METHOD OF PREVENTING COCHLEAR DISEASE, DISORDER OR CONDITION USING (+)-AZASETRON

Abstract

Disclosed is a method for treating ear disorders, including the administration of (+)-azasetron, or a pharmaceutically acceptable salt and/or solvate thereof.

Claims

1. A method of prevention of a cochlear disease, disorder or condition in a subject in need thereof, wherein said method comprises the administration to the subject of a composition comprising (+)-azasetron, of Formula (R)-I: ##STR00006## or a pharmaceutically acceptable salt and/or solvate thereof, and at least one pharmaceutically acceptable excipient.

2. The method according to claim 1, wherein the composition comprises at least a 90:10 w/w mixture of (+)-azasetron of Formula (R)-I:(−)-azasetron, or pharmaceutically acceptable salts and/or solvates thereof.

3. The method according to claim 1, wherein the pharmaceutically acceptable salt of (+)-azasetron of Formula (R)-I is selected from the group consisting of: (+)-azasetron besylate, (+)-azasetron malate, and (+)-azasetron hydrochloride.

4. The method according to claim 1, wherein the composition is administered at a dose ranging from about 0.01 mg to about 100 mg.

5. The method according to claim 1, wherein the composition is administered at a dose ranging from about 20 to about 100 mg of free-base equivalent per day.

6. The method according to claim 1, wherein the composition is administered systemically.

7. The method according to claim 1, wherein the composition is administered orally.

8. The method according to claim 1, wherein the composition is administered locally.

9. The method according to claim 1, wherein the cochlear disease, disorder or condition is sensorineural hearing loss, cochlear ototoxicity, cochlear excitotoxic-inducing occurrence, cochlear hyperacusis, or cochlear tinnitus.

10. The method according to claim 1, wherein the cochlear disease, disorder or condition is ototoxic compound-induced hearing loss.

11. The method according to claim 1, wherein the cochlear disease, disorder or condition is platinum-induced hearing loss.

12. The method according to claim 1, wherein the cochlear disease, disorder or condition is cisplatin-induced hearing loss.

13. The method according to claim 1, wherein the subject is diagnosed with cancer.

14. The method according to claim 1, wherein the subject is diagnosed with cancer and is awaiting the receipt of, or is receiving platinum-based chemotherapy.

15. The method according to claim 1, wherein the subject is diagnosed with cancer and is awaiting the receipt of, or is receiving cisplatin, carboplatin, oxaliplatin or a combination thereof.

16. The method according to claim 1, wherein the subject is diagnosed with cancer and wherein the composition is administered before, during and/or after platinum-based chemotherapy.

17. The method according to claim 1, wherein the subject is diagnosed with cancer and wherein the composition is administered before, during and/or after cisplatin-, carboplatin- or oxaliplatin-based chemotherapy or a combination thereof.

18. The method according to claim 1, wherein the cochlear disease, disorder or condition is sudden sensorineural hearing loss, noise-induced hearing loss, or age-related hearing loss.

19. The method according to claim 1, wherein the composition is an immediate release composition or a sustained release composition.

20. The method according to claim 1, wherein the composition is an orodispersible composition.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0221] FIG. 1 is a set of three graphs comparing the concentration (nM) of the racemic and two enantiomers of azasetron in the perilymph, the inner ear, and the peripheral plasma (tail plasma) after oral administration, at termination time-point. Average per time-point subgroups within treatment groups (+/−SE).

[0222] FIG. 2 is a graph comparing the effects of the racemic and two enantiomers of azasetron on auditory brainstem response (ABR) threshold shifts in a sensorineural hearing loss (SSNHL) model.

[0223] FIG. 3 is a set of two graphs showing the effect of the (+) enantiomer of azasetron at different doses on auditory brainstem response (ABR) threshold shifts and distortion product otoacoustic emission (DPOAE) amplitude loss in a SSNHL model. Statistical analyses were performed using 2-way analysis of variance (ANOVA) (treatment group, stimulus frequency) with Holm-Sidak post-test versus vehicle controls. A P value of <0.05 was statistically significant.

[0224] FIG. 4 is a set of two graphs showing the effect of azasetron racemate treatment against acoustic trauma by oral administration.

