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Aim: Nowadays, there is a great interest in the therapeutic potential of sigma1 receptor ligands for treating different CNS pathologies. Our previous investigations led to identify (R)-RC-33 as a potent and selective S1R agonist. Results: Herein, we report the gram-scale synthesis, pharmacokinetic profile and CNS distribution of (R)-RC-33 in the mouse to determine the most suitable dosage schedule for in vivo administration. For comparative purposes, the same experiments were also performed with PRE-084, the most widely used S1R agonist commonly in pharmacological experiments. Discussion: (R)-RC-33 shows a similar pharmacokinetic profile and a better CNS distribution when compared with PRE-084. Conclusion: (R)-RC-33 may be a promising candidate for in vivo studies in animal models of neurodegenerative diseases.

Multiple Sclerosis (MS) is a neuroinflammatory and immune-mediated chronic disease of the Central Nervous System which progressively damages the axonal myelin sheath, leading to axonal transmission impairment and to the development of neurological symptoms. Most MS cases are characterized by a relapsing-remitting course, and current therapies rely only on the use of immunomodulating drugs which are, however, unable to reverse disease progression. Among the newly proposed alternative therapies, Mesenchymal Stem Cells (MSCs) are considered suitable for MS treatment due to their capacity to modulate the immune response and to modify the pattern of the released cytokines. So far, encouraging results have been obtained with the administration of MSCs before disease onset, mainly in animal models of acute Experimental Autoimmune Encephalomyelitis (EAE) in which MSCs were able to reduce inflammation, thus ameliorating also the disease’s clinical symptoms. On the contrary, only a very small number of studies have investigated the effect of MSCs on relapsing-remitting models of the disease. Here, we investigated the therapeutic potential of MSC administration, both before and after the disease’s onset, in an animal model of MS represented by Dark Agouti rats affected by chronic Relapsing-Remitting EAE. Our results demonstrated that in chronic Relapsing-Remitting EAE the administration of MSCs after the clinical disease’s appearance is able to completely abrogate the relapsing phase and to strongly reduce spinal cord demyelination. These encouraging results have demonstrated that MSCs can provide a protective and reparative strategy for MS treatment.

1-Deoxysphingolipids (1-deoxySLs) are atypical neurotoxic sphingolipids that are formed by the serinepalmitoyltransferase (SPT). Pathologically elevated 1-deoxySL concentrations cause hereditary sensory and autonomic neuropathy type 1 (HSAN1), an axonal neuropathy associated with several missense mutations in SPT. Oral L-serine supplementation suppressed the formation of 1-deoxySLs in patients with HSAN1 and preserved nerve function in an HSAN1 mouse model. Because 1-deoxySLs also are elevated in patients with type 2 diabetes mellitus, L-serine supplementation could also be a therapeutic option for diabetic neuropathy (DN). This was tested in diabetic STZ rats in a preventive and therapeutic treatment scheme. Diabetic rats showed significantly increased plasma 1-deoxySL concentrations, and L-serine supplementation lowered 1-deoxySL concentrations in both treatment schemes (P < 0.0001). L-serine had no significant effect on hyperglycemia, body weight, or food intake. Mechanical sensitivity was significantly improved in the preventive (P < 0.01) and therapeutic schemes (P < 0.001). Nerve conduction velocity (NCV) significantly improved in only the preventive group (P < 0.05). Overall NCV showed a highly significant (P = 5.2E-12) inverse correlation with plasma 1-deoxySL concentrations. In summary, our data support the hypothesis that 1-deoxySLs are involved in the pathology of DN and that an oral L-serine supplementation could be a novel therapeutic option for treating DN.

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.

Cisplatin, paclitaxel and bortezomib are effective chemotherapy drugs in cancer treatment. However, they share severe peripheral neurotoxicity (PN) as one of their major dose-limiting side effects, often impairing cancer patients' quality of life and sometimes being permanent. Even if preclinical oncology is largely based on the use of immune-deficient mice, rodent models used to study the chemotherapy-induced PN are available only in immune-competent animals. In this study we characterized for the first time the PN induced by these chemotherapies through neurophysiological, behavioral, morphological and morphometric studies in athymic nude mice, a commonly employed strain in the preclinical oncology. The animals, divided into four groups, were chronically treated with cisplatin, paclitaxel or bortezomib once or twice a week for 4 or 6. weeks or were left untreated. These schedules were tolerated, neurotoxic and in the range of antineoplastic effectiveness. Despite similarities, differences in the features of PN were evident if compared with immune-competent models under comparable regimens of treatment. The results of this study may provide a basis for future combined analysis of antineoplastic and neurotoxic effects of chemotherapy in the same animals.

Cisplatin, paclitaxel and bortezomib are anticancer drugs widely employed in the treat-ment of different solid tumours even though peripheral neurotoxicity represents a major limitation in their clinical use. During the last decades many rat and mouse models of chronic chemotherapy-induced peripheral neurotoxicity (CIPN) have been characterized from the clinical, pathological, neurophysiological and behavioural point of view. These models were based on immune-competent animals, however immune-deficient mice are mainly used in preclinical oncology. . In this respect, the development of immune-deficient mice models could represent a basis for the concurrent investigation of the mechanisms of both anticancer drug efficacy and neurotoxicity in animals implanted with human derived cancer. Moreover, in the same models, neuroprotective and non-interfering strategies could be better studied. In this study we established the feasibility of new immune-deficient murine models of pe-ripheral neurotoxicity induced by three anticancer drugs. Forty-eight athymic nude mice were randomized in 4 groups of 12 animals, three were treated respectively with cisplatin, paclitaxel and bortezomib, and one was left untreated. All animals were followed up for 6 weeks. They were examined at baseline, week 4 and 6 for neurophysiological functions and behavioural tests, whilst morphological and mor-phometric analysis were performed on dorsal root ganglia (DRG) and peripheral nerves collected after 4 and 6 weeks of treatment. The results of the study demonstrate that athymic nude mice show CIPN features similar to those of conventional models even if some differences must be remarked as the pro-longed time of treatment required to develop a chronic neuropathy. The characterization of this new mice model of CIPN will allow studies of antineoplastic and neurotoxic effects in the same animal.

