Trans-Tympanic Drug Delivery for the Treatment of Ototoxicity

Podsumowanie

We present a technique for localized administration of drugs through the trans-tympanic route into the cochlea. Drug delivery through this route would not interfere with the anti-cancer efficacy of the chemotherapeutic drugs such as cisplatin.

Streszczenie

The systemic administration of protective agents to treat drug-induced ototoxicity is limited by the possibility that these protective agents could interfere with the chemotherapeutic efficacy of the primary drugs. This is especially true for the drug cisplatin, whose anticancer actions are attenuated by antioxidants which provide adequate protection against hearing loss. Other current or potential otoprotective agents could pose a similar problem, if administered systemically. The application of various biologicals or protective agents directly to the cochlea would allow for high levels of these agents locally with limited systemic side effects. In this report, we demonstrate a trans-tympanic method of delivery of various drugs or biological reagents to the cochlea, which should enhance basic science research on the cochlea and provide a simple way of directing the use of otoprotective agents in the clinics. This report details a method of trans-tympanic drug delivery and provides examples of how this technique has been used successfully in experimental animals to treat cisplatin ototoxicity.

Wprowadzenie

The peripheral auditory system is exquisitely sensitive to drugs such as cisplatin and aminoglycoside antibiotics. Cisplatin is a widely used chemotherapeutic agent for the treatment of a variety of solid tumors, such as ovarian, testicular, and head and neck cancers. Ototoxicity experienced with the use of this drug is dose-limiting and quite common, affecting 75-100% of patients treated¹. Other drugs, such as carboplatin and oxaliplatin, have emerged as alternatives to cisplatin^2,3,4,5, but their usefulness is limited to a few cancers.

Early studies have shown the critical role of reactive oxygen species (ROS) in mediating the ototoxicity produced by cisplatin and aminoglycosides. Subsequent studies showed that the NOX3 isoform of NADPH oxidase is the primary source of ROS in the cochlea, and is activated by cisplatin^6,7. The generation of ROS compromises the antioxidant buffering capacity of cells, leading to increased lipid peroxidation of cellular membranes⁸. Furthermore, cisplatin increases the production of hydroxyl radicals which generate highly toxic aldehyde 4-hydroxynonenal (4-HNE), an initiator of cell death^9,10. Based on these findings, several antioxidants have been examined for the treatment of cisplatin ototoxicity. These include N-acetyl cysteine (NAC), sodium thiosulfate (STS), amifostine, and D-methionine. However, a major concern of antioxidant therapy is that these antioxidants could reduce cisplatin chemotherapeutic efficacy when administered systemically¹¹ through the interaction of cisplatin with thiol groups in the antioxidant molecules.

In view of these problems with antioxidant therapy, the goal of this study was to examine the trans-tympanic route of delivering antioxidants and other drugs to the cochlea to reduce hearing loss. The trans-tympanic route of drug and short interfering (si) RNA, described below, appears particularly promising.

Protokół

Male Wistar rats were handled in accordance with the National Institutes of Health animal use guidelines and a protocol approved by the Southern Illinois University School of Medicine Laboratory Animal Care and Use Committee. Auditory brainstem response (ABR) was performed on rats while under anesthesia prior to drug administration and 72 h after to verify the effect of the trans-tympanic drug delivery.

1. Auditory Brainstem Response (ABR)

Note: ABR measurements were collected using the auditory testing equipment and software. ABR represents evoked potentials or high-frequency waves generated by the eighth cranial nerve (wave I and II) and other higher auditory brain stem structures including anteroventral cochlear nucleus (wave III), lateral lemniscus (wave IV), and inferior colliculus (wave V). These waves are differentiated depending on the latency. ABR measurements were performed as described previously¹².