[0225] FIG. 5 is a set of two graphs showing auditory brainstem response (ABR) threshold shifts (A) and distortion product otoacoustic emission (DPOAE) amplitude loss (B) following cisplatin infusion after 14 days of oral treatment with placebo (n=4) or 26.4 mg/kg (+)-azasetron besylate (n=5).

[0226] FIG. 6 is a graph showing the survival of cochlear outer hair cells following cisplatin infusion after 14 days of oral treatment with placebo (n=4) or 26.4 mg/kg (+)-azasetron besylate (n=5).

[0227] FIG. 7 is a set of two graphs showing auditory brainstem response (ABR) threshold shifts (A) and distortion product otoacoustic emission (DPOAE) amplitude loss (B) following cisplatin infusion after 14 days of oral treatment with placebo (n=6), 6.6 mg/kg (+)-azasetron besylate (n=7) or 13.2 mg/kg (+)-azasetron besylate (n=5).

[0228] FIG. 8 is a graph showing the survival of cochlear outer hair cells following cisplatin infusion after 14 days of oral treatment with placebo (n=5), 6.6 mg/kg (n=5) or 13.2 mg/kg (n=5) (+)-azasetron besylate treatment.

[0229] FIG. 9 is a set of two graphs showing the effect of (+)-azasetron besylate (5.6 mg/kg; n=16) versus placebo (n=18) on unilateral vestibular dysfunction induced by intratympanic kainic injection over time (in hours). A, Spontaneous nystagmus. B, Roll angle deviation.

EXAMPLES

[0230] The present invention is further illustrated by the following examples.

Example 1: Pharmacokinetics Study

Material and Methods

Test Animals Assignment to the Study

[0231] The 72 male Wistar rats received on 28 Jan. 2016 from Janvier (Janvier, Le Genest-St-Isle) were assigned to the study. They were divided in 3 groups of 24 animals each, one group per test article. Each group was divided in 7 subgroups according to the 6-termination time-points.

[0232] 72 Wistar rats were subjected to oral single dose administration of the test article, racemic azasetron hydrochloride, azasetron hydrochloride (+) or azasetron hydrochloride (−), and sacrificed at different time-points to assess the exposure of the test article in the inner ear, perilymph, and plasma. [0233] Group racemic azasetron: 24 male Wistar, 4 rats per time-point: 10 mg/kg azasetron HCl dissolved in NaCl 0.9%. Administrated as 5 mL/kg p.o.; [0234] Group azasetron (+): 24 male Wistar, 4 rats per time-point: 10 mg/kg (+)-azasetron HCl dissolved in NaCl 0.9%. Administrated as 5 mL/kg p.o.; [0235] Group azasetron (−): 24 male Wistar, 4 rats per time-point: 10 mg/kg (−)-azasetron HCl dissolved in NaCl 0.9%. Administrated as 5 mL/kg p.o.

[0236] There were 6 termination time-points for each test article. At time-points 15/30/60/120/240/480 minutes after administration: peripheral plasma from the tail was sampled. The animals were sacrificed by i.v. administration of 0.25 mL/rat of a 160 mg/mL pentobarbital solution. Following sacrifice, the heart blood and both tympanic bullas (left and right) were sampled.

Sample Preparation for Bio-Analytics

Perilymph and Inner Ear Samples

[0237] Both tympanic bullas (left and right) were collected, however, only the left one was used for the perilymph and inner ear analysis. The right bulla was stored at −20° C. for future needs when required. For animal IDs 1 and 4, the right bulla was used for the sampling of perilymph and inner ear.

[0238] The left bulla was dissected fresh. Briefly, the bulla was opened via the auditory opening to reveal the bone surrounding the cochlea after which the tip of the bone was removed with forceps and a 10 μL pipette tip was inserted to withdraw perilymph.

[0239] Upon exposure of the tip of the cochlea, 2 μL of perilymph (fluid) could be recovered from the bulla. The perilymph was brought to 10 μL by the addition of 8 μL water, and then extract via the addition of 30 μL of acetonitrile. After that, the samples were centrifuged and the supernatant was transferred to a glass vial appropriate for the HPLC-MS auto-sampler.

