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F. A. Selaimen et al.
reiterate that the patient’s consent is necessary for recording
and storing the images.
We believe that, regardless of the technology used to
acquire otoscopy images, it should be performed by physicians, preferably otolaryngologists, for two main reasons:
rst, the visualization of the tympanic membrane is not
always easily accessible. Due to the presence of earwax,
crusts, discharge, or even anatomical variations of the external auditory canal, access to the tympanic membrane can be
arduous and require some experience. The second reason is
that the manipulation of specic structures that may be present in the external auditory meatus may indicate injuries that
should not be manipulated outside the surgical setting. An
example is pulsatile aural polyps that can correspond to paragangliomas, highly vascularized lesions.
For now, we are unaware of portable devices that the
patient can use to perform remote ENT appointments using
telemedicine. Due to the singularities of the otorhinolaryngological examination, which requires specic instruments
and adequate lighting, we believe that face-to-face consultations will still be essential in the short and medium term for
adequate diagnoses.
However, teleconsultation is just one feature of telemedicine. The possibility of discussing cases among colleagues,
remotely evaluating consultancies for other doctors, and
even distance teaching has dramatically improved in recent
years. All these possibilities for exchanging information and
knowledge are of paramount importance for the evolution of
otorhinolaryngology, especially because some diseases are
more typical of specic populations. The possibility of
exchanging knowledge with professionals from various parts
of the world brings many benets, especially for patients.
References
1. da Costa SS, Rosito LPS, Dornelles C, Sperling N.The contralateral ear in chronic otitis media. Arch Otolaryngol Head Neck Surg.
2008;134(3):290–3.
2. Rosito LPS, Silveira Netto LF, Teixeira AR, da Costa
SS. Classication of cholesteatoma according to growth patterns.
JAMA Otolaryngol Head Neck Surg. 2016;142(2):168–72. https://
doi.org/10.1001/jamaoto.2015.3148.
3. Selaimen FA, Rosito LPS, da Silva MNL, de Souza Stanham
V, Sperling N, da Costa SS. Tympanic membrane perforations: a
critical analysis of 1003 ears and proposal of a new classication
based on pathogenesis. Eur Arch Otorhinolaryngol. 2022;279:1277.
https://doi.org/10.1007/s00405- 021- 06776- 8.
4. Comunello E, Von Wangenheim A, Heck V Jr, Dornelles C, Costa
SS. A computational method for the semi-automated quantitative
analysis of tympanic membrane perforations and tympanosclerosis.
Comput Biol Med. 2009;39(10):889–95. https://doi.org/10.1016/j.
compbiomed.2009.07.002.
Non-surgical Strategies toRestore
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Middle Ear Aeration
BernardArs andDominiqueEstève
17
The main function of the middle ear, provided by the
tympano- ossicular system, is the initial phase of the
mechanical- electrical transduction of sound energy of atmospheric origin. The quality of this function requires a perfect
and permanent balance between the pressure of the endotympanic gas and the atmospheric pressure. This balance is
ensured by a system called the isobaric system of the middle
ear cleft [1–4]. (cfr. to the chapter “Regulated balance of
pressure variations in the temporal pneumatic spaces”).
The most important function of the middle ear cleft isobaric system is to prevent the development of a state of endotympanic gas depression. This is, moreover, the main
pathogen responsible for the lytic and progressive lesions
encountered in chronic otitis media.
Technique Necessary fortheDiagnosis
ofDysregulation ofPressure Variations
intheMiddle Ear Cleft: theTubomanometry
Tubomanometry allows the functional exploration of the isobaric system of the middle ear cleft by measuring the active
gas transport of the rhinopharynx into the middle ear cleft
(Fig.17.1) after the tubal opening caused by a dysbaric event
[5–8] (Table17.1).