1. Anesthetize rats with a mixture of 90 mg/kg ketamine and 17 mg/kg xylazine via intraperitoneal injection. Confirm the depth of anesthesia via toe-pinch reflex. Apply ophthalmic lubrication (or mineral oil drops) to both eyes to prevent the eyes from drying and avoid ulceration during anesthesia.
2. Place the rat in the prone position on a heating pad (37 °C) inside of a controlled acoustical booth. Audiology testing equipment is located outside and next to the booth.
3. Insert stainless steel electrodes accordingly: the ground electrode in the rear flank, the positive electrode between the two ears directly atop the skull, and the negative electrodes below the pinna of each ear.
4. Apply acoustic stimuli using High-Frequency Transducers as a 5 ms tone burst at 8, 16, and 32 kHz. Determine stimulus intensities as decibel sound pressure level (dB SPL). This begins at 10 dB SPL and reaches 90-dB SPL with a 10-dB step size. Calibrate the sound intensities to a maximum output of 90-dB SPL using an impulse precision sound level meter.
   Note: The test result gives an indication of the integrity of the peripheral hearing organ, the cochlea, and the aforementioned auditory structures. The waveforms are generated within 15 ms of stimulus input and the latency of the waves depends on conduction time, which in turn is regulated by three critical factors: volume of the brain, the intensity, and the frequency of the sound. Auditory evoked potentials were recorded using software provided by the manufacturer.
5. Consider the minimum intensity which can evoke two different waveforms (II and III) of 0.5 µV amplitude as threshold prominently.
   Note: Threshold shift represents the difference in the threshold measured after treatment compared with the threshold obtained prior to treatment.

2. Trans-Tympanic Injections

1. To administer the drug trans-tympanically, place the rat in the left lateral decubitus position.
   1. Insert 2.5 mm disposable ear specula into the ear canal.
   2. With the use of a surgical scope, position the specula so that the tympanic membrane is visible.
   3. Using a 29 G X ½, 0.5 mL insulin syringe, draw up 50 µL of the [R]-N-phenyl isopropyl adenosine (R-PIA) solution (1 µM), 8-Cyclopentyl-1,3-dipropylxanthine (DPCPX) solution (3 µM), or siRNA solution (0.9 µg) to inject (5 units).
   4. Use the specula to direct the needle to the anterior inferior region of the tympanic membrane.
   5. Poke a hole through the membrane with the needle and administer the drugs mentioned in 1.2.3. Allow the rat to rest in this position for 15 min.
      Note: 50 µL should be the adequate volume to fit behind the tympanic membrane. No fluid should be in the ear canal after administration.
   6. Place the rat in the right lateral decubitus position and repeat steps 1.2.1 through 1.2.5 for administration in the other ear.
2. For rats receiving cisplatin intraperitoneally, place the rat in the supine position on a heating pad at 37 °C.
   1. Using a 21 G X 3/4 butterfly needle (12" length tubing), draw up cisplatin (11 mg/kg, 1 mg/mL solution in sterile phosphate buffer saline [PBS]).
   2. Using a syringe pump, administer cisplatin (1 mg/mL) via intraperitoneal injection over 30 min.
      Note: For a 250-g rat, the volume would be 2.75 mL at a rate of approximately 0.1 mL per min.
3. Continue to monitor the depth of anesthesia throughout these procedures. Once cisplatin administration is complete, place the rat back in the cage in the prone position, making sure there is nothing to obstruct its breathing.
4. Monitor the rat until fully recovered.

3. Cochlea Dissection and Decalcification

1. Following the final ABR (after 72 h), anesthetize the rat with a mixture of 90 mg/kg ketamine and 17 mg/kg xylazine via intraperitoneal injection. Confirm anesthesia with a toe-pinch reflex. Euthanize the rat via decapitation.
2. Dissect out the temporal bone as described previously¹³.
3. Place cochlea in 4% paraformaldehyde in 1x PBS solution in a 7-mL glass scintillation vial (completely covering the cochlea). Refrigerate overnight at 4 °C. Remove paraformaldehyde and wash with 1x PBS at room temperature.
4. Remove PBS and fill the tube completely with a 120-mM solution of EDTA (pH 7.3). Place on a rotator at room temperature and allow decalcification for 2 to 3 weeks, changing the EDTA solution daily.

