Therapeutic efficiency of the APAF‐1 antagonist LPT99 in a rat model of cisplatin‐induced hearing loss

Dear Editor, Cis-diammine-dichloroplatinum[II] (cisplatin) is a potent and widely used chemotherapeutic agent with significant efficacy against several forms of cancer in adults and children. Unfortunately, cisplatin has multiple adverse effects and causes irreversible hearing loss (HL) in a large proportion of patients, which severely impacts their quality of life.1 Here, we confirm that treatment with LPT99, a small molecule inhibitor of the apoptosome, preserves hearing levels in a rat model of cisplatin-induced HL and prevents apoptosis in cisplatin-exposed HEI-OC1 cells. These data are encouraging and support the potential of LPT99 for the prevention of the secondary effects of cisplatin for HL. Cisplatin primarily induces damage to outer (OHC) and inner hair cells and to spiral ganglion neurons, and longterm retention of cisplatin in the cochlea contributes to its irreversible ototoxic effects.2 Once inside the cell, cisplatin forms a highly reactive complex that induces DNA damage, activates death receptor pathways, and causes mitochondrial dysfunction, triggering reactive oxygen species generation, oxidative stress, and cytochrome c release. Cytochrome c binds with apoptotic protease activating factor 1 (APAF-1) forming the apoptosome, which recruits and activates caspase-9 and -3 to execute apoptosis3 (Figure 1A). Antioxidants and antiapoptotic drugs have been tested as otoprotectants in experimental models and clinical trials4 (Figure 1A), but their lack of specificity and efficacy aremajor limitations andnone are currently approved for the treatment of cisplatin-induced HL. Accordingly, more effective agents are urgently needed, preferably formulated for local (intratympanic) administration to avoid interfering with the antineoplastic activity of cisplatin. As a central component of the apoptosome and a regulator of apoptosis, APAF-1 is a key pharmacological target that has been validated in experimental models.5 Orzáez et al.6 described a series of 2,5-piperazinedione derivatives (Patent US9040701B2) with APAF-1 inhibitory