Ethoxyquin (EQ) is a synthetic potent antioxidant approved by Federal Drug Administration for use in animal food in order to protect it against lipid peroxidation and stabilize fat soluble vitamins. It was found that diet supplemented with EQ allowed mice to live longer than littermates. Antimutagenic effect was observed in mice and rats treated with cancer chemotherapy. However, these observations were never carried out to human studies and the primary use of EQ is still as a supplement in animal food. EQ should be considered a potential neuroprotective drug against platinum-based chemotherapy, a cornerstone of the current antineoplastic treatment limited by the onset of peripheral nervous system dysfunction in cancer patients. In fact platinum-induced oxidative stress contributes to dorsal root ganglia (DRG) and peripheral nerves damage. Intriguing preliminary in vitro data demonstrated that EQ can prevent platinum toxicity on primary DRG culture. In this study we tested in vivo neuroprotective properties of EQ through Nerve Conduction Velocities (NCV) studies, morphological and morphometrical analysis of DRG, caudal and sciatic nerves and behavioral assessment of neuropathic pain. Cisplatin (i.p, 2 mg/Kg, twice weekly) and/or EQ (i.p, 75 micrograms/kg, daily) were administered in female Wistar rats for four weeks. When co-administered, EQ was injected 1 hour after cisplatin. A group of control animals was left untreated. At the third week of treatment, cisplatin alone determined piloerection, kyphosis and hypokinesia while co-treated animals had only piloerection. Neurophysiological measurements showed that cisplatin induced functional abnormalities in caudal nerve evident as a significant decrease in NCV (p<0.001 vs controls) while, if EQ was co-administered, the NCV impairment was significantly prevented (p<0.05 cisplatin alone vs cisplatin+EQ). Similarly, cisplatin-induced mechanical allodynia (p<0.05 vs controls) and thermal hypoalgesia (p<0.01 vs

Cisplatin is an anticancer drug employed for the treatment of several solid tumours, including testicular, ovarian, breast and lung cancers. However, treatment side effects remain an unsolved problem. Some of the patients treated with cisplatin, after a cumulative dose of 300 mg/m2, develop a painful peripheral neuropathy (PPN) characterized by distal paresthesias, numbness, sensory ataxia and neuropathic pain. Since a different degree of the PPN severity suggests an individual variation in the drug toxicity response, the understanding of the genomics underlying PPN would lead to new therapeutic targets and to early diagnostic screening. In this study we assessed the severity of cisplatin-induced PPN in six mouse strains using Nerve Conduction Velocities (NCV), Neurometer studies, morphological analysis of DRG, caudal and sciatic nerves and lumbar spinal cord, morphometrical analysis of DRG and behavioral assessment of mechanical allodynia. Cisplatin 4 mg/Kg was intraperitoneally administered twice a week for 4 weeks in CD1, Balb-c, C57BL6, FVB, DBA and AJ mice. Cisplatin induced a significant impairment of caudal and digital NCV in all except FVB and DBA mice. Moreover, cisplatin administration resulted in a significant decrease in the response to mechanical stimulation in Balb-c, C57BL6, CD1 and DBA starting from the first week of treatment while AJ and FVB never develop mechanical allodynia. Neurometer analysis, that allows a quantitative assessment of the functionality of the three major subpopulations of sensory nerve fibers (A-delta, A-beta and C), showed that large myelinated fibers (A-beta) were affected by cisplatin in Balb-c and CD1, small myelinated (A-delta) only in Balb-c while small unmyelinated fibers (C) were unaffected only in FVB and AJ mice. The morphometrical analysis revealed a nucleolar and somatic atrophy of DRG sensory neurons of CD1 but not of FVB-treated mice. Taken together, these results demonstrate that CD1 and FVB mice were the most and

Bortezomib (BTZ) is a proteasome inhibitor used as first-line therapy for multiple myeloma. However, its administration induces the development of severe painful peripheral neuropathy (PPN). This painful condition is an important medical need since the available treatments are actually ineffective. We recently described a mice model of PPN that shares most of the conditions found in patients treated chronically with BTZ (Carozzi et al., 2013). In fact, BTZ determines dysfunction of all fiber types in sensory nerves and, at least in mice, alters the electrical activity of the spinal dorsal horn neurons. This alteration of the basal electrophysiological activity induces also relevant changes in the central nociceptive transmission. In this work we characterize the neuroprotective effects of an imidazoline receptor 2 ligand (CR4056) able to allosterically inhibit the activity of monoamine oxidase-A, a key enzyme in the regulation of neuropathic pain. Wistar rats were treated with BTZ 0.20 mg/kg, 3 times a week for 8 weeks (i.v). Then CR4056 was orally administered in a curative schedule at 6 mg/kg, once a day, for 2 weeks. Gabapentin (100 mg/kg, daily, p.o.) and buprenorphine (28,8µg/kg, daily, s.c.) were used as internal analgesic standards. At the end of both BTZ and analgesic treatments, we measured the caudal and sciatic nerve conduction velocity (NCV), the morphological/morphometrical alterations in the caudal nerve and the neuropathic pain development. BTZ treatment induced a significant impairment of sensory, but not motor NCV, slight hyperalgesia, significant mechanical allodynia and clearing of myelinated fibers in the caudal nerves. After two weeks of follow up animals did not spontaneously recover functional, morphological and behavioral abnormalities while the 2 weeks-treatment with CR4056 (but not with gabapentine and buprenorphine) significantly resolved BTZ-induced mechanical allodynia. Results obtained show that CR4056 produces a marked analgesic effect

