1. Background

Ototoxicityis a well-known side effect of the anti-neoplastic agent cisplatin. Recently,clinicians have advocated the use of otoacoustic emissions (OAEs) to measurecisplatin-induced ototoxicity as they not only provide a quick and objectivemeans of assessing cochlear function, they can also identify ototoxicityearlier than conventional pure tone audiometry. There are reports of reductionof OAE amplitude following cisplatin chemotherapy in cancer patients.

Animal studies attempting to investigate themechanism(s) of cisplatin ototoxicity have used doses of cisplatin that aredifficult to compare with those used clinically in humans. The extrapolation offindings from these animal studies to humans has, therefore, been difficult.Furthermore, individual variability in susceptibility to cisplatin-inducedototoxicity has been reported in animal experiments by some researchers. Whilststudies relating plasma concentrations of total platinum to ototoxic changesunsuccessfully attempted to explain this variability, there have beensuggestions that it could possibly be explained by inter-individual differencesin plasma concentrations of filterable rather than total platinum (Pt).

2. Aims of the study

In the present study, a dog model was used todelineate the early effects of cisplatin on OAEs, and to investigate therelationship between cisplatin ototoxicity and pharmacokinetics of platinum inindividual animals.

The dog has several advantages over smallmammalian species (rats, guinea pigs and gerbils) in that they receive routinecisplatin chemotherapy at levels similar to those administered to humans for thetreatment of a variety of carcinomas (1), and they are suitable for multipleblood sampling for plasma platinum (Pt) analysis because of their relativelylarge blood volume. In addition, valid OAE responses have been recorded in themusing commercially available equipment (2, 3,4).

3. METHODS

3.1 Subjects

Five healthymixed breed dogs were used (see table 1).Only dogs that had intact external ear canal, normal middle ear function,

ABR thresholds equalto or less than 20 dB nHL and r

eplicableOAE recordings were included in the study.

Table 1            Demographicdetails of dogs

3.2 Audiological Measurements

Bilateralotoscopy and tympanometry were performed to rule out external and middle eardisease and dysfunction. Baseline click-evoked auditory brainstem response(ABR) and otoacoustic emission (OAE) measurements were recorded 2 days prior tocisplatin infusion. These measurements were repeated 2 days and 4 days aftercisplatin administration and compared with baseline measurements

3.3 Procedure

OAEs were measuredusing the ILO’92 Otoacoustic emission analyzer system.

Both click-evoked TEOAEs (from both the user ordefault setting [US] and the quickscreen programs [QS]) and tone-evoked 2f1-f2DPOAEs were recorded. 2f1-f2 DPOAEs were recorded to two different stimuluslevel combinations: 65/55 dB SPL and 55/55 dB SPL.

The magnitude of TEOAEs, the amplitude of DPOAEs (fromthe DP-grams) and the DPOAE thresholds (based on the input/output function)were used in the analysis of results.

OAE and ABRmeasurements (6) were performed while the dogs were anaesthetized.

Each dog was administered a single dose of 100 mg/m²of cisplatin intravenously by slow infusion.

3.4Platinum analysis

                   Totalplasma cisplatin concentration was determined on 0.1ml plasma, while thefilterable fraction (the pharmacologically active form of the drug) wasmeasured by centrifuging 0.4 ml of plasma in ultra-filtration (UF) filters(Millipore Ultrafree 10000 NMWL, Bedford, MA, USA) for 30 min at ~ 1,600 g at 4 ºC. All samples were stored at-70ºC until assayed for Pt content. The plasma samples were diluted 10-fold inhigh purity water with the addition of 100 ug/L of indium (In) as the internalstandard and then vortexed to achieve solution homogeneity in readiness for Ptanalysis. Inductively coupled plasma-mass spectrometry (ICP-MS) based on amethod reported by Taylor et al. (5),was used to determine Pt concentration. The instrument was a VG PlasmaQuad (VGElemental, Winsford, UK) in standard configuration with a Meinhard nebuliserand a double pass, water-cooled borosilicate spray chamber. All ICP-MSmeasurements were determined in peak-jumping mode by selection of mass Pt(33.8% natural abundance) together with In (95.7% natural abundance) as theinternal standard. An external calibration technique using matrix-matched standardscoupled with internal standardisation was employed for quantification. With thevolumes of sample available, the minimum quantifiable Pt concentration (MQC)was set to 25 mg/L inplasma and the plasma ultrafiltrate, and 0.1mg/g dry mass of tissue(coefficient of variation, CV% <5%).

3.5 Pharmacokinetics

                   Plasmaconcentrations of total Pt as well as filterable Pt were plotted against timeto visualise the changes in concentration over the 48 h period. Theserum-concentration time data for filterable Pt (the pharmacologically activespecies) were fitted to a bi-exponential model using the NONMEM program. Dataobtained during the infusion period were incorporated in the kinetic analysis.

3.6Post-mortem OAE measurements

Dogs were euthanased using an intravenous injection of Lethabarb (325 mg/ml ofpentobarbitone sodium)

.TEOAEs using the US and QS programs and DP-grams to55/55 dB SPL and 55/45 dB SPL stimulus primaries were measured 10 minutes afterdeath to ascertain the biological validity of OAE responses.

