Drug delivery systems for the treatment of sensorineural hearing loss

Sensorineural hearing loss is one of the most common disabilities in our society. Experimentally, many candidates for therapeutic molecules have been discovered. However, the lack of safe and effective methods for drug delivery to the cochlea has been a considerable obstacle to clinical application. Local application of therapeutic molecules into the cochlea has been used in clinic and in animal experiments. Advances in pharmacological technology provide various drug delivery systems via biomaterials, which can be utilized for local drug delivery to the cochlea. Recent studies in the field of otology have demonstrated the potential of synthetic and natural biomaterials for local drug delivery to the cochlea. Although problems still remain to be resolved for clinical application, introduction into clinical practice of these controlled-release systems may be reasonable because of their certain advantages over previous methods.     

腺病毒携带EGFP基因经完整圆窗膜转导豚鼠耳蜗的实验研究

目的研究腺病毒携带目的基因经完整圆窗膜途径耳蜗转导的可行性及安全性，为内耳基因治疗提供实验基础和理论依据。方法20只白色红目豚鼠，术前及术后分别行听性脑干反应（ABR）检查。实验组（12只）以重组腺病毒携带的增强型绿色荧光蛋白基因（enhancedgreenfluorescentprotein，EGFP），对照组（8只）以人工外淋巴液注入豚鼠圆窗龛内。分别于术后5天、14天取双侧耳蜗标本做基底膜铺片，耳蜗冰冻切片观察。结果于圆窗龛内注入腺病毒携带目的基因的转导方法对听力无明显影响。转染耳蜗及对侧耳蜗内目的基因呈广泛表达。5天组表达产物最高，14天组逐渐降低。对照组耳蜗未见EPFP表达。结论于圆窗龛内注入腺病毒携带目的基因转导的方法对耳蜗无明显毒害作用，且能够将目的基因成功转导至双侧耳蜗组织并广泛表达。     

经完整圆窗膜途径EGFP基因在大鼠耳蜗中的表达

目的 研究经完整圆窗膜途径进行耳蜗基因转导的可行性及安全性，为内耳基因治疗提供实验基础和理论依据。方法 24只SD大鼠术前及术后分别行听性脑干反应(ABR)检查。实验组(18只)用阳离子脂质体携带的增强型绿色荧光蛋白(enhanced green fluoresent protein，EGFP)基因，对照组(6只)用生理盐水，注入置于圆窗龛处的明胶海绵内。分别于术后3、7、14天取双侧耳蜗标本做基底膜铺片观察。结果 于圆窗龛处放置明胶海绵的转导方法对听力无明显影响。转染耳蜗呈明显的绿色荧光。3天组表达产物最高，7天组逐渐降低，14天组更弱。对侧及对照组耳蜗均未见荧光表达。结论 于圆窗龛处放置明胶海绵进行基因转导的方法对听力没有影响，且能够成功转染耳蜗组织，是一种行之有效的方法。     

adenoviral-mediated hath1-egfp gene transfer into guinea pig cochlea through intact round window membrane

Objective To study expression of adenovira1-mediated Hathl-EGFP gene in the guinea pig cochlea after transfer through intact round window membrane (RWM), and to assess its effects on hearing. Methods Twenty adult guinea pigs were used, of which: 12 were surgically inoculated with AdHath1-EGFP in the bony groove of round window niche, and 8 with artificial perilymph. Auditory brainstem response(ABR) thresholds were determined in all animals before and 5 days after surgery. On post-surgery day 5 and day 14, animals were sacrificed and whole mounts of cochlea and fro zensections were examined. Results ABR tests showed no significant change of hearing after the surgery.Strong fluorescence staining in the cochleae was seen in Ad-Hathl-EGFP groups. The highest levels of gene expression were seen in the post-surgery day 5 group with tittle decrease on post-surgery day 14.The contralateral cochlea and those in the control groups were free of fluorescence staining. Conclusion The transgenic Hath1-EGFP can be effectively delivered into the inner ear through intact RWM, in an atraumatic manner.     