Even if mortality rates of genitourinary cancers patients are declining due to earlier diagnosis and increasingly aggressive antineoplastic regimens, treatment-related complications that severely compromise the patients’ quality of life remain an unsolved problem. Patients treated with cisplatin often develop painful peripheral neuropathy that becomes evident after a cumulative dose of 300 mg/m2. Cisplatin produces a symptomatic and clinically detectable sensory peripheral neuropathy characterized by distal paresthesias and numbness. In advanced stages, it may progress to severe neuropathic pain and sensory ataxia. The fact that patients receiving cisplatin differentially develop neuropathy suggests an individual variation in the drug toxicity response. Moreover, understanding the genomics of CIPN would lead to new therapeutic targets and should be useful for a molecular diagnostic screening. In this study we assessed the severity of CIPN in six mouse strains using Nerve Conduction Velocities (NCV) and Neurometer studies, morphological analysis of DRG, caudal and sciatic nerves and lumbar spinal cord and behavioral assessment of mechanical allodynia. Once the characteristics of painful peripheral neuropathy have been established in the different strains, a genome-wide association study will be performed to elucidate the genetic determinants of the variability in developing peripheral neuropathy. Cisplatin 4 mg/Kg was intraperitoneally administered twice a week for 4 weeks in CD1, Balb-c, C57BL6, FVB, DBA and AJ mice. Cisplatin induced a significant impairment of caudal and digital NCV in AJ, Balb-c, C57BL6 and CD1 mice but not in FVB and DBA mice. Neurometer analysis, that uses neuroselective electrical stimuli to perform a quantitative assessment of the functionality of the three major subpopulations of sensory nerve fibers (A-delta, A-beta and C) revealed that large myelinated fibers (A-beta) were affected by cisplatin in Balb-c and CD1 mice starting from the 2nd

Bortezomib (BTZ), a selective proteasome inhibitor, is an antitumor drug used to treat multiple myeloma. As a side effect, BTZ determines a painful peripheral neuropathy (PPN), often refractory to management. Chronic BTZ administration in female Wistar rats induced a PPN in which the development of mechanical allodynia is associated to an increase in the expression of TRPV1 and CGRP in the dorsal root ganglia (DRG) and spinal cord dorsal horn. In this study we examine the possible role of two standard analgesics, Gabapentin (Gaba) and buprenorfin (Bupre), in modulating the expression of TRPV1 and CGRP in the DRG and spinal cord of rats chronically treated with BTZ. To this aim, female Wistar rats were treated with BTZ 0.20 mg/kg, 3 times a week for 8 weeks (i.v.). Then Gaba (100 mg/kg, daily, p.o.) and Bupre (28,8 µg/kg, daily, s.c.) were orally administered in a curative schedule for 2 weeks. The expression of TRPV1 and CGRP, a neuropeptide involved in nociceptive neurotransmission, was examined in L4-L5 DRG and spinal cord segments by means of western blot (WB) and immunohistochemistry. WB analysis did not show any statistically significant change of protein levels in DRG and spinal cord. Instead, chronic BTZ treatment followed by a 2 weeks follow-up period affected TRPV1 expression by inducing an increase in the proportion of TRPV1-like immunoreactive (LI) DRG neurons, whereas did not affect the percentage of CGRP-LI neurons. Labeled perikarya were mostly of small- and medium-size. No BTZ-induced changes in immunolabeling were appreciable in the dorsal horn of the spinal cord. Treatment with Gaba and Bupre reduced levels of TRPV1 protein in spinal cord homogenates, whereas had no effect on DRG protein levels. None of the analgesics appeared to reverse in a statistically significant way the BTZ-induced increase of labelled DRG neurons nor induced any change in the detectability of positive innervation in the spinal cord dorsal horn.

Bortezomib (BTZ) is a chemotherapy drug that shows a high antineoplastic activity against multiple myeloma and some type of solid tumours. This drug acts by inhibiting protein degradation by the proteasome; however its clinical use is limited by the onset of a severe peripheral neuropathy associated with neuropathic pain. Different mechanisms may underlie the development of peripheral neuropathy among which the proteasome inhibition and the alteration of microtubule’s stability. To study these mechanisms we used a well-characterized rat model of BTZ-induced peripheral neuropathy in which BTZ was administered in Wistar rats at the dose of 0.20 mg/kg three times/week for 8 weeks. To confirm the onset of peripheral neuropathy we performed neurophysiological measures of the caudal nerve conduction velocity and the morphological analysis at the light microscope of the sciatic nerve and the dorsal root ganglia. After one single dose of BTZ and at the end of the 8 weeks-period of treatment, the level of proteasome inhibition was evaluated by the proteasome activity assay in blood mononuclear cells, sciatic nerve and brain by fluorimetric assay. Furthermore, the alteration of microtubule’s stability was examined in sciatic nerve by comparing the distribution of acetylated alpha-tubulin between polymerized and soluble fractions by western blot experiments. When BTZ was injected in a single acute dose we observed the recovery of the proteasome activity within 24 hours from the drug administration while when the drug was chronically administered, the proteasome activity remained suppressed. These different results were probably due to a cumulative effect of chronic administration of BTZ. Moreover, at the end of the treatment we observed the increase of acetylated alpha-tubulin in the polymerized fraction in sciatic nerves of treated animals as compared with control. This study suggests a potential explanation for the development of peripheral neuropathy induced by chronic admi

