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Chemotherapy-induced peripheral neuropathy (CIPN) is a frequent and potentially irreversible adverse event of cytotoxic chemotherapy. We evaluate whether sensory neurons derived from induced pluripotent stem cells (iPSC-DSN) can serve as human disease model system for chemotherapy induced neurotoxicity. Sensory neurons differentiated from two established induced pluripotent stem cell lines were used (s.c. BIHi005-A https://hpscreg.eu/cell- line/BIHi005-A and BIHi004-B https://hpscreg.eu/cell-line/BIHi004-B, Berlin Institute of Health Stem Cell Core Facility). Cell viability and cytotoxicity assays were performed, comparing susceptibility to four neurotoxic and two non-neurotoxic drugs. RNA sequencing analyses in paclitaxel vs. vehicle (DMSO)treated sensory neurons were performed. Treatment of iPSC-DSN for 24 h with the neurotoxic drugs paclitaxel, bortezomib, vincristine and cisplatin led to a dose dependent decline of cell viability in clinically relevant IC50 ranges, which was not the case for the non-neurotoxic compounds doxorubicin and 5-fluorouracil. RNA sequencing analyses at 24 h, i.e. before paclitaxel-induced cell death occurred, revealed the differential expression of genes of neuronal injury, cellular stress response, and sterol pathways in response to 1 mu M paclitaxel. Neuroprotective effects of lithium chloride co-incubation, which were previously shown in rodent dorsal root ganglia, could be replicated in human iPSC-DSN. Cell lines from the two different donors BIHi005-A and BIHi004-B showed different responses to the neurotoxic treatment in cell viability and cytotoxicity assays.

Additional file 1: Figure S1. Supplementary material and methods and supplementary figure 1.