The measurements are taken at three intensities of the
dysbaric event (30, 40, and 50 mbar); this makes it possible
to determine the value of the opening latency index (R-value),
which reects the latency between the application of pressure in the rhinopharynx and the measurement of a pressure
change in the external auditory canal in relation to the opening of the tube. This latency describes the function of the
bro-cartilaginous Eustachian tube. Three results are possi-
B. Ars (*)
University of Namur, Namur, Belgium
Temporal Bone Foundation, Brussels, Belgium
D. Estève
Manosque, France
®
ble: early opening (R≤1), late opening (R>1), or no opening (R not measurable).
In normal subjects, this opening latency, because it results
from a reex phenomenon [9–11], has a xed value, whatever the intensity of the stimulation for a given middle ear
cleft. This means that the variability between the three stimulation levels (30, 40, and 50 mbar), which is translated mathematically by the variance of the index R (VarR), is very
small.
The results of the tubomanometry examination are presented on a so-called expert system, which combines two
parameters: the average index R (MoyR) carried on the horizontal axis and the variance of its index (VarR) carried on the
vertical axis. The expert system comprises seven ordered
zones numbered from 1 to 7, making it possible to characterize the reactivity of the middle ear cleft isobaric system as a
function of the position of this middle ear cleft on the expert
system. This position in the expert system also makes it possible, depending on the normal or pathological state of the
middle ear cleft examined, to determine rehabilitative or
therapeutic behavior.
In the case of a normal middle ear cleft, tubomanometry
tests the ability of the ear to respond to a dysbaric situation
that may be the cause of the constitution of a middle ear
barotrauma. In this context, the ear can only occupy four of
the seven areas of the expert system (Fig.17.2).
In the case of a middle ear cleft with chronic otitis media,
the tubomanometry tests the same function as in the case of
a normal middle ear cleft. This function can be more or less
altered during chronic otitis media, and the degree of dysfunction depends on the intensity of the endotympanic gas
depression and the time during which the middle ear cleft is
subjected to this situation of endotympanic gas depression.
The ear with chronic otitis media can occupy one of the
seven zones of the expert system (Fig.17.3), evolving over
time (Fig.17.3).
This reects a progressive gradation of the pathology,
from zone 1 (diseased ear but whose functionality of the isobaric system of the middle ear cleft is still considered normal)
© The Author(s), under exclusive license to Springer Nature Switzerland AG 2023
M. V. Goycoolea et al. (eds.), Textbook of Otitis Media, https://doi.org/10.1007/978-3-031-40949-3_17
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B. Ars and D. Estève
Fig. 17.1 Specimen tubomanometry curve for a normal subject (right
ear with intact tympanic membrane, stimulus at 30mbar, early eustachian tube opening). Lower curve (yellow)=change in rhinopharyngeal pressure over time: (1) C1: soft palate closed, start of reex
swallowing phase and of increasing pressure in rhinopharynx. (2) C2:
rhinopharyngeal pressure reaches stimulus level (30mbar). The C2–C1
phase lasts less than 0.30s. (3) C2–C3: isometric contraction of soft
palate muscle. This phase lasts 2s and (4) relaxation of soft palate muscle and return to baseline rhinopharyngeal pressure. Upper curve
Table 17.1 Value of MoyR and VarR depending on the area of the
expert system and belonging to the group of normal ears or those with
chronic otitis media
Zone 1 Zone 2 Zone3 Zone 4
Normal ears MoyR 0.68 1.53 1.86 No Ears
VarR 0.02 0.09 0.66 No Ears
Ears+COM MoyR 1.00 1.40 1.74 3.94
VarR 003 0.07 1.15 6.65
VarR variance of the index R, MoyR average index R
up to zone 7 (total areexia zone, acquired in chronic otitis
media). The pathological evolution takes place over time and
is marked by a joined increase in the two parameters retained
(MoyR and VarR), in the case of chronic otitis media, with a
very large increase in the VarR parameter, and this from the
entry of the middle ear cleft into zone 3 of the expert system
on (Table17.1).