[0240] Following recovering of the perilymph, the bones of the inner ear and cochlea components were removed with fine forceps and added to a tared Eppendorf tube. The collection was weighed and brought to 10 mg with the addition of water and placed on ice until extraction. 30 μL of acetonitrile was added and the tubes were incubated in an ultrasound bath, after which they were centrifuged and the supernatant was transferred to a glass vial appropriate for the HPLC-MS auto-sampler. In the process or removing the bones of the inner ear, they were finely distributed in the media and broken by forceps. (Due to the very low volume of the tissue and its fine structure, separate grinding is considered risky vs the potential losses to plastic and media. The materials are very fine in nature and the bone particles are small thus providing only short interfaces to media. The absence of fat and the incubation in solvent is intended to provide sufficient solubility of active ingredient).

[0241] The acetonitrile used in the sample preparation for bio-analysis contained the internal standard for the HPLC-MS analysis.

Tail Plasma

[0242] 30 μL acetonitrile including the internal standard were added to each 10 μL plasma sample. After adding the acetonitrile, the samples were mixed by vortex and further centrifuged. The supernatant was transferred to a glass vial appropriate for the HPLC-MS auto-sampler.

Heart Plasma

[0243] The plasma samples from the heart blood are stored at −20° C. for future optional analysis when needed.

LC-MS/MS Apparatus

[0244] The LC system comprised an Agilent 1260 liquid chromatography equipped with a binary pump and a column oven, together with a PAL HTS-xt auto-sampler. It is linked to the AB SCIEX TRIPLE QUAD™ 4500 (triple-quadrupole) instrument with an ESI interface and a switching valve integrated (Mass spectrometric analysis). The data acquisition and control system were created using Analyst Software from Applied Biosystems Inc.

[0245] The test article, azasetron hydrochloride, used for the standard curve was provided by the Sponsor. Water and acetonitrile were HPLC grade. All other solvents and chemicals were analytical grade or better.

Results

Plasma Kinetic and Distribution to Inner Ear of Azasetron Following a Single Oral Dose of Racemic Azasetron, (+)-Azasetron Hydrochloride and (−)-Azasetron Hydrochloride in Wistar Rats

[0246] This study was conducted to compare exposure to azasetron following administration of a single oral dose (10 mg/kg) of racemic azasetron hydrochloride, (+)-azasetron hydrochloride and (−)-azasetron hydrochloride. The concentration of the test articles in the inner ear samples were analyzed by HPLC-MS and quantified by comparison with a known concentration standard curve (Table 1).

TABLE-US-00001 TABLE 1 Concentration (ng azasetron free-base/g tissue) of racemic azasetron, (+)-azasetron and (−)-azasetron in the inner ear tissue at termination time-point. Average per time-point subgroups within treatment groups, and standard error (SE). Average SE Dose Inner ear Inner ear Treatment (mg/kg) Route Time (ng/g) (ng/g) Racemic azasetron 10 p.o. 15 25 13 30 61 13 60 63 3 120 35 9 240 37 2 480 7 2 (+)-azasetron 10 p.o. 15 57 29 30 86 50 60 93 34 120 79 21 240 52 8 480 6 0 (−)-azasetron 10 p.o. 15 29 15 30 27 16 60 8 1 120 46 11 240 21 6 480 3 1

[0247] The concentration of the racemic and two enantiomers of azasetron hydrochloride in the perilymph, the inner ear and the peripheral plasma is represented in FIG. 1 and Table 2.

TABLE-US-00002 TABLE 2 (AUC) Area under the curve (nM min) of the azasetron free-base pharmacokinetics. AUC nM min Tail Perilymph Inner ear plasma Dose AUC nM AUC nM AUC nM Treatment (mg/kg) Route Time min min min Racemic 10 p.o 15 1099 712 10078 azasetron 30 1965 2019 15807 60 2729 5319 18556 120 3811 8394 22450 240 7026 12260 45507 480 9243 14889 64797 Total AUC 25873 43592 177194 (+)- 10 p.o 15 1161 1225 5897 azasetron 30 1783 3059 11548 60 3102 7643 21012 120 7719 14739 37299 240 13642 22527 66332 480 13504 19844 68681 Total AUC 40910 69036 210768 (−)- 10 p.o 15 304 614 1217 azasetron 30 613 1189 2037 60 898 1484 3092 120 2179 4631 10183 240 4482 11560 24941 480 3425 8351 23978 Total AUC 11901 27829 65448

[0248] Following administration of a single equivalent oral dose of each compound, greater exposure (total AUC) to azasetron was observed in the perilymph, inner ear, and plasma after administration of (+)-azasetron than that observed after administration of racemic azasetron or (−)-azasetron.

