Pharmacokinetic Studies of ADAC after Systemic Administration

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Chapter 3 Effects of ADAC on Impulse Noise Injury after Local Drug Administration

Introduction

This chapter describes the studies investigating ADAC pharmacokinetics after local (intratympanic) administration and the effect of locally administered ADAC on cochlear injury induced by exposure to impulse noise. Since the analytical methodology was well established in the previous study, the pharmacokinetics of ADAC in cochlear tissue following local (intratympanic) administration was determined using the RP-HPLC method. The sensitivity of this method was considered to be sufficient to detect presumably high concentrations of ADAC in cochlear tissues after local administration. The method was validated in cochlear tissue in accordance to the US FDA guidelines on bioanalytical method for linearity, selectivity/specificity, precision and accuracy (US Department of Health and Human Services FDA, 2001). This chapter also examined the potential otoprotective effect of ADAC in rats exposed to impulse noise after local (intratympanic) drug administration. Thewere measured functionally using auditory brainstem responses and quantitative histology (hair cell and spiral ganglion neuron counts).

Materials and Methods

Experimental Outline

The first part of the chapter was designed to determine the pharmacokinetics of ADAC (1 mM) in cochlear tissue of adult Wistar rats (6-8 weeks) after intratympanic administration. The cochleae were removed at intervals after drug administration (Table 19A), and the homogenised cochlear tissues were analysed by RP-HPLC. In the second part of the chapter, the local administration route was used in order to assess the potential otoprotective effects of ADAC in rats exposed to impulse noise (Table 19B). Adult Wistar rats (6-8 weeks) were exposed to 75 pairs of acute impulse noise at 155 dB SPL for 75 seconds (1 second interval). A single dose of ADAC was administered locally (intratympanically) 6 hours after noise exposure. Functional outcomes were measured using ABR, while cochlear injury was assessed by evaluating HC and SGN survivals. The procedures of drug preparation, RP-HPLC instrumentation, RP-HPLC conditions, cochlear extraction and purification, pharmacokinetic analysis, auditory brainstem responses, equipment and preparation of animals, ABR recording and threshold determination, impulse noise exposure, spiral ganglion neuron counts, and statistical analysis were described in Chapter 2.

RP-HPLC Validation Procedure

The assay was validated in rat plasma in accordance to the US FDA guidelines on bioanalytical method for linearity, selectivity/specificity, precision and accuracy (US Department of Health and Human Services FDA, 2001). Selectivity/specificity was examined by extracting blank homogenised cochlear tissue samples from three different rats to look for any endogenous peaks that could interfere with the ADAC peak. Accuracy and precision were evaluated by analysing the matrix spike samples at two concentration levels: low QC (16.67 µg/mL) and high QC (50 µg/mL). Five replicates were examined per QC concentration. Accuracy was calculated by comparing the detected concentration with the true spiked concentration in plasma. Precision was expressed as relative standard deviations (RSD %). For RP-HPLC, calibration standards of 10 concentrations in the range 0.05 to 150 µg/mL were prepared using vehicle (0.9% saline) solution, whilst for LCMS/MS, another 10 concentrations ranging from 0.01 to 150 ng/mL were prepared. Calibration curves obtained in this study included a blank sample, five non-zero samples including the lower limit of quantification (LLOQ). LLOQ was evaluated on the signal to noise ratio of 5:1 with precision and accuracy within 20% of the nominal value. Linearity was assessed by preparing calibration curves and plotting the peak area size of ADAC against its concentration.

Animals

Male Wistar rats (6-8-weeks old; average weight of 250 g) were supplied by the Vernon Jansen Unit (an animal facility at the University of Auckland). All experimental procedures described in this study were approved by the University of Auckland Ethics Committee (AEC number: R935) in accordance with the Animal Welfare Act 1999. The animals had free access to food and water throughout the experiment. In the RP-HPLC study, 12 animals were randomly assigned to the group. In the functional and quantitative histology study, the animals were randomly allocated to one of the two experimental groups (control vehicle group, n = 8; ADAC 100 µM group, n = 7). Table 19B summarises the experimental groups in this study.