This stage (zone 3 of the expert system) is characterized
by central neuronal damage, which reects a loss of sensitivity to endotympanic gas depression, in relation to the physiological capacities of adaptation and accommodation of
(red) = changes in EAC pressure over time: (1) P1: opening of
Eustachian tube. (2) P2: end of rising pressure phase in external auditory canal (P1–P2). P2–P3: pressure remains constant in the absence of
change in tympanic membrane position. (3) P3: closure of Eustachian
tube. (4) Middle ear emptying phase, beginning at P3. (5) Note that P3
is directly below C3, indicating that the tube remains open as long as a
difference exists between nasopharyngeal pressure and atmospheric
pressure, and demonstrating continual transmission of pressure status
information to the integration center
barosensitive neuronal structures. This sensory loss, if left
unchecked, can increase over time, causing the middle ear
cleft to shift to the right on the expert system. The entry of
the middle ear cleft into zone 5 of the expert system results
in an absence of response that initially concerns the lowest
stimulation intensity, that is, 30 mbar (Fig.17.4), responsible
for the appearance of a grade I neurosensory scotoma
(Fig.17.4).
The ultimate evolution of this state is the absence of
response to the three stimulation intensities (ear in zone 7 of
the expert system), a stage corresponding to a grade III neurosensory scotoma.
In the event of chronic otitis media, we can schematize
the therapeutic attitude according to the position of the middle ear cleft on the expert system:
– Zone 1: zone of absolute normality of the middle ear cleft
isobaric system. The ear with chronic otitis media should
be monitored (tubomanometry at 12 or 18 months).
Treatments aimed at reducing inammation of the middle
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Fig. 17.2 Distribution of middle ears of a cohort of normal subjects on
the tubomanometry expert system. Zone 1: zone of optimum efciency
of the isobaric system of the middle ear cleft. There is no risk of barotrauma for the middle ears located in this area. Zone 2: slight isolated
increase in the MoyR parameter. Low risk of barotrauma for the middle
ears located in this area. Zone 3: slight coupled increase in the parameters, MoyR and VarR.A signicant risk of barotrauma for the middle
Fig. 17.3 Distribution of a
cohort of middle ears with
chronic otitis media on the
tubomanometry expert system
ear is located in this area. Zone 7: strong increase in the MoyR parameters. Zone of so-called physiological areexia, with a certain risk of
barotrauma for the middle ears contained in this zone. The presence of
a normal middle ear cleft in one of the above four areas of the expert
system is a genetically determined trait, and this trait is xed, that is, it
is nonevolving over time in the absence of pathogenic elements. If necessary, it can be modied by the application of a specic rehabilitation
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B. Ars and D. Estève
Fig. 17.4 Chronic oFFs media, result of a right ear: the value of R
becomes dependent on the intensity of sFmulaFon, and there is a parFal
anesthesia of the isobaric system of the middle ear, revealing the pres-
ear cleft mucosa and the state of endotympanic gas
depression should be initiated to avoid or minimize an
unfavorable outcome.
– Zone 2: tubomanometry monitoring every 6months and
treatments aimed at reducing inammation of the middle
ear cleft mucosa and the state of endotympanic gas
depression.
– Zone 3: existence of neuronal damage, beginning but cer-
tain. This condition requires tubomanometry monitoring
every 3months or the decision to initiate specic treatment depending on the position in the area and clinical
data. Entry into this state is irreversible in the absence of
specic treatment.
– Zone 4: last stage before entering the neurosensory sco-
toma. Decision of a specic treatment to avoid crossing a
course that would require a longer specic treatment.
ence of neurosensory scotoma (here of grade I) and resulFng in an alteraFon of the tubal opening more marked for low sFmulaFon values
– Zone 5, zone 6, and zone 7: zones of grade I, II, and III
neurosensory scotoma. Specic treatment decision.
The aim of the specic treatment is to bring the affected
ear back into zone 1.
The isobaric system of the middle ear cleft is based on the
existence of a complex reex neurosensory loop. If the reex
pathway is a guarantee of speed and efciency, the physiological characteristics of its components have the disadvantage of allowing the deterioration of their performances in
the event of persistent depressive stimulation by endotympanic gas. This explains the progressive neurological impairment detected by tubomanometry in the event of chronic
otitis media, which is irreversible spontaneously at a certain
stage and may worsen to the state of areexia (neurosensory
scotoma).