Example 2: Activity of (+)-Azasetron Besylate on Noise-Induced Hearing Loss

Material and Methods

Animals

[0249] All experiments were performed using 7-week-old male Wistar rats (CERJ, Le Genest, France) in accordance with the French Ministry of Agriculture regulations and European Community Council Directive no. 86/609/EEC, OJL 358. The rats were fed a standard diet ad libitum and maintained on a 12 hours' light-dark cycle.

Audiometry

[0250] Auditory Brainstem Reponses (ABR) and Distortion Product Otoacoustic Emssions (DPOAE) were recording using a RZ6 Auditory Workstation (Tucker-Davis Technologies, Alachua, Fla., USA) with animals deeply anesthetized using 90 mg/kg ketamine and 10 mg/kg xylazine and placed on a 35° C. recirculating heating pad inside a sound attenuating cubicle (Med Associates Inc., St. Albans, Vt., USA) throughout the experiment.

[0251] For ABR recordings, three stainless steel needle electrodes were placed subdermally over the vertex, the right mastoid and right hind leg of each animal. Tone-pips (5 msec duration presented at a rate of 21/s) at 8, 16 and 24 kHz were delivered to the right ear using a calibrated MF-1 speaker in closed-field configuration (Tucker-Davis Technologies, Alachua, Fla., USA) at attenuating intensity until no reproducible response could be recorded. Close to the ABR threshold, the responses to 1000 acoustic stimuli in 5 dB steps were averaged. Responses were low-pass filtered at 3 kHz.

[0252] DPOAEs were recorded and using ER10B+ Low Noise DPOAE microphone (Etymotic Research, Inc., Elk Grove Village, Ill., USA) with acoustic stimuli delivered by two calibrated MF-1 speakers in closed-field configuration (Tucker-Davis Technologies, Alachua, Fla., USA). DPOAEs were recorded at fixed stimulus levels (L1=L2=70 dB SPL), with an f2/f1 ratio of 1.2. Responses were recorded at 4, 8, 16, 24 and 32 kHz.

Acoustic Trauma

[0253] Animals were exposed to 120 dB octave band noise (8-16 kHz) for 2 hours in groups of 4 rats, placed in individual compartments of a custom built circular cage placed on a 30-cm diameter platform rotating at 3 turns/minute (Aqila Innovation, Valbonne, France). The calibrated octave band noise generated by the RZ6 SigGen software was further amplified by a Crown D-75 amplifier in bridge mode (Crown Audio, Elkhart, Ind., USA) and delivered by four Beyma CP16 compression tweeters (Acustica Beyma S.L., Moncada, Valencia, Spain) positioned 39 cm above the rotating platform, each 10 cm from the platform center.

Results

Comparative Effect Ofintraperitoneal Administration Ofracemic Azasetron, (+)-Azasetron and (−)-Azasetron on Ear Disorder

[0254] The ability of the racemic and two enantiomers of azasetron hydrochloride to reduce SSNHL after acoustic trauma was evaluated in a randomized, vehicle-controlled study with 7 weeks old male Wistar rats. Rats were randomized to receive 4.22 mg/kg of the racemic, two enantiomers of azasetron or vehicle control (physiological saline) by intra-peritoneal injection immediately after acoustic trauma, followed by daily dosing for a total of 14 days. Two days prior to acoustic trauma, baseline audiometric readings using auditory brainstem response (ABR) (8, 16, and 24 kHz) were recorded for each animal. Animals were exposed to 120-dB octave-band noise (8-16 kHz) for 2 hours to induce acoustic trauma.

[0255] Results for auditory threshold shift are provided in FIG. 2.

[0256] Data show that the (+) enantiomer reduced ABR threshold shifts more than racemic azasetron or (−)-enantiomer across the tested frequencies when compared to the vehicle control group.