Bortezomib (BTZ) is an effective antineoplastic drug for the treatment of multiple myeloma. Its clinical use induce the development of a peripheral neuropathy characterized by sensory alterations and neuropathic pain. Rat models of BTZ-induced peripheral neuropathy had been established. However, since only a few cancer cell lines induce the development of cancer in the rat, these models don’t represent the most effective way to study, at the same time, the antineoplastic activity and the neurotoxic effects of BTZ. Here, immunodeficient SCID mice were s.c injected with RPMI8266 human myeloma cells. Three weeks after tumour injection, mice were i.v treated with BTZ 1 mg/kg once a week for five weeks. The tumour volume was assessed as a measure of BTZ antitumor activity; the mechanical nociceptive threshold and nerve conduction velocities were measured to assess the neuropathic pain and the toxic effect of BTZ on the peripheral nervous system, respectively. Starting from the first administration, BTZ 1 mg/Kg was able to block tumour-growth but inducing the development of mechanical allodynia and an impairment of nerve functions. Interestingly, myeloma itself seemed to be able to induce mild functional alterations of peripheral nerves. This mouse model and treatment schedule allowed the study of the antineoplastic activity and of the neurotoxic effects of BTZ at the same time. Moreover, a preliminary evaluation of the effect of myeloma itself on the peripheral nervous system was assessed. This animal model should be used in the preclinical discovery of new neuroprotective as well as of analgesic compounds. This work was partially sponsored by POR della Lombardia OB. 2 FSE 2007-2013, Asse IV - Sviluppo del Capitale Umano

Bortezomib is an antineoplastic agents that is often used to treat the multiple myeloma and of some lymphomas. Although its well-known antitumor activity, its effectiveness is limited by the highly incidental development of severe peripheral neuropathy (BIPN). This neuropathy is characterized by dysesthesia, numbness and a painful sensations. In order to obtain a pre-clinical model to improve our understanding of BIPN molecular pathways, we performed an rat model in which bortezomib (0,20 mg/kg) was administered three times weekly for eight weeks, followed by a four-week follow up period. At the end of the treatment period, we assessed nerve conduction velocity (NCV) and pathological changes in caudal nerve, dorsal root ganglia (DRGs), and sciatic nerve. Afterwards, we verified the involvement of proteasome inhibition and also we evaluated the microtubule stability in sciatic nerve by comparing the distribution of acetylated tubulin between polymerized (P) and soluble (S) fractions by western blot experiments. The neurophysiological evaluation demonstrated a reduction in NCV both at eight weeks and after the follow up period: at the pathologic analysis, caudal nerves were the most affected structures, whereas DRGs showed a vacuolization in the cytoplasm in sensory neurons and of satellite cells, although the sciatic nerves evidenced a mild axonopathy. Proteasome activity assay was performed on peripheral blood mononuclear cells (PBMCs), sciatic nerve and brain: PBMCs and sciatic nerve recovered quickly in the acute setting, while maintaining strong inhibition until 48 hours after last injection when the drug was chronically administered. Moreover, at six and eight weeks of treatment, we observed the increase of acetylated alpha-tubulin in the polymerized fraction in sciatic nerves of BTZ-treated animals as compared with control, that returning at baseline during follow up period. In conclusion, our results demonstrated that BTZ is able to induce a toxic effect on pe

Cisplatin (CDDP), Taxol (TAX) and Bortezomib (BZ) are chemotherapeutic drugs commonly employed in clinical practice for the treatment of solid and haematological tumors. Their clinical use is limited by the development of a peripheral neuropathy characterized by sensory alterations, pain and, in part, by motor and autonomic dysfunctions. Several rat models had been performed to study and describe the mechanisms of the peripheral neurotoxicity induced by these drugs. However, since only few cancer cell lines are able to induce the development of cancer in the rat, we focused our attention on mice models to allow the combined study of the antineoplastic activity and of the neurotoxic effects of the anticancer compounds. Moreover, different mice strains should be useful for different kind of studies. Here we investigated the interactions between the mice genotype and their drug response to determine the susceptibility to the development of chemotherapy-induced peripheral neuropathy. To this aim, we investigated the neurophysiological and neuropathological alterations induced by the chronic treatment with different chemotherapy drugs in female Balb/c, CD1 and c57 mice and in male c57 and FVB mice. Mice were injected with Cisplatin (2, 4 mg/Kg, ip, 2qw), or Taxol (50, 70, 80 mg/Kg, iv, 1qw) or Bortezomib (0.4, 0.8 mg/Kg, iv, 2qw) for a period of 4-6 weeks. At the end of the treatment the nerve conduction velocities (NCVs) were determined in the caudal and in the digital nerves and the dorsal root ganglia (DRG) and sciatic nerves collected for the neuropathological analysis; behavioural tests were performed to study if these drugs are able to induce allodynia or hyperalgesia. In all mice models we observed that TAX and CDDP induced a significant alteration in body weight at the end of the treatment. All the drugs induced a significant reduction in the caudal and digital NCV in Balb/c and CD1 mice. TAX and BZ caused the axonal degeneration of the sciatic nerves while CDDP

