Blood molecular biomarkers for chemotherapy-induced peripheral neuropathy: From preclinical models to clinical practice.

Chemotherapy-induced peripheral neuropathy (CIPN) has long been recognized as a clinically significant issue in patients treated with antineoplastic drugs. This common long-term toxic side-effect which negatively impacts the outcome of the disease can lead to disability and have detrimental effects on patients' quality of life. Since axonal injury is a prominent feature of CIPN, responsible for several sensory symptoms, including pain, sensory loss and hypersensitivity to mechanical and/or cold stimuli in the hands and feet, neurophysiological assessments remain the gold standard for clinical diagnosis of CIPN. Given the large impact of CIPN on cancer patients, there is increasing emphasis on biomarkers of adverse outcomes in safety assessment and translational research, to prevent permanent neuroaxonal damage. Since the results on reliable blood molecular markers for axonal degeneration are still controversial, here we provide a brief overview of blood molecular biomarkers used for assessing and/or predicting CIPN in preclinical and clinical settings.

Bortezomib-induced peripheral neurotoxicity in human multiple myeloma-bearing mice.

The proteasome inhibitor bortezomib is an antineoplastic drug mainly used for the treatment of multiple myeloma (MM). Despite its effectiveness, bortezomib clinical use is often limited by the onset of peripheral neuropathy (BiPN). To better understand the mechanisms of BiPN several rat and mice models have been proposed, but no studies in MM-bearing animals allowing to test the antitumor activity of the selected schedules and the role of MM by itself in peripheral nervous system damage have been reported to date. Here, we carried out a study using immunodeficient C.B-17/Prkdcscid (SCID) mice injected with RPMI8266 human MM cells and treated with bortezomib 1 mg/kg once a week for five weeks. Animals were assessed with neurophysiological, behavioral and pathological methods and tumor volume measurement was performed along the study. At the end of the study BiPN was evident in bortezomib-treated animals, and this neurotoxic effect was evident using a schedule able to effectively prevent tumor growth. However, neurophysiological and pathological evidence of MM induced peripheral nervous system damage was also reported. This model based on MM-bearing animals is more reliable in the reproduction of the clinical setting and it is, therefore, more suitable than the previously reported models of BiPN to study its pathogenesis. Moreover, it represents an optimal model to test the efficacy of neuroprotective agents and at the same time their non-interference with bortezomib antineoplastic activity.

Chemotherapy-induced peripheral neuropathy: What do we know about mechanisms?

Cisplatin, oxaliplatin, paclitaxel, vincristine and bortezomib are some of the most effective drugs successfully employed (alone or in combinations) as first-line treatment for common cancers. However they often caused severe peripheral neurotoxicity and neuropathic pain. Structural deficits in Dorsal Root Ganglia and sensory nerves caused symptoms as sensory loss, paresthesia, dysaesthesia and numbness that result in patient' suffering and also limit the life-saving therapy. Several scientists have explored the various mechanisms involved in the onset of chemotherapy-related peripheral neurotoxicity identifying molecular targets useful for the development of selected neuroprotective strategies. Dorsal Root Ganglia sensory neurons, satellite cells, Schwann cells, as well as neuronal and glial cells in the spinal cord, are the preferential sites in which chemotherapy neurotoxicity occurs. DNA damage, alterations in cellular system repairs, mitochondria changes, increased intracellular reactive oxygen species, alterations in ion channels, glutamate signalling, MAP-kinases and nociceptors ectopic activation are among the events that trigger the onset of peripheral neurotoxicity and neuropathic pain. In the present work we review the role of the main players in determining the pathogenesis of anticancer drugs-induced peripheral neuropathy.

Bortezomib treatment produces nocifensive behavior and changes in the expression of TRPV1, CGRP, and substance P in the rat DRG, spinal cord, and sciatic nerve.

