Lithium for use in the treatment of antibody drug conjugate (ADC)-induced neuropathy

Abstract

Provided are methods of treating and preventing Antibody drug conjugate (ADC)- induced neuropathy using effective amounts of lithium.

Description

METHODS FOR TREATING ADC-INDUCED NEUROPATHY RELATED APPLICATIONS

[0001] This application claims the benefit of priority to U.S. Provisional Application No. 63 / 626,850, filed January 30, 2024, the entire contents of which are incorporated herein by reference. BACKGROUND

[0002] Antibody drug conjugates (ADCs) are a class of small molecule anticancer agents that consist of cytotoxic drugs conjugated to antibodies via chemical linkers. These agents are designed to specifically target tumor cells while minimizing effects arising from systemic exposure. There are hundreds of ADCs in development and at least fifteen are approved by the FDA for the treatment of various cancers.

[0003] The auristatin class of tubulin modulator such as monomethyl auristatin E (MMAE) and monomethyl auristatin E (MMAF) are the most commonly used payloads for constructing ADCs. Despite their promise, nervous system toxicity associated with the use of these types of ADCs is quite common. MMAE-based ADCs, for example, are known to cause peripheral neuropathy (PN) such that treatment discontinuation and / or dose reduction frequently occurs. Although hypotheses exist, a definitive correlation between the use of MMAE-based ADCs and peripheral neuropathy has yet to be realized. Nonetheless, given that cancer rates are still on the rise, and because ADCs with Auristatin payloads have significantly improved cancer outcomes over current treatments, the need to reduce nervous system effects arising from ADC-based chemotherapies remains. SUMMARY

[0004] We previously showed that lithium (Li) was effective in decreasing calcium signaling in peripheral neurons of paclitaxel (PTX)-treated mice. See e.g., Mo et al., FASEB J, 2012 Nov; 26(11):4696-709. Although PTX and monomethyl auristatin E (MMAE) share antitumor mechanisms through microtubule disruption, we identified that unlike PTX, which increases calcium release from intracellular stores, MMAE binds to a distinct portion of NCS1 and consequently decreases calcium release instead of promoting calcium surges. See e.g., FIGs.4 and 6 and the exemplification section below. These data suggests that the mechanism of action of MMAE is not the same as PTX in neurons and, therefore, the events leading to PTX and MMAE-induced neuropathy are unique. Despite these differences, we surprisingly found that lithium may also be effective in preventing MMAE-inducedneuropathy. See e.g., the results shown in FIG.2, where the addition of 1 mM lithium chloride (LiCl) to MMAE treated zebrafish embryos resulted in higher survival rates in comparison to embryos which were treated with MMAE or LiCl alone.

[0005] In MMAE-induced cognitive impaired mice, lithium pretreatment was found to prevent cognitive impairment (see FIGs.11A to 11E), and lithium pretreatment did not interfere with the antitumor effect of MMAE (see e.g., the exemplification section below).

[0006] Provided herein, therefore, are methods of using lithium to treat or prevent ADC- induced neuropathy and / or cognitive impairment. BRIEFDESCRIPTION OF THEFIGURES

[0007] FIG.1 shows the LC50for zebrafish embryos treated with MMAE at 50% epiboly.

[0008] FIG.2 shows the survival rate of zebrafish embryos treated with MMAE, LiCl, and the combination of MMAE and LiCl.

[0009] FIG.3A shows intracellular calcium changes in representative SK-N-AS cells (using Fluo-4 / AM) before and during carbachol stimulation after a 4-hour treatment with 0.2 µM MMAE, 0.2 µM MMAE + 1 m< LiCl, or 1 mM LiCl.

[0010] FIG.3B shows a comparison of the area under curve (AUC) for the changes in intracellular calcium control SK-N-AS cells, after a 4-hour treatment as in panel A (n =177- 215 cells, N = 3 independent replicates; ****p<0.0001 and ns p>0.05).

[0011] FIG.3C shows baseline for the changes in intracellular calcium control SK-N-AS cells, after a 4-hour treatment as in panel A (n =177-215 cells, N = 3 independent replicates; ****p<0.0001 and ns p>0.05).

[0012] FIG.3D shows representative fluorescence traces showing changes in intracellular calcium over time after 4-hour treatment as in panel A.

[0013] FIG.3E is a representative Western blot showing NCS1 and GAPDH (a loading control) levels after 4-hour treatment with three concentrations of MMAE.

[0014] FIG.3F is a quantification of all Western blots of NCS1 and GAPDH in SK-N- AS cells as shown in panel E (N = 3 independent replicates).

[0015] FIG.3G shows intracellular calcium changes in representative SK-N-AS cells (using Fluo-4 / AM) before and during carbachol stimulation in response to acute treatment with 0.2 µM MMAE (T - treatment, C - carbachol stimulation)

[0016] FIG.3H is a comparison of the area under curve (AUC) for the intracellular calcium signaling between control and acutely treated with MMAE cells (n = 125-131 cells, N = 3 independent replicates; ****p<0.0001 and ns p>0.05).

[0017] FIG.3I shows the baseline for the intracellular calcium signaling between control and acutely treated with MMAE cells (n = 125-131 cells, N = 3 independent replicates; ****p<0.0001 and ns p>0.05).

[0018] FIG.4A shows the PTX and MMAE binding sites with tubulin.

