Compositions and methods for assessing and treating chemotherapy-induced peripheral neuropathy

Abstract

Methods and compositions are provided for assessing a subject's susceptibility to chemotherapy-induced neuropathy (CIPN) comprising obtaining one or more measures of GSH recycling-dependent antioxidant activity of the subject's red blood cells.

Description

[Background technology]

[0001] Chemotherapy-induced peripheral neuropathy (CIPN) is a disabling side effect of platinum-based chemotherapy such as cisplatin, oxaliplatin, and carboplatin. Although this side effect does not occur in all patients, those who do experience this side effect are at risk for lifelong chronic neuropathy.

[0002] Cancer survivorship issues have grown in parallel with the positive outcomes of cancer treatment. As a result, increased attention has been paid to the diagnosis, prognosis, and treatment of CIPN cases. The prevalence of CIPN resulting from various antitumor drugs and their doses varies widely. Indeed, while approximately 70% of patients undergoing chemotherapy develop CIPN during the first month of treatment, 20-30% of these patients transition to chronic CIPN after 6 months of chemotherapy cessation. Importantly, symptoms of these delayed complications may not only persist for months, but may also progressively worsen. Cases in which mild neuropathy worsens or develop new forms of CIPN are referred to as “coasting.” This disorder poses a great challenge to clinicians, as no chemotherapy drugs have been administered and patients may be cancer-free at the time of coasting, but still suffer from neuropathy caused by their earlier cancer treatment.

[0003] While pharmacological approaches have been developed for prevention or treatment, it is also essential to develop compositions and methods to identify patients who are at highest risk for chronic CIPN. Summary of the Invention

[0004] The inventors have discovered a method for assessing a patient's susceptibility to chronic chemotherapy-induced peripheral neuropathy (CIPN) by assessing the level of oxidative stress in the subject's bloodstream before, during, and after administration of a chemotherapy agent. Thus, the inventors have demonstrated a novel use of assays for the detection of oxidative stress and toxicity in blood, and use such measurements to optimize a patient's treatment or chemotherapy regimen.

[0005] In one aspect, provided herein is a method of assessing susceptibility to or severity of chemotherapy-induced neuropathy (CIPN) in a subject to which a chemotherapeutic agent is administered, the method comprising: (a) obtaining a baseline measurement of glutathione (GSH) recycle activity in one or more biological samples comprising red blood cells (RBCs) obtained from the subject prior to a first administration of the chemotherapeutic agent; (b) obtaining a first measurement of GSH recycle activity in one or more biological samples comprising RBCs obtained from the subject after the first administration of the chemotherapeutic agent, but prior to a second administration of the chemotherapeutic agent; (c) obtaining a second measurement of GSH recycle activity in one or more biological samples comprising RBCs obtained from the subject after the second administration of the chemotherapeutic agent, but prior to a third administration of the chemotherapeutic agent; (d) obtaining a third measurement of GSH recycle activity in one or more biological samples comprising RBCs obtained from the subject after the third administration of the chemotherapeutic agent, but prior to a fourth administration of the chemotherapeutic agent; and (e) obtaining an average of the baseline measurement, the first measurement, the second measurement, and the third measurement.

[0006] In one aspect, provided herein is a method of assessing susceptibility to or severity of chemotherapy-induced neuropathy (CIPN) in a subject to which a chemotherapeutic agent has been administered, the method comprising: (a) obtaining a first measure of GSH recycle activity in one or more biological samples comprising RBCs obtained from the subject after a first administration of a chemotherapeutic agent but prior to a second administration of the chemotherapeutic agent; and (b) obtaining a first measure of GSH recycle activity in one or more biological samples comprising RBCs obtained from the subject after a third administration of the chemotherapeutic agent but prior to a third administration of the chemotherapeutic agent. (c) obtaining a second measurement of GSH recycling activity in one or more biological samples comprising RBCs obtained from the subject prior to the fourth administration of the agent; and (c) obtaining an average value of the baseline measurement, the first measurement, and the second measurement.

