aesar, except as noted. Risedronate
monosodium was a kind gift from Warner Chilcott (formerly
P&G Pharmaceuticals). Triethylamine (TEA) was distilled
from KOH, and dioxane and THF were distilled from sodium.
15-Amino-4,7,10,13-tetraoxopentadecanoic acid 4 was pur-
chased as the N-Boc derivative from Chem-Impex Interna-
tional, Inc. Other reagents were used as supplied by the
manufacturer. Flash chromatography purification was per-
formed using a Teledyne CombiFlash Rf+ Lumen system.
Thin layer chromatography (TLC) was performed on Merck
Silica Gel 60 F254 plates, and the developed plates were
visualized under a UV lamp at 354 nm. HPLC separations were
F
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performed on a Rainan Dynamax Model SD-200 system with a
Rainan Dynamax absorbance detector Model UV-DII. NMR
spectra were recorded on either Varian Mercury 400, Varian
VNMRS-500, or Varian VNMRS-600 spectrometer. Chemical
shifts are expressed as ppm downfield from a solvent residual
(1H, 13C) peak or an external standard (H3PO4, 31P). LC-MS
analyses were performed on a Thermo-Finnigan LCQ DECA
XP MAX Ion Trap LC/MS/MS spectrometer operated in ESI
mode.
13C NMR (101 MHz, DMSO-d6) δ 192.81, 167.27, 158.41,
158.11, 143.29, 139.00, 137.98, 135.43, 130.15, 129.57, 128.91,
128.62, 128.51, 128.27, 128.17, 128.04, 123.84, 115.66, 105.40,
74.38, 70.63.
4-(7,8-Bis(benzyloxy)-4-oxo-4H-chromen-2-yl)benzoic
Acid (10). To a solution of 9 (242 mg, 0.5 mmol) in 15 mL of
DMSO was added iodine (12.7 mg, 0.1 equiv), and the mixture
was heated to 120 °C. After stirring for 12 h, the reaction
mixture was cooled to rt and quenched with ice cold water. A
slightly yellow precipitate appeared, that was filtered. Extraction
with EtOAc was performed, and the organic layer was washed
with saturated Na2S2O3 and then brine. The solvent was
removed by evaporation, and 10 was collected as a slight yellow
solid residue and combined with the yellow precipitate, total
yield 133.4 mg (55%).
Synthesis of the DHF Conjugate, 5. 2,3,4-Trisbenzylox-
yacetophenone (7). In a dry 100 mL round-bottom flask, 1.7 g
(10 mmol) of 6 was added to a stirred suspension of 6.0 g (35.0
mmol, 3.5 equiv) of benzyl bromide, 8.0 g (58.0 mmol) of
potassium carbonate, and 0.25 g (1.5 mmol) of potassium
iodide in 20 mL of anhydrous DMF. The reaction mixture was
heated to 50 °C and stirred for 8 h. After cooling to rt, solid
potassium carbonate was filtered, and the filtrate was diluted
with 100 mL of ethyl acetate. The resulting solution was
washed with water and brine and then dried over Na2SO4. The
crude product was purified by column chromatography on silica
gel using a gradient of 0−50% ethyl acetate in hexane: white
solid after evaporation of solvent, 3.53 g (80%).
1H NMR (400 MHz, DMSO-d6) δ 8.03 (s, 4H), 7.77 (d, J =
9.0 Hz, 1H), 7.54−7.50 (m, 2H), 7.47−7.29 (m, 9H), 7.01 (s,
1H), 5.35 (s, 2H), 5.19 (s, 2H).
13C NMR (101 MHz, DMSO-d6) δ 177.02, 162.30, 156.17,
150.61, 137.47, 136.77, 136.04, 129.99, 126.18, 118.53, 79.86,
79.53, 79.20, 75.69, 71.03.
4-(7,8-Dihydroxy-4-oxo-4H-chromen-2-yl)benzoic Acid
(2). 69 mg of 10 and a catalytic amount of 10% Pd/C (0.1
equiv) were stirred in 15 mL of a 1:1 v/v mixture of
MeOH:THF. The reaction mixture was heated to 40 °C,
flushed with N2 gas (3×), and stirred overnight under H2 at
atmospheric pressure. Solids were removed by filtration, and
the filtrate was evaporated at reduced pressure. The solid
residue was recrystallized from methanol to give 10.6 mg of 2
(25%).