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Technique Necessary fortheReeducational
Management ofDysregulation ofPressure
Variations intheMiddle Ear Cleft:
TheKinetube
This state of plasticity, which is based on the important neuronal contingent of the isobaric system of the middle ear
cleft (neuroplasticity), is, however, not unambiguous: if a
pejorative evolution is possible, an evolution toward improvement is also possible, based on the fact that every reex of
the organism is re-educable. This is the basis of a rehabilitative treatment of chronic otitis media whose tubomanometry
shows that it has crossed the threshold of zone 3 of the expert
system. This specic treatment is called Kinétube® [5,
12–14].
The Kinetube is a portable device similar to the calibrated
pressure generator of the tubomanometer, that is, it delivers
a succession of positive pressures in the rhinopharynx during
swallowing, simulating high-amplitude dysbaric events (30,
40, and 50 mbar).
Indications forTreatment withKinetube
– Normalization of dysfunctions of the isobaric system of
the middle ear cleft observed by tubomanometry in the
context of chronic otitis media. The prescription of
Kinetube treatment will take place after the eradication of
a cholesteatoma, but ideally before any tympanoplasty
indicated outside of a cholesteatoma, in order to guaran-
tee the best chance of success for this surgical procedure.
– Rehabilitation of an isobaric system of the middle ear
cleft that is innately incapable (i.e., in normal subjects
without an otological history) to prevent the occurrence of
barotrauma.
– Rehabilitation of the velar musculature in the event of
infantile swallowing with an operated or irradiated soft
palate.
Methods ofTreatment withKinetube:
– The treatment is carried out at home, one session per day
with 15 swallows at 30 mbar, 15 swallows at 40 mbar, 15
swallows at 50 mbar.
– Six days a week.
– By specifying never to exceed the prescribed doses, and
to respect a period of at least 20h between two sessions.
Failure to comply with these instructions would generate
the absence of results to treatment, due to the exhaustion
and nonrenewal of the neurotransmitters at the origin of
the meaningful message to the barostat of the middle ear
cleft isobaric system.
– The duration of treatment will be at least 3months, up to
6months, rarely more.
– In France: rental of the device in pharmacies.
Criteria forAssessing theResult
Clinical, audiometric, and tubomanometric evolution (tubomanometry control at 1, then 3months; possibly at 6months).
More rarely: middle ear cleft CT scan.
Visualization of the effects of treatment with Kinetube
(Figs.17.5 and 17.6).
Contraindications toTreatment withKinetube
– Children under the age of seven
– Subjects with chronic otitis media and for whom it is sus-
pected that they will not be able to complete the treatment
(due to its duration): lack of motivation, insufcient intellectual development, trisomy 21.
– An acute inammatory episode in the ENT sphere (rhini-
tis, rhinopharyngitis, angina, otitis, etc.) is a temporary
contraindication.
Fig. 17.5 109 ears with chronic oFFs media (in pink). In blue, 111
normal ears, which are the “model” to reach for ears with chronic oFFs
media
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Fig. 17.6 109 ears with chronic oFFs media before (in pink) and the
same (in green) aSer 21days of treatment with Kinetube. In blue, 111
normal ears, which are the “model” to be reached for ears with chronic
oFFs media
Conclusion
Tubomanometry®* is a means of exploring the capacity of
the bro-cartilaginous Eustachian tube to prevent (or not)
barotrauma of the middle ear. It has been shown that this
capacity is very frequently more or less strongly disturbed in
cases of chronic otitis media, which makes tubomanometry a
powerful tool for exploring this pathology, in particular for
detecting and grading the neuronal involvement synonymous
with sensory hypo or anesthesia, vis-à-vis the endotympanic
gas depression. This explains the evolution toward severe
forms of chronic otitis media, in particular tympanic retraction pockets and cholesteatoma.