[0007] In another aspect, provided herein is a method of assessing susceptibility to chemotherapy-induced neuropathy (CIPN) or predicting the severity of CIPN in a subject to which a chemotherapeutic agent is administered, the method comprising: (a) obtaining a baseline measure of glutathione (GSH) recycle activity in one or more biological samples comprising red blood cells (RBCs) obtained from the subject prior to a first administration of a chemotherapeutic agent; and (b) obtaining (i) a measure of GSH recycle activity in one or more biological samples comprising RBCs obtained from the subject after a first administration of a chemotherapeutic agent but prior to a second administration of the chemotherapeutic agent, (ii) a measure of GSH recycle activity in one or more biological samples comprising RBCs obtained from the subject after a second administration of a chemotherapeutic agent but prior to a third administration of the chemotherapeutic agent, or (iii) a measure of GSH recycle activity in one or more biological samples comprising RBCs obtained from the subject after a third administration of a chemotherapeutic agent but prior to a fourth administration of the chemotherapeutic agent, wherein a decrease in GSH recycle activity predicts an increase in susceptibility to CIPN or the severity of CIPN.

[0008] In yet another aspect, provided herein is a method of reducing susceptibility to CIPN following treatment with a chemotherapeutic agent, the method comprising: (a) obtaining a baseline measure of glutathione (GSH) recycle activity in one or more biological samples comprising red blood cells (RBCs) obtained from the subject prior to a first administration of the chemotherapeutic agent; (b) obtaining a first measure of GSH recycle activity in one or more biological samples comprising RBCs obtained from the subject after the first administration of the chemotherapeutic agent, but prior to a second administration of the chemotherapeutic agent; and (c) obtaining a first measure of GSH recycle activity in one or more biological samples comprising RBCs obtained from the subject after the second administration of the chemotherapeutic agent, but prior to a third administration of the chemotherapeutic agent. (d) obtaining a second measurement of GSH recycle activity in one or more biological samples comprising RBCs obtained from the subject after a third administration of a chemotherapeutic agent but before a fourth administration of the chemotherapeutic agent; (e) obtaining an average of the baseline measurement, the first measurement, the second measurement, and the third measurement, where an average of 1.85 or less indicates susceptibility to or severity of CIPN; and (f) modifying the subject's treatment regimen and / or administering a treatment for CIPN to reduce susceptibility to or severity of chemotherapy-induced neuropathy (CIPN).

[0009] In yet another aspect, provided herein is a method of reducing susceptibility to CIPN following treatment with a chemotherapeutic agent, the method comprising: (a) obtaining a first measure of GSH recycle activity in one or more biological samples comprising RBCs obtained from the subject after a first administration of the chemotherapeutic agent but before a second administration of the chemotherapeutic agent; (b) obtaining a second measure of GSH recycle activity in one or more biological samples comprising RBCs obtained from the subject after a third administration of the chemotherapeutic agent but before a fourth administration of the chemotherapeutic agent; (c) obtaining an average value of the first and second measures, wherein an average value of 1.8 or less indicates susceptibility to CIPN or severity of CIPN; and (d) modifying the subject's treatment regimen and / or administering a treatment for chemotherapy-induced neuropathy (CIPN) to reduce susceptibility to or severity of CIPN.

[0010] In yet another aspect, provided herein is a method of reducing susceptibility to CIPN following treatment with a chemotherapeutic agent, the method comprising: (a) measuring levels of glutathione (GS) in one or more biological samples comprising red blood cells (RBCs) obtained from the subject prior to a first administration of the chemotherapeutic agent. H) obtaining a baseline measure of recycle activity; and (b) obtaining (i) a measure of GSH recycle activity in one or more biological samples comprising RBCs obtained from the subject after a first administration of a chemotherapeutic agent but before a second administration of the chemotherapeutic agent, (ii) a measure of GSH recycle activity in one or more biological samples comprising RBCs obtained from the subject after a second administration of a chemotherapeutic agent but before a third administration of the chemotherapeutic agent, or (iii) a measure of GSH recycle activity in one or more biological samples comprising RBCs obtained from the subject after a third administration of a chemotherapeutic agent but before a fourth administration of the chemotherapeutic agent, where a decrease in GSH recycle activity is predictive of increased severity of or susceptibility to CIPN; and (c) modifying the subject's treatment regimen and / or administering a treatment for CIPN to reduce susceptibility to or severity of chemotherapy-induced neuropathy (CIPN).