[0019] FIG.4B shows the NCS1 portion of FIG.4A where PTX and MMAE bind to different sites.

[0020] FIG.4C shows the effects of taxol or MMAE on the interaction between NCS1 and the IP3R.

[0021] FIG.5A shows a representative immunofluorescence image with SK-N-AS cells treated with 0.2 uM MMAE, 0.2 uM MMAE + 1 mM LiCl or 1 mM LiCl and stained for alpha-tubulin.

[0022] FIG.5B is a graph showing microtubule length (n = 26-30 cells, N = 2 individual replicates; ****p<0.0001).

[0023] FIG.6 is a model outlining the molecule differences between PTX and MMAE on neuronal signaling.

[0024] FIG.7 shows the percentage impact on embryo survival using a combination of MMAE with LiCl vs MMAE with NaCl.

[0025] FIG.8 shows the percentage impact on embryo survival using a combination of PTX with LiCl.

[0026] FIG.9 shows the lack of impact on cells motility as measured by wound closure following the use of MMAE with LiCl.

[0027] FIG.10A shows a comparison of the area under curve (AUC) for the changes in intracellular calcium control Neuro-2A cells, after a 4-hour treatment with 0.2 µM MMAE, 0.2 µM MMAE + 1 m< LiCl, or 1 mM LiCl.

[0028] FIG.10B is a representative Western blot showing NCS1 and GAPDH (a loading control) levels after 4-hour treatment with three concentrations of MMAE.

[0029] FIG.10C is a quantification of all Western blots of NCS1 and GAPDH in Neuro- 2A cells.

[0030] FIG.11A is Visual representation of the Novel Object Recognition Test protocol described in the Exemplification section.

[0031] FIG.11B shows the training sessions for the Novel Object Recognition Test showed no statistical difference between the two objects, suggesting no preference for either object.

[0032] FIG.11C are heat maps from training sessions demonstrating the relative time spent exploring each object.

[0033] FIG.11D shows both the saline (n=13) and lithium (n=14) treatment groups were able to identify which object was changed (p<0.001; p<0.0001), whereas the MMAE group (n=15) expressed no significant difference between familiar and novel objects (p>0.05). The LiCl+MMAE group (n=14) showed the ability to differentiate the objects, indicating that lithium pretreatment protects the mice from cognitive impairment induced by MMAE.

[0034] FIG.11E shows heat maps from the Novel Object Recognition Test demonstrating the relative time of exploration for each object.

[0035] FIG.12A show the licking time upon capsaicin injection was significantly different in the MMAE group (n=5) when compared to both the saline (n=4) or lithium (n=5) groups (p<0.001), suggesting loss of peripheral sensitivity to pain. Lithium pretreatment (n=4) prevented nociceptive impairment caused by MMAE-based chemotherapy, with licking time comparable to the control (p>0.05).

[0036] FIG.12B is a comparison of Capsaicin Test results after 7 and 28 days of treatment termination showed no significant difference, indicating that the nociceptive impairment induced by MMAE is sustained up to 28 days and the protection by lithium pretreatment is maintained.

[0037] FIG.12C is where Von Frey Test showed a significant difference between MMAE group and both saline or lithium groups (p<0.001), indicating the development of allodynia. Lithium pretreatment was able to prevent the allodynia, showing no statistical difference in the response threshold compared to the control (p<0.001). For this experiment, groups were composed with 7 animals.

[0038] FIG.12D shows the quantification of the diameter of the ex vivo DRG axons showed reduction in myelin width in the MMAE group compared to the saline or lithium groups (p<0.0001). Lithium administration was able to prevent the reduction, with myelin width comparable to the control (p>0.05).

[0039] FIG.13A is time course of tumor size during the indicated treatments, showing that lithium does not harm the antitumor activity of MMAE.

[0040] FIG.13B is size of excised tumors after indicated treatments. MMAE and LiCl+MMAE (n=7 each group) had a reduction in tumor, with size final comparable to the saline (n=5) treated animals (p<0.01).

[0041] FIG.14A shows the tumor volume size in MDA-MB-468 xenograft model over time following treatment with using Padcev, alone or in combination with lithium.

[0042] FIG.14B shows the tumor growth inhibition in MDA-MB-468 xenograft model using Padcev, alone or in combination with lithium. DETAILED DESCRIPTION

[0043] In one aspect, provided herein is a method of treating or preventing antibody drug conjugate (ADC)-induced peripheral neuropathy in a subject undergoing anticancer treatment with at least one ADC, comprising administering to the subject an effective amount of lithium (Li). Also provided herein is the use of an effective amount of lithium for treating or preventing ADC-induced peripheral neuropathy in a subject. Also provided herein is the use of an effective amount of lithium for the manufacture of a medicament for treating or preventing ADC-induced peripheral neuropathy in a subject. Further provided herein is a pharmaceutical composition comprising an effective amount of lithium for treating or preventing ADC-induced peripheral neuropathy in a subject.

[0044] As used herein, “antibody drug conjugate (ADC)-induced neuropathy” refers to neuropathy caused from administering an ADC.

[0045] The term “neuropathy” refers to both “peripheral neuropathy” and “cognitive impairment”. Peripheral neuropathy refers to damage that affects the peripheral nervous system and cognitive impairment refers to damage that causes a deficit in neurocognitive domains.