The pharmacokinetic profiles of dexamethasone and methylprednisolone concentration in perilymph and plasma following systemic and local administration

CONCLUSION: Both methylprednisolone (MTH) and dexamethasone (DEX) could successfully and effectively penetrate the round window membrane (RWM) into perilymph. RWM topical application and otocyst infusion with MTH and DEX result in high perilymph drug concentrations and low plasma levels. An intratympanic administration schedule for DEX or MTH could be carried out twice daily. OBJECTIVE: To explore the pharmacokinetics of DEX and MTH in the inner ear fluids and plasma following systemic and local administration. MATERIALS AND METHODS: Three routes of administration of steroids were used in guinea pigs: intracardial injection, otocyst infusion and RWM topical application by granule gelfoam soaked with steroids. Samples of blood or perilymph of the scala tympani were collected at 1-6 h after administration. High-performance liquid chromatography was used to assay concentrations of steroids. RESULTS: Both the topical application and infusion administration resulted in a significantly higher concentration of steroids in perilymph than intracardial injection. The level of steroids in the perilymph reached a peak at 1-2 h after dosing, and this was maintained at a relatively high level for several hours. The intracardial injection with steroids yielded very low perilymph levels at all sampling times after administration.     

Transsynaptic delivery of nanoparticles to the central auditory nervous system

CONCLUSION: Silica nanoparticles may serve as a nonviral delivery system to the sensory hair cells, spiral ganglion cells within the cochlea, and the vestibular organ, as well as the cochlear nucleus. OBJECTIVES: At present there are no targeted therapeutics for inner ear disease. A variety of viral vector systems have been tested in the inner ear with variable efficacy but they are still not regarded as safe systems for inner ear delivery. Nanoparticles are a nonviral method of delivering a variety of macromolecules that potentially can be used for delivery within the auditory system. In this study, we evaluated the distribution and safety of nanoparticles in the inner ear. MATERIALS AND METHODS: Cy3-labeled silica nanoparticles were placed on the round window membrane of adult mice. Hearing thresholds were determined after nanoparticle delivery by auditory brainstem responses (ABRs). Distribution of particles was determined by histological evaluation of the cochlea, vestibular organs, and brain stem. RESULTS: Fluorescent microscopy demonstrated Cy3-labeled nanoparticles signals in the sensory hair cells and the spiral ganglion neurons of both the treated and contralateral inner ears. Additionally, the distal part of the central auditory pathway (dorsal cochlear nucleus, superior olivary complex) was found to be labeled with the Cy3-linked silica nanoparticles, indicating a retrograde axonal transport. No hearing loss or inflammation was noted in the treated cochlea.     

Substance distribution in a cochlea model using different pump rates for cochlear implant drug delivery electrode prototypes

Several studies using animals have shown the protective effects of neurotrophic factors (NF) on spiral ganglion cells (SGC). This is of particular importance since the number of SGCs is considered to be among the factors defining the efficacy of cochlear implants. A device for local inner ear treatment is therefore of great interest. As described previously, we modified a Contour(TM) cochlear implant electrode, to examine the inbuilt canal to be used for fluid release [Paasche, G., Gibson, P., Averbeck, T., Becker, H., Lenarz, T., St?ver, T., 2003. Technical report: modification of a cochlear implant electrode for drug delivery to the inner ear. Otol. Neurotol. 24, 222-227]. In the present study, three different electrode prototypes with openings of the delivery channel at various locations along the electrode array were examined to determine distribution of dye in a cochlea model over time. We compared dye delivery with: (a) release of the dye at the tip, (b) release of the dye at the tip and the side of the electrode, and (c) release of the dye only at the side of the electrode (6 mm from the tip). A mechanical pump was used to drive the system at pump rates of 100, 10, and 1 microl/h. Dye concentration changes along the length of the whole cochlea were investigated. Mean values for all experimental conditions show that the distribution along the array is fastest with two outlets whereas the distribution via a single outlet at the side of the electrode array is not considered to be sufficient. The established experimental setup provides the possibility of investigating prototypes of a fluid based drug delivery system for the treatment of inner ear pathologies in combination with electrical stimulation.     

