Alessandra Gilardini

Bortezomib-induced peripheral neurotoxicity: A neurophysiological and pathological study in the rat

Bortezomib is a new proteasome inhibitor with a high antitumor activity, but also with a potentially severe peripheral neurotoxicity. To establish a preclinical model and to characterize the changes induced on the peripheral nerves, dorsal root ganglia (DRG) and spinal cord, bortezomib was administered to Wistar rats (0.08, 0.15, 0.20, 0.30 mg/kg/day twice [2q7d] or three times [3q7d] weekly for a total of 4 weeks). At baseline, on days 14, 21 and 28 after the beginning the treatment period and during a 4-week follow-up period sensory nerve conduction velocity (SNCV) was determined in the tail of each animal. Sciatic nerve, DRG and spinal cord specimens were processed for light and electron microscope observations and morphometry. At the maximum tolerated dose bortezomib induced a significant reduction in SNCV, with a complete recovery at the end of the follow-up period. Sciatic nerve examination and morphometric determinations demonstrated mild to moderate pathological changes, involving predominantly the Schwann cells and myelin, although axonal degeneration was also observed. Bortezomib-induced changes were also observed in DRG and they were represented by satellite cell intracytoplasmatic vacuolization due to mitochondrial and endoplasmic reticulum damage, closely resembling the changes observed in sciatic nerve Schwann cells. Only rarely did the cytoplasm of DRG neurons has a dark appearance and clear vacuoles occurring in the cytoplasm. Spinal cord was morphologically normal. This model is relevant to the neuropathy induced by bortezomib in the treatment of human malignancies and it could be useful in increasing our knowledge regarding the mechanisms underlying bortezomib neurotoxicity.

Protective Effect of Erythropoietin and Its Carbamylated Derivative in Experimental Cisplatin Peripheral Neurotoxicity

Purpose: Antineoplastic drugs, such as cisplatin (CDDP), are severely neurotoxic, causing disabling peripheral neuropathies with clinical signs known as chemotherapy-induced peripheral neurotoxicity. Cotreatment with neuroprotective agents and CDDP has been proposed for preventing or reversing the neuropathy. Erythropoietin given systemically has a wide range of neuroprotective actions in animal models of central and peripheral nervous system damage. However, the erythropoietic action is a potential cause of side effects if erythropoietin is used for neuroprotection. We have successfully identified derivatives of erythropoietin, including carbamylated erythropoietin, which do not raise the hematocrit but retain the neuroprotective action exerted by erythropoietin. Experimental Design: We have developed previously an experimental chemotherapy-induced peripheral neurotoxicity that closely resembles CDDP neurotoxicity in humans. The present study compared the effects of erythropoietin and carbamylated erythropoietin (50 μg/kg/d thrice weekly) on CDDP (2 mg/kg/d i.p. twice weekly for 4 weeks) neurotoxicity in vivo. Results: CDDP given to Wistar rats significantly lowered their growth rate (P < 0.05), with slower sensory nerve conduction velocity (P < 0.001) and reduced intraepidermal nerve fibers density (P < 0.001 versus controls). Coadministration of CDDP and erythropoietin or carbamylated erythropoietin partially but significantly prevented the sensory nerve conduction velocity reduction. Both molecules preserved intraepidermal nerve fiber density, thus confirming their neuroprotective effect at the pathologic level. The protective effects were not associated with any difference in platinum concentration in dorsal root ganglia, sciatic nerve, or kidney specimens. Conclusions: These results widen the spectrum of possible use of erythropoietin and carbamylated erythropoietin as neuroprotectant drugs, strongly supporting their effectiveness.