Protective Effect of Oral Administration of (+)-Azasetron Post-Trauma

[0257] The ability of the (+)-enantiomer of azasetron hydrochloride to reduce SSNHL after acoustic trauma was evaluated in a randomized, vehicle-controlled study in male Wistar rats, 7 weeks of age. Rats were randomized to receive 5, 10, or 20 mg/kg of the (+)-enantiomer of azasetron hydrochloride or vehicle control (physiological saline) by oral gavage immediately after acoustic trauma, followed by daily dosing for a total of 14 days. Two days prior to acoustic trauma, baseline audiometric readings using auditory brainstem response (ABR) (8, 16, and 24 kHz) and distortion-product otoacoustic emissions (DPOAE) (4, 8, 16, 24, and 32 kHz) were recorded for each animal. Post-trauma audiometry recordings were conducted at 14 days.

[0258] Results for auditory threshold shifts are provided in FIG. 3.

[0259] Data show that 10 and 20 mg/kg of the (+) enantiomer significantly reduced ABR threshold shifts by a mean of 35.1% and 30.5%, respectively, from baseline to day 14, indicating a mean 22.3 and 19.4 dB improvement in hearing, respectively. When compared to the vehicle control group, 10- and 20-mg/kg doses of the (+) enantiomer demonstrate a treatment effect that reduces the hearing loss (average) from moderately severe (56 to 70 dB) to mild/moderate (26 to 40 dB/41 to 55 dB) based on American Speech Language Hearing Association (ASLHA) criteria (ASLHA, 2015).

[0260] Otoacoustic emissions were evaluated to further understand the effect of the (+) enantiomer on hearing loss. Using animals from the acoustic trauma study, otoacoustic emissions were evaluated following administration of 5, 10, or 20 mg/kg of the (+) enantiomer.

[0261] The (+) enantiomer (20 mg/kg) significantly reduced DPOAE amplitude loss from baseline to day 14 by a mean of 51.2%, suggesting significantly reduced loss of outer hair cells in the cochlea (FIG. 3).

Effect of Oral Administration Ofracemic Azasetron

[0262] The ability of the racemic azasetron hydrochloride by oral administration to reduce SSNHL after acoustic trauma was also evaluated. 10 mg/kg of azasetron or vehicle control (physiological saline) were administered to rats by oral gavage immediately after acoustic trauma (same protocol as above), followed by daily dosing for a total of 14 days.

[0263] A small tendency towards a protective effect of the racemic azasetron hydrochloride was observed on ABR thresholds and DPOAE amplitude loss (FIG. 4), but unlike treatment with the same dose of (+)-azasetron (FIG. 3), it did not reach statistical significance.

Example 3: Activity of (+)-Azasetron Besylate on Cisplatin-Induced Hearing Loss and Ototoxicity

[0264] The activity of (+)-azasetron besylate to protect against cisplatin ototoxicity induced hearing loss was evaluated by assessing effects on ABR threshold shift and DPOAE amplitude loss after slow intravenous cisplatin infusion in female Wistar rats (8 mg/kg cisplatin, 30-minute infusion). After baseline audiometry, rats were randomly assigned to receive daily placebo or (+)-azasetron besylate treatment for 14 days initiated 15 minutes before cisplatin administration. ABR threshold shifts and DPOAE amplitude loss were evaluated on day 14 (D14).

[0265] In an initial study, animals were divided into 2 groups: Placebo (n=4) and 26.4 mg/kg (+)-azasetron besylate (corresponding to 18.2 mg/kg (+)-azasetron free-base, n=5), both administered by oral gavage for a total of 14 days. Two days after baseline audiometry (ABR and DPOAE measurements), oral treatment was initiated in both groups 15 minutes before 8 mg/kg cisplatin infusion during 30 minutes under isoflurane anesthesia. On D14, ABR and DPOAE measurements were performed to evaluate ABR threshold shifts and DPOAE amplitude loss relative to baseline and the temporal bones of each animal were subsequently preserved with PFA fixation for histology (FIG. 5).

[0266] Daily oral treatment with 26.4 mg/kg significantly reduced both ABR threshold shifts (˜10-23 dB, up to 79% reduction) (FIG. 5A) and DPOAE amplitude loss (˜3.5-15 dB, up to 78% reduction) (FIG. 5B) at D14 after cisplatin infusion compared to the placebo treated group.