1H NMR (400 MHz, chloroform-d) δ 7.50 (d, J = 8.8 Hz,
1H), 7.47−7.19 (m, 15H), 6.81 (d, J = 8.9 Hz, 1H), 5.16 (s,
2H), 5.15 (s, 2H), 5.06 (s, 2H), 2.52 (s, 3H).
13C NMR (101 MHz, chloroform-d) δ 198.29, 156.70,
137.12, 136.17, 128.72, 128.64, 128.53, 128.51, 128.32, 128.23,
128.14, 127.52, 127.04, 125.67, 108.83, 76.37, 75.60, 70.91,
31.10.
(E)-4-(3-Oxo-3-(2,3,4-tris(benzyloxy)phenyl)prop-1-en-1-
yl)benzoic Acid (8). Compound 7, 476 mg (1.09 mmol), was
dissolved in 50 mL of MeOH, and 15 mL of 20% KOH was
added. The suspension was heated to 70 °C and stirred until
the compound was completely dissolved. 207 mg of 4-
carboxybenzaldehyde (1.38 mmol, 1.3 equiv) was added.
After stirring for 8 h, the mixture was cooled to rt and filtered.
The pH of the filtrate was adjusted to pH 4 with acetic acid,
and the filtrate was extracted with ethyl acetate. The organic
layer was washed with water and brine, dried over Na2SO4, and
evaporated, and the residue was purified by column
chromatography (0−50% methanol in dichloromethane, 0.5%
trifluoroacetic acid), giving 395 mg (64%) of a white solid.
1H NMR (600 MHz, chloroform-d) δ 9.56 (bs), 8.03 (d, J =
8.5 Hz, 2H), 7.63 (s, 2H), 7.58 (d, J = 8.8 Hz, 1H), 7.49−7.27
(m, 14H), 7.24−7.13 (m, 3H), 6.88 (d, J = 8.9 Hz, 1H), 5.19
(s, 2H), 5.12 (s, 2H), 5.09 (s, 2H).
1H NMR (400 MHz, DMSO-d6) δ 13.25 (s, 1H), 10.34 (s,
1H), 9.51 (s, 1H), 8.26 (d, J = 8.7 Hz, 2H), 8.08 (d, J = 8.7 Hz,
2H), 7.40 (d, J = 8.7 Hz, 1H), 7.09−6.86 (m, 2H).
LC-MS: tR = 1.85 min, calcd for C16H9O6− [M − H]− 297.04
m/z, found 297.11 m/z.
2,5-Dioxopyrrolidin-1-yl 4-(7,8-Bis(benzyloxy)-4-oxo-4H-
chromen-2-yl)benzoate (11). In a dry 50 mL flask 10 (198.9
mg, 0.42 mmol), 101.6 mg of EDC·HCl (0.53 mmol, 1.25
equiv), and 64.6 mg of N-hydroxysuccinimide (0.56 mmol, 1.33
equiv) in 30 mL of anhydrous THF were heated to 40 °C and
stirred overnight. The solvent was removed, and the residue
was washed with isopropyl alcohol and ether and dried under
vacuum, giving 179.6 mg of the product (75%).
1H NMR (400 MHz, DMSO-d6) δ 8.25−8.13 (m, 4H), 7.78
(d, J = 9.0 Hz, 1H), 7.58−7.25 (m, 11H), 7.11 (s, 1H), 5.36 (s,
2H), 5.20 (s, 2H), 2.91 (s, 4H).
1-Amino-3,6,9,12-tetraoxapentadecan-15-oic Acid (4). In
a 25 mL flask, 40.3 mg (0.11 mmol) of the N-tBoc amide of 4
in 3 mL of DCM was treated with 3 mL of trifluoroacetic acid.
After stirring for 2 h, the solvent was removed, and completion
13C NMR (101 MHz, chloroform-d) δ 189.90, 171.24,
141.73, 140.97, 136.53, 136.13, 130.55, 130.35, 129.25, 128.73,
128.68, 128.38, 128.30, 128.19, 127.55, 126.98, 126.56, 109.32,
77.00, 70.97.
1
of N-Boc deprotection was verified by H NMR.
1H NMR (400 MHz, acetone-d6) δ 12.27 (s, 1H), 7.49 (s,
1H), 3.92−3.81 (m, 2H), 3.80−3.56 (m, 14H), 3.38 (h, J = 5.7
Hz, 2H), 2.58 (t, J = 30.4, 6.1 Hz, 2H).