4. Cryosectioning

1. Upon the completion of decalcification, completely immerse the cochlea in the following solutions (7 mL each) for 24 h at 4 °C: 10% sucrose, 20% sucrose, 1:1 mixture of 20% sucrose, and optimal cutting temperature (OCT) embedding compound.
2. Place fresh OCT compound to fill and completely cover the cochlea in a 15 mm x 15 mm x 5 mm disposable embedding mold. Orient the cochlea on its side so that it is parallel to the bottom of the embedding mold, as described by Whitlon et al.¹⁴
3. Immediately place the mold on dry ice to solidify the OCT and store at -80 °C overnight.
4. Immerse microscope slides in 0.01% Poly-L Lysine at room temperature for 30 min. Remove the slides, do not rinse, and allow to dry overnight. Use these slides for cryosections.
5. Remove the cochlea in OCT from the -80 °C freezer and place it on dry ice. Using a sharp microtome blade (L x W: 80 mm x 8 mm, thickness 0.25 mm, cutting angle of 34 °), section the OCT blocks at 10 µm using a cryostat at -30 °C. Place two sections per slide.
6. Refrigerate slides at 4 °C when finished.

5. Immunohistochemistry

1. Place the slides in a glass microscope slide staining dish on a slide rack. Fill the dish with 350 mL of 1x PBS and wash the slides for 5 min three times at room temperature.
2. Remove the slides from the dish and dry off the area surrounding the tissue using a dry wipe, being sure not to dry off the tissue.
   1. Using a liquid blocking pen, draw a circle around the tissue section.
3. Block the tissue for 1 h at room temperature by adding 150 µL of blocking solution containing 10% normal (donkey) serum, 1% nonionic detergent, and 1% BSA in 1x PBS.
4. Tap off excess blocking solution and incubate the tissue overnight at 4 °C in a humidified chamber with 150 µL of 10% normal (donkey) serum, 0.1% nonionic detergent, and primary antibody (see the Note below) in 1x PBS.
   Note: For the following antibodies, these dilutions were used: For Figure 2 and 3, p-STAT1 Ser⁷²⁷ 1:300. For Figure 4, p-STAT1 Ser⁷²⁷, 1:100 and TRPV1 1:100.
5. Place the slides in the glass microscope slide staining dish on a slide rack. Fill the dish with 350 mL of 1x PBS and wash slides for 5 min three times at room temperature.
6. Incubate with the secondary antibodies diluted in a solution containing 0.01% nonionic detergent and 10% normal (donkey) serum in 1x PBS for 2 to 3 h at room temperature in a humidified chamber in the dark.
   Note: For the following secondary antibodies, these dilutions were used: For Figure 2 and 3, Donkey Anti-Rabbit IgG 1:600. For Figure 4, Rhodamine (TRITC) Donkey Anti-Rabbit IgG 1:500 and Donkey Anti-Goat IgG 1:500.
7. Place the slides in the glass microscope slide staining dish on a slide rack. Fill the dish with 350 mL of 1x PBS and wash slides for 5 min three times at room temperature.
8. Tap excess liquid off the slide, and dry the slide with a dry wipe around the tissue. Mount slides by adding one drop of the mounting agent with DAPI directly onto the tissue. Slowly place the coverslip on top, ensuring that no bubbles are formed underneath, and allow the slides to cure overnight at room temperature in the dark. Store slides at 4 °C.
9. Image slides using confocal microscopy. Use lasers for imaging as follows: UV laser for DAPI, 488 nm laser for Donkey Anti-Rabbit IgG, and 543 nm laser for rhodamine TRITC.

Wyniki

ABR responses measured in rats at three days following cisplatin administration showed a significant elevation in thresholds. Elevation of these thresholds was significantly reduced in rats administered with trans-tympanic [R]-N-phenylisopropyladenosine (R-PIA), adenosine A₁ receptor agonist¹⁵, prior to cisplatin. The specificity of the action of R-PIA at the adenosine A₁ receptor was demonstrated by the observation ...

Dyskusje

The trans-tympanic administration route allows for localized delivery of drugs and other agents to the cochlea which could otherwise produce significant systemic side effects if administered systemically. This method of drug administration allows rapid access of drugs to the site of action at significantly higher doses than would be achieved through the systemic route. Results presented here and published previously showed that trans-tympanic administration of [R]-N-phenyl isopropyl adenosine (R-PIA) pr...