[0046] ADCs are known in the art and include small molecules composed of an antibody (e.g., a monoclonal antibody) linked to a biologically active cytotoxic (anticancer) payload via a chemical linker. ADC payloads include tubulin modulators, DNA damaging agents, and the like. In one aspect, the payload on the ADCs described herein is a tubulin modulator.

[0047] Tubulin modulators are chemical structures which alone, or as part of an ADC, inhibit or promote polymerization of microtubules. Tubulin modulators include, but are not limited to auristatins such as MMAE and MMAF, maytansinoid derivatives such as DM2 and DM4, tubulysins, and halichondrin. In one aspect, the payload on the ADCs described herein is an auristatin. In another aspect, the payload on the ADCs described herein is MMAE or MMAF. In another aspect, the payload on the ADCs described herein is MMAE. In oneaspect, the ADC described herein is selected from brentuximab vedotin, polatuzumab vedotin, enfortumab vedotin, belantamab mafodotin, disitamab vedotin, tisotumab vedotin, telisotuzumab bedotin, and zilovertamab vedotin. In another aspect, the ADC described herein is selected from brentuximab vedotin, polatuzumab vedotin, and enfortumab vedotin. In another aspect, the ADC described herein is brentuximab vedotin.

[0048] It will be understood that unless otherwise indicated, the administration and use of lithium described herein includes administering lithium prior to, concurrently with, or after administration of the ADC. Thus, simultaneous administration is not necessary for therapeutic purposes. In one aspect, however, lithium is administered concurrently with the ADC. In another aspect, lithium is administered prior to administering the ADC. For example, lithium may be administered at least 30 min, at least an hour, or at least 3 hours before administering the ADC.

[0049] In one aspect, the lithium used in the present methods is in the form of a pharmaceutically acceptable salt. Such salt forms include, but are not limited to, lithium salts such as lithium chloride, lithium carbonate, lithium citrate, lithium sulfate, and lithium orotate, among others. In one aspect, the lithium used in the present methods is lithium chloride.

[0050] Also provided herein is the use of delivery systems comprising an effective amount of at least one ADC described herein and an effective amount of lithium. In certain aspects, the delivery system has the ability to deliver the at least one ADC and the lithium at different times.

[0051] The terms “subject” and “patient” may be used interchangeably, and mean a mammal in need of treatment, e.g., companion animals (e.g., dogs, cats, and the like), farm animals (e.g., cows, pigs, horses, sheep, goats and the like) and laboratory animals (e.g., rats, mice, guinea pigs and the like). Typically, the subject is a human in need of treatment.

[0052] As used herein, the terms “treatment,” “treat,” and “treating” refer to reversing, alleviating, or inhibiting the progress of ADC-induced neuropathy, or one or more symptoms thereof. The terms “prevent,” “preventing,” and “prevention” refer to the prevention of the onset or recurrence of ADC-induced neuropathy. In one aspect, “prevent,” “preventing,” and “prevention” refer to the prevention of the onset of ADC-induced neuropathy.

[0053] An “effective amount” or “therapeutically effective amount” refers to an amount of lithium described herein that will treat or prevent ADC-induced neuropathy e.g., a dosage of between 0.01 - 100 mg / kg body weight / day. In one aspect, the effective amount of lithium used in the present methods is an amount sufficient to treat or prevent ADC-inducedneuropathy, but not in an amount that is sufficient to have a therapeutic impact on mood or psychological disorders. As such, in certain aspects, the effective amount of lithium described herein excludes amounts of lithium that are useful for treating mood and / or psychological diseases and disorders. In other aspects, however, the effective amount of lithium used in the present methods may be an amount sufficient to treat or prevent ADC-induced neuropathy and also have a therapeutic impact on mood or psychological disorders such as treating bipolar disorder.

[0054] It should be noted that a specific dosage and treatment regimen for any particular patient will depend upon a variety of factors, including the activity of the specific compound employed, the age, body weight, general health, sex, diet, time of administration, rate of excretion, drug combination, and the judgment of the treating physician and the severity of the particular disease being treated. EXEMPLIFICATION

[0055] The representative examples that follow are intended to help illustrate the present disclosure, and are not intended to, nor should they be construed to, limit the scope of the invention. Protective Role of Lithium from MMAE Exposure in Zebrafish Embryos

[0056] Materials and Methods

[0057] Zebrafish colonies were maintained in Yale University School of Medicine in accordance with standard protocols (Whitlock et al., Development.2000;127(17):3645-53), the guidelines of and protocols approved by Yale University Institutional Animal Care and Use Committee (protocol number 2021-10778). Wild type fish of the TU / AB background were placed in breeding tanks overnight with a divider. The divider was removed in the early morning and embryos were collected from natural spawning approximately 2 hours post fertilization (hpf). Embryos were subsequently placed in embryo media (5mM NaCl, 0.17mM KCl, 0.33mM CaCl2, 0.33mM MgSO4 and 0.1% (w / v) methylene blue) and monitored hourly until the 50% epiboly stage.

[0058] All drug treatments occurred at the 50% epiboly stage. Embryos were first treated with varying concentrations of paclitaxel (PTX; Cayman Chemicals) and Monomethyl Auristatin E (MMAE; Cayman Chemicals) to determine lethal concentration 50 (LC50). Subsequent drug treatments used the LC50. DMSO concentrations were 1% in all treatments. Drugs were washed from embryos at 24 hpf and replaced with drug-free embryo media. In alltreatments, embryos were observed at 24 hpf, 48 hpf, 72 hpf, 96 hpf, and 120 hpf for morphological changes.