(E)-4-(3-(3,4-Bis(benzyloxy)-2-hydroxyphenyl)-3-oxoprop-
1-en-1-yl)benzoic Acid (9). In a 100 mL round-bottom flask,
395 mg (0.69 mmol) of 8 was dissolved in 20 mL of toluene,
followed by the addition of trifluoroacetic acid (TFA) (5:1
toluene:TFA v/v). After stirring for 1.5 h, the solvent was
evaporated, and the product was purified by flash column
chromatography (0−50% methanol in dichloromethane, 0.5%
trifluoroacetic acid) giving 242 mg of a white solid (73%).
1H NMR (500 MHz, DMSO-d6) δ 13.18 (s, 1H), 8.22−8.08
(m, 2H), 8.06−7.95 (m, 4H), 7.87 (d, J = 15.4 Hz, 1H), 7.51−
7.21 (m, 11H), 6.85 (d, J = 9.2 Hz, 1H), 5.30 (s, 2H), 5.00 (s,
2H).
1-(4-(7,8-Bis(benzyloxy)-4-oxo-4H-chromen-2-yl)phenyl)-
1-oxo-5,8,11,14-tetraoxa-2-azaheptadecan-17-oic Acid (12).
In a 25 mL flask, the pH of a solution of 29.1 mg of 4 (0.11
mmol) in 1 mL of anhydrous DMF was made basic by a few
drops of freshly distilled TEA and treated dropwise with a
solution of 61.6 mg (0.107 mmol, 1 equiv) of 11 in 2.5 mL of
anhydrous DMF. The resulting mixture was stirred for 6 h at rt.
The solvent was removed by evaporation under vacuum, and
the residue was dissolved in 10 mL of chloroform. The organic
G
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Article
layer was washed with 30 mL 0.1 M HCl (3×), followed by
brine, and then dried over sodium sulfate. Removal of the
solvent by evaporation left the product as a solid, 17.2 mg
(21.5%), which was used in the next reaction without further
purification.
1H), 6.90 (d, J = 8.5 Hz, 1H), 6.61 (s, 1H), 4.41 (dd, J = 13.2,
9.7 Hz, 1H), 4.21 (s, 1H), 3.85−3.02 (m, 26H), 2.91 (d, J =
22.6 Hz, 4H), 2.73 (s, 3H), 2.46 (t, J = 5.9 Hz, 2H).
31P NMR (162 MHz, D2O) δ 15.6−16.7 (br, 2P).
ESI-MS: calcd for C37H46N3O18P2− 882.23 m/z; found [M −
1H NMR (400 MHz, CDCl3) δ 7.92−7.79 (m, 4H), 7.47−
7.21 (m, 11H), 7.06 (d, J = 9.0 Hz, 1H), 6.84 (s, 1H), 5.21 (s,
2H), 5.14 (s, 2H), 3.74−3.49 (m, 18H), 2.51 (t, J = 5.8 Hz,
2H).
2H]− 882.4 m/z.
In Vitro Spiral Ganglion Neurite Outgrowth Model. P4
CBA/CaJ pups were euthanized, and cochleae were dissected
from the temporal bone as previously described.48 The stria
vascularis and the sensory epithelium were then removed,
exposing the modiolus containing SGN from the modiolus.
The SGN population was sharply dissected horizontally and
vertically, resulting in 4 pieces.
2,5-Dioxopyrrolidin-1-yl-1-(4-(7,8-bis(benzyloxy)-4-oxo-
4H-chromen-2-yl)phenyl)-1-oxo-5,8,11,14-tetraoxa-2-aza-
heptadecan-17-oate (13). In a 25 mL flask, 17.2 mg of 12
(0.024 mmol) in 5 mL of THF was treated with 2 equiv of N-
hydroxysuccinimide (6.25 mg, 0.054 mmol) and with EDC·
HCl (14.55 mg, 0.076 mmol). The reaction mixture was stirred
overnight. After the solvent was removed by evaporation, the
residue was dissolved in 15 mL of chloroform. The organic
layer was washed with 30 mL of water, followed by brine, and
then dried over sodium sulfate. Evaporation of the solvent left
16.0 mg of 13 (82%), which was used in the next step without
further purification.
A 4 well dish plate (CellStar) was prepared by adding
sterilized glass coverslips to the wells. Plates were covered with
a 1:10 dilution of Matrigel (Corning) for 10 min at 37 °C for
SGN outgrowth experiments. Right before plating, plates were
washed with HBSS (ThermoFisher Scientific). Each dissected
SGN was plated onto the glass plate with the tissue was plated
50 μL of culture medium [DMEM/F12 (GIBCO), N2
(ThermoFisher), B27 (ThermoFisher), 50 μg/mL Ampicillin
(Sigma-Aldrich), 1:300 Fungizone (250 ug/mL, GIBCO), and
1:100 Hepes (1 M GIBCO)]. The tissues adhered overnight at
37 °C, 5% CO2. All drugs were kept as stock solutions in
DMSO at 400 μM and stored at −20 °C. After attachment was
confirmed under the microscope, SGNs were treated in
darkness with drugs diluted in culture medium at a final
concentration of 400 nM. Treatments consisted of Risedronate,
7,8-DHF, Ris-DHF, or DMSO alone (control). Cultures were
kept at 37 °C, and drug medium was exchanged every 24 h.
After 48 h, the samples were fixed with 4% paraformaldehyde
for immunohistochemical analysis. Cultures were permeabilized
and blocked with blocking solution (15% goat serum and 0.3%
Triton X-100 in PBS), and primary antibodies for neurons
mouse TuJ1 (1:500, Biolegend, #801201) were diluted in
antibody solution (10% goat serum and 0.1% Triton X-100 in
PBS) and applied to cultures overnight at 4 °C. After several
washing steps with 1× PBS, fluorescent secondary antibody
goat anti-mouse Alexa Fluor 568 (1:500, ThermoFisher, #A-
11004) was diluted in antibody medium and incubated for 1 h
at rt. Nuclei were stained with DAPI (Thermo Fisher). Tissues
were then visualized with Leica SP8 confocal microscopy.
In Vitro Hydroxyapatite Outgrowth Assays. HA
nanopowder (Sigma-Aldrich) (10 mg) was suspended in 1
mL of culture medium and passed through a 40 μm sterile filter
to remove agglutinated nanoparticles. All samples, each
containing ∼500 μg of nanopowder, were prepared in darkness
due to light sensitivity of the compounds. Five 1 mL
suspensions of HA with medium containing 8 nM of FAM-
Ris (1:50 relative to the other compounds added) as
fluorescent label were prepared. Risedronate, Ris-DHF,
DMSO alone, and DHF (Abcam) was added to each dilution
at a final concentration of 400 nM. After brief vortexing, the
samples were incubated at 37 °C.
1H NMR (400 MHz, CDCl3): δ 7.89−7.78 (m, 5H), 7.44−
7.21 (m, 10H), 7.11 (s, 1H), 7.05 (d, J = 9.0 Hz, 1H), 6.70 (s,
1H), 5.21 (s, 2H), 5.14 (s, 2H), 3.71 (t, J = 6.4 Hz, 2H), 3.66−
3.51 (m, 16H), 2.75 (m, 6H).
ESI-MS: calcd for C45H46N2O13Na+ 845.29 m/z; found [M +
Na]+ 845.4 m/z
2,5-Dioxopyrrolidin-1-yl-1-(4-(7,8-dihydroxy-4-oxo-4H-
chromen-2-yl)phenyl)-1-oxo-5,8,11,14-tetraoxa-2-azahepta-
decan-17-oate (14). In a 25 mL flask, 16.0 mg of 13 (0.019
mmol) in 20 mL of 1:1 v/v THF:ethanol was treated with 30%
w/w of Pd catalyst (1:1 w/w Pd/C:Pd(OH)2/C, 5.0 mg). The
reaction mixture was frozen, pumped out, thawed, and flushed
with N2 (3×) and H2 (1×) before being stirred overnight under
1 atm of H2. The catalyst was filtered off, and the solvent was
removed, giving 14 in quantitative yield, which was used in the
next step without further purification.
1H NMR (400 MHz, CD3OD) δ 8.04 (d, J = 8.4 Hz, 2H),
7.91 (d, J = 8.5 Hz, 2H), 7.54 (d, J = 8.7 Hz, 1H), 6.91 (d, J =
8.8 Hz, 1H), 6.73 (s, 1H), 3.64−3.53 (m, 18H), 2.84−2.72 (m,
6H).
ESI-MS: calcd for C31H34N2O13Na+ 665.20 m/z; found [M +
Na]+ 665.5 m/z.