Kinétube®* is a therapeutic device designed and used in
the context of chronic otitis media based on data from the
physiology of the middle ear cleft isobaric system and the
pathophysiology of chronic otitis media, which makes it possible to eradicate it in children as well as in adults and allows
a return to a normal physiological situation.
*Tubomanometer® and Kinétube® are manufactured by:
DTF médical—Diffusion technique française. 13 rue de la
Presse—CS 60132. F42003 SAINT-ETIENNE cedex 1—
France. (dtf@dtf.fr).
B. Ars and D. Estève
Acknowledgments We wish to thank Professor Jean Lebacq for thoroughly reviewing the manuscript.
References
1. Estève D, Dubreuil C, Della Vedova C, Normand B, Lavieille
JP, Martin C.Physiology and physiopathology of the Eustachian
tube function: interest of tubomanometry. J Français d’ORL.
2001;50:233–42.
2. Estève D, Vecellio L, Lavieille JP.Middle ear cleft pressure regulation in health and disease. Sustained approach for a central regulating system: the middle ear isobaric system. In: Chronic otitis media.
Pathogenesis-oriented therapeutic management. Amsterdam:
Kugler Publications; 2008. p.213–25.
3. Salburgo F, Garcia S, Lagier A, Estève D, Lavieille JP, Montava
M.Histological identication of neuropharyngeal mechanoreceptors. Eur Arch Otorhinolaryngol. 2016;273:4127–33.
4. Alshukry A, Lagier C, Della Vedova C, Salburgo F, Lavieille JP,
Montava M.The effects of hypoxia on middle ear pressure regulation. High Alt Med Biol. 2020;21(1):99–104.
5. Estève D, Dubreuil C, Della Vedova C, Normand B, Martin C.
Évaluation par tubomanométrieâ de la fonction d’ouverture tubaire
et de la réponse tympanique chez le sujet normal et chez le sujet
porteur d’une otite séro-muqueuse chronique. Comparaison des
résultats. J Français d’ORL. 2001;5(50):223–32.
6. Estève D.Tubomanometry and pathology. In: Fibrocartilaginous
eustachian tube—middle ear cleft. Amsterdam: Kugler Publications;
2003. p.159–75.
7. Ars B, Dirckx JJ. Tubomanometry. In: Fibrocartilaginous eustachian tube—middle ear cleft. Amsterdam: Kugler Publications;
2003. p.151–8.
8. Collin M, Coulange M, Devèze A, Montava M, Estève D, Lavieille
JP.Middle ear barotrauma due to thinopharyngeal scar tissue: tubomanometry diagnostic and therapeutic contribution. Rev Laryngol
Otol Rhinol. 2012;133:157–61.
9. Eden A.Neural connections between the middle ear, eustachian
tube and brain: implication for the reex control of middle ear aeration. Ann Otol. 1981;90:566–9.
10. Eden A, Gannon P. Neural control of middle ear aeration. Arch
Otolaryngol Head Neck Surg. 1987;113:133–7.
11. Martin C, Karkas A, Prades JM.Tubotympanic system functioning.
Eur Ann Otorhinolaryngol Head Neck Dis. 2017;134:177–84.
12. Estève D. Re-adaptation of the baroreceptors of the brocartilaginous eustachian tube and middle ear cleft by the mean of
the Kinétube®. In: Fibrocartilaginous eustachian tube—middle ear
cleft. Amsterdam: Kugler Publications; 2003. p.203–17.
13. Estève D. Rehabilitative management of the imbalance of pressure variations in the middle ear cleft. In: Chronic otitis media.
Pathogenesis-oriented therapeutic management. Amsterdam:
Kugler Publications; 2008. p.341–9.
14. Barbut J.Place de la réhabilitation tubaire dans la prise en charge
des otites moyennes chroniques. Thèse de Doctorat en médecine de
l'Université Aix-Marseille. 2014. pp.1–126.