[0059] Results

[0060] The LC50for zebrafish embryos treated with MMAE at 50% epiboly was in line with the LC50for mammalian cells (FIG.1). Subsequent experiments included treatment with either 0.2 µM MMAE, 1 µM PTX, 1 mM LiCl, 0.2 µM MMAE + 1 mM LiCl, or 1 µM PTX + 1 mM LiCl from 5.25 hours post fertilization (hpf) to 24 hpf. Embryos were then monitored twice daily for changes in morphology, development, and survival for 4 days post treatment (dpt). In general, we noted that embryos that had core endpoints of lethality, such as gastrulation arrest, coagulation, or degrees of tail detachment typically did not survive past 24 hpf (18.75 hpt).

[0061] Treatment with 0.2 µM MMAE resulted in approximately half of the embryos dying at 4 dpt, compared to control (untreated) embryos which had a survival rate of 85% (FIG.2). With the addition of 1mM LiCl to the MMAE treated embryos, there was a significant increase in the number of embryos that survived. LiCl treatment on its own did not alter survival in the control group. To ensure that the results were not due to osmolarity differences, the experimental protocol was repeated with NaCl, which has a similar ionic profile to LiCl. NaCl treatment did not significantly impact embryo survival. See FIG.7. The increased survival in the presence of LiCl suggests that LiCl has a protective role in mitigating the lethal toxicity of MMAE exposure. These experiments were also performed with paclitaxel (PTX) instead of MMAE and similar results were observed, i.e., LiCl protected the embryos from toxicity resulting from PTX exposure. See FIG.8. Intracellular Calcium Signaling

[0062] To provide insight on the underlying role of MMAE on neuronal action, and to compare results from earlier studies using the tubulin stabilizer paclitaxel (PTX), the following experiments were conducted.

[0063] Materials and Methods

[0064] Cell maintenance: Primary experiments were done using human neuroblastoma SK-N-AS (ATCC, CRL-2137). Some assays were done using the human neuroblastoma SHSY-5Y (ATTC, CRL-2226) cell line or mouse neuroblastoma Neuro-2A cell line (ATCC, CCL-131). All cell lines were cultured in DMEM:F12 with 10% FBS, 1% Pen Strep, 1% MEM, 1% L-glutamate and 1% sodium pyruvate. Cells were maintained at 37ºC and 5% CO2.

[0065] Western blots: 100,000 Neuro-2A cells were plated on a 6-well plate and used after 5 days. Cells were treated with 0.2, 0.5 and 1.0 µM MMAE for 4 hours. After treatment cells were lysed with Mammalian Protein Extraction reagent (M-PER; Thermo Scientific) and 1x Protease Inhibitor (Chemcruz). Lysate was treated with 4x NuPage LDS loading buffer (Invitrogen) and 10x NuPage sample reducing agent (Invitrogen) using the protocol provided by the company. Lysed samples were added to a 4-12% Bis-Tris gel (Invitrogen), a 1X MES SDS running buffer (diluted in water from a 20X Invitrogen solution) was used and samples were run in the cold at 150V for 55 minutes. Proteins in the gel were transferred to a nitrocellulose membrane using a transfer buffer (2% methanol; 1% 10x transfer buffer - glycine - 3.8 mol.L-1; Tris Base 0.5 mol.L-1) at 100V for 2 hours. Membranes were cut and covered with 1x Tris-Buffered Saline with 1% Casein (Bio-Rad) for 1 hour to block non- specific binding sites. After blocking, membranes were washed with TBST (0,2% Tween 20 in 10x TBS -Tris / HCl - 0.2 mol.L-1; NaCl - 1.5 mol.L-1; pH = 7.6) and incubated with primary antibodies overnight in a cold room with constant agitation. After incubation, membranes were washed with TBST and incubated with the appropriate secondary antibody for 1 hour at room temperature (RT) in the dark. Membranes were imaged using a Li-Cor Odyssey DLx (Li-Cor) and ImageJ was used for image analysis. The mean gray value in each band of interest was determined and the result was represented as the intensity of NCS1 band divided by the intensity of GAPDH band (used as a loading control) and normalized using control values.

[0066] Calcium Signaling: 20,000 SK-N-AS or Neuro-2A cells were plated on a 24-well glass bottom plate and used after 5 days. Cells were treated with 0.2 µM MMAE, 1 mM lithium chloride and both drugs together 4 hours before the experiment or immediately before starting the experiment. Intracellular calcium concentration was measured using 6 µM fluo- 4 / AM, added 30 minutes before imaging and incubated at 36ºC and 5% CO2. The cells were monitored using a 20X magnification lens, at a rate of 1 frame / second, on a Leica SP8 confocal microscope (Leica), exciting at 488 nm and collecting emitted light at 505 nm. These cells were perfused by calcium-free HEPES media (NaCl, 130.0 mmol.L-1; KCl, 5.0 mmol.L-1; EGTA, 1.0 mmol.L-1; MgCl -1 -1 2,12.5mmol.L ; KH2PO4, 1.2 mmol.L ; MgSO4, 1.0 mmol.L-1; HEPES, 19.7mmol.L-1; glucose, 5.0 mmol.L-1; pH 7.4) and stimulated with 20 µM carbachol. See e.g., Timmins et al., Oncologist 2021, 26(5):366-374; and Haussmann et al., Neurosci Lett 2021, 760:136044. Fluorescence changes were analyzed using ImageJ software, manually tracing cells and measuring the mean gray value, after normalization by the initial 30 seconds fluorescence, without stimulation, (F0) and were expressed as (F-F0).

