Neuroprotective effect of alogliptin on oxaliplatin-induced peripheral neuropathy in vivo and in vitro.

Oxaliplatin is a platinum-based antineoplastic drug commonly used for treating colorectal, gastric, and pancreatic cancer. However, it frequently causes peripheral neuropathy as dose-limiting toxicity and is lacking a strategy for prevention. Alogliptin, a dipeptidyl peptidase 4 (DPP-4) inhibitor, is an oral antidiabetic drug. Previous studies have shown that DPP-4 inhibitors have pleiotropic effects, including neuroprotection. In this study, we investigated the effects of alogliptin on oxaliplatin-induced peripheral neuropathy using in vitro and in vivo models. In PC12 cells, alogliptin attenuated neurite disorders induced by oxaliplatin and cisplatin. The repeated injection of oxaliplatin caused mechanical allodynia and axonal degeneration of the sciatic nerve in rats. These neuropathies were ameliorated by co-administration of alogliptin. Moreover, alogliptin did not attenuate tumor cytotoxicity of oxaliplatin in the cultured colon, gastric, or pancreatic cancer cell lines and tumor-bearing mice. These findings suggest that alogliptin may be beneficial for preventing oxaliplatin-induced peripheral neuropathy.


Discussion
It is conceivable that neurodegeneration, such as axonopathy, neuronopathy, or myelinopathy, are responsible for oxaliplatin-dependent chronic neuropathy [7][8][9] . In the present study, we examined the protective property of alogliptin on oxaliplatin-induced peripheral neuropathy focused on axonopathy. In the cultured cell model, neurite outgrowth was analyzed as an index of axonopathy. Treatment with oxaliplatin, cisplatin, paclitaxel, or bortezomib shortened neurite outgrowth of PC12 cells. Alogliptin prevented only oxaliplatin-and cisplatin-induced neurite shortening. Consequently, alogliptin may exert a neuroprotective effect selectively for platinum drugs.
We also evaluated neurotoxicity using a behavioral and histological approach with the peripheral neuropathy model rats. In the von Frey test, oxaliplatin, paclitaxel and bortezomib caused mechanical allodynia. Co-administration of alogliptin inhibited the behavioral alteration induced by oxaliplatin: meanwhile alogliptin improved neither paclitaxel-nor bortezomib-induced behavioral alteration. In the histological study, axonal degeneration of the sciatic nerve was induced by repeated infusion of oxaliplatin. This means axonopathy and is in line with our in vitro results.
Our previous reports indicated an association between mechanical allodynia and the degeneration of axons 18,19 . Axonal degeneration induced by oxaliplatin was alleviated by co-treatment with alogliptin. These results indicate that alogliptin ameliorates oxaliplatin-induced chronic peripheral neuropathy by inhibiting neurodegeneration. Furthermore, the influence on the anticancer activity of oxaliplatin was assessed, and no impeding was observed in the cultured carcinoma cells or tumor cell-implanted mice. Hence, alogliptin is unlikely to weaken the antineoplastic efficacy of oxaliplatin.
It remains to be seen how alogliptin prevents oxaliplatin-induced peripheral neuropathy. DPP-4 inhibitors exert their activity principally by increasing the level of GLP-1. Vildagliptin suppressed the development of diabetic neuropathy by activation of GLP-1 signals 20 . Exenatide, a GLP-1 receptor agonist, inhibited oxaliplatin-induced neurodegeneration in cultured cells and rats 17 . Thus, we do not rule out the possibility that GLP-1 contributes to the antineuropathic effect of alogliptin. Conversely, GLP-1 receptor antagonist did not reverse the neuroprotective effect of alogliptin in our examination. Thus, it is thought that not only GLP-1 but also other mechanisms play a role in the neuroprotective effect of alogliptin on oxaliplatin-induced neuropathy. Many reports have shown that DPP-4 inhibitors have effects independent of GLP-1. DPP-4 is a serine exopeptidase that cleaves X-proline dipeptides from the N-terminus of polypeptides 21 . This enzyme is expressed ubiquitously and has various substrates, such as pituitary adenylate cyclase activating polypeptide (PACAP) and neuropeptide Y, in addition to GLP-1 22 . Some studies have reported these peptides have neuroprotective effects on the neurotoxicity of platinum in cultured cells and animals 23,24 . Moreover, DPP-4 inhibitors have also been reported to have antioxidant effects 25,26 . Many studies have shown that oxidative stress plays a role in oxaliplatin-induced neuropathy [27][28][29] . Taken together, the neuroprotective peptides and antioxidant effect, aside from GLP-1, might play a role in the neuroprotective effect of alogliptin on oxaliplatin-induced neuropathy. Interestingly, in our previous study, oxaliplatin and cisplatin, but not paclitaxel and bortezomib, down-regulated superoxide dismutase (SOD) activities in PC12 cells 30 , which means that the oxidative stress plays major roles in the neurotoxicity caused by platinum rather than by the other anticancer drugs. This mention might be a reason why alogliptin showed the neuroprotective effects specific to platinum.

Animals. We used male
Von Frey test for mechanical allodynia. The von Frey test was conducted to assess the effect of alogliptin on mechanical allodynia. The test was performed on days 0 (pre), 4, 11, 18, and 25 for oxaliplatin model, on days 0, 4, 8, 11, and 15 for paclitaxel and bortezomib models. Each test was conducted before drug administration. Each rat was adapted in a wire mesh box for 30 min before the test. The method of von Frey test was described in a previous report 18 . The plantar withdrawal thresholds for stimulations by von Frey filaments (Aesthesio; DanMic Global. LLC., San Jose, CA, USA) were determined using a modified up-down method. Each experiment was performed in five to seven animals per group.
Assessment of sciatic nerve axonal degeneration. Sciatic nerves were harvested from three rats per group anesthetized with sevoflurane (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan) on day 30. Sciatic nerves were embedded in Epon after fixing in glutaraldehyde (2%), followed by sucrose (8%) substitution. The slice samples were stained with toluidine blue 18 . Each section was evaluated using light microscopy (CKX41SF; Olympus Co.). The axon circularity was calculated as a quantitative index of axonal degeneration 37 . The quantitative data were acquired from 4,061-4,919 fibers of three animals per group.
Tumor cytotoxicity assay in cultured cell lines. C-26, HCT116, MKN45, and MIA PaCa-2 cells were seeded in collagen-coated 24-well plates (Thermo Fisher Scientific Inc.) at a density of 2.0 × 10 4 cells/well. On the following day, cells were treated with 10 µM oxaliplatin and 0.1-100 nM alogliptin for 24 h. Cell viability was assessed using the WST-8 method (Cell Counting Kit-8; Dojindo Laboratories, Kumamoto, Japan). Each experiment was performed in four wells per group.

Statistical analysis.
The results are expressed as the median ± quartile values or the mean ± standard error of the mean. Statistical analyzes were performed using a one-way analysis of variance followed by the Games-Howel test or the Tukey-Kramer test (Statview; Abacus Concepts, Berkeley, CA, USA) for the experiments whose sample sizes were less than 30 or more than 30, respectively. A probability level of P < 0.05 was accepted as statistically significant.

Data availability
The data that support the findings of this study are available from the corresponding author upon reasonable request.