To investigate neurochemical changes associated with bortezomib-induced painful peripheral neuropathy (PN), we examined the effects of a single-dose intravenous administration of bortezomib and a well-established "chronic" schedule in a rat model of bortezomib-induced PN. The TRPV1 channel and sensory neuropeptides CGRP and substance P (SP) were studied in L4-L5 dorsal root ganglia (DRGs), spinal cord, and sciatic nerve. Behavioral measures, performed at the end of the chronic bortezomib treatment, confirmed a reduction of mechanical nociceptive threshold, whereas no difference occurred in thermal withdrawal latency. Western blot analysis showed a relative increase of TRPV1 in DRG and spinal cord after both acute and chronic bortezomib administration. Reverse transcriptase-polymerase chain reaction revealed a decrease of TRPV1 and CGRP mRNA relative levels after chronic treatment. Immunohistochemistry showed that in the DRGs, TRPV1-, CGRP-, and SP-immunoreactive neurons were mostly small- and medium-sized and the proportion of TRPV1- and CGRP-labeled neurons increased after treatment. A bortezomib-induced increase in density of TRPV1- and CGRP-immunoreactive innervation in the dorsal horn was also observed. Our findings show that bortezomib-treatment selectively affects subsets of DRG neurons likely involved in the processing of nociceptive stimuli and that neurochemical changes may contribute to development and persistence of pain in bortezomib-induced PN.

The role of glutamate in diabetic and in chemotherapy induced peripheral neuropathies and its regulation by glutamate carboxypeptidase II.

Glutamate has been implicated in the pathogenesis of several diseases on the central nervous system, but recent studies have also suggested that it can be involved also in the onset and course of peripheral neuropathies. Given the increasing evidence of this possibility, several attempts have been performed in order to modulate its activity. Among them, glutamate carboxypeptidase II (GCP II) inhibition demonstrated promising results in different models of peripheral nerve damage, including diabetic and toxic neuropathies.

Expression, distribution and glutamate uptake activity of high affinity-excitatory aminoacid transporters in in vitro cultures of embryonic rat dorsal root ganglia.

Glutamate is the major mediator of excitatory signalling in the mammalian central nervous system, but it has recently been shown to play a role in the transduction of sensory input at the periphery and in peripheral neuropathies. New advances in research have demonstrated that rat peripheral sensory terminals and dorsal root ganglia (DRG) express molecules involved in glutamate signalling, including high-affinity membrane-bound glutamate transporters (GLAST [glutamate aspartate transporter], GLT1 [glutamate transporter 1], EAAC1 [excitatory aminoacid transporter 1]) and that alterations in their expression and/or functionality can be implicated in several models of peripheral neuropathy, neuropathic pain and hyperalgesia. Here we describe, through immunoblotting, immunofluorescence assays and ß-counter analysis of [(3)H] l-glutamate uptake, the expression, distribution and activity of the glutamate transporters in in vitro cultures of embryonic dorsal root ganglia sensory neurons, sensory neurons+satellite cells and satellite cells. In this work we demonstrated that glutamate transporters are expressed in all cultures with a peculiar pattern of distribution. Even if GLAST is strongly detected in satellite cells, it is slightly expressed also in sensory neurons. GLT1 immunostaining is very weak in DRG neurons, but it was evident in the satellite cells. Finally, EAAC1 is localized in the soma and in the neuritis of sensory neurons, while it is not detectable in satellite cells. Moreover, all the cell cultures showed a strong sodium-energy-dependent glutamate uptake activity and it is more marked in neurons alone or in co-culture with satellite cells compared to satellite cells alone. Finally, we show that the complete or partial pharmacological inhibition of glutamate transporters virtually completely or partially abolish glutamate uptake in all cell culture. These results, that demonstrate that functionally active glutamate transporters can be studied in dorsal root ganglia cell cultures, provide further evidence for a role of glutamatergic transport in the peripheral nervous system and will be useful for testing whether any changes occur in in vitro models of peripheral nervous system damage.

In vivo comparative study of the cytotoxicity of a liposomal formulation of cisplatin (lipoplatin™).