Bortezomib-induced painful neuropathy in rats: a behavioral, neurophysiological and pathological study in rats

Bortezomib is a proteasome inhibitor showing strong antitumor activity against many tumors, primarily multiple myeloma. Bortezomib‐induced neuropathic pain is the main side effect and the dose‐limiting factor of the drug in clinical practice. In order to obtain a pre‐clinical model to reproduce the characteristic pain symptoms in bortezomib‐treated patients, we developed an animal model of bortezomib‐induced nociceptive sensory neuropathy. In this study, bortezomib (0.15 or 0.20mg/kg) was administered to Wistar rats three times/week for 8 weeks, followed by a 4 week follow‐up period. At the end of the treatment period a significant decrease in weight gain was observed in the treated groups vs. controls, and hematological and histopathological parameters were evaluated. After the treatment period, both doses of bortezomib induced a severe reduction in nerve conduction velocity and demonstrated a dose‐cumulative effect of the drug. The sensory behavioral assessment showed the onset of mechanical allodynia, while no effect on thermal perception was observed.

Proteasome Inhibition: A Promising Strategy for Treating Cancer, but What About Neurotoxicity?

The inhibition of protein degradation through the ubiquitin-proteasome pathway is a recently developed approach to cancer treatment which extends the range of cellular targets for chemotherapy. This therapeutic strategy is very interesting since the proteasomes carry out the regulated degradation of unnecessary or damaged cellular proteins, a process that is dysregulated in many cancer cells. Based on this hypothesis, the proteasome complex inhibitor bortezomib was approved for use in multiple myeloma patients by the US Food and Drug Administration (FDA) in 2003 and by the European Medicines Agency (EMEA) in 2004, and several new drugs with the same target and, sometimes, mechanism of action are currently under development. Interestingly, proteasome inhibitors have now also been tested in combination chemotherapy for the treatment of several solid tumors and it is likely that there will be more generalized use of these compounds in the near future. Despite its remarkable effectiveness, which led to it being rapidly approved for clinical use, some concern has been raised regarding the safety of bortezomib (and in general of proteasome inhibitors) since reduced degradation of damaged proteins has been postulated as being the basic mechanism of severe neurological diseases affecting the central nervous system. While this concern has not been confirmed by the clinical course of treated patients, from the first Phase I studies, it emerged that peripheral sensory neurotoxicity was one of the major dose-limiting toxicities. The main results from the use of proteasome inhibition in cancer chemotherapy and the implications for treatment on the nervous system will be reviewed.

Myelin structure is unaltered in chemotherapy-induced peripheral neuropathy

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.

Cationic liposomes target sites of acute neuroinflammation in experimental autoimmune encephalomyelitis.

The binding selectivity of charged liposomes to the spinal cord of rats affected by experimental autoimmune encephalomyelitis (EAE), a model of multiple sclerosis, was investigated. Positively and negatively charged liposomes were injected into the tail vein of rats, and blood/brain barrier (BBB) targeting was determined by confocal microscopy as a function of the temporal evolution of the inflammatory response. Accumulation in spinal cord endoneural vessels was observed for cationic, but not for anionic, liposomes, and only in EAE but not in healthy rats. The overall binding efficacy paralleled the severity of the clinical score, but targeting was observed already before clinical manifestation of inflammation. Preferential binding of positively charged liposomes in the course of acute EAE can be ascribed to subtle changes of BBB morphology and charge distribution in a similar way as for the binding of cationic particles to proliferating vasculature in chronic inflammation and angiogenesis. Our findings suggest that vascular changes related to increased binding affinity for cationic particles are very early events within the inflammatory reaction in acute EAE. Investigation of cationic vascular targeting can help to shed further light on these occurrences, and, potentially, new diagnostic and therapeutic options may become available. In neuroinflammatory diseases, cationic colloidal carrier particles may enable intervention at affected BBB by an approach which is independent from permeability increase.

Peripheral Neurotoxicity of Platinum Compounds

The peripheral neuropathies induced by antineoplastic drugs are an important side effect of cancer treatment having a relevant impact on the field of research at the present time. Platinum compounds are the most widely used anti-cancer drugs and their neurotoxicity on the peripheral nervous system seems to be defined, using both in vitro and in vivo experimental models, by specific molecular pathways that involve programmed cell death. The goal of research is the understanding of the neurotoxic mechanisms of platinum compounds in order to develop neuroprotective agents that can ameliorate the quality of life of cancer patients.