[0067] Immunofluorescence: 20,000 SK-N-AS cells were plated on a 24-well plate and used after 5 days growing. Cells were treated with 0.2 µM MMAE, 1 mM lithium chloride, or both drugs together for 4 hours. After treatment, cells were fixed with 4% paraformaldehyde (Santa Cruz) for 15 min at RT. After washing with 1x Phosphate-Buffered Solution (PBS; Gibco) three times, the cells were permeabilized with 0.3% Triton X-100 in PBS for 15 min, washed with PBS, and blocked in 1% bovine serum albumin (BSA) in PBS for 1 hour at RT. After blocking, samples were incubated with the primary antibody diluted in PBS (1:500) overnight at 4°C. After washing in PBS for 1 hour, the samples were stained in the dark for 1 hour at RT with the secondary antibody which was diluted in PBS (1:500), rhodamine- phalloidin for F-actin, and nucleus dye Hoechst. After samples were washed in PBS they were imaged using a Leica SP8 confocal microscope, exciting at 488 nm and collecting emitted light at 505 nm, using a 40X magnification lens, 1x zoom. Quantification of microtubule length was performed with Fiji (ImageJ) and an ImageJ plugin “Ridge Detection”. See e.g., Boehmerle et al., Proc Natl Acad Sci U S A 2007, 104(26):11103- 11108.

[0068] Max intensity projection was generated based on the fluorescent confocal images of individual cells, followed by this sequence: “Gaussian Blur, sigma=0.8 > Subtract Background”. The processed microtubule images were then measured using “Ridge Detection” for the total microtubule length. For the parameter settings in “Ridge Detection”, the “Line width” was set to “2 micrometer”. “High Contrast” was manually adjusted within the range of “80-120”, depending on the overall intensity of the image. “Sigma” was set to 1.08, and “Upper Threshold” was set to 8.33 for the “Mandatory_parameters”.

[0069] Results

[0070] MMAE treatment yielded a significant reduction (p < 0.0001) in intracellular calcium response following a 4-hour exposure (FIG.3A-D). The area under the curve (AUC) for the MMAE-treated cells was decreased by 30% when compared to control cells (1108±650 a.u. for control cells, 780±466 a.u. for MMAE-treated cells). Baseline calcium, measured as the resting fluorescence, also was reduced 25% in MMAE-treated cells (a pixel value for control of 3.2±1.6 and for MMAE of 2.4±0.8). Lithium addition restored the calcium response to levels similar to controls (AUC = 1128±555 a.u. in MMAE+LiCl treatment (p > 0.9999)) although, different as expected, did not recover the baseline calcium (Baseline = 2.7±1.1 in MMAE+LiCl treatment (p<0.0001)).

[0071] This response to MMAE treatment suggested an impact on NCS1-dependent signaling similar to that observed with PTX treatment (see e.g., WO 2012 / 149267), wherechronic treatment with PTX (6-12 hours) reduced calcium signaling as a consequence of reduced NCS1 levels. However, Western blot assays did not confirm the presence of this reduction in NCS1 levels after MMAE treatment for 4 hours (FIG.3E-F), even with the decreasing of intracellular calcium signaling. This lack of effect on NCS1 levels was observed with several concentrations of MMAE (0.2, 0.5 and 1 µM MMAE; FIG.3E). A similar lack of effect on NCS1 levels was also observed in a second cell line, Neuro-2A cells. See FIGs.10A-10C.

[0072] To understand the maintained NCS1 levels, we examined the acute response to drug treatment. For PTX there is a two-step process. First, there is an immediate increase in intracellular calcium. Subsequently, this increased calcium turns on calpain to cleave NCS1 with a long-term effect of decreased calcium signaling. In contrast, acute MMAE treatment results in a 66% reduction in the calcium response (p < 0.0001), where the AUC was 741±462 a.u. in the control cells and 249±236 a.u. in the MMAE treated cells (Figure 2G-J). These findings show an influence of MMAE treatment on the calcium signaling pathway, but without NCS1 degradation by calpain.

[0073] Nonetheless, lithium, as used previously for prevention of PTX-induced changes in calcium signaling (see e.g., WO 2012 / 149267) had the same effect in MMAE-treated cells. Our findings show that lithium addition prevents changes to calcium signaling (the AUC in control cells = 1108±650 and in MMAE + LiCl treated cells = 1228±690 a.u.; p = 0.3198) (FIG.3A-D). These results were unexpected and led us to investigate differences at the molecular level. Molecular Docketing