Materiały

Name	Company	Catalog Number	Comments
Ketathesia (100 mg/ml) 10 ml	Henry Schein	56344	Controlled substance
AnaSed Injection/Xylazine (20 mg/ml) 20 ml	Henry Schein	33197
2.5 mm disposable ear specula	Welch Allyn	52432
Surgical Scope	Zeiss
29 G X 1/2 insulin syringe	Fisher Scientific	14-841-32	Can be purchased through other vendors
cis-Diammineplatinum(II) dichloride	Sigma Aldrich	P4394	TOXIC - wear proper PPE
Harvard 50-7103 Homeothermic Blanket Control Unit	Harvard Apparatus	Series 863
Excel International 21 G X 3/4 butterfly needle	Fisher	14-840-34	Can be purchased through other vendors
BSP Single Speed Syringe Pump	Brain Tree Sci, Inc	BSP-99
Pulse Sound Measurement System	Bruel & Kjaer	Pulse 13 software
High-Frequency Module	Bruel & Kjaer	3560C
1/8″ Pressure-field Microphone —-Type 4138	Bruel & Kjaer	bp2030
High Frequency Transducer	Intelligent Hearing System	M014600
Opti-Amp Power Transmitter	Intelligent Hearing System	M013010P
SmartEP ABR System	Intelligent Hearing System	M011110
Disposable Subdermal EEG Electrodes	CareFusion	019-409700
16% Formaldehyde, Methanol-free	Fisher Scientific	28908	TOXIC - wear proper PPE
7 mL Borosilicate Glass Scintillation Vial	Fisher Scientific	03-337-26	Can be purchased through other vendors
EDTA	Fisher Scientific	BP118-500	Can be purchased through other vendors
Sucrose	Fisher Scientific	S5-500	Can be purchased through other vendors
Tissue Plus OCT Compound	Fisher Scientific	4585
CryoMolds (15 mm x 15 mm x 5mm)	Fisher Scientific	22-363-553	Can be purchased through other vendors
Microscope Slides (25mm x 75mm)	MidSci	1354W	Can be purchased through other vendors
Coverslips (22 x 22 x 1)	Fisher Scientific	12-542-B	Can be purchased through other vendors
Poly-L-Lysine Solution (0.01%)	EMD Millipore	A-005-C	Can be purchased through other vendors
HM525 NX Cryostat	Thermo Fischer Scientific	956640
MX35 Premier Disposable Low-Profile Microtome Blades	Thermo Fischer Scientific	3052835
Wheaton™ Glass 20-Slide Staining Dish with Removable Rack	Fisher Scientific	08-812
Super Pap Pen Liquid Blocker	Ted Pella, Inc.	22309
Normal Donkey Serum	Jackson Immuno Research	017-000-121	Can be purchased through other vendors
TritonX-100	Acros	21568	Can be purchased through other vendors
BSA	Sigma Aldrich	A7906	Can be purchased through other vendors
Phospho-Stat1 (Ser727) antibody	Cell Signaling	9177
VR1 Antibody (C-15)	Santa Cruz	sc-12503
DyLight 488 Donkey anti Rabbit	Jackson Immuno Research	711-485-152	Discontinued
DyLight 488 Donkey anti Goat	Jackson Immuno Research	705-485-003	Discontinued
Rhodamine (TRTIC) Donkey anti Rabbit	Jackson Immuno Research	711-025-152	Discontinued
ProLong® Diamond Antifade Mountant w/ DAPI	Thermo Fisher	P36971
(−)-N6-(2-Phenylisopropyl)adenosine	Sigma Aldrich	P4532
8-Cyclopentyl-1,3-dipropylxanthine	Sigma Aldrich	C101
siRNA pSTAT1	Qiagen	Custome Made	Kaur et al. 201120
siRNA NOX3	Qiagen	Custome Made	Kaur et al. 201120
Scrambled Negative Control siRNA	Qiagen	1022076	Kaur et al. 201120