Drug Delivery Across theIntact
https://t.me/medicina_free
Tympanic Membrane: Methods,
Mechanisms andPotential Impact
ArwaKurabi, MollyCooper, EmilySereno,
andAllenF.Ryan
18
Diseases oftheMiddle Ear
The middle ear (ME) is an air-lled cavity enclosed by the
temporal bone. The impermeable tympanic membrane (TM)
separates the outer ear from the ME, while the oval and
round windows connect to the inner ear. The eustachian tube
(ET) connects the nasopharynx to the ME and functions to
equalize ME pressure to atmospheric changes. When there is
ET dysfunction, symptoms such as earache, congestion, discomfort, and dizziness may occur [1].
ME infections (otitis media ([OM]), a serious disease of
childhood, are the most common reason for physician visits
and surgery in children [2, 3]. While most children recover
quickly from acute OM, 10–15% of US children experience
chronic or recurrent disease. Moreover, in many developing
countries with limited access to medical care, undertreated
OM progresses to chronic supurrative OM, the most serious
form, which is estimated to cause 28,000 annual deaths and
to be responsible for half of the world’s burden of handicapped hearing loss [4, 5], making it the world’s leading
hearing loss cause.
Since acute OM typically resolves spontaneously, watchful waiting is recommended for children over 2years of age.
Systemic antibiotics are recommended for OM in children
under two and for more complicated chronic or recurrent
OM in older children [6]. However, systemic antibiotics can
cause signicant gastrointestinal side effects [7] and the
A. Kurabi (*) · M. Cooper · E. Sereno
Department of Surgery/Otolaryngology, UCSD School of
Medicine, San Diego, CA, USA
e-mail: akurabi@ucsd.edu
A. F. Ryan
Department of Surgery/Otolaryngology, UCSD School of
Medicine, San Diego, CA, USA
Department of Neurosciences, UCSD School of Medicine,
San Diego, CA, USA
San Diego VA Healthcare System, La Jolla, CA, USA
exposure of off-target organisms throughout the body [8].
Moreover, in up to 15% of children, little or no systemically
administered antibiotic reaches the ME [9]. Local antibiotic
delivery to the ME can alleviate these issues, but it requires
breaching the TM, which must be performed by a trained
surgeon. Local ME delivery is typically performed when
tympanostomy tubes are surgically inserted (considered
invasive since the TM is breached). Ciprodex drops or other
drug formulations are administered locally into the ME
through tubes [10].
Cholesteatoma, in which keratinocytes invade the ME and
often grow aggressively, can damage both the ME and inner
ear and, in the worst case, progress intracranially [11].
Surgical removal of cholesteatoma tissue, the only treatment,
often fails to remove all of the cholesteatoma. Remnants can
continue to grow, leading to the need for further surgery [12].
Drugs to inhibit tissue growth, which have been proposed as
potential treatments [13], have yet to be applied to this
disease.
In both OM and cholesteatoma, local drug delivery could
be benecial. Noninvasive delivery would be especially useful, as it would not require surgery. Moreover, when access
to ENT surgeons is limited, it would provide a practical
means of local ME treatment. In this review, we describe our
recent efforts to discover and validate peptides that can cross
the intact TM into the ME while carrying large cargo. These
peptides can be developed to deliver drugs and drug packages. As well, we provide a summary of other recent progress in the eld of noninvasive intratympanic drug delivery
and some of the available approaches.
Phage Display Reveals Rare Peptides that Can
Cross theIntact TM
Many tissue barriers possess mechanisms by which select
substances can be transported. We reasoned that the TM
might harbor such a capacity. We also felt that transport
would involve the recognition, by cellular receptors on the
© The Author(s), under exclusive license to Springer Nature Switzerland AG 2023
M. V. Goycoolea et al. (eds.), Textbook of Otitis Media, https://doi.org/10.1007/978-3-031-40949-3_18
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A. Kurabi et al.
external surface of the TM, of biomolecules. Since many
biologically relevant molecules are proteins, we used bacteriophage display to screen a library of peptides to identify
amino acid sequences that could cross the TM [14]. Phage
display is a high-throughput tool for the identication of
molecules with specic characteristics [15].