PURPOSE: Cisplatin is one of the most effective cytotoxic agents in the treatment of solid malignancies, but its use is limited by several side effects. Among them, peripheral neurotoxicity can be dose limiting. A liposomal formulation of cisplatin, Lipoplatin™, was developed to reduce the systemic toxicity of cisplatin but without preventing its efficacy. The aim of this study was to use an animal model to establish, through a multimodal approach, whether chronic treatment with two different schedules of Lipoplatin™, selected within the range of its anticancer effective dose, is less neurotoxic than cisplatin administration. METHODS: Female Wistar rats were treated intraperitoneally with cisplatin at a dose of 4 mg/kg or with Lipoplatin™ at doses delivering 12 or 24 mg/kg of cisplatin once weekly for 4 weeks. General toxicity was assessed by daily observation, body weight change, hematological and blood chemistry analysis, and histopathology of liver and kidney. The onset of peripheral neurotoxicity was assessed by measuring tail nerve conduction velocity (NCV), morphological and morphometric analysis of dorsal root ganglia (DRG), and morphological analysis of the sciatic nerve. RESULTS: Cisplatin induced a statistically significant reduction in body weight, the development of renal failure, and impairment in NCV with pathological alterations in the DRG and sciatic nerve. By contrast, Lipoplatin™ was markedly less nephrotoxic, and no significant weight gain reduction was observed in animals treated with both doses of the drug. Moreover, the lowest dose induced less severe damage to the peripheral nervous system with a moderate decrease in NCV and mild pathological alterations in DRG and the sciatic nerve. CONCLUSIONS: The results suggest that Lipoplatin™ 12 mg/kg is less neurotoxic than cisplatin 4 mg/kg, thus opening up the possibility of using this new formulation in future studies where its anticancer activity and the peripheral neurotoxicity will be assessed in parallel.

Neurophysiological and neuropathological characterization of new murine models of chemotherapy-induced chronic peripheral neuropathies.

Cisplatin, paclitaxel and bortezomib belong to some of the most effective families of chemotherapy drugs for solid and haematological cancers. Epothilones represent a new family of very promising antitubulin agents. The clinical use of all these drugs is limited by their severe peripheral neurotoxicity. Several in vivo rat models have reproduced the characteristics of the peripheral neurotoxicity of these drugs. However, since only a very limited number of cancer types can be studied in immunocompetent rats, these animal models do not represent an effective way to evaluate, at the same time, the antineoplastic activity and the neurotoxic effects of the anticancer compounds. In this study, we characterized the neurophysiological impairment induced by chronic chemotherapy treatment in BALB/c mice, a strain suitable for assessing the activity of anticancer treatments. At the end of a 4-week period of treatment with cisplatin, paclitaxel, epothilone-B or bortezomib, sensory and sensory/motor nerve conduction velocities (NCV) were determined in the caudal and digital nerves and dorsal root ganglia (DRG) and sciatic nerves were collected for histopathological analysis. The electrophysiological studies revealed that all the compounds caused a statistically significant reduction in the caudal NCV, while impairment of the digital NCV was less severe. This functional damage was confirmed by the histopathological observations evidencing axonal degeneration in the sciatic nerve induced by all the drugs associated with pathological changes in DRG induced only by cisplatin and bortezomib. These results confirm the possibility to use our models to combine the study of the antineoplastic activity of anticancer drugs and of their toxic effects on the peripheral nervous system in the BALB/c mouse strain.

The role of oxidative stress and anti-oxidant treatment in platinum-induced peripheral neurotoxicity.