[0267] In a follow-up study with lower doses of (+)-azasetron besylate, animals were divided into 3 groups: Placebo (n=6), 6.6 mg/kg (+)-azasetron besylate (corresponding to 5 mg/kg (+)-azasetron free-base, n=7) and 13.2 mg/kg (+)-azasetron besylate (corresponding to 10 mg/kg (+)-azasetron free-base, n=5), both administered by oral gavage for a total of 14 days. Two days after baseline audiometry (ABR and DPOAE measurements), oral treatment was initiated in both groups 15 minutes before 8 mg/kg cisplatin infusion for 30 minutes under isoflurane anaesthesia. On D14, ABR and DPOAE measurements were performed to evaluate ABR threshold shifts and DPOAE amplitude loss relative to baseline (FIG. 7).

[0268] Both 6.6 mg/kg (+)-azasetron besylate (˜23-29 dB, up to 65% reduction) and 13.2 mg/kg (+)-azasetron besylate (22-29 dB, up to 73% reduction) daily oral treatment significantly reduced ABR threshold shifts compared to placebo (FIG. 7A) and the reduction of DPOAE amplitude loss by 6.6 mg/kg (+)-azasetron besylate (1.5-19 dB, up to 78% reduction) also reached statistical significance (FIG. 7B).

[0269] To evaluate the effect of (+) azasetron on outer hair cells (OHC) survival after cisplatin administration at an ototoxic dose, cochleograms of surviving OHCs were constructed from whole-mount fixed cochleae. In placebo treated animals, 14 days after cisplatin administration the mean number of surviving OHCs per 200 μm segment had decreased significantly, starting at 40-60% distance from the apex and fell to −10 cells or less in the basal turn. The number of surviving OHCs was significantly higher in animals having received 14 days of (+) azasetron treatment (p<0.001; FIGS. 6 and 8) and while hair cell loss was still observed, it was more gradual throughout the medial and basal turns, with conservation of up to 11-fold more OHCs in the basal turn of the cochlea, where the apoptotic effect was most prominent.

[0270] These data support the capacity of (+)-azasetron besylate to provide a significant benefit as a treatment against cisplatin induced ototoxicity, as demonstrated using functional audiometric measures of hearing (ABR threshold shift reduction, DPOAE amplitude loss reduction) corresponding to recognized clinical outcomes (pure tone audiograms, otoacoustic emissions) supported by histological demonstration of improved survival of cochlear outer sensory hair cells in cochleograms constructed from fixed tissue samples.

Example 4: Activity of (+)-Azasetron Besylate on Vestibular Dysfunction

[0271] The capacity of (+)-azasetron besylate to protect against lesions in the peripheral vestibular system was assessed using the kainate model of peripheral excitotoxic injury. Following unilateral, transtympanic injection of the excitotoxic kainic acid leading to the swelling, retraction and uncoupling of synapses between vestibular sensory hair cells and primary afferent neurons, baseline recordings of spontaneous nystagmus (pathological eye movements caused by a deficit of the vestibuloculomotor reflex) and postural deviations are performed before randomization to treatment groups. Recordings were repeated daily for 3 days, then again at D6 and D13 after lesion induction.

[0272] Following lesion induction and baseline recordings at t=1 h, female Long-Evans were randomized to receive intraperitoneal injections of placebo (n=18) or 5.6 mg/kg (+)-azasetron besylate daily for 3 days (t=1/24/25 h). Recording of spontaneous nystagmus and postural deviations were repeated at 2/4/6/24/25/48/49 hours and D6/D13 after initial lesion induction (FIG. 9).

[0273] (+)-azasetron besylate treatment significantly improved spontaneous nystagmus after excitotoxic peripheral vestibular lesion (p=0.048, 6-48% improvement), particularly in the early phase before central compensation of the vestibulooculomotor reflex deficits, already after the first administration. An early as well as lasting significant treatment effect of (+)-azasetron besylate administration (p<0.001, 30-62% improvement) was seen in the reduction of the roll angle deviation (postural head tilt) after excitotoxic peripheral vestibular lesion.

[0274] These data demonstrate the treatment benefit of (+)-azasetron besylate for reducing lesion induced deficits of the peripheral vestibular system of the inner ear.