Efficiency of a transtympanic approach to the round window membrane using a microendoscope

There has been increasing interest in cochlear drug delivery through the round window membrane (RWM). However, placing drugs on the RWM is difficult because of anatomical barriers. We examined the efficacy of a microendoscope for a transtympanic approach to the RWM. We evaluated the visibility of the RWM using four approaches: transtympanic microendoscopic, transtympanic microscopic, transmastoid microendoscopic, and transmastoid microscopic in ten human temporal bones. For the transtympanic approach, we made a fenestration (2 × 1 mm) in the postero-inferior quadrant of the tympanic membrane. For the transmastoid approach, conventional posterior hypotympanotomy was performed. The transtympanic microendoscopic approach enabled visualization of the RWM in all specimens, whereas the transtympanic microscopic approach only permitted visualization in three specimens. Through the transmastoid approach, the RWM was visible in all specimens using either a microendoscope or a microscope. The transtympanic microendoscopic approach can be utilized for cochlear drug delivery through the RWM.     

Cochlear pharmacokinetics with local inner ear drug delivery using a three-dimensional finite-element computer model

HYPOTHESIS: Cochlear fluid pharmacokinetics can be better represented by three-dimensional (3D) finite-element simulations of drug dispersal. BACKGROUND: Local drug deliveries to the round window membrane are increasingly being used to treat inner ear disorders. Crucial to the development of safe therapies is knowledge of drug distribution in the inner ear with different delivery methods. Computer simulations allow application protocols and drug delivery systems to be evaluated, and may permit animal studies to be extrapolated to the larger cochlea of the human. METHODS: A finite-element 3D model of the cochlea was constructed based on geometric dimensions of the guinea pig cochlea. Drug propagation along and between compartments was described by passive diffusion. To demonstrate the potential value of the model, methylprednisolone distribution in the cochlea was calculated for two clinically relevant application protocols using pharmacokinetic parameters derived from a prior one-dimensional (1D) model. In addition, a simplified geometry was used to compare results from 3D with 1D simulations. RESULTS: For the simplified geometry, calculated concentration profiles with distance were in excellent agreement between the 1D and the 3D models. Different drug delivery strategies produce very different concentration time courses, peak concentrations and basal-apical concentration gradients of drug. In addition, 3D computations demonstrate the existence of substantial gradients across the scalae in the basal turn. CONCLUSION: The 3D model clearly shows the presence of drug gradients across the basal scalae of guinea pigs, demonstrating the necessity of a 3D approach to predict drug movements across and between scalae with larger cross-sectional areas, such as the human, with accuracy. This is the first model to incorporate the volume of the spiral ligament and to calculate diffusion through this structure. Further development of the 3D model will have to incorporate a more accurate geometry of the entire inner ear and incorporate more of the specific processes that contribute to drug removal from the inner ear fluids. Appropriate computer models may assist in both drug and drug delivery system design and can thus accelerate the development of a rationale-based local drug delivery to the inner ear and its successful establishment in clinical practice.     

Pharmacokinetics of caroverine in the inner ear and its effects on cochlear function after systemic and local administrations in Guinea pigs

Caroverine, an N-methyl-D-aspartate and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor antagonist, has been shown to protect the inner ear from excitotoxicity and to be effective in the treatment of cochlear synaptic tinnitus. Local administration of caroverine on the round window membrane (RWM) could be a more effective means of administration to avoid systemic side/adverse effects. The present study investigates the pharmacokinetics of caroverine in the perilymph, cerebrospinal fluid (CSF) and plasma following intravenous and local applications at different dosages. High-performance liquid chromatography was used to determine the drug concentrations. Our results show much higher caroverine concentrations in the perilymph with lower concentrations in CSF and plasma following local applications compared with systemic administration. Auditory brainstem responses were measured to evaluate the changes in auditory function. The effects on hearing were transient and fully reversible 24 h after local caroverine applications. The findings suggest that local application of caroverine on the RWM for the treatment of excitotoxicity-related inner ear diseases, such as tinnitus and noise-induced hearing loss, might be both safe and more efficacious while avoiding high blood and CSF caroverine levels seen with systemic administration.     

Comparison of the protective efficacy of neurotrophins and antioxidants for vibration-induced trauma