Platinum-based anticancer drugs are a cornerstone of the current antineoplastic treatment. However, their use is limited by the onset of peripheral nervous system dysfunction, which can be severe and persistent over a long period of time. Among the several hypothesis proposed to explain this side effect, evidence is increasing that dorsal root ganglia (DRG) oxidative stress can be an important pathogenetic mechanism and, possibly, a therapeutic target to limit the severity of platinum-induced peripheral neurotoxicity but preserving the anticancer effectiveness. In fact, DRG energy failure has been suggested as a result of mitochondrial DNA-platinum binding and several antioxidant drugs have been tested in pre-clinical experiments and clinical trials. In this review, an update on the current knowledge on the relationship existing between oxidative stress and platinum drugs peripheral neurotoxicity will be given.

Regression of diabetic complications by islet transplantation in the rat.

AIMS/HYPOTHESIS: Type 1 diabetes is a chronic disease leading to complications such as peripheral neuropathies, nephropathy and cardiovascular disease. Pancreatic islet transplantation is being extensively investigated for blood glucose control in animals and in human type 1 diabetic patients, but the question of whether it can reverse long-term diabetic complications has not been fully explored. We investigated the effects of islet transplantation on diabetic complications in a rat model of streptozotocin-induced diabetes. METHODS: Three groups of rats were used: healthy controls, diabetic and diabetic rats transplanted with microencapsulated islets at 2 months after diabetes induction, when neuropathy was detectable by a decrease in tail nerve conduction velocity (NCV) and impaired nociceptive thresholds. Blood glucose levels and body weight were measured weekly. The variables considered were: thermal (hot plate test) and mechanical sensitivity (Randal-Selitto paw withdrawal test), NCV and Na+, K+-ATPase activity in the sciatic nerve. At the end of the experiments hearts were removed for morphometric determination and myocyte number, and kidneys removed for histological examination. RESULTS: Islet transplantation in diabetic rats induced normoglycaemia in a few days, accompanied by a rapid rise in body weight and amelioration of impaired nociceptive thresholds, as well as normalisation of NCV and Na(+), K(+)-ATPase, which were both about 25% below normal in diabetic rats. Myocyte loss was reduced (-34%) by islet transplantation and the observed mild kidney damage of diabetic rats was prevented. CONCLUSIONS/INTERPRETATION: Besides controlling glycaemia, transplantation of microencapsulated pancreatic islets induced almost complete regression of neuropathy and prevented cardiovascular alterations.

Neuroprotective effects of a ligand of translocator protein-18 kDa (Ro5-4864) in experimental diabetic neuropathy.

Peripheral neuropathy represents an important complication of diabetes involving a spectrum of structural, functional and biochemical alterations in peripheral nerves. Recent observations obtained in our laboratory have shown that the levels of neuroactive steroids present in the sciatic nerve of rat raised diabetic by a single injection of streptozotocin (STZ) are reduced and that, in the same experimental model, treatment with neuroactive steroids, such as progesterone, testosterone and their derivatives show neuroprotective effects. On this basis, an interesting therapeutic strategy could be to increase the levels of neuroactive steroids directly in the nervous system. With this perspective, ligands of translocator protein-18 kDa (TSPO) may represent an interesting option. TSPO is mainly present in the mitochondrial outer membrane, where it promotes the translocation of cholesterol to the inner mitochondrial membrane, and, as demonstrated in other cellular systems, it allows the transformation of cholesterol into pregnenolone and the increase of steroid levels. In the diabetic model of STZ rat, we have here assessed whether treatment with Ro5-4864 (i.e., a ligand of TSPO) could increase the low levels of neuroactive steroids in sciatic nerve and consequently to be protective in this experimental model. Data obtained by liquid chromatography-tandem mass spectrometry show that treatment with Ro5-4864 was able to significantly stimulate the low levels of pregnenolone, progesterone and dihydrotestosterone observed in the sciatic nerves of diabetic rats. The treatment with Ro5-4864 also counteracted the impairment of NCV and thermal threshold, restored skin innervation density and P0 mRNA levels, and improved Na+,K+-ATPase activity. In conclusion, data here reported show for the first time that a TSPO ligand, such as Ro5-4864, is effective in reducing the severity of diabetic neuropathy through a local increase of neuroactive steroid levels.