The clinical use of cisplatin (CDDP) is often associated with a severe neurotoxicity that represents the dose-limiting factor in therapy. In the past, several rat models has been accomplished to study the mechanisms involved in peripheral neuropathy induced by chronic CDDP administration and they highlight its toxic effects particularly in the dorsal root ganglia (DRG) sensory neurons that are smaller in sizes. Nevertheless, since only a limited number of cancer cell lines are able to induce the development of cancer in rat, we decided to use different murine models that allow a future combined study of the antineoplastic activity and the neurotoxic effects of CDDP. To achieve this aim, we evaluated the neurophysiological, neuropathological and morphometrical alterations induced by chronic CDDP treatment in female Balb/c, CD1 and Hola Hsd NuNu mice. Balb/c mice were treated intraperitoneally with CDDP 4mg/Kg twice a week for 4 weeks, while CD1 and Hola Hsd NuNu mice were treated with the same schedule followed by 2 weeks of follow-up. The body weight was measured once a week while at the end of the treatment we evaluated the nerve conduction velocities (NCVs) in the caudal and digital nerves and we collected DRG for the morphological and morphometrical analysis. We observed in all mice models a significant decrease in the body weight and a significant reduction in the caudal and digital NCV. This nerve functional damage was confirmed by morphological observations in DRG showing an increase in multinucleolated neurons and in nucleolar eccentricity and by the presence of nucleolar segregation. Moreover the morphometrical analysis demonstrated that CDDP affects neurons through a significant decrease in the size of somatic and nucleolar area in Balb/c mice and also in nuclear size in CD1 mice while the Nu/Nu mice showed only a significant decrease in the nuclear area. These results can confirm that the chosen schedule is able to induce peripheral neuropathy at rather di

Since peripheral sensory neuropathy is the major, clinically relevant side effect of sagopilone we investigated the general and peripheral neurotoxicity of sagopilone administered intravenously with different doses (1.2 and 2.4 mg/kg) and schedules in 48 Wistar rats and we performed in parallel a pharmacokinetic/pharmacodynamic (PK/PD) study. A trend toward a different peripheral neurotoxicity could be assessed after 2 weeks of treatment (bolus > 30-min infusion > 3-h infusion) with both doses of sagopilone. Although sagopilone concentrations in peripheral nerve tissue above 100 ng/g were associated with a reduction in nerve conduction velocity (NCV), a clear dose-dependence of this reduction on the level of systemic exposure to sagopilone was not observed. The PK/PD evaluation revealed no consistent effect of the infusion duration on serum PK parameters or the PD read-out NCV. Sagopilone concentrations in brain, sciatic nerve, liver, and kidney were higher after bolus compared to infusion, but there were no influence of infusion duration on these concentrations. No correlation between sagopilone concentrations in any organ/tissue with NCV changes was detected. This study evidences that the PD of sagopilone is not the main determinant of the onset and severity of sagopilone-induced peripheral neurotoxicity in the investigated clinically-relevant dose range, thus indicating that further investigation might identify neuronal-specific mechanisms of action able to drive a focused strategy to prevent peripheral neurotoxicity without reducing the anticancer effectiveness of the epothilones.

1-[(2-adamantyl)amino]acetyl-2-cyano-(S)-pyrrolidine, monohydrochloride (PKF275-055), a vildagliptin analog, is a novel, selective, potent, orally bioavailable, and long-acting dipeptidyl peptidase IV inhibitor. We studied the effect of PKF275-055 administration on the prevention, protection, and treatment of diabetic neuropathy in the streptozotocin-induced diabetic rat. PKF275-055 improved body and muscle weight. Oral glucose tolerance tests showed a marked improvement in glucose metabolism under all treatment schedules. When tested in prevention and protection experiments, PKF275-055 completely averted the decrease of Na +/K +-ATPase activity and partially counteracted the nerve conduction velocity (NCV) deficit observed in untreated diabetic rats but had no effects on abnormal mechanical and thermal sensitivity. When used in a therapeutic setting, PKF275- 055 induced a significant correction in the alteration in Na +,K +- ATPase activity and NCV present in untreated diabetics. Diabetic rats developed mechanical hyperalgesia within 2 weeks after streptozotocin injection and exhibited significantly longer thermal response latencies. It is noteworthy that PKF275-055 treatment restored mechanical sensitivity thresholds by approximately 50% (p < 0.01) and progressively improved the alteration in thermal responsiveness. In conclusion, PKF275-055 showed an anabolic effect, improved oral glucose tolerance, and counteracted the alterations in Na +,K +-ATPase activity, NCV, and nociceptive thresholds in diabetic rats. The present data support a potential therapeutic effect of PKF275-055 in the treatment of rodent diabetic neuropathy.