BACKGROUND: Patients undergoing temporal bone surgery or subjects working with vibrating tools may develop vibration-induced hearing loss (VHL). The aim of this study was to characterize the effects of pretreatment with N-acetylcysteine (NAC) or the neurotrophic factors, brain-derived neurotrophic factor (BDNF) and ciliary neurotrophic factor (CNTF), on VHL in an animal model. METHODS: Trauma to the cochlea was created with a vibrating probe placed on the bone of the external ear canal. BDNF and CNTF(Ax1) were delivered into the cochlea with mini-osmotic pumps. NAC was delivered into the cochlea by round window membrane (RWM) injection, by RWM permeation, or by oral administration. Hearing was evaluated with electrocochleography (ECoG). RESULTS: For control animals, vibration resulted in an average immediate threshold shift of 42 +/- 26 dB. NAC provided no protective benefit in animals subjected to VHL, regardless of the delivery method, with average threshold shifts varying from 38 to 56 dB across groups. NAC injection through the round window membrane was toxic, causing a ECoG threshold shift of >25 dB. In BDNF+CNTF(Ax1)-treated animals, immediate hearing loss was similar to that in control animals. There was a trend of threshold recovery by 1 day after vibration; however, the improvement was not statistically significant, nor was there a significant difference in 1-day thresholds across groups. CONCLUSIONS: Local infusion of BDNF and CNTF(Ax1) may enhance the rate of recovery from VHL, compared to control animals. In contrast, NAC had no effect on VHL, and when delivered by RWM injection, was actually toxic to the inner ear.     

The pharmacokinetic profiles of dexamethasone and methylprednisolone concentration in perilymph and plasma following systemic and local administration

Conclusion. Both methylprednisolone (MTH) and dexamethasone (DEX) could successfully and effectively penetrate the round window membrane (RWM) into perilymph. RWM topical application and otocyst infusion with MTH and DEX result in high perilymph drug concentrations and low plasma levels. An intratympanic administration schedule for DEX or MTH could be carried out twice daily. Objective. To explore the pharmacokinetics of DEX and MTH in the inner ear fluids and plasma following systemic and local administration. Materials and methods. Three routes of administration of steroids were used in guinea pigs: intracardial injection, otocyst infusion and RWM topical application by granule gelfoam soaked with steroids. Samples of blood or perilymph of the scala tympani were collected at 1–6 h after administration. High-performance liquid chromatography was used to assay concentrations of steroids. Results. Both the topical application and infusion administration resulted in a significantly higher concentration of steroids in perilymph than intracardial injection. The level of steroids in the perilymph reached a peak at 1–2 h after dosing, and this was maintained at a relatively high level for several hours. The intracardial injection with steroids yielded very low perilymph levels at all sampling times after administration.     

Fibroblast-mediated delivery of GDNF induces neuronal-like outgrowth in PC12 cells

HYPOTHESIS: Recombinantly modified cells deliver neurotrophic factors with the capacity to induce differentiation and the outgrowth of neurites of rat pheochromocytoma cells 12 (PC12) serving as a neuronal model. BACKGROUND: The benefit of cochlea implant (CI) is depending, among other factors, on the number of surviving spiral ganglion neurons (SGN). Studies have shown that the external application of neurotrophic factors in combination with electrical stimulation increases the survival rate of SGN after ototrauma. Therefore, functionalization of electrodes with recombinantly modified cells providing neurotrophic factors to the SGN for inducing survival mechanisms may be an approach to realize drug delivery to the cochlea. METHODS: Murine NIH3T3 cells were recombinantly modified with an infectious lentiviral monocistronic and bicistronic system to synthesize glial cell line-derived neurotrophic factor and the green fluorescent protein. Free glial cell line-derived neurotrophic factor from the supernatant of the modified NIH3T3 cells was added to rat PC12, and the neuronal-like outgrowth was determined for 10 days. RESULTS: A significant neuronal-like outgrowth appeared as early as Day 3 after the application of the supernatant. CONCLUSION: The results indicate that the established in vitro model represents a powerful basic model for determining signal pathways between neuronal-like processing PC12 cells and cellular drug delivery systems.     

Canalostomy is an ideal surgery route for inner ear gene delivery in big animal model

Abstract

Background: Inner gene therapy offers great promises as a potential treatment for hearing loss.

Aims/objectives: One of the critical determinants of the success of inner ear gene therapy is to find a delivery method which results in consistent transduction efficiency of targeted cell types while minimizing hearing loss.

Material and methods: Surgery was performed only in the right ear of each Bama miniature pig, and the left ear served as a control. The gene delivery to inner ear via round window membrane (RWM) and posterior semicircular canal (PSC) approach was performed with the viral vector AAV1-CMV-GFP.

Results: The gene delivery through RWM and the PSC (canalostomy) is able to perfuse the inner ear.

Conclusions and significance: The easy anatomic identification of the PSC, as to RWM, as well as minimal manipulation of the temporal bone required, make this surgical approach an attractive option for inner ear gene delivery in big animal model.     

Feasibility of inner ear gene transfer after middle ear administration of an adenovirus vector

BACKGROUND: Several groups demonstrated in animal experiments that gene transfer is a feasible tool for inner ear intervention. Various approaches for inoculation of vectors have been successfully used for inner ear gene therapy. One possible way to reduce the risk of hearing loss following the opening of the cochlea for application of the vector into the perilymphatic space is to deliver vectors through the round window. This study was designed to determine whether middle ear application of an adenoviral vector is a feasible approach to inoculate vectors and lead to transduction of cells in the inner ear. METHODS: A unilateral middle ear application of an adenoviral vector was performed in 4 guinea pigs directly on the round window membrane (RWM) and in 4 additional animals by placing a cotton patch soaked with the vector solution on the RWM. The expression of a reporter gene (lacZ) was used to localize vector-transduced cells. RESULTS: Only one out of 8 animals showed cochlear expression of the reporter gene, whereas all 8 animals showed strong lacZ expression in the middle ear mucosa, in the RWM and in the mucosa surrounding the stapes. CONCLUSION: Our results indicate that the RWM presents a close barrier, almost completely preventing the adenovirus to diffuse into the perilymphatic space. Therefore middle ear application of an adenoviral vector cannot be used to induce inner ear gene transfer. However, middle ear application of a viral vector may be useful for developing treatment for diseases of the middle ear mucosa.     

Transsynaptic delivery of nanoparticles to the central auditory nervous system

Conclusion. Silica nanoparticles may serve as a nonviral delivery system to the sensory hair cells, spiral ganglion cells within the cochlea, and the vestibular organ, as well as the cochlear nucleus. Objectives. At present there are no targeted therapeutics for inner ear disease. A variety of viral vector systems have been tested in the inner ear with variable efficacy but they are still not regarded as safe systems for inner ear delivery. Nanoparticles are a nonviral method of delivering a variety of macromolecules that potentially can be used for delivery within the auditory system. In this study, we evaluated the distribution and safety of nanoparticles in the inner ear. Materials and methods. Cy3-labeled silica nanoparticles were placed on the round window membrane of adult mice. Hearing thresholds were determined after nanoparticle delivery by auditory brainstem responses (ABRs). Distribution of particles was determined by histological evaluation of the cochlea, vestibular organs, and brain stem. Results. Fluorescent microscopy demonstrated Cy3-labeled nanoparticles signals in the sensory hair cells and the spiral ganglion neurons of both the treated and contralateral inner ears. Additionally, the distal part of the central auditory pathway (dorsal cochlear nucleus, superior olivary complex) was found to be labeled with the Cy3-linked silica nanoparticles, indicating a retrograde axonal transport. No hearing loss or inflammation was noted in the treated cochlea.     

Math1基因内耳导入径路的探索研究

目的研究腺病毒携带Math1-EGFP基因经完整圆窗膜途径及鼓阶打孔途径导入耳蜗后对听功能和转导效率的影响,为内耳基因治疗提供实验基础和理论依据。方法健康成年白色红目豚鼠40只,雌雄不限,体重250—300g。随机分成四组,完整圆窗膜组12只,鼓阶打孔组12只,各组分别设对照8只。实验组（24只）导入重组腺病毒携带的Math1基因及增强型绿色荧光蛋白基因（enhanced green fluorescent protein,EGFP）,对照组（16只）导入人工外淋巴液,所有动物均以左耳作为导入耳。术前及术后分别行听性脑干反应（ABR）检查。分别于术后5天、14天取双侧耳蜗标本做基底膜铺片观察基因表达情况。结果完整圆窗膜组导入耳ABR阈值,术后5天各频率与术前比较无显著性差异（P〉0．05）;鼓阶打孔组导入耳ABR阈值,术后5天在2kHz、4kHz与术前比较无差异（P〉0．05）,8kHz较术前增高（P〈0．05）,16kHz、20kHz较术前明显增高（P〈0．01）,术后14天在16kHz、20kHz较术后5天时明显好转（P〈0．01）,但较术前仍有增高（P〈0．05）。转导成功率鼓阶打孔组为91．6％,优于完整圆窗膜组的50％。两种转导途径对目的基因在耳蜗内的表达部位和表达时间没有显著影响。结论完整圆窗膜途径及鼓阶打孔途径在转导成功率及听功能保护方面各有优劣。完整圆窗膜途径因其对耳蜗的损伤极小,在临床应用方面具有更好的发展前景。     

Multi-frequency tympanometry in experimentally-induced cochlear lesions in chinchillas and guinea pigs

OBJECTIVES: To establish that susceptance-conductance tympanograms at a probe-tone frequency of 2 kHz reflects the status of the annular ligament (AL) and through it of the cochlea. METHODS: Experimental study in 5 chinchillas and 22 guinea pigs. Six validating experiments were used: blockages of the stapes and of the round window membrane (RWM), fistula of the RWM, fluid removal from the cochlea, injection of saline in the scala tympani (ST) and acoustic trauma (AT). Quantitative data (mean values of Y226, FR, Y2000, G2000 and B2000) and shape of the curves were analyzed before and immediately after lesions were done. RESULTS: Guinea pig was the most convenient provided bulla was vented and the same tip was used along the experiments. Only the shape of the curves are discriminant: 1/a supplementary sharp peak, centered around negative pressures, is observed in Y/G tympanograms in every case of RWM fistulas and in some case of AT. 2/injection of saline into ST induces immediate and reproducible Y2000, G2000, et B2000 curves modifications. 3/RWM and stapes blockages provoke foreseeable stiffening and sharpening of the tympanograms at 2 kHz. 4/on the contrary, fluid removal from the cochlea induces multiple peaks curves. CONCLUSIONS: Experimentally-induced modifications at the AL either direct (stapes blockage) or indirect by AT or decrease/increase of pressure load at the cochlear interface at the footplate result in noticeable, constant, reproducible changes of curves registered at 2 kHz. The stapes behaves both as the plotter of the curves and the interpreter of the inner ear pressure.     

Efficacy of gene transfer through the round window membrane: an in vitro model

The viral vector-transgene soaked gelatin-sponge method has been shown to be successful in mediating transgene expression across an intact round window membrane (RWM) in mouse in vivo. However, there are many confounding factors which make it difficult to evaluate the role of the RWM in gene transfer. We have created an in vitro model to test the feasibility of gene delivery through an intact RWM. The round window including the bony niche of a CD1 mouse was removed under an operating microscope and fixed with adhesive on the base of a petri dish through which a hole had been drilled. Toluidine blue was injected into the niche containing a hyaluronic acid ester sponge against the round window membrane. The niche was closed with a fascia. A plastic tube containing PBS was fixed on the opposite side, from where the samples were collected at different time points. The concentration of toluidine blue was evaluated spectrophotometrically. An adenoviral vector containing green fluorescent protein (GFP) marker gene was injected into the niche. Samples were collected from the opposite side at different time points. The presence of the vector was studied with GFP PCR. We also modulated the permeability of the RWM by treating it with clinically applicable detergents, histamine or silver nitrate. Silver nitrate and trichloracetic acid caused destruction of the surface epithelium of the RWM as shown by light microscopy. Both toluidine blue and adenoviral vectors passed through the RWM in a time-dependent fashion. RWM cells expressed GFP after Ad-GFP treatment. The permeability of the RWM was decreased after treatment with different detergents, histamine or silver nitrate. RWM offers an atraumatic route to the inner ear. Compared with more invasive gene delivery methods, this technique represents a safer and clinically more viable route of cochlear gene delivery.     

Simulation of application strategies for local drug delivery to the inner ear

Local, rather than systemic, drug delivery to the inner ear is becoming more widely used to treat inner ear disorders. While many substances are undergoing preclinical and clinical studies, it is equally important to develop appropriate drug delivery systems. Pharmacokinetic studies are technically demanding in animals and almost impossible in humans. Computer simulations have helped establish the basic principles of drug distribution in the inner ear. The distribution of methylprednisolone in the guinea pig cochlea has been simulated for different drug delivery systems based on kinetic parameters established in prior studies. Results were compared for different rates of drug clearance from the middle ear. Absolute and relative drug levels in the perilymph were highly dependent on how long the drug remained in the middle ear. For a brief (30 min) application, the basal to apical drug gradient was higher than for longer delivery times. These findings show that controlling middle ear drug clearance is of critical importance.