[0011] In another aspect, provided herein is a method for assessing susceptibility to chemotherapy-induced neuropathy (CIPN) or the ability of a treatment to alter the severity of CIPN in a subject, the method comprising: (a) obtaining a baseline measure of glutathione (GSH) recycle activity in one or more biological samples comprising red blood cells (RBCs) obtained from the subject prior to a first administration of a chemotherapeutic agent; (b) obtaining a first measure of GSH recycle activity in one or more biological samples comprising RBCs obtained from the subject after the first administration of the chemotherapeutic agent, but before a second administration of the chemotherapeutic agent; and (c) obtaining a first measure of GSH recycle activity in one or more biological samples comprising RBCs obtained from the subject after the second administration of the chemotherapeutic agent, but before a second administration of the chemotherapeutic agent. (d) obtaining a second measurement of GSH recycle activity in one or more biological samples comprising RBCs obtained from the subject prior to a third administration of the chemotherapeutic agent; (e) obtaining an average of the baseline measurement, the first measurement, the second measurement, and the third measurement, wherein an average of 1.85 or less indicates susceptibility to or severity of CIPN; and (f) administering a treatment for CIPN to the subject.

[0012] In another aspect, provided herein is a method for assessing susceptibility to chemotherapy-induced neuropathy (CIPN) or the ability of a treatment to alter the severity of CIPN in a subject, the method comprising: (a) obtaining a first measure of GSH recycle activity in one or more biological samples comprising RBCs obtained from the subject after a first administration of a chemotherapeutic agent but before a second administration of the chemotherapeutic agent; (b) obtaining a second measure of GSH recycle activity in one or more biological samples comprising RBCs obtained from the subject after a third administration of a chemotherapeutic agent but before a fourth administration of the chemotherapeutic agent; (c) obtaining an average value of the baseline measure, the first measure, and the second administration, where an average value of 1.8 or less indicates susceptibility to CIPN or severity of CIPN; and (d) administering a treatment for CIPN to the subject.

[0013] In another aspect, provided herein is a method for assessing susceptibility to chemotherapy-induced neuropathy (CIPN) or the ability of a treatment to alter the severity of CIPN in a subject, the method comprising: (a) obtaining a baseline measure of glutathione (GSH) recycle activity in one or more biological samples comprising red blood cells (RBCs) obtained from the subject prior to a first administration of a chemotherapeutic agent; and (b) obtaining a measure of GSH recycle activity in one or more biological samples comprising RBCs obtained from the subject after (i) the first administration of a chemotherapeutic agent, but prior to a second administration of the chemotherapeutic agent, (ii) a measure of GSH recycle activity in one or more biological samples comprising RBCs obtained from the subject after the second administration of a chemotherapeutic agent, but prior to a third administration of the chemotherapeutic agent, or (iii) a measure of GSH recycle activity in one or more biological samples comprising RBCs obtained from the subject after the third administration of a chemotherapeutic agent, but prior to a fourth administration of the chemotherapeutic agent, (c) administering to the subject a treatment for CIPN.

[0014] Other aspects and advantages of these methods are further described in the detailed description that follows. [Brief description of the drawings]