Chemotherapy-induced neuropathy (CIN) is a common and serious side effect of many chemotherapeutic drugs including paclitaxel. CIN is the number one reason to reduce or discontinue treatment, which directly affects patients’ chance for survival. Despite the well-defined time point of damage, knowledge of patients at risk, and several published preclinical prevention strategies, no prevention of CIN is currently clinically available for patients. Major challenges for successful clinical translation of preclinical data include lack of reproducibility and randomization, small sample sizes and insufficient statistical tests. We thus propose to conduct a rigorous multi-center replication trial. We and others identified pivotal steps in the pathophysiology of paclitaxel-induced neuropathy, which can be targeted by preventive medication: (1) Entrance of paclitaxel into sensory neurons via organic anion–transporting polypeptide proteins, which can be targeted with nilotinib, (2) intracellular induction of calcium dyshomeostasis, which can be modified with lithium ions, and (3) a complex neuro-immune interaction orchestrated by the cytokine IL-6, which can be blocked with IL-6 neutralizing antibodies. In the proposed research, we will use a two step approach to confirm the optimal dose which will then be used in a clinically meaningful mouse breast cancer xenograft model to test and compare safety and efficacy. The design of our study will allow controlling major confounding factors and help to determine which strategy can then be pursued in a clinical proof-of-concept study., Experimental Intervention Dose confirmation study: PINPRICS-DC In the first phase of the study, neuropathy is induced with paclitaxel 12 x 20 mg/kg body weight (BW) intraperitoneally (i.p.) with 3 injections/week over the course of 4 weeks plus either: Nilotinib i.p.: 20 mg/kg BW, 100 mg/kg BW, 500 mg/kg BWLithium carbonate i.p.: 2.6 mg/kg BW, 12.8 mg/kg BW, 64 mg/kg BWAnti-mouse-IL-6 IgG (MAB406) i.p.: weekly 1 mg/kg BW, 5 mg/kg BW, 25 mg/kg BW Prevention study: PINPRICS-PS Induction of neuropathy by paclitaxel 12 x 20 mg/kg body weight (BW) intraperitoneally (i.p.) with 3 injections/week over the course of 4 weeks plus either: Nilotinib i.p.Lithium carbonate BW i.p. Anti-mouse-IL-6 IgG (MAB406) i.p. Dose: Lowest tolerated dose yielding an optimal effect as obtained in the dose confirmation study. Control intervention (pos./neg.): Animals treated with paclitaxel 12 x 20 mg/kg BW i.p. and vehicle of preventive substances will serve as negative control groupAnimals treated with vehicle of paclitaxel and vehicle of preventive substances will serve as positive control group. Duration of intervention: 4 weeks Follow-up: 2 weeksIn and exclusion criteria: Dose confirmation study (PINPRICS-DC): Number of experimental groups: 9 (3 drugs, 3 doses/drug) Key inclusion criteria: Female sex (Breast cancer is a predominantly female condition)Strain: BALB/cAge: 12 – 16 weeksTreatment with 12 x 20 mg/kg BW paclitaxel + neuroprotective substance as described above Key exclusion criteria: As in the main trial Prevention study (PINPRICS-PS): Number of experimental and control groups: 3 experimental and 2 control groups Key inclusion criteria: Female sex (Breast cancer is a predominantly female condition)Strain: BALB/cAge: 12 – 16 weeksTransplantation of 4T1 breast cancer cells into the mammary fat padTreatment with 12 x 20 mg/kg BW paclitaxel + neuroprotective or control intervention Key exclusion criteria: Weight loss > 20% of baselineTumor diameter > 15 mmGross abnormalities in behavior as defined by the UKCCCR (e.g. apathy, aggression, difficulty breathing) Consideration of external validity: The experimental model mimics the situation of breast cancer patients receiving a dose-dense neoadjuvant paclitaxel therapyOutcome: Primary efficacy endpoint: Quantification of nerve damage measured by changes in tail nerve sensory nerve action potential amplitude (SNAP)Key secondary endpoint(s):Safety: Changes in tumor sizeFunctional: Changes in mechanical withdrawal threshold Mechanistic: Changes in myelinated nerve fiber density in the sciatic nerveChanges in intraepidermal nerve fiber densityChanges in invading macrophages in the dorsal root ganglia Randomization and blinding: Upon arrival of the animals in the animal facility, unequal block randomization will be performed to determine group and cage allocation. Experimenters are fully blinded as they will apply number coded trial medication, which they receive from another participating site. All animals receive standardized handling., Chemotherapy-induced neurotoxicity constitutes an unmet and relevant medical need. Extensive literature research was carried out in open access databases (PubMed, Clinicaltrials.gov. Cochrane library etc.), but also the GlobalData database. In terms of clinical prevention and treatment trials, there is a positive phase III study for duloxetine in the symptomatic treatment of CIPN, but prevention and causal treatment trials with a number of strategies (among others vitamin E, glutathione, acetyl-L-carnitine) failed . In summary, treatment options for CIPN presently consist of (often-insufficient) symptomatic treatments and no preventive interventions exist. Based on published results and data from our consortium, we identified several pivotal steps in the pathophysiology of PTX-induced neuropathy, which can be targeted by a preventive co-medication: 1. Entrance of PTX into sensory neurons via OATP, 2. Intracellular induction of calcium dyshomeostasis and 3. Complex neuro-immune interaction mediated by the cytokine IL-6. In the proposed research, we will use a clinically meaningful mouse model to test and compare safety and efficacy of modulating the pathophysiology at these targets.

Chronic inflammatory demyelinating polyradiculoneuropathy (CIDP) is an autoimmune disorder, which causes progressive sensory and motor deficits and often results in severe disability. Knockout of the co-stimulatory protein CD86 in mice of the non-obese diabetic background (NoD.129S4-Cd86 tm1Shr /JbsJ) results in the development of a spontaneous autoimmune peripheral polyneuropathy (SAPP). We used this previously described transgenic model to study the effects of the sphingosine-1-phosphate receptor agonist fingolimod on SAPP symptoms, functional and electrophysiological characteristics. Compared to two control strains, knockout of CD86 in NOD mice (CD86−/− NOD) resulted in progressive paralysis with distinct locomotor deficits due to a severe sensory-motor axonal-demyelinating polyneuropathy as assessed by electrophysiological measurements. We started fingolimod treatment when CD86−/− NOD mice showed signs of unilateral hind limb weakness and continued at a dose of 1 mg/kg/day for eight weeks. We did not observe any beneficial effects of fingolimod regarding disease progression. In addition, fingolimod did not influence the functional outcome of CD86−/− NOD mice compared to vehicle treatment nor any of the electrophysiological characteristics. In summary, we show that fingolimod treatment has no beneficial effects in autoimmune polyneuropathy, which is in line with recent clinical data obtained in CIDP patients.