Cisplatin (CDDP) and Bortezomib (BTZ) are two antineoplastic drugs commonly used against different solid tumors and multiple myeloma, respectively. Although their efficacy, the clinical employment of these chemoterapeutics is associated with a severe neurotoxicity that represents the dose-limiting factor in therapy. Previous well established rat models evidenced the toxic effects of CDDP particularly on the dorsal root ganglia (DRG) sensory neurons that are smaller in the somatic, nuclear and nucleolar sizes. To investigate the role of CDDP and BTZ on the peripheral nervous system we used three mice models: female Balb/c were treated with CDDP 4mg/Kg (ip, 2qwx4) and with BTZ 0,8 mg/Kg (iv, 2qwx4), while female Hola Hsd NuNu and CD1 mice were treated with CDDP 4mg/Kg (ip 2qwx4 followed by 2 weeks of follow-up) and with BTZ 0,8 mg/Kg (iv, 2qwx6). The aim of this study was to perform a morphometrical quantification of cisplatin and bortezomib-induced toxic effects on the DRG neurons in the different mice strains. Three L4-L5 DRG/animals were collected, fixed and resin-embedded; 1 micro-sections stained with toluidine blu were analyzed with a computer-assisted image analyzer (Image J NIH sotware). In randomly selected sections the somatic, nuclear and nucleolar sizes of DRG sensory neurons were measured on at least 200 DRG neurons/animal. The morphometrical analysis of DRG showed that the administration of CDDP affects neurons causing a decrease in the size of somatic and nucleolar area in the Balb/c and CD1mice while the Nu/Nu strain showed only a decrease in the nuclear area. By contrast, the administration of BTZ induced an increase in the size of somatic, nuclear and nucleolar area in the Balb/c mice while only nucleolar area was increased in Nu/Nu and CD1 mice. The results obtained with the administration of CDDP seems to confirm the previous studies in rat, showing the toxic role of CDDP on neurons of DRG. Since the BTZ-induced increase in the cellular sizes seems

Sagopilone is the first fully synthetic epothilone entered in clinical investigation for the treatment of solid tumors. Since peripheral sensory neuropathy is its major side effect, this study was aimed to provide a preclinical model to evaluate factors that might modulate this side effect. We investigated the general toxicity and the peripheral neurotoxicity of sagopilone administered with different doses and schedules in Wistar rats and we performed a PK/PD study. The aims of the study were to establish the effect of different infusion times (i.v. bolus, i.v. 30-min infusion, and i.v. 3-hr infusion) over a 4-week treatment period; to evaluate the effect on nerve conduction velocity (NCV), to assess whether these schedules have an impact on peripheral neurotoxicity; to allow PK/PD modelling during the first 24 hours after infusion After 2 weeks, a trend toward a different peripheral neurotoxicity (bolus > 30-min infusion > 3-hr infusion) could be assessed at the pathological and neurophysiological levels with both doses, but this trend was no longer present after 4 weeks. When sagopilone concentrations were measured in brain, sciatic nerve, liver and kidney a linear relationship with the PK parameters (AUC(0-24h), Cmax) was observed. Sagopilone concentrations in sciatic nerve above 100 ng/g 24 hr after the 4th administration were associated with a reduction in NCV. A clear dose-dependence of NCV reduction on the level of systemic exposure was not observed but there was a time- dependence with more severe effects after 4 weeks. Both infusion duration of sagopilone had no consistent effect on PK parameters and on NCV; however, bolus was associated with higher AUC. Although lowest mean NCV values were observed at highest AUC values after 2 weeks, this relationship between AUC or Cmax and NCV impairment was no longer evident at the end of the study. Cmax and AUC values had a significant correlation across the study, therefore, the Cmax – NCV relationship was very

Objective: Bortezomib, a potent and selective proteasome inhibitor, is the first line treatment of relapsed, refractory multiple myeloma. One of the most commonly reported adverse reactions induced by this drug, is a peripheral neurotoxicity characterized by sensory and often painful neuropathy representing the major reason for a dose reduction and discontinuation of life-saving therapy. The use of currently available drugs for the treatment of neuropathic pain is largely empirical, and their mechanisms of action are not completely understood. CR4056 is a promising analgesic drug that interacts with imidazoline-2 receptors, and inhibits the activity of monoamine oxidases. Aim of this study was to evaluate if the new analgesic compound CR4056 could prevent (preventive schedule) or reverse (curative schedule) the neuropathic pain elicited by bortezomib in a rat model of chronic painful peripheral neuropathy . Methods: Neuropathy was induced in female Wistar rats by intravenous administration of Bortezomib (0.20 mg/kg, 3 times a week). CR4056 was orally administered at 6 mg/kg, once a day, in both preventive and curative schedules. In the preventive schedule animals were dosed with bortezomib and CR4056 starting from day 1 till week 8 or 10. In the curative schedule, established neuropathic animals were treated with CR4056 either from week 6 or week 8 till week 10. The sensory neuropathy was evaluated by tail nerve conduction velocity (NCV) at week 6, 8 and 10 after the first challenge with bortezomib. The development of neuropathic pain behaviour (mechanical allodynia) was followed by mechanical testing on day 1, at week 6, 8, 9 and 10. General toxicity, evaluated as body weight changes, was monitored twice a week Results: Bortezomib induced a severe reduction in NCV and a significant mechanical allodynia at all the indicated time points. Administration of CR4056, neither in preventive nor in curatives schedules, affected the NCV impairment. Conversely, CR4056 treatme

Bortezomib (BTZ) is a highly effective and widely used antineoplastic drug for the treatment of multiple myeloma. Despite its effectiveness, the use of this proteasome inhibitor is limited by its toxicity and in particular peripheral neurotoxicity is a major and clinically-relevant problem and an unsolved issue. The administration of Acetyl-L-Carnitine (ALC) in rat models of different peripheral neuropathies induced by other antineoplastic drugs has evidenced a neuroprotective effect of ALC. To investigate the neuroprotective role of ALC, we used a multiple myeloma-bearing animal model in which scid mice were iv treated with BTZ 1 mg/kg (q7dx5) or ALC 200 mg/kg/daily (qdx5/wx5w) alone or in co-treatment. Mice were s.c. injected into the right flank with 10x106 cell/0.1 mL/mouse of RPMI-8226 multiple myeloma. Treatments started three days after tumor injection. Aim of our study was to evaluate if the co-administration of ALC was able to prevent the peripheral neurotoxicity induced by BTZ chronic treatments. The results of this study evidenced that the administration of BTZ was able to induce significant neurophysiological changes vs. vehicle-treated animals in conduction velocity in the caudal and digital nerves and in potential amplitude in the digital nerve. The same results were observed in nerve conduction velocities in the BTZ+ALC treated mice, while the potential amplitude in the digital nerve was not significantly different from vehicle-treated animals in BTZ+ALC mice. Moreover, the administration of BTZ induced evident pathological changes vs. vehicle-treated animals in the caudal sciatic nerve or dorsal root ganglia (DRG). The same changes were observed in the BTZ+ALC treated mice, although in the caudal nerve an evident trend toward a less severe effect of BTZ administration was present in the animals co-treated with ALC. In conclusion, although not all the parameters investigated in this study evidenced a positive effect, a neuroprotective activity of ALC

PURPOSE: Alterations in mRNA for myelin proteins are reported in animal models of chemotherapy-induced peripheral neuropathies (CIPN); however, ultrastructural changes in aldehyde-fixed and plastic-embedded myelin are not evident by electron microscopy. Therefore, we used X-ray diffraction (XRD) to investigate more subtle changes in myelin sheath structure from unfixed nerves. EXPERIMENTAL DESIGN: We used in vivo chronic animal models of CIPN in female Wistar rats, administering cisplatin (CDDP 2mg/kg, i.p. twice/week), paclitaxel (PT 10mg/kg, i.v. once/week) or bortezomib (0.20mg/kg, i.v. three times/week) over a total period of 4weeks. Animal weights were monitored, and tail nerve conduction velocity (NCV) was determined at the end of the treatments to assess the occurrence of peripheral neuropathy. Sciatic nerves were collected and the myelin structure was analyzed using electron microscopy (EM) and XRD. RESULTS: All the rats treated with the chemotherapy agents developed peripheral neuropathy, as indicated by a decrease in NCV values; however, light and electron microscopy indicated no severe pathological alterations of the myelin morphology. XRD also did not demonstrate significant differences between sciatic nerves in treated vs. control rats with respect to myelin period, relative amount of myelin, membrane structure, and regularity of membrane packing. CONCLUSIONS: These results indicate that experimental peripheral neuropathy caused by CDDP, PT, and bortezomib-which are among the most widely used chemotherapy agents--does not significantly affect the structure of internodal myelin in peripheral nerve.

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, LipoplatinTM, 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 LipoplatinTM, selected within the range of its anticancer effective dose, is less neurotoxic than cisplatin administration.

Purpose: Cisplatin is one of the most effective cytotoxic agent 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, LipoplatinTM, was developed to reduce the systemic toxicity of cisplatin but preventing its efficacy. Aim of this study was to test in an animal model through a multimodal approach if chronic treatment with two different schedules of LipoplatinTM selected in the range of its anticancer effective dose is less neurotoxic than cisplatin administrations. Methods: Female Wistar rats were treated intraperitoneally with cisplatin at the dose of 4mg/kg or with LipoplatinTM 12 and 24mg/kg once weekly for 4 times. General toxicity was assessed by daily observation, body weight change, hematological and blood chemistry analysis and histopathology of liver and kidney. The onset of the peripheral neurotoxicity was assessed using tail nerve conduction velocity (NCV), morphological and morphometrical analysis of dorsal root ganglia (DRG) and by morphological analysis of sciatic nerve. Results: Cisplatin induced a statistically significant reduction in body weight, the development of renal failure and the impairment in NCV with pathological alterations in the DRG and sciatic nerve. By contrast no significant weight gain reduction was observed in animals treated with both doses of LipoplatinTM. Moreover the lowest dose induced a less severe damage to the peripheral nervous system with a moderate decrease of NCV and mild pathological alterations of DRG and sciatic nerve. Conclusions: The results suggest that LipoplatinTM 12mg/kg is less neurotoxic than cisplatin 4mg/kg, thus opening the possibility to use this new formulation in future studies where its anticancer activity and the peripheral neurotoxicity will be assessed in parallel. This study was supported by “Fondazione Banca del Monte di Lombardia” Regulon supported this study exclusi

Cisplatin, oxaliplatin, paclitaxel, epothilone-B and bortezomib represent some of the most employed treatments in colon, genitourinary cancers and multiple myeloma. Nevertheless, their clinical use is often associated to the development of a peripheral neuropathy characterized by sensory alterations, pain and, in part, by motor and autonomic dysfunctions. Several rat models of chemotherapy-induced peripheral neuropathy (CIPN) had been established in the past to describe the mechanisms of its development and pathogenesis revealing that all chronic chemotherapy regimens cause neurophysiological impairments and alterations in peripheral nerves and DRG. However, since only few cancer cell lines induce the development of cancer in the rat, this model doesn’t represent the most effective way to study, at the same time, the antineoplastic activity and the neurotoxic effects of the anticancer compounds. Here we report a neurophysiological and neuropathological characterization of new immunocompetent (IC) and immunodeficient (ID) murine models of chronic CIPN. Balb-c (IC) and Hsd Athymic nude nu/nu (ID) mice were treated with cisplatin (2, 4 mg/Kg), or paclitaxel (50, 70, 80 mg/Kg) or oxaliplatin (3.5 mg/Kg), or epothilone-B (2, 4 mg/Kg), or bortezomib (0.4, 0.8 mg/Kg), for a 4-6 week-period. Sensory/motor and sensory nerve conduction velocities (NCVs) were determined in the caudal and the digital nerves. DRG and sciatic nerves were collected for neuropathological and morphometrical analysis. The electrophysiological studies revealed that in Balb-c mice all compounds determined a significant reduction in caudal NCV, and at a lesser extent, in the digital NCV. These functional damages were confirmed by morphological observations describing an axonal degeneration in the sciatic nerves induced by paclitaxel, epothilone-B and bortezomib and alterations in DRG induced by platinum compounds and bortezomib. Platinum compounds determined also a quantitative reduction- in- size of DR

Peripheral tissue injury can alter somatic sensory pathways, resulting in behavioural hypersensitivity and increased responses to pain caused by noxious stimuli (hyperalgesia) and normally innocuous stimuli (allodynia). The absence of knowledge concerning mechanism of chemotherapy- induced neuropathic pain has hindered the development of new treatment strategies. The anticancer drug bortezomib (BTZ) is a inhibitor of the 26S proteasome mainly used for treatment of multiple myeloma. BTZ modulates protein metabolism leading to the apoptosis of malignant cells. However BTZ induces a painful neuropathy that cause a significant impairement of patient’s quality of life. The use of adjuvant for neuropathic cancer pain is largely empirical and their mechanisms of action is not completely know. Gabapentin is a structural analogue of gamma-aminobutyric acid (GABA) and is a promising anticonvulsant drug with an analgesic effect in neuropathic pain. The aim of this study was to evaluate through neurophysiological, histological and behavioural methods the effect of BTZ long-term administration on the rat peripheral nervous system. Furthermore the analgesic effect of gabapentin was evaluated. BTZ was administrated for 8 weeks at doses of 0.20 mg/kg [3q7d] i.v. In a second experimental phase, half of the animals were treated with gabapentin (100 mg/kg for 4 weeks daily) and half of the animals were left untreated. General toxicity was monitored twice a week by body weight measure. The neurotoxicity was evaluated by tail nerve conduction velocity (NCV) measures at 8 and 11 weeks after the begin of the experiment. Mechanical allodynia was assessed by Dynamic test at baseline, at 56, 57, 67, 70, 77, 84 days of treatment. BTZ induced a significant reduction in body weight after 8 weeks (p<0.05 vs CTRL) that was no longer significant at the end of follow-up period. Electrophysiological evaluation at the 8 weeks of treatment evidenced a significant reduction of NCV (p<0.0001 vs. CTRL).

Introduction: CR4056 is a novel imidazoline-2 receptor ligand endowed with low affinity MAO inhibition properties. Objectives: Aims of this study was to test with different methods the anti-nociceptive effect of CR4056 in rat models of neuropathic pain, i.e. Bortezomib(Btz)-induced neuropathic pain, streptozotocin-induced diabetes and chronic constriction injury (CCI). Methods: In the Btz model (0.20mg/kg 3qwx8 iv) and in the experimental diabetes model CR4056 was administered at 6-60mg/kg po. In the CCI mononeuropathy model CR4056 was administered at 10-80 mg/kg po. Nerve conduction velocity (NCV), dynamic, Randall-Selitto, plantar and von Frey tests were used to assess the effect of CR4056. Results: After 8 weeks of treatment, Btz induced a severe reduction in NCV and this impairment was not reversed by CR4056. The behavioral assessment showed a significant mechanical allodynia in rats treated wit Btz for 8 wks and the daily administration of CR4056 significantly reduced this condition. This effect was persistent along the entire period of administration after Btz withdrawal (2 weeks). In the models of streptozotocin-induced diabetes and CCI, treatment with CR4056 significantly attenuated mechanical and thermal hyperalgesia, while CR4056 had no effect on mechanical allodynia in both experimental models. Conclusions: These results promote the use of CR4056 as a potential treatment in the chronic neuropathic pain therapy.

The anticancer drug bortezomib (BTZ) is a reversible 20S proteasome inhibitor used in the treatment of multiple myeloma and certain lymphomas. BTZ-induced peripheral neuropathy (BIPN) is a widely recognised dose-limiting side effect of this drug that can influence the patient’s quality of life. Few data are available about the mechanisms underlying BTZ neurotoxicity. In this work we used different animal models to investigate the BTZ-induced peripheral nervous system (PNS) impairment. In a preliminary study we used Wistar rats treated with BTZ 0.20 mg/kg 3qwX4. In order to mimic the clinical long-term use of BTZ, and to reproduce the typical pain symptoms in BTZ-treated patients, we designed a second experimental model in which we extended the treatment phase up to 8 weeks, preserving the drug dose. In both rat models we demonstrated an axonopathy, but only through the second ones we assessed the severity and the time-course of BTZ-induced neuropathic pain. We then set up a third study in order to evaluate the neurotoxicity and the antitumor activity of BTZ in the same in vivo model. Since only few cancer cell lines are able to induce the development of cancer in immunocompetent rats, we decided to use Balb/c (immunocompetent) and Hsd Athymic nude nu/nu (immunodeficient) murine models of chronic BIPN. BTZ was admistered 0.4 or 0.8 mg/kg, for 4 or 6 weeks. In both models we investigated the sensory/motor and sensory nerve conduction velocity (NCV) in the caudal and digital nerves. In all models BTZ decreased the NCV, indicating a neurophysiological impairment; this damage was confirmed by morphological alterations in the PNS. These results suggest that all the models mentioned above are usefull to reproduce the BIPN. Furthemore, mice model will allow us to study the neurotoxicity induced by BTZ and its activity against cancer of human and murine origin in the same in vivo model. Supported in part by a research grant from the “Fondazione Banca del Monte di Lombardia”