Establishment of an optimized guinea pig model of cisplatin-induced ototoxicity.

Background: Cisplatin is among the most effective antineoplastic agents and has revolutionized the treatment of many cancer diseases. However, one of its serious side effects is a progressive and irreversible hearing loss, occurring in both adults and children. For the development of otoprotective therapies that prevent this side effect, cisplatin-induced hearing loss animal models are indispensable. Due to the high toxicity of cisplatin, the establishment of such animal models is a difficult and time-consuming task. Here we introduce the detailed protocol of a sophisticated guinea pig model with a sufficient and permanent hearing loss induced by cisplatin. This manuscript is intended to provide guidance in the development of future cisplatin guinea pig models which may reduce the mortality rate of the animals and help to gain more reproducible results. Methods: Pigmented and unpigmented guineapigs were treated with an intravenous single application of 8 mg/kg cisplatin under general anesthesia. An extensive and long-term intensive care protocol consisting of scheduled application of fluids, antiemetics, analgesics, glucose and supportive feeding among others, was used to ensure wellbeing of the animals. Hearing tests were performed prior to and 5 days after cisplatin application. Animals were then euthanized. Results: The ABR audiometry 5 days after cisplatin application revealed a hearing threshold ranging from 70 dB to 90 dB in the frequencies from 1 kHz to 32 kHz respectively.All animals presented a good health condition despite the treatment with cisplatin. Discussion: The introduced care protocol in this manuscript is intended to serve as a guidance for the establishment of a stable guinea pig model for short- and long-term investigation regarding the inner ear and its protection in the frame work of cisplatin-induced damage.

A Novel Preparation Technique for Human Nasal Respiratory Mucosa to Disclose Its Glycosylation Pattern for Bioadhesive Drug Delivery.

To shed some light on glycotargeting as a potential strategy for nasal drug delivery, a reliable preparation method for human nasal mucosa samples and a tool to investigate the carbohydrate building blocks of the glycocalyx of the respiratory epithelium are required. Applying a simple experimental setup in a 96-well plate format together with a panel of six fluorescein-labeled lectins with different carbohydrate specificities allowed for the detection and quantification of accessible carbohydrates in the mucosa. As confirmed by binding experiments at 4 °C, both quantitatively by fluorimetry and qualitatively by microscopy, the binding of wheat germ agglutinin exceeded that of the others by 150% on average, indicating a high content of N-acetyl-D-glucosamine and sialic acid. Providing energy by raising the temperature to 37 °C revealed uptake of the carbohydrate-bound lectin into the cell. Moreover, repeated washing steps during the assay gave a slight hint as to the influence of mucus renewal on bioadhesive drug delivery. All in all, the experimental setup reported here for the first time is not only a suitable approach to estimating the basics and potential of nasal lectin-mediated drug delivery but also meets the needs for answering a broad variety of scientific questions involving the use of ex vivo tissue samples.

The impact of calcium phosphate on FITC-BSA loading of sonochemically prepared PLGA nanoparticles for inner ear drug delivery elucidated by two different fluorimetric quantification methods.

Although therapeutically active proteins are highly efficacious, their content in protective nanoparticles is often too low to elicit adequate plasma levels. A strategy to increase protein loading is the in-situ generation of calcium phosphate as a protein adsorbent. To verify this approach, a highly sensitive and reliable fluorimetric method for quantification of incorporated fluorescein-labelled bovine serum albumin (FITC-BSA) as a model protein drug was developed. Dequenching the fluorescein label by pronase E, which digests the protein backbone, and dissolving the nanoparticle matrix in acetonitrile enabled FITC-BSA quantification in the nanogram per milliliter range. This test was confirmed by a second assay involving alkaline hydrolysis of FITC-BSA and the matrix. Nanoparticles prepared with calcium phosphate contained 40 µg FITC-BSA/mg and nanoparticles without calcium phosphate only 15 µg FITC-BSA/mg, representing a 2.7-fold increase in model protein loading. In this work the nanoparticle preparation procedure was optimized in terms of size for administration in the inner ear, but the range of applications is not limited.

Evaluation of Levels of Triamcinolone Acetonide in Human Perilymph and Plasma After Intratympanic Application in Patients Receiving Cochlear Implants: A Randomized Clinical Trial.

Importance: The use of intratympanically applied steroids is of increasing interest. Consequently, research has focused on finding an ideal drug that diffuses through the round window membrane and can be retained in the perilymph. Objective: To compare levels of triamcinolone acetonide (TAC) in perilymph and plasma after intratympanic injection. Design, Setting, and Participants: This randomized clinical trial included 40 patients receiving cochlear implants at a single tertiary care center in Vienna, Austria. Patients were randomized to 1 of 4 treatment groups receiving 1 of 2 intratympanic doses of TAC (10 mg/mL or 40 mg/mL) at 1 of 2 approximate time points (24 hours or 1 hour) before sampling the perilymph. Inclusion was carried out between November 2017 and January 2020, and data were analyzed in December 2020. Interventions: All patients underwent intratympanic injection of TAC. During cochlear implantation, perilymph and plasma were sampled for further analysis. Main Outcomes and Measures: Levels of TAC measured in perilymph and plasma. Results: Among the 37 patients (median [range] age, 57 [26-88] years; 18 [49%] men) included in the analysis, TAC was present at a median (range) level of 796.0 (46.4-7706.7) ng/mL. In the majority of patients (n = 29; 78%), no drug was detectable in the plasma after intratympanic injection. Levels above the limit of detection were less than 2.5 ng/mL. The 1-factorial analysis of variance model showed lower TAC levels in the group that received TAC, 10 mg/mL, 24 hours before surgery (median, 271 ng/mL) compared with the group that received TAC, 10 mg/mL, 1 hour before surgery (median, 2877 ng/mL), as well as in comparison with the groups receiving TAC, 40 mg/mL, 24 hours before surgery (median, 2150 ng/mL) and 1 hour before surgery (median, 939 ng/mL). The 2-factorial analysis of variance model showed lower TAC levels in the group receiving TAC, 10 mg/mL, 24 hours before surgery than the group receiving TAC, 10 mg/mL, 1 hour before surgery, and higher TAC levels in the group receiving TAC, 40 mg/mL, 24 hours before surgery compared with the group receiving TAC, 10 mg/mL, 24 hours before surgery. Patients with thickening of the middle ear had statistically significantly higher plasma levels (median, 1.4 ng/mL vs 0 ng/mL) and lower perilymph levels (median, 213.1 ng/mL vs 904 ng/mL) than individuals with unremarkable middle ear mucosa. Conclusions and Relevance: In this randomized clinical trial, TAC was shown to be a promising drug for intratympanic therapies, with similar levels in perilymph 1 hour and 24 hours after injection (distinctly in the groups receiving the 40 mg/mL dose). There was also minimal dissemination to the plasma, especially in patients with unremarkable middle ear mucosa. Trial Registration: ClinicalTrials.gov Identifier: NCT03248856.

Metabolite profiling of natural substances in human: in vitro study from fecal bacteria to colon carcinoma cells (Caco-2).

Flavonoids, including anthocyanins, are polyphenolic compounds present in fruits, vegetables and dietary supplements. They can be absorbed from the intestine to the bloodstream or pass into the large intestine. Various bacterial species and enzymes are present along the entire intestine. The aim of the present work was to investigate the intestinal metabolism of selected dietary polyphenol and polyphenol glycosides (quercetin, cyanidin-3-O-glucoside, cyanidin-3-O-galactoside, and delphinidin-3-O-galactoside) by human fecal bacteria. Moreover, the metabolism of metabolites formed from these compounds in human colon carcinoma cells (Caco-2) was also point of the interest. Test compounds were added to fresh human stool in broth or to Caco-2 cells in medium and then incubated for 6 or 20 h at 37°C. After incubation, samples were prepared for LC/MS determination. Main metabolic pathways were deglycosylation, hydrogenation, methylation, hydroxylation, and decomposition. 2,4,5-trihydroxybenzaldehyde, as a metabolite of cyanidin glycosides, was detected after incubation for the first time. Metabolites formed by fecal bacteria were further glucuronidated or methylated by intestinal enzymes. This metabolite profiling of natural compounds has helped to better understand the complex metabolism in the human intestine and this work also has shown the connection of metabolism of natural substances by intestinal bacteria followed by metabolism in intestinal cells.

Intratympanic application of poloxamer 407 hydrogels results in sustained N-acetylcysteine delivery to the inner ear.

N-acetylcysteine is a thiol-containing antioxidant, which has shown otoprotective effects in in vitro as well as in vivo models of cisplatin-induced hearing loss. Systemic administration of antioxidants, however, is associated with the major potential drawback of interference with the tumoricidal effect of cisplatin. This therapeutic limitation can be overcome by local intratympanic injection of the antioxidant N-acetylcysteine, which results in very restricted systemic uptake of the drug, whilst intracochlear drug levels are substantially higher. Furthermore, osmolality and pH properties of formulations for intratympanic injection need to be controlled, as they impact the fraction of drug crossing the barriers of the inner ear and could potentially damage middle and inner ear structures. This study focused on (i) the evaluation of concentration-time profiles of N-acetylcysteine in perilymph, cerebrospinal fluid and plasma after intratympanic administration, (ii) the influence of the dosage form, i.e. a thermoreversible poloxamer 407 hydrogel versus a solution, on N-acetylcysteine pharmacokinetics, and (iii) the development of a pH- and osmolality-adjusted formulation for intratympanic N-acetylcysteine delivery. 49 female albino guinea pigs were randomized into two treatment groups, receiving either a single intratympanic injection of a 4% N-acetylcysteine poloxamer 407 hydrogel or a 4% N-acetylcysteine solution. 8 animals served as untreated controls. N-acetylcysteine levels in perilymph, cerebrospinal fluid and plasma were monitored over a period of 24 h. Samples were taken at 1, 3, 6, 12 and 24 h (poloxamer 407 hydrogel group) and 1, 6 and 24 h (solution group) post injection, and analysed by high performance liquid chromatography-tandem mass spectrometry. Intratympanic application of the 4% N-acetylcysteine poloxamer 407 hydrogel resulted in a 4-fold larger perilymph area under the concentration-time curve (0-24 h) than topical administration of the equally concentrated N-acetylcysteine solution but in similar plasma N-acetylcysteine levels. N-acetylcysteine concentrations in the cerebrospinal fluid were below the level of detection (5 ng/ml) in both treatment groups. N-acetylcysteine-containing formulations applied to the middle ear were isohydric and osmolality was reduced by up to 200 mosmol/kg compared to equally concentrated formulations used in previous studies. In summary, we were able to demonstrate that the intratympanic injection of thermoreversible poloxamer 407 hydrogels increases and sustains N-acetylcysteine delivery to the inner ear. Given the low plasma N-acetylcysteine levels after topical application and the physiological pH and osmolality of the hydrogel, the risk of compromising the antineoplastic effects of cisplatin therapy and of local side effects is minimal.

Long-term effects and potential limits of intratympanic dexamethasone-loaded hydrogels combined with dexamethasone-eluting cochlear electrodes in a low-insertion trauma Guinea pig model.

Cochlear implantation has become the most effective hearing restoration method and is one of the great advances in modern medicine. Early implants have been continuously developed into more efficient devices, and electro-acoustic stimulation is increasingly expanding the indication criteria for cochlear implants to patients with more residual hearing. Therefore, protecting the cochlear structures and maintaining its intrinsic capacities like residual hearing has become more important than ever before. In the present study, we aimed to assess the long-term protective effects of a dexamethasone-eluting electrode combined with the preoperative intratympanic application of a dexamethasone-loaded thermoreversible hydrogel in a cochlear implant guinea pig model. 40 normal-hearing animals were equally randomized into a control group receiving an unloaded hydrogel and a non-eluting electrode, a group receiving a dexamethasone-loaded hydrogel and a non-eluting electrode, a group receiving an unloaded hydrogel and a dexamethasone-eluting electrode and a group receiving both a dexamethasone-loaded hydrogel and a dexamethasone-eluting electrode. Residual hearing and impedances were investigated during a period of 120 days. Tissue response and histological changes of cochlear structures were analyzed at the end of the experiments. Treatment with dexamethasone did not show a significant protective effect on residual hearing independent of treatment group. Although the majority of the cochleae didn't exhibit any signs of electrode insertion trauma, a small degree of tissue response could be observed in all animals without a significant difference between the groups. Foreign body giant cells and osteogenesis were significantly associated with tissue response. Hair cells, synapsin-1-positive cells and spiral ganglion cells were preserved in all study groups. Cochlear implantation using a dexamethasone-eluting electrode alone and in combination with a dexamethasone-loaded hydrogel significantly protected auditory nerve fibers on day 120. Post-implantation impedances were equal across study groups and remained stable over the duration of the experiment. In this study we were able to show that use of a dexamethasone-eluting electrode alone and in combination with preoperative application of dexamethasone-loaded hydrogel significantly protects auditory nerve fibers. Furthermore, we have shown that a cochlear implantation-associated hearing threshold shift and tissue response may not be completely prevented by the sole application of dexamethasone.

Sustained-Release Triamcinolone Acetonide Hydrogels Reduce Hearing Threshold Shifts in a Model for Cochlear Implantation with Hearing Preservation.

INTRODUCTION: In recent years, the preservation of residual hearing has become a major factor in patients undergoing cochlear implantation (CI). In studies attempting to pharmaceutically improve hearing preservation rates, glucocorticoids (GCs) applied perioperatively in many institutions have emerged as a promising treatment regimen. Although dexamethasone is most commonly used and has been applied successfully by various research groups, recently pharmacological properties have been reported to be relatively unsuitable for topical delivery to the inner ear. Consequently other glucocorticoids merit further evaluation. The aim of this study was therefore to evaluate the otoprotective effects of the topical application of a sustained-release triamcinolone acetonide (TAAC) hydrogel in CI with hearing preservation. METHODS: Normal-hearing pigmented guinea pigs were randomized into a group receiving a single dose of a 6% TAAC poloxamer 407 hydrogel, a group receiving a 30% TAAC hydrogel and a control group. All hydrogel applications were performed 1 day prior to CI. After a cochleostomy was drilled, a specifically designed silicone electrode was inserted into the scala tympani for 5 mm. Frequency-specific compound action potentials of the auditory nerve (0.5-32 kHz) were measured pre- and directly postoperatively as well as on days 3, 7, 14, 21, and 28. Finally, temporal bones were harvested for histological evaluation. RESULTS: Application of the TAAC hydrogels resulted in significantly reduced hearing threshold shifts in low, middle and high frequencies and improved spiral ganglion cell survival in the second turn of the cochlea. Outer hair cell numbers in the basal and second turn of the cochlea were slightly reduced after TAAC application. CONCLUSION: In summary, we were able to demonstrate functional benefits of a single preoperative application of a TAAC hydrogel in a guinea pig model for CI, which persisted until the end of the observational period, that is, 28 days after surgery.

Evaluation of Sustained-Release Steroid Hydrogels in a Guinea Pig Model for Noise-Induced Hearing Loss.

The otoprotective effects of thermoreversible poloxamer 407 hydrogels containing dexamethasone or triamcinolone acetonide were evaluated in an animal model of noise-induced hearing loss. Seven days after noise exposure, hearing threshold shifts at 16 kHz were significantly reduced in the 6% dexamethasone group (p < 0.05). Even though no significant differences in hair cell counts were found, histological analysis revealed a significantly higher spiral ganglion cell density in the first turn of the cochlea in this group (p < 0.05). No otoprotective effects were observed after the application of the triamcinolone acetonide hydrogels. As the findings of this study indicate potential otoprotective effects of sustained topical dexamethasone delivery in the setting of noise-induced hearing loss, this strategy merits further evaluation.

Effects of sustained release dexamethasone hydrogels in hearing preservation cochlear implantation.

It has been shown that glucocorticoids reduce the hearing threshold shifts associated with cochlear implantation. Previous studies evaluated the administration of glucocorticoids immediately before surgery or the repeated pre- or perioperative systemic application of glucocorticoids. The aim of this study was to evaluate the effects of a sustained release dexamethasone hydrogel in hearing preservation cochlear implantation. To address this issue, a guinea pig model of cochlear implantation was used. 30 normal hearing pigmented guinea pigs were randomized into a group receiving a single dose of a dexamethasone/poloxamer407 hydrogel one day prior to surgery, a second group receiving the hydrogel seven days prior to surgery and a control group. A silicone cochlear implant electrode designed for the use in guinea pigs was inserted to a depth of 5 mm through a cochleostomy. Compound action potentials of the auditory nerve (frequency range 0.5-32 kHz) were measured preoperatively, directly postoperatively and on postoperative days 3, 7, 14, 21 and 28. Following the last audiometry, temporal bones were harvested and histologically evaluated. Dexamethasone hydrogel application one day prior to surgery resulted in significantly reduced hearing threshold shifts at low, middle and high frequencies measured at postoperative day 28 (p < 0.05). Application of the hydrogel seven days prior to surgery did not show such an effect. Dexamethasone application one day prior to surgery resulted in increased outer hair cell counts in the cochlear apex and in reduced spiral ganglion cell counts in the basal and middle turn of the cochlea, a finding that was associated with a higher rate of electrode translocation in this group. In this study, we were able to demonstrate functional benefits of a single preoperative intratympanic application of a sustained release dexamethasone hydrogel in a guinea pig model of cochlear implantation.