[0074] Materials and Methods

[0075] Molecular docking: Docking to NCS1 (PDB ID 1g8i) was performed with four different predictive programs (CB-Dock2, HADDOCK 2.4 (High Ambiguity Driven protein- protein DOCKing), and AutoDock Vina) (see e.g., Mo et al., FASEB J 2012, 26(11):4696- 4709; Pourmohammadi et al., Basic Clin Pharmacol Toxicol 2012, 110(3):231-237; and Nguyen et al., Mol Neurodegener 2021, 16(1):41) to determine the binding sites for paclitaxel (PTX; CID 36314) and Monomethyl Auristatin E (MMAE; CID 11542188). Predicted interaction residues were compiled, and the binding sites with the greatest residue hits were used. All docking was analyzed with PyMol 2.5. Analysis of the binding sites on tubulin were performed with PyMol using the established crystallized structures for auristatin-tubulin (PDB ID 7JFR) and paclitaxel-tubulin (PDB ID 3J6G).Immunoprecipitation

[0076] Results

[0077] To understand the unexpected difference in cellular response to PTX and MMAE, we turned to structural studies. There are established crystallized structures of tubulin, PTX, and MMAE. Using PyMol, a comparison of the binding site for each of the drugs to tubulin showed that PTX and MMAE both bound to the same chain of tubulin (FIG.4A). Even though the binding sites of these drugs are slightly different (FIG.4A), they both alter microtubule formation.

[0078] In contrast, although there are crystal structures of NCS1 alone (PDB ID 1g8i), there are no established crystallized structures of NCS1 bound to PTX or MMAE. Therefore, predictive docking software packages were used to identify potential binding sites of these drugs to NCS1. To locate optimal binding site(s) for PTX and MMAE on NCS1, we used AutoDock Vina, HADDOCK, and CB-Dock2. These packages allowed testing of a number of parameters. AutoDockVina evaluates van der Waals, hydrogen bonding, and electrostatic interactions while considering ligand conformational flexibility to predict binding sites effectively. In contrast, the HADDOCK package integrates experimental data and bioinformatics information to guide docking, emphasizing solvent accessibility and energy calculations to predict protein-ligand binding sites. See Dominguez et al., J Am Chem Soc. 2003;125(7):1731-7. CB-Dock2 employs shape and pharmacophore matching, emphasizing molecular shape complementarity and functional group interactions for accurate prediction of protein-ligand binding sites.

[0079] The top 10 structure predictions from each server were analyzed in PyMol. For both the NCS1-PTX complex and the NCS1-MMAE complex, predicted sites of binding were analyzed residue by residue. If a residue was listed as a potential binding site on NCS1 for the drug, the residue was given a score of 1. This was repeated for each set of predictions from each software, and the result was a list of top residue “hits,” which were used to color NCS1 residues in PyMol as a visual representation of the hotspots. From these, the most frequently predicted residues were chosen for analysis, which revealed that MMAE had the majority of residue hits near the H5 and H6 regions of NCS1 and PTX had the majority of residue hits near H4 and H7 regions of NCS1.

[0080] PTX and MMAE were then added to the crystal structure of NCS1 to show the sites where each of the drugs received the highest number of respective residue hits (i.e., their predicted “hotspots” on NCS1). Contrary to what we expected, residues in the hydrophobic pocket of NCS1 were predicted as binding sites for only MMAE. See FIG.4B.

[0081] Although there were a few hits for PTX binding in the hydrophobic pocket, there were notably more residue hits for PTX directly opposite the binding pocket. This suggests that PTX and MMAE bind to different sites on NCS1, where MMAE lies in the binding pocket and PTX is on the opposite side of NCS1. It is noteworthy that the binding site for MMAE overlaps with the binding site required for NCS1 binding to the inositol trisphosphate receptor (ITPR) suggesting that MMAE and ITPR compete for binding to NCS1. See e.g., Nguyen LD, Petri ET, Huynh LK, Ehrlich BE: Characterization of NCS1-InsP3R1 interaction and its functional significance. J Biol Chem 2019, 294(49):18923-18933.

[0082] A previous report showed that PTX increases NCS1 binding to the ITPR (Boehmerle W, Splittgerber U, Lazarus MB, McKenzie KM, Johnston DG, Austin DJ, Ehrlich BE: Paclitaxel induces calcium oscillations via an inositol 1,4,5-trisphosphate receptor and neuronal calcium sensor 1-dependent mechanism. Proc Natl Acad Sci U S A 2006, 103(48):18356-18361]). Microtuble Disruption

[0083] Material and Methods

[0084] Scratch assay: 60,000 SK-N-AS or SHSY-5Y cells were plated on 24-well plates and used after attaining approximately 90% confluence (3-4 days after plating). We pretreated the cells for 4 hours with 0.2 µM MMAE, 1mM LiCl, or both drugs. After 4 hours, the drugs were removed, cells were washed with 1x PBS, and a scratch was made with a P200 tip. Images of the scratch were taken using a Leica DMi1 bright-field microscope with 5x magnification every 24 hours for 3 days, and analyzed to assess the area of scratch using ImageJ software. Values were normalized by area on day 0, and expressed as percentage of wound closure

[0085] Results

[0086] The protective effects of lithium on the MMAE-induced changes in calcium signaling pathway did not correlate with any impact on microtubule disruption, as was previously reported for PTX (see Benbow et al., J Biol Chem.2012;287(45):37907-16). Immunofluorescence analysis of tubulin showed that lithium administered with MMAE has the same effect as treatment with MMAE only (p = 0.92), with microtubule length equal to 170±104 µm in MMAE treatment, and 121±71 µm in MMAE + lithium treatment (FIG.5A- B). Also, lithium treatment alone does not impact microtubule formation (p = 0.99), with the microtubule length of 858±430 µm in the lithium treatment case and 837±381 µm in the control case.