[0015] [Figure 1] A table containing grading criteria for nervous system disorders is provided. [Diagram 2] Results from an analysis comparing patient age with mean CIPN grade are shown. [Diagram 3] Results from an analysis of Chemotox scores comparing patient gender and time of year are shown. [Figure 4] Results are shown from an analysis that determined the Chemotox score versus mean CIPN grade for patients utilizing a minimum of four different collection time points. [Diagram 5]Figure 1 shows the ability to predict grade 3 vs. grade 0 CIPN using patient baseline / pretreatment Chemotox score. [Figure 6] FIG. 1 shows the ability to predict grade 3 versus grade 0 CIPN using patient Chemotox scores determined after the first treatment but before the second treatment ("Pre2"). [Figure 7] FIG. 1 shows the ability to predict grade 3 versus grade 0 CIPN using patient Chemotox scores obtained from a series of four time points. [Figure 8] A graph containing the ROC curve is shown demonstrating the superiority of the approach utilizing multiple Chemox scores during treatment over the approach using only baseline / pretreatment assessments in predicting CIPN. The total number of patients used in the analysis was 260 (N=260). Using the average value of the first four subject samplings, the ROC approaches 0.8. [Figure 9A] Shown are the mean Chemotox scores versus CIPN severity for patients using Chemotox scores obtained at four different time points (A) or two different time points (B). [Figure 9B] Shown are the mean Chemotox scores versus CIPN severity for patients using Chemotox scores obtained at four different time points (A) or two different time points (B). [Figure 10] Graphs are provided showing examples of intra-patient variability and relative change in Chemotox scores during treatment. [Figure 11] Using Chemotox scores obtained from four time points, we show the accuracy in predicting grade 3 CIPN versus grade 0 in patients receiving taxol-containing therapy (A) or oxaliplatin / FOLFOX combination therapy (B). [Figure 12]Shown are eight well strips utilized in the high throughput assays provided herein that can be loaded (up to 12) into a frame that holds samples during incubation, centrifugation, and absorbance reading steps. [Figure 13] 1 shows results obtained from parallel analysis of samples using the high-throughput plate-based assay provided herein and the tube-based method previously described. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0016] Methods and compositions described herein are directed to assessing susceptibility to chemotherapy-induced peripheral neuropathy (CIPN) in a subject and using that assessment to create or modify a treatment regimen, including the administration of chemotherapeutic agents.

[0017] The inventors have demonstrated that measurements of the antioxidant glutathione (GSH) recycling in blood are consistent with It has been shown to predict CIPN or the severity of CIPN in elephants. Specifically, it has been demonstrated that the time course of changes in a patient's blood before and after the administration of chemotherapy can be used to identify patients at elevated risk for CIPN. Specifically, patients are at increased risk for CIPN if their baseline levels of GSH recycling capacity remain reduced and do not rebound during multiple cycles of chemotherapy.

[0018] Without wishing to be bound by theory, platinum-based treatments cause a burst of reactive oxygen species (ROS) that can induce structural changes in peripheral nerves, including neuronal, axonal, and / or myelin damage. Glutathione is a natural endogenous antioxidant and plays a key role in reduction-oxidation (redox) homeostasis. It is hypothesized that the alterations in GSH recycling capacity that occur in some patients after chemotherapy are predictive of CIPN for the following reasons: Reactive oxygen species (ROS) are known to destroy various tissue and cellular components. Neuropathy is caused by damage to ion channels, microtubules, dorsal root ganglia, small nerve fibers, mitochondria, and myelin, and ROS are known to damage mitochondria, myelin sheets, and calcium ion homeostasis. GSH is an important antioxidant in the neutralization of ROS. When ROS are neutralized by GSH, the latter are oxidized to a dimeric form (GSSG), which is required for recycling to its reduced monomeric form (GSH) that can neutralize more ROS. Thus, tissue damage can occur if more ROS are produced than there is GSH available to neutralize them. It is unclear why some patients are able to efficiently recycle GSSG and maintain GSH levels during chemotherapy at levels sufficient to quench ROS, while others are not.

[0019] The inventors concluded that a patient's risk of CIPN may reflect naturally occurring individual differences in the ability to recycle GSH so that free radicals are efficiently removed during the period when chemotherapy is administered to cancer patients. Redox homeostasis maintained by GSH recycling can be used as a biomarker assayed during chemotherapy to predict the risk of chronic CIPN in individuals.

[0020] GSH activity is known to be a crucial determinant of cells' ability to survive the burst of free radicals generated by chemotherapy treatment. Therefore, efficient GSH recycling is essential for cells to detoxify ROS and minimize tissue damage. The present inventors have identified a link between the redox homeostasis maintained by glutathione (GSH) and susceptibility to chronic CIPN in subjects.