Bacteriophages are bacterial viruses that, upon infecting
their targets, replicate in large numbers, followed by release
and continued bacterial infection. For phage display, each
phage is engineered to express a different molecule, which is
fused to the free end of a lamentous capsular protein [16,
17]. With a sufciently large library of phages, each express-
ing a different molecule, this provides rapid combinatorial
power to identify molecules of interest.
We initially screened a 7-mer peptide M13 phage library
(PhD-C7C™, New England Biolabs, Cambridge, MA,
USA), with 2.5×108 unique peptide sequences in the library.
OM was induced in rats by ME injection with nontypeable
Haemophilus inuenzae (NTHi). After 48h, when OM was
well established, a library aliquot containing four copies on
average of each peptide clone was applied to the TM in saline
for 2h. The external canal was rinsed and the ME contents
harvested, after which bacteriophage were tittered. The
resulting phages were amplied and screened for three additional rounds. However, there was no evidence of library
“collapse” to a small number of peptides that would indicate
selection for transport across the TM.
We felt it possible that the peptide length chosen was too
short to interact with a possible transport mechanism. We
therefore switched to a library expressing peptides that are
12 amino acids in length (Ph.D-12TM Phage Display Peptide
Library; New England Biolabs), expressed on the free end of
the M13 pIII protein. Five copies per phage of this lamentous, capsular protein mediate the recognition of and binding
to the Escherichia coli hosts of the phage. The expressed
peptides were thus well positioned to contact TM cells. Ten
phages expressing one of 109 random peptides, thus totaling
1010 phages, were applied to the TMs of NTHI-infected rat
MEs as above. Two screening strategies were employed.
First, sequential biopanning was used to screen for
phage that rst bound to, then internalized into, and nally
crossed the TM.To accomplish this, the library was applied
to TMs for 1h, after which the external canal was extensively rinsed to remove any phage that had not bound to
TMs. The TMs were harvested and homogenized. The
homogenate was then applied to E. coli, the bacterial target
of the M13 phage, and any phage present infected and multiplied within. These phages were harvested and used to
repeat the binding screen two additional times. The resultant “TM-binding” library was then applied to rat TMs for
1h. The TMs were again rinsed with saline, but then a low
pH wash was applied to remove any phage bound to the TM
surface. The TMs were then dissected from the M bulla,
homogenized, applied to E. coli, and the phage was harvested. Repeated twice, this process yielded a
“TM-penetrating” library, which was again applied to the
TMs of NTHi-infected rats for 1h. After extensive external
canal rinsing with saline and low pH, the contents of the
ME were harvested, used to infect E. coli, phage harvested,
and the process repeated twice. From the resultant
“TM-transiting” library, 30 colonies were selected for DNA
sequencing to identify the peptide inserts. Each phage
clone and corresponding peptide sequence were given a
sequential name Binding-Penetrating-Transiting (BPT).
For the second strategy, the unselected 12-mer peptide
library was applied to the TMs of NTHi-infected MEs for
1h, and the external canal was rinsed with saline and low
pH.The ME contents were then used to infect E. coli, and the
process was repeated twice. From the resultant library, 30
phage clones were selected and sequenced. The phage clones
were given names with a TMT (TM transmigrating)
acronym.
For strategy 1, seven unique BPT peptide phages were
present more than once among the 30 sequenced phages,
suggesting efcient crossing of the TM, labeled BPT1–7.
Strategy 2 yielded ve unique peptides present multiple
times in the sequence sample, labeled TMT1–5. These 12
sequences were aligned to determine sequence relationships
(Fig.18.1). Four peptides formed a group characterized by
all or part of STKx consensus motif within the sequence.
Five peptides formed a second group that exhibited all or
part of a PxxP motif. Two peptides aligned separately but
were more closely related to the STKx group than the PxxP
group.
Analysis of the peptides using SAROTUP (Scanner and
Reporter of Target Unrelated Peptides; [18]) found that the
peptides are novel, not having been reported from earlier
phage display studies. However, the MimoBlast tool [19]
revealed that the BPT-1 sequence was identied in a biopanning study [20] as part of a human single-chain variable fragment (HuScFv) that targets the dengue virus envelope protein
and blocks its infectivity.
To compare transport rates, 1010 of each trans-TM phage
was applied to the TMs of NTHi-infected rat MEs for 1h.
Phage recovery was then used to assess trans-TM transport
efciency. A phage not expressing a peptide (wild type
[WT]) was controlled for possible contamination. All the
peptide-bearing phages were recovered in amounts signicantly greater than those of the WT phage. While this validated the success of our biopanning strategies, it was clear
that the different phages crossed the TM with different
efciencies.
18 Drug Delivery Across theIntact Tympanic Membrane: Methods, Mechanisms andPotential Impact
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Fig. 18.1 Amino acid sequences of pIII extension peptides recovered
from phage display biopanning against the tympanic membrane for
transport into the middle ear. (Left) Amino acids (aa) are colored by
side-chain functional characteristics and show a high degree of diversity. Blue is basic aa, red is acidic aa, orange is aromatic aa, yellow is
Trans-TM Phage Transport Is Active
To investigate the mechanism of phage transport, we varied
the time of TM exposure, phage concentration, temperature,
and oxygen level. When time on the TM was increased from
1–4 h, ME phage recovery increased exponentially [14].
Increasing phage concentration proportionally also increased
recovery. The sensitivity of transport to oxygen deprivation
and temperature was evaluated exvivo using intact ME bullae. Transport was observed up to 6h after death, but not at
later times. At bulla temperature of 4 °C, transport was
almost completely eliminated. These results strongly suggest
that trans-TM transport occurs via an active, not passive,
mechanism.
Additional Amino Acids Can Increase Transport
Given that 7-mer peptides were too short to mediate trans TM transport, we reasoned that 12 amino acids might not be
sufcient for optimal interaction with the TM transport
mechanisms. We therefore used two 12-mer trans-TM phage
with different transport rates, TMT-2 and TMT-3, as backbones for the construction of two new phage libraries. For
each phage, we added six random amino acids to the free end
of the peptide [21]. We exposed each library briey (15min)
to the TMs of infected rat MEs. After three rounds of selection, we sequenced 30 phages recovered from the ME for
each library and tested phages with multiple copies in the
sample for transport rate. For TMT-2, which had a low transport rate, the best 6-mer extension doubled ME recovery.
sulfur-containing aa, polar hydroxylic aa are light red, polar amidic aa
are light blue, and green is proline. (Right) The phylogenic tree
sequence analysis reveals any potential homology between the peptides
and divides them into two groups with shared common motifs:
“ST(K/R)T” and “PxxP”
However, for TMT-3, a 12-mer with a high transport rate, the
best 18-mer showed an eightfold increase.
Trans-TM Peptide Phage Do Not Damage
theMiddle or Inner Ear
Obviously, the usefulness of peptides for drug delivery
depends on their safety. To evaluate trans-TM phage middle and inner ears, we applied 1010 WT, TMT1, TMT2,
TMT3, or TMT4 phage in saline into normal rat MEs for 1,
2, or 3 days via transbullar injection. A saline injection
was used as a control. ME morphology was evaluated on
day 3. Auditory brainstem response (ABR) thresholds
were measured before and at 1, 2, and 3 days after ME
injection. ME morphology was comparable across all
treatments. ABR thresholds were increased by approximately 20dB in all groups at day 1, presumably due to the
presence of uid in the ME.Thresholds for all groups had
recovered to normal by day 3 [22]. Titers of perilymph on
day 3 indicated that transport of phage into the inner ear
was minimal. The results indicate that trans-TM phages
are safe as a potential drug delivery technique, at least in
the short term.
Peptides Mediate Transport Across
theHumanTM
Since trans-TM peptides mediate the transport of bacteriophage, which are nearly 1μm in length, they seem likely to
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