activity through binding to the APAF-1 caspase recruitment and nucleotide-binding oligomerization domains, which impair the conformational change required for its oligomerization and, consequently, inhibit the activation of procaspase-9 and the induction of apoptosis. These molecules attenuated cisplatin-induced apoptosis in HEI-OC1 cells and the loss of neuromasts in zebrafish, demonstrating that APAF-1 inhibition is effective against cisplatin-associated ototoxicity. 6 We investigated the efficacy of LPT99 (Patent US10561736B1), a second-generation derivative with similar APAF-1 inhibitory activity but better pharmacological properties, in preventing cisplatininduced ototoxicity using in vitro and in vivo models.
The addition of 1 μM LPT99 to cisplatin-treated HEI-OC1 cell cultures 7 significantly improved cell survival (p < 0.05) across the cisplatin concentration ranging from 1 to 4 μg/ml ( Figure 1B, a). Flow cytometry analysis of annexin V/propidium iodide staining indicated that LPT99 alone did not trigger apoptosis, but significantly reduced the percentage of total apoptotic cells (early and late apoptosis) in co-treated cultures as compared with cisplatin alone (p < 0.001) ( Figure 1B, b). Activated caspase-3 levels were also lower in cells co-treated with cisplatin and LPT99 ( Figure 1B, c and d).
LPT99 was formulated in cyclodextrin and Poloxamer 407 hydrogels to achieve sustained delivery, and was administered intratympanically 8 in rats. Dosing of LPT99/cyclodextrin (50-200 μM) provided high LPT99 cochlear levels (Figure 2A, a) and had no effect on hearing thresholds even 30 days after administration (Figure 2A,  b). Similarly, dosing of LPT99/Poloxamer 407 (100-797 μM) reached the cochlea, was detectable up to 14 days after injection, and had no effect on hearing, supporting its tolerability at the tested doses ( Figure 2B). LPT99 was undetectable in plasma at all-time points and doses studied (quantification limit 5 ng/ml). We evaluated the efficacy of LPT99 by monitoring the auditory brainstem response (ABR) before and 3 days after bilateral F I G U R E 1 (A) General mechanisms of cisplatin-induced cytotoxicity. Cisplatin enters into the cell by passive diffusion or by active uptake through specialized copper transporters. Once in the cytosol, cisplatin forms a highly reactive complex that activates several pathological molecular processes (red ovals) including reactive oxygen species (ROS) production, DNA damage, BAX translocation to the mitochondria, and cytochrome c (Cyt c) release. Apoptosome formation is triggered by Cyt c binding to the apoptotic protease activating factor 1 (APAF-1) in the cytosol along with caspase-9 and -3 (Cas-9 and Cas-3) recruitment. Apoptosis inhibitors and other blocking molecules (green ovals) have been tested as otoprotective candidates. Abbreviations: CRT1, copper receptor transporter 1; NOX-3, NADPH oxidase 3; ER, endoplasmic reticulum. (B) LPT99 suppresses cisplatin-induced apoptosis in HEI-OC1 cells. (a) Cell viability in HEI-OC1 cells untreated (control, white bar), challenged with cisplatin (1-5 μg/ml, red bars) or co-treated with LPT99 (1 μM, green bars), and cultured for 24 h. Data are expressed as % relative to the control (100% viability) and shown as mean ± SEM of two independent experiments, performed in quadruplicate. (b) Apoptosis in HEI-OC1 cells untreated (control, white bars), challenged with cisplatin (3 μg/ml, red bars) or co-treated (1 μM, green bars) with LPT99 and incubated for 24 h. Data are expressed as the percentage of apoptotic cells (early, late or total) in the plate and expressed as mean ± SEM of two independent experiments, performed in triplicate. (c) Microphotographs of HEI-OC1 cells untreated (control) or treated with LPT99 (1 μM), cisplatin (4 μg/ml) or both for 24 h, and immunostained for activated caspase-3 (green) and phalloidin (red). Scale bar 20 μm. Images are representative of triplicate samples from two independent experiments and quantified in (d). (d) Active caspase-3 staining of HEI-OC1 cells untreated (control, white bar), challenged with cisplatin (red) and co-treated (green) or not with LPT99, expressed as the percentage of relative to the cisplatin group. Statistical differences were assessed by one-way ANOVA and Student's t-test, or the Kruskal-Wallis and Mann-Whitney U test, for normal or nonnormal data, respectively. Differences were considered significant for *p < 0.05, **p < 0.01, ***p < 0.01 intratympanic administration of LPT99 in cyclodextrin (Figure 2A, c) or in Poloxamer 407 ( Figures 2C and 3) and intraperitoneal infusion of cisplatin (10 mg/kg). Analysis of the untreated group revealed that 86% of rats exhibited HL following cisplatin administration, whereas normal hearing was observed in 50%, 58%, and 70% of rats in the groups co-treated with 50, 100, or 478 μM LPT99/Poloxamer 407, respectively ( Figure 2C). LPT99 treatment also notably reduced the percentage of animals with moderate and profound HL ( Figure 2C). Co-administration of LPT99 reduced the cisplatin-induced threshold shifts for clicks (∼50% reduction) and pure tones (∼60%, 45%, and 35% reduction for 8, 16, and 28-40 kHz). Significant dosedependent differences were found in threshold shifts between LPT99/Poloxamer 407-treated and nontreated rats, with the best protection profile observed at a dose between 100 μM and 478 μM LPT99 ( Figure 3A and B). Cisplatin-induced changes in the latency and amplitude of the ABR waves were also attenuated by LPT99 ( Figure 3C and D). The LPT99 dosing scheme improved hearing thresholds in a range similar to that reported for other otoprotective drugs, such as a p53 inhibitor, 9 and improved upon those reported for the intracochlear perfusion of inhibitors of JNK, caspases-3, 9, and 8 and cathepsin B. 10 The protective effects of LPT99/Poloxamer 407 were further confirmed by assessing cochlear cytoarchitecture (Figure 4). Cisplatin is known to induce substantial OHC loss in the basal and middle turns. Rats co-treated with LPT99 showed better preservation of the organ of Corti and spiral ganglion ( Figure 4A-F, J, and L). The protective effect of LPT99 against cisplatin-induced OHC loss was further confirmed by the preserved E-cadherin and F-actin expression ( Figure 4G, K, M, and Q) and the higher expression levels of prestin, a motor OHC protein ( Figure 4H). LPT99-co-treated cochleae also showed less dilatation of strial microvasculature, particularly in the middle turn and exhibited higher levels of Kir4.1, a marker of intermediate cells ( Figure 4I and O). Because the stria vascularis is the entry point of cisplatin into the cochlea and a long-term accumulation site, it has emerged as an important target to prevent cisplatin ototoxicity. 2 Further studies are needed to demonstrate a direct effect of LPT99 on strial cells.
In conclusion, LPT99 exerts dose-dependent protective effects on auditory function and cochlear cytoarchitecture in cisplatin-treated rats. LPT99 also protects HEI-OC1 cells against cisplatin-induced apoptosis, although further mechanistic studies will be needed to fully establish the participation of APAF-1. Overall, our findings highlight the role of APAF-1 as a pharmacological target for new drugs and point to LPT99 as a potential prophylactic local treatment for cisplatin-induced HL.

A C K N O W L E D G M E N T S
We thank our colleagues of the Neurobiology of Hearing Group for critical comments and sharing unpublished information. We thank the Non-Invasive Neurofunctional Evaluation and Genomic facilities (Institute for Biomedical Research "Alberto Sols" CSIC-UAM) and the Histology facility (National Biotechnology Center, CSIC) for technical support. This work was supported by a SPIRALTH-CIBERER ER17PE12 grant to SMC. SMC and LRR hold CIBERER contracts. BC, AMC, and JMB contracts were supported in part by projects from FP7-PEOPLE-2013-IAPP-TARGEAR and from MINECO/FEDER SAF2017-86107-R to IVN.

C O N S E N T F O R P U B L I C AT I O N
All authors read and approved the final manuscript. The data that support the findings of this study are available on request from the corresponding author. The data are not publicly available due to privacy or ethical restrictions.

C O N F L I C T O F I N T E R E S T
SMC has received research support from Spiral Therapeutics and IVN has received compensation for consulting. The remaining authors declare that they have no significant competing financial, professional, or personal interests that might have influenced the performance or presentation of the work described in this manuscript.