[0021] At present, there are no approved pharmacological means to treat or prevent CIPN. Only dose reduction, delay, or cessation of potentially life-saving chemotherapy can limit the onset and progression of CIPN. Meanwhile, many cancer patients and oncologists are increasingly concerned about the risk of CIPN, and at the same time, research continues to be conducted to reduce or prevent this side effect. Therefore, there is a high interest in identifying patients at highest risk and appropriately modifying or adding treatment regimens to eliminate CIPN or reduce its severity.

[0022] As used herein, the term "chemotherapy-induced peripheral neuropathy" or "CIPN" refers to or describes symptoms resulting from damage to peripheral nerves in patients who are or have been treated with chemotherapy. Depending on the affected nerve, symptoms may include, but are not limited to, tingling ("pins and needles"), pain, burning, decreased sensation, increased sensitivity to touch, temperature, pressure, or pain, loss of feeling (which may be numbness or decreased ability to sense pressure, touch, hot, or cold), difficulty using fingers to lift or hold objects, objects falling from hands, loss of balance, stumbling or staggering. Symptoms of CIPN include difficulty walking due to pain, more pain than usual in response to pressure or temperature (e.g., cold sensitivity), muscle shrinkage, muscle weakness, difficulty swallowing, constipation, difficulty urinating, blood pressure changes, and changes in nerve conduction velocity with reduced or lost reflexes. A grading scale can be used to assess the severity of CIPN (see, e.g., FIG. 1). See also Zhang et al. Biomed Rep. 2017 Mar;6(3):267-271, which is incorporated herein by reference.

[0023] As used herein, "hyperalgesia" refers to increased sensitivity to pain, which may be caused by damage to nociceptors or peripheral nerves (i.e., neuropathy). The term refers to temporary and permanent hyperalgesia, and includes both primary hyperalgesia (i.e., pain sensitivity arising directly in damaged tissue) and secondary hyperalgesia (i.e., pain sensitivity arising in non-damaged tissue surrounding the damaged tissue). The term includes, but is not limited to, hyperalgesia caused by neuropathy caused by drug toxicity, neuropathy resulting from drug toxicity, or neuropathy otherwise associated with drug toxicity. In some embodiments, the hyperalgesia is caused by chemotherapy-induced peripheral neuropathy.

[0024] In one aspect, the method for assessing susceptibility to CIPN comprises assaying a biological sample of a mammalian subject, comprising red blood cells (RBCs), for the level of oxidative stress.Oxidative stress is essentially the imbalance between the production of free radicals and the body's ability to counteract or detoxify their harmful effects through neutralization by antioxidants.In one embodiment, the assay is carried out by assessing the GSH recycling activity of RBCs in the sample as an indicator of the quality of the antioxidant response to oxidative stress.This assay is based on the discovery that the decrease in the GSH recycling capacity of RBCs of a subject during chemotherapy (i.e., the relative decrease in the ability to survive high oxidative stress) is inversely correlated with the increased risk of the subject developing CIPN.

[0025] According to the methods described herein, a biological sample is obtained from a mammalian subject before and after administration of a chemotherapeutic agent. "Biological sample" or "sample" as referred to herein means a biological fluid that contains red blood cells. In certain embodiments, the biological sample is whole blood. In certain embodiments, the sample is another fluid that contains RBCs. In certain embodiments, the sample is diluted. In certain embodiments, the sample is a concentrated sample.

[0026] "Patient" or "subject" as used herein means a mammalian animal, including humans, veterinary or livestock animals, farm animals or pets, and animals typically used in clinical research (such as mice and rats). More specifically, the subject of these methods and compositions is a human. In certain embodiments, the subject has cancer.