[0087] The protective effects of lithium on the MMAE-induced changes in calcium signaling pathway also did not correlate with any impact on cell motility, as measured with a scratch assay. The cell motility was impacted similarly in the MMAE and MMAE + LiCl, with a smaller percentage of wound closure comparing to control and lithium only in all days of analysis, with wound closure on the 3rd day equal to 51±5% in control cells, 51±17% in lithium only treated cells, 14±6% in MMAE and 22±6% in MMAE + LiCl cells. A similar lack of effect on microtubule assembly was observed in a second cell line, SHSY-5Y cells. See FIG.9. These results show that the addition of lithium is unlikely to impact MMAE treatment in subjects.

[0088] All statistics were performed in GraphPad Prism 10. For comparisons across two groups, unpaired t-test was used. For comparisons across three or more groups, one-way ANOVA with Tukey multiple comparisons test was used. Differences were considered significantly different when the p-value was smaller than 0.05. All bar plots are represented as mean ± standard deviation (SD) and p-values are expressed using: *p<0.05, **p<0.01,***p<0.001 or ****p<0.0001, if p>0.05 is not represented on graphs. MMAE-Induced Chemotherapy-Induced Peripheral Neuropathy (CIPN) and Chemotherapy-Induced Cognitive Impairment (CICI) Model

[0089] Studies were conducted to examine the effects of lithium pretreatment in an MMAE-induced CIPN and CICI mouse model. See e.g., Itaborahy et al., “Effective Prevention of Chemotherapy Induced Peripheral Neuropathy: Lithium Pretreatment Mitigates Nerve Damage”, the Federal University of Minas Gerais (UFMG) Summit, September 2024. In these studies, mice received intraperitoneal injections of lithium chloride, MMAE, or a combination of lithium chloride and MMAE with the lithium chloride being administered 1 hour before injection of MMAE. Injections occurred every other day for a period of 8 days.

[0090] Effects on Short-Term Memory Impairment

[0091] To determine whether MMAE administration induced cognitive impairment, the studies used the known Novel Object Recognition (NOR) test, targeting short-term memory acquisition. This series of tests also evaluated the effectiveness of lithium co-administration as a strategy to prevent MMAE-induced cognitive impairment. During the training phase, mice showed no preference for either object (p>0.05) (see FIG.11B and 11C). During the testing phase, control mice receiving only saline or lithium showed a significant preference for the novel object (p>0.001). In contrast, mice receiving MMAE showed no preference for either object, indicating impaired short-term memory acquisition (p>0.05) (see FIG.11D and 11E). When pretreated with lithium, however, animals in which MMAE was administeredmaintained the preference for the novel object. These results support that MMAE decreased short-term memory acquisition in mice and that lithium pretreatment prevents this cognitive impairment.

[0092] Effects on Peripheral Neuropathy

[0093] To evaluate the degree of diminished peripheral sensibility caused by chemotherapy, the studies used the nociceptive test based on capsaicin application. In this test, the status of pain-associated peripheral sensitivity was assessed in the treated mice. Animals exhibited an immediate licking / shaking response after intradermal injection of capsaicin. Mice treated with MMAE showed a diminished licking time (73.6 ± 5.0 s) compared to a group receiving only saline injections (104.7 ± 6.3 s; p<0.001) (see FIG.12A). The pretreatment with lithium was able to prevent MMAE-induced loss of nociception (102.8 ± 12.7 s; p<0.001). The decreased response in the capsaicin test and its prevention by lithium were maintained 28 days after the treatment ended (see FIG.12B). When lithium was administered alone, the licking time was comparable with the control group (102.8 ± 8.19 s) (see FIG.12A) suggesting lithium alone does not alter the nociception response.

[0094] Allodynia, another common effect of chemotherapy-induced peripheral neuropathy, was assessed using the Von Frey Monofilaments test. MMAE injections provoked a reduction in the mechanical threshold (3.5 ± 0.3) compared to the control group (4.2 ± 0.1; p<0.001) (see FIG.12C). These results show that MMAE treatment invoked a hypersensitivity to the stimuli. Co-administration of lithium completely prevented MMAE- induced mechanical allodynia, presenting a response similar to the control group (4.3 ± 3290.1; p>0.05). These results indicate that both allodynia and loss of nociception were prevented by lithium co-administration with MMAE. A histological measure for assessing neuronal integrity is with myelin width. Lithium administration was able to prevent the MMAE-induced reduction in myelin width, where myelin width was comparable to the control (p>0.05) (Fig 12D).

[0095] Lithium Treatment Does Not Weaken the Antitumor Effect of MMAE

[0096] Lithium showed a positive and promising response in its ability to prevent both peripheral and central neuropathies (see FIGs.11B – 12D). Nevertheless, it is also important to establish that the pretreatment with lithium does not interfere in the antitumor effect of MMAE. Therefore, an in vivo experiment was performed to induce xenograft tumors and then to measure and analyze the differences in tumor size after treatment among the groups.