Local Drug Administration

For intra-tympanic ADAC administration, animals were anaesthetised with ketamine (25 mg/kg) and Domitor® (0.5 mg/kg) delivered intraperitoneally. The pedal withdrawal reflexes were used to determine the level of anaesthesia. Once the animal was anaesthetised to a surgical level, it was positioned lying on its side and the tympanic membrane visualised under the Leica Wild M3Z dissection microscope. A Hamilton syringe (50 µL) filled with 30 µL freshly prepared ADAC or vehicle solution was anchored to a Narishige MM-3 micromanipulator mounted on GJ-8 stand. During the drug administration, the syringe needle was carefully lowered inside the ear canal towards the middle ear cavity. After piercing the tympanic membrane, ADAC was injected close to the RWM. ADAC was also injected into the contralateral ear of the animal after 30 min. At the end of the experiment, the animals were euthanized with an anaesthetic overdose (pentobarbitone, 90 mg/kg, i.p.) and both sides of the cochleae were collected.

Drug Preparation for Local Administration

Intratympanic drug injections were also used to investigate the otoprotective effects of adenosine amine congener (ADAC). ADAC was mixed with poloxamer-407 powder (Sigma-Aldrich), resulting in a solution which is in a liquid state below room temperature, but converts to a viscous gel at normal body temperature. This allowed for prolonged drug release, which is a significant advantage compared to aqueous formulations (Wang et al., 2009; Bogosanovic, 2015). A frozen stock solution of ADAC (2 mM) was thawed and prepared by diluting with vehicle solution (0.4% 1 M HCl in 0.9% Saline), then diluted to a designated concentration (100 µM) for intratympanic injection. Vehicle and drug solutions were mixed with 17% w/w poloxamer-407 powder, placed on ice and left for approximately one hour to allow the poloxamer-407 powder to fully dissolve. As ADAC is light sensitive, all solutions were covered in tin foil and left in the dark. Once poloxamer-407 powder was fully dissolved, solutions were aliquoted in sterile Eppendorf tubes and frozen at -20°C for later use. The procedure of local administration was described in 3.2.4 .

Analysis of Hair Cell Survival

Cochleae obtained from noise-exposed and control animals were fixed in 4% PFA as described previously, and decapsulated under the microscope. The lateral wall, tectorial membrane, and Reissner’s membrane were removed. The organ of Corti was then separated from the modiolus, with focus on preserving the entire length of the organ of Corti. Wholemounts of the apical, middle, and basal turns were placed in a 24 well-plate for further processing. Wholemounts of the organ of Corti were then permeabilised with 1% Triton X-100 for one hour, and washed with 0.1 M PBS (3 x 20 mins). Tissues were then incubated with phalloidin – Alexa Fluor 488 (Invitrogen) dissolved in 0.1 M PBS for one hour in the dark. Phalloidin is a high-affinity filamentous actin (F-actin) probe which labels cuticular plates and stereocilia of the sensory hair cells. Following three washes in 0.1 M PBS, tissues were mounted onto microscope glass sides with Citifluor AF1 mounting medium containing PBS and glycerol, covered with a coverslip and sealed with nail polish. Slides were imaged with an Axiocam camera controlled by NIS-elements BR 2.30 software at 20x magnification. Images of hair cells were taken with a laser wavelength of 488 nm for the detection of Alexa Fluor 488 fluorescence. The hair cell in all animals were displayed by ImageJ. The images of the entire length of the cochlea were taken, and the number of missing hair cells was counted in each turn (apical, middle and basal) and presented as percentage of the total number of hair cells.

Chapter 1 Literature Review
1.1 Anatomy of the Ear
1.2 Auditory Transduction
1.3 Auditory Neurotransmission
1.4 Noise Induced Hearing Loss (NIHL) and its Causes
1.5 Experimental Treatments for NIHL
1.6 Adenosine Receptor Signalling in the Cochlea
1.7 Adenosine Receptors and NIHL
1.8 Treatment of NIHL with Adenosine Receptor Agonists
1.9 Thesis Objectives
Chapter 2 Pharmacokinetic Studies of ADAC after Systemic Administration
2.1 Introduction
2.2 Materials and Methods
2.3 Results
2.4 Discussion
Chapter 3 efects of ADAC on Impulse Noise Injury after Local Drug Administration
3.1 Introduction
3.2 Materials and Methods
3.3 Results
3.4 Discussion
3.5 Limitations and Future Directions
Chapter 4Conclusions and General Discussion

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A thesis submitted in fulfillment of the requirements for the degree of Doctor of Philosophy in Physiology University of Auckland, 2016