1-(1-(4-(7,8-Dihydroxy-4-oxo-4H-chromen-2-yl)phenyl)-
20-hydroxy-1,17-dioxo-5,8,11,14-tetraoxa-2,18-diazahenico-
san-21-yl)-3-(2-hydroxy-2,2-diphosphonoethyl)pyridin-1-
ium (5). 14 (17.0 mg, 0.026 mmol) in 800 μL of THF was
added dropwise to 34.4 mg of 15 (0.060 mmol, 2.3 equiv) in
800 μL of water (pH first adjusted to 8.3 with solid sodium
carbonate), and the reaction mixture was stirred overnight. The
solvent was removed by evaporation, and the residue was
dissolved in water. The crude product was purified by reversed-
phase (RP) HPLC on a C18 column (21.2 mm × 250 mm, 5
μm, 100 Å pore size), flow rate 8.0 mL/min, using 10% MeCN
0.1 M TEAC (pH 7.0) as buffer A and 75% MeCN 0.1 M
TEAC (pH 7.8) as buffer B with the gradient increased to 40%
of buffer B over 25 min, followed by elution with 100% of
buffer B for 75 min. The desired product was collected (3.0 mg,
13%) and redissolved in a 70% MeOH, 0.1 M ammonium
acetate buffer (pH 5.0) (UV, 260 nm).
After 1 h, the particles were centrifuged at 2000 rpm for 2
min, and the supernatant was removed. The residues were
washed 4× with fresh medium. HA pellets were then
resuspended in 50 μL of 1:5 Matrigel/medium mix (Corning).
The Matrigel-HA solution was then spread onto round glass
coverslips and placed in the incubator at 37 °C to set for 15
min. SGNs were plated onto the solidified Matrigel-HA
suspension for 48 h, and outgrowth was analyzed as described
above.
1H NMR (400 MHz, D2O): δ 8.82 (s, 1H), 8.60 (dd, J =
26.4, 7.0 Hz, 2H), 8.27 (d, J = 6.9 Hz, 2H), 8.08 (d, J = 7.5 Hz,
2H), 8.01−7.86 (m, 1H), 7.39 (s, 1H), 7.26 (d, J = 8.6 Hz,
H
DOI: 10.1021/acs.bioconjchem.8b00022
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Article
In Vitro Spiral Ganglion Neurite ANA12 Outgrowth
Assay. SGN samples were dissected and plated as described
above. After the tissues had adhered to the plate for 5 h at 37
°C, 5% CO2, SGN samples receiving ANA12 had media
changed to 50 μL of culture medium containing ANA12
(Tocris Bioscience) at a final concentration of 10 nM. Control
samples were incubated overnight without ANA12. ANA12 was
stored as stock solution in DMSO at 10 μM at −20 °C. All
tissues in both plates were incubated overnight at 37 °C, 5%
CO2.
After 24 h, all cultures received 400 nM of DHF, Ris, Ris-
DHF, or DMSO alone (control) as described above. SGNs in
the ANA12 assay plate additionally received 10 nM ANA12 in
culture medium. Cultures was kept at 37 °C, and media was
changed every 24 h. After 48 h, the cells were fixed,
permeabilized, stained, and evaluated with confocal microscopy
as described above.
Synaptic Ribbon Quantification and Colocalization
Analysis. Colocalization image analysis was performed by an
observer blinded to sample identity. Image stacks were
imported to Amira v6.0. Isosurface Rendering and Image
Orthoprojections functions were used to create 3D renderings
of the image stacks for counting as previously described.5,6,49
CONCLUSIONS
■
A rationally designed conjugate of 7,8-DHF (1), a TrkB
agonist, linked to a bone mineral-targeting bisphosphonate, Ris-
DHF (5), was synthesized in nine steps and found to retain the
ability of DHF to support SGN neurite outgrowth between
cochlear hair cells and spiral ganglion neurons. Strikingly, the
ability of Ris-DHF to promote spiral ganglion neurite
outgrowth is enhanced following adsorption to hydroxyapatite,
which mimics bone mineral. Finally, we have shown that Ris-
DHF promotes regeneration of ribbon synapses between hair
cells and spiral ganglion neurons using an in vitro excitotoxic
assay. Taken together, these findings provide strong preliminary
evidence that cochlea-targeted delivery of a neurotrophic
agonist linked to a BP is a promising strategy to regenerate
ribbon synapses following noise damage or aging in the
mammalian cochlea.
