Dexamethasone concentration gradients along scala tympani after application to the round window membrane

Abstract

Hypothesis: Local application of dexamethasone-21-dihydrogen-phosphate (Dex-P) to the round window (RW) membrane of guinea pigs produces a substantial basal-apical concentration gradient in scala tympani (ST) perilymph.

Background: In recent years, intratympanically applied glucocorticoids are increasingly being used for the treatment of inner ear disease. Although measurements of intracochlear concentrations after RW application exist, there is limited information on the distribution of these drugs in the inner ear fluids. It has been predicted from computer simulations that substantial concentration gradients will occur after RW application, with lower concentrations expected in apical turns. Concentration gradients of other substances along the cochlea have recently been confirmed using a sequential apical sampling method to obtain perilymph.

Methods: Dexamethasone-21-dihydrogen-phosphate (10 mg/ml) was administered to the RW membrane of guinea pigs (n = 9) in vivo for 2 to 3 hours. Perilymph was then collected using a protocol in which 10 samples, each of approximately 1 mul, were taken sequentially from the cochlear apex into capillary tubes. Dexamethasone-21-dihydrogen-phosphate concentration of the samples was analyzed by high-performance liquid chromatography. Interpretation of sample data using a finite element model allowed the longitudinal gradients of Dex-P in ST to be quantified.

Results: The Dex-P content of the first sample in each experiment (dominated by perilymph from apical regions) was substantially lower than that of the third and fourth sample (dominated by basal turn perilymph). These findings qualitatively demonstrated the existence of a concentration gradient along ST. After detailed analysis of the measured sample concentrations using an established finite element computer model, the mean basal-apical concentration gradient was estimated to be 17,000. Both absolute concentrations of Dex-P in ST and the basal-apical gradients were found to vary substantially.

Conclusion: The existence of substantial basal-apical concentration gradients of Dex-P in ST perilymph were demonstrated experimentally. If the variability in peak concentration and gradient is also present under clinical conditions, this may contribute to the heterogeneity of outcome that is observed after intratympanic application of glucocorticoids for various inner ear diseases.

Fig. 1

Sequential apical sampling: A: Cartoon drawing of the view to the opened guinea pig otic bulla through a ventrolateral surgical approach showing the tympanic membrane (TM) and the cochlea (I, II, III: cochlear turns) and drug application pipette (AP) in the RW niche. After removal of the mucosa, cyanoacrylate glue (Aesculap, Tuttlingen, Germany) was applied to the apex and a silicone “cup” (two-part silicone elastomer sealant, KWIK-Cast, WPI, Sarasota, USA) was constructed (1). B: Cross section of the cochlear apex with the layer of cyanoacrylate (CA) and silicone over the bony surface covering the perilymphatic space (PL). C: At the end of the application (2), the pump was stopped and the RWM niche was carefully wicked dry avoiding any contact with the round window membrane (RWM). When the cochlear apex is perforated, perilymph is slowly expelled, driven by cerebrospinal fluid pressure. D: The expelled fluid was collected in calibrated glass capillaries (Blaubrand^® intraMARK, BRAND GmbH, Wertheim, Germany) used as sampling pipettes (SP), (3).

Fig. 2

Left: When the cochlear apex is perforated, cerebrospinal fluid (CSF) is slowly expelled through the cochlear aqueduct entering at the base of scala tympani (black arrow). Sequential samples taken from the apex contain perilymph from different regions (–4) from scala tympani. During the drug application and the sampling process substance is also partitioned and repartitioned to and from other compartments like the modiolus, scala vestibule, endolymphatic space and lateral wall (dotted arrows). Middle/Right: Each curve shows the measured Dex-P concentrations in 10 samples taken sequentially from the cochlear apex. The first 4 samples (grey area) contain mainly perilymph from different regions of scala tympani (1–4 on left panel). The higher concentration of the 3^rd and 4^th samples (basal region) relative to the first sample (apical region) indicates a significant basal-apical concentration gradient along ST. The following samples (numbers 5 to 10) mainly contain cerebrospinal fluid which has replaced and diluted ST perilymph during the sampling process. The measured Dex-P levels in these later samples can be explained by repartitioning of the drug from other cochlear tissues back into scala tympani. A: absolute concentrations, B: normalized to concentration in the 4^th (most basal) sample.

Fig. 3

Concentrations of the first four samples taken in each experiment shown as absolute values (A), on a logarithmic scale (B), and normalized to the concentration in sample number 4 (C). The distance along scala tympani plotted is that of the mid-point of the estimated region of origin (half the sample volume apical and half the volume basal to the location), as indicated by the insert in panel A. Despite the limited spatial resolution based on the four samples, all graphs clearly demonstrate the existence of significant basal-apical concentration gradients.

Fig. 4

Left: Interpretation of sample concentrations using a computer model: An example of a best fit between measured and simulated sample concentrations is shown. Middle and right: Dex-P gradients along scala tympani derived from simulations of each experiment: Concentration gradients are plotted on an absolute concentration scale (A) and a logarithmic scale (B).

Fig. 5

At the time of sampling part of the applied prodrug (Dex-P) was converted into the active moiety (Dex). The sample concentration courses between the active moiety (Dex) and the applied prodrug (Dex) were compared in three experiments (A, B, C). Although concentrations for Dex were lower, the sample concentration course followed the one for Dex-P.