4. RESULTS

4.1 DPOAEthreshold shifts following cisplatin infusion

            The ANOVA forrepeated measures among days showed a significant difference (p<0.05) forDPOAE thresholds at 6 kHz (see Table 2 and Figure 1). Post-hoc, pairwise comparisonby the Least Significantly Different (LSD) test revealed a significantdifference between DPOAE thresholds at pre-infusion and 4 days post infusion(p<0.05), and between 2 days post-infusion and 4 days post-infusion(p<0.01)[ see Table 2].

Table 2:

            Mean and SD of DPOAE thresholdsrecorded pre-infusion and 2 and 4 days post-infusion of cisplatin

* Significant decreaseon ANOVA for repeated measures

Figure  1:                     Boxplots showing the distribution of DPOAE thresholds at 6 kHz (L1/L2 = 55/55 dBSPL) at pre-infusion and 2 and 4 days post infusion of cisplatin. The lines atthe top and bottom of box (whiskers) represent the largest and the smallestvalues. The shaded area of the rectangular box represents the inter-quartilerange (IQR) and the horizontal line in the box represents the median.

 4.2 Platinum Analysis

                   The plasma concentration-timeprofile of free and total Pt in individual dogs over 48 hr followingadministration of cisplatin is shown in Figure 2a to 2e.  The concentrations offilterable Pt as well as the total plasma Pt reached a peak at 20 min aftercommencing the cisplatin infusion. Filterable Pt displayed log-linear decaywith a rapid initial phase (5 dogs, mean half-life 6 hr; range 2.9-7.0 hr)followed by a slower, second phase (4 dogs, mean life 15.8 h, range 7.98-19.4h). In dog 2, data could only be obtained for the initial phase because of avery rapid rate of Pt elimination.

Figurea: Dog 1

                                                Figure b: Dog 2

                                                 Figure c:  Dog 3

                                                Figure d: Dog 4

                                                Figure e: Dog 5

Figure 2:Plasmaconcentration levels of total and filterable Pt in dog 1 to dog 5 (Figures a toe) over 48 hours following the infusion of 100 mg/m² of cisplatin.O=Total Pt, ·=FilterablePt     

4.3 Post-mortem OAE and ABR measurements

Post-mortem TEOAEs(using US and QS programs) and DP-grams recorded to 55/55 dB SPL and 55/45 dBSPL primaries 10 min after death were absent.

This demonstrated that the OAE responses recorded inthe present study were indeed biological in origin and that they reflected themore active process of the cochlea.

5.Conclusion

                   Inconclusion, the present study found that DPOAEs, in particular DPOAE thresholdsmeasured to L1/L2:55/55 dB stimulus primaries at 6 kHz , have the potential tobe used as a clinical measure to detect early cisplatin-induced ototoxicity. Inaddition, the present study revealed that the variation in the elevation ofDPOAE thresholds between animals could not be attributed to variations inplasma concentrations of filterable Pt. However, there are limitations in thispreliminary study that need to be addressed in future studies. First, a largernumber of dogs may be needed to provide a more accurate assessment of theinter-animal variability in OAE and ABR responses. Second, the frequency rangeof the OAE measurements in the present study was limited to 6kHz. Whilst thiswas a limitation of the commercially available equipment used in the study,future studies employing equipment (be it a customized system or a commerciallyavailable one) that would allow collection of OAE data at frequencies up to 20kHz in order to capture any early ototoxic effects of cisplatin at the higherfrequencies are warranted. Finally, cisplatin is administered every 3 or 4weeks cyclically to dogs in veterinary oncology clinics [14] whereas in thepresent study only the effects of a single of such high doses of cisplatin werestudied. Future studies may benefit by addressing any cumulative effects ofrepeated clinical doses of cisplatin on OAEs.

6.References:

1. Shapiro, W. (1989). Cisplatin chemotherapy. In Kirk RW, Bonagura JD (Eds). Current Veterinary Therapy. 10, Small Animal Practice (pp.497-502). Philadelphia: WB Saunders, 497-502.
2. Sims, M.H., Rogers, R.K., & Thelin, J.W. (1994). Transiently evoked otoacoustic emissions in dogs. Progress in Veterinary Neurology, 5, 49-56.
3. Sockalingam, R, Charles, B, Murdoch, B (1999). The role of otoacoustic emissions in the identification and monitoring of cisplatin-induced ototoxicity.  The Journal of Audiological Medicine, 8(1), 1-17.
4. Sockalingam, R, Filippich, L, Sommerlad, S., Murdoch, B., & Charles, B. (1998). Transient-evoked and 2F1-F2 distortion product otoacoustic emissions in dogs: Preliminary findings. Audiology & Neuro-Otology, 3, 373-385.
5. Taylor, A., Branch, S, Crews, H..M., & Halls, D.J. (1993). Atomic Spectrometry - Clinical and biological materials, foods and beverages. Journal of Atomic Spectrometry, 8, 79-113.
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