Neurite Tracing Analysis. Neurite tracing was performed
in blinded fashion. Confocal microscopy z-stack images were
analyzed in 3D space with ImageJ and Fiji Software (version
444 K 1.51n NIH public domain). Using the “Analyze-Set
Scale” function, the pixel unit of neurite length measurement
was set in micrometers. Images were rendered with
segmentation function, and Neurite tracer function was applied
choosing the starting point at the SGN cell bodies. This
allowed for a tracing of the neurite path in 3D through each z-
stack in the image file, resulting in a compiled skeleton render
of all measured neurites. Only those neurites contained entirely
within the image were analyzed. Single neurite lengths and
average outgrowth length were then compared between drug
treatments.
In Vitro Cochlear Synaptopathy Model. For OC
explants, P4 CBA/CaJ pups were euthanized, and the cochlea
were harvested in similar fashion as described above. The stria
vascularis was removed, and the OC was left attached to the
modiolus. The middle turn was dissected and separated into 2
pieces using microscissors. The tissue was plated on Laminin
(20 μg/mL, 37 °C overnight, ThermoFisher) and poly-L-
ornithine 0.01% (Millipore, 2 h at 37 °C) pretreated 4-well
culture dishes with glass coverslips. Explants attached overnight
in culture medium as described above. After attachment was
confirmed microscopically, explants were treated in darkness
with 0.5 mM KA (Abcam) diluted in culture medium for 2 h.
An untreated explant was kept as negative control, and a second
explant treated with KA only was kept as positive control. KA
medium was removed, and after washing with prewarmed
medium, explants were treated in darkness with Ris 400 nM,
DHF 400 nM, or Ris-DHF 400 nM diluted in medium. After 24
h of exposure to drug, explants were fixed and permeabilized as
described above. Presynaptic ribbon synapses were stained with
CtBP2 (IgG1, mouse, 1:200, #612044, BD Biosciences), while
the postsynaptic neural synapse portion was labeled with
PSD95 (IgG2a, mouse, 1:50, #75-028, Neuromab). Nerve
fibers were stained with Neurofilament (NF-H, chicken,
1:2500, #AB5539, Millipore), and hair cells were labeled with
Myosin VIIa (Myo7a, rabbit, #25-6790, Proteus). Alexa Fluor
antibodies (Invitrogen, 1:500) were used for fluorescent
secondary labeling (goat anti-mouse IgG1 488, goat anti-
mouse IgG2a 568, goat anti-rabbit 647, goat anti-chicken 647).
Explants were imaged with a Leica SP8 confocal microscope,
visualizing the entire hair cell with the synaptic pole, at a z-step-
size of 0.25 mm.
ASSOCIATED CONTENT
■
S
* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.bioconj-
chem.8b00022.
Chromatography traces and NMR and MS spectra with
experimental details for the preliminary synthetic studies
(PDF).
AUTHOR INFORMATION
■
Corresponding Authors
*E-mail: david_jung@meei.harvard.edu.
*E-mail: mckenna@usc.edu.
ORCID
Charles E. McKenna: 0000-0002-3540-6663
Author Contributions
J.S.K., K.N., and C.H. contributed equally. C.E.M. and D.H.J.
conceived the project and wrote the manuscript. C.E.M. and
B.A.K. designed the synthetic work, which was carried out by
K.N. D.H.J. and A.S.E. designed the biological studies, which
were carried out by C.H., J.S.K., and N.K. All authors have
given approval to the final version of the manuscript.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank Ms. Inah Kang for her invaluable assistance in the
preparation of this manuscript and Prof. M. Charles Liberman
for initial guidance in 3-D imaging of synapses. This work was
supported by the American Academy of Otolaryngology-Head
and Neck Surgery Herbert Silverstein Otology and Neuro-
tology Research Award, the American Otological Society
Research Grant, and by a grant from the National Institute of
Deafness and other Communicative Disorders, R01 DC007174.
I
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ABBREVIATIONS
■
SNHL, sensorineural hearing loss; SGN, spiral ganglion
neuron; DHF, 7,8-dihydroxyflavone; TrkB, tropomyosin
receptor kinase B; BP, bisphosphonate; RWM, round window
membrane; KA, kainic acid; OC, organ of Corti; Ris,
risedronate; Zol, zoledronate; Ris-DHF, risedronate-DHF
conjugate; HA, hydroxyapatite
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■
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