[0097] The group treated only with MMAE and the group pretreated with lithium before MMAE administration presented shrinkage of the xenograft tumor during the experiment(FIG.13A) and the tumor size of these groups after finishing the treatment (0.8 ± 0.3 mm3 / g and 0.7 ± 0.7 mm3 , respectively) was significantly smaller than the control group (1.6 ± 0.6 mm3 ; p<0.05) (Fig 13B). DAPI and mkate2 fluorescence, together with Ki-67 staining, were used to confirm the injected cells were present and proliferating in the tumor. These results indicate that lithium administration does not harm the antitumor effect of MMAE treatment.

[0098] Lithium Treatment Does Not Weaken the Antitumor Effect of Padcev

[0099] Animals and Tumor Implantation

[0100] Groups of eight (8) female BALB / c nude mice weighing 18 ± 2 grams (g; 7 - 8 weeks old) were used. The mice were subcutaneously (SC) implanted with viable human TNBC MDA-MB-468 cells at 1 x 107 cells per mouse with 50% Matrigel in 0.2 mL / mouse volume at the right flank. Treatment was initiated when grouped mean tumor volume reached 100-150 mm3, denoted as Day 1 upon randomization.

[0101] Compound Administration

[0102] Padcev®(2 or 5 mg / kg) or Vehicle-1 (0.9% saline), was injected intravenously (IV) every other day for a total of four doses (QOD x 4 doses) on Days 1, 3, 5, and 7. During the studies, the animals were housed socially under constant temperature, humidity, and a 12- h light-dark cycle.

[0103] Lithium (12.8 mg / kg) or Vehicle-2 (0.9% saline), was dosed intraperitoneally (IP) at 1 h before Padcev® or Vehicle-1 administration on Days 1, 3, 5, and 7.

[0104] Tumor Volume Measurement and Calculation

[0105] The tumor volume was measured with a caliper and calculated by tumor volume = length x (width)2 x 0.5. Significant difference in tumor volume (p<0.05) is assessed with two-way ANOVA followed by Tukey’s multiple comparison by comparing treatment groups with vehicle control on each measurement day. The tumor volume was then used for calculations of Tumor growth inhibition (TGI) and T / C values, defined as the ratio of the mean tumor volume for the treated versus control group. TGI was calculated for each group using the formula: TGI (%) = [1-(Tn-T1) / (Cn-C1)] ×100; Tnis the average tumor volume of a treatment group on a given day, T1is the average tumor of the treatment group on the first day of treatment, Cnis the average tumor volume of the vehicle control group on the same day with Tn, and C1is the average tumor volume of the vehicle group on the first day of treatment. T / C value was calculated for each group using the formula: T / C (%) = T / C x 100; T is the tumor volume of treatment group on a given day, C is the tumor volume of vehicle control group on the same day with T. The optimal value is the minimal %T / C ratio reflectingthe maximal tumor growth inhibition achieved. A %T / C value ≤ 42% compared to that of the vehicle control group is considered significant anti-tumor activity following the guidelines of National Cancer Institute (NCI).

[0106] The group treated only with Padcev (two concentrations) and the group pretreated with lithium before Padcev administration presented showed shrinkage of the xenograft tumor during the experiment (FIG.14A and 14B). These results show that lithium had no impact on the efficacy of Padcev to shrink tumors.

[0107] Although we have described a number of embodiments, it is apparent that our basic examples may be altered to provide other embodiments that utilize the compounds and methods of this invention. Therefore, it will be appreciated that the scope of this invention is to be defined by the appended claims rather than by the specific embodiments that have been represented by way of example.

[0108] The contents of all references (including literature references, issued patents, published patent applications, and co-pending patent applications) cited throughout this application are hereby expressly incorporated herein in their entireties by reference. Unless otherwise defined, all technical and scientific terms used herein are accorded the meaning commonly known to one with ordinary skill in the art.

Claims

Listing of Claims:

1. A method of treating or preventing antibody drug conjugate (ADC)-induced neuropathy in a subject, comprising administering to the subject an effective amount of lithium (Li).

2. The method of claim 1, wherein said neuropathy is peripheral neuropathy.

3. The method of claim 1, wherein said neuropathy is cognitive impairment.

4. The method of Claims 1-3, wherein the subject is currently undergoing anticancer treatment with an ADC.

5. The method of Claim 4, wherein the ADC comprises a tubulin modulator payload.

6. The method of Claim 4 or 5, wherein the ADC comprises an auristatin payload.

7. The method of any one of Claims 4 to 6, wherein the ADC comprises monomethyl auristatin E (MMAE) or monomethyl auristatin F (MMAF) as the payload.

8. The method of any one of Claims 4 to 7, wherein the ADC comprises MMAE as the payload.

9. The method of any one of Claims 4 to 8, wherein the ADC is selected from brentuximab vedotin, polatuzumab vedotin, enfortumab vedotin, belantamab mafodotin, disitamab vedotin, tisotumab vedotin, telisotuzumab vedotin, and zilovertamab vedotin 10. The method of any one of Claims 4 to 9, wherein the ADC is selected from brentuximab vedotin, polatuzumab vedotin, and enfortumab vedotin.

11. The method of any one of Claims 4 to 10, wherein the lithium is administered concurrently with the ADC.

12. The method of any one of Claims 4 to 10 wherein the lithium is administered prior to the ADC.

13. The method of any one of Claims 1 to 12, wherein the lithium is in the form of a pharmaceutically acceptable salt.

14. The method of any one of Claims 1 to 13, wherein the lithium is lithium chloride.

Images