[0027] "Carrier" as used herein refers to, for example, a diluent, matrix, adjuvant, preservative (e.g., Thimersol, benzyl alcohol), antioxidant (e.g., ascorbic acid, sodium metabisulfite), solubilizer (e.g., Tween 80, Polysorbate 80), emulsifier, buffer (e.g., Tris HCl, acetate, phosphate), antimicrobial agent, bulking substance (e.g., lactose, mannitol), excipient, auxiliary, or vehicle in which the active agent of the present invention may be maintained. Carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable, or synthetic origin. Water or saline as well as aqueous dextrose and glycerol solutions can also be used as carriers.

[0028] "Chemotherapeutic agent," as used herein, refers to any compound (including derivatives thereof) that can be used in the treatment of cancer. Chemotherapeutic agents (e.g., anticancer agents) are well known to those of skill in the art. These chemotherapeutic agents include, but are not limited to, anthracenediones (anthraquinones) (such as anthracyclines (e.g., daunorubicin (daunomycin; rubidomycin), doxorubicin, epirubicin, idarubicin, and valrubicin)), mitoxantrone, and pixantrone; platinum-based agents (e.g., cisplatin, carboplatin, oxaliplatin, satraplatin, picoplatin, nedaplatin, triplatin, and lipoplatin); tamoxifen and its metabolites (4-hydroxytamoxifen (afimoxifen)); tamoxifen (Campan) and N-desmethyl-4-hydroxytamoxifen (endoxifen); taxanes (such as paclitaxel (Taxol) and docetaxel); alkylating agents (such as nitrogen mustards (mechlorethamine (HN2), cyclophosphamide, ifosfamide, melphalan (L-sarcolysin), and chlorambucil); ethylenimines and methylmelamines (such as hexamethylmelamine, thiotepa, alkyl sulfonates (such as busulfan), nitrosoureas (carmustine (BCNU), lomustine (CCNLJ), semustine (methyl-CCN--U), and streptozoein (streptozotocin); triazenes (such as decarbazine (DTIC; dimethyltriazenoimidazole carboxamide); antimetabolites (such as folic acid analogs, such as methotrexate (amethopterin)); pyrimidine analogs, such as fluorouracil (5-fluorouracil; 5-FU), floxuridine (fluorodeoxyuridine; FUdR), and cytarabine (cytosine arabinoside)); analogs and related inhibitors, such as mercaptopurine (6-mercaptopurine; 6-MP), thioguanine (6-thioguanine; 6-TG), and pentostatin (2'-deoxycofonnycin); natural products, such as vinca alkaloids, such as vinblastine (VLB) and vincristine, epipodophyllotoxins, such as etoposide and teniposide, and antibiotics, such as dactinomycin (actinomycin D), bleomycin, plicamycin (mithramycin), and mitomycin (mitomycin Q);These include enzymes (such as L-asparaginase); biological response modifiers (such as interferon alpha); substituted ureas (such as hydroxyurea); methylhydrazine derivatives (such as procarbazine (N-methylhydrazine; MIH)); adrenal cortex suppressants (such as mitotane (o,p'-DDD) and aminoglutethimide); analogs thereof, derivatives thereof, and combinations thereof. Still other cytostatic chemotherapeutic agents known in the art may also be useful in the methods described herein. See, for example, the chemotherapy described in U.S. Pat. No. 9,186,357 (incorporated by reference);

[0029] In certain embodiments, the method includes administering to the subject a chemotherapeutic agent that is a taxane, a platinum compound, optionally oxaliplatin or cisplatin, a vinca alkaloid, thalidomide, an epothilone, eribulin, ipilimumab, pembrolizumab, nivolumab, or bortezomib.

[0030] As used herein, the term "cancer" refers to or describes a physiological condition in a mammal that is typically characterized by uncontrolled cell proliferation. In one embodiment, the term "cancer" refers to any cancer characterized by the presence of a solid tumor. In another embodiment, the cancer is a hematological cancer. As referred to herein, cancer includes, but is not limited to, melanoma, breast cancer, brain cancer, colon / rectal cancer, lung cancer, ovarian cancer, adrenal cancer, anal cancer, bile duct cancer, bladder cancer, bone cancer, endometrial cancer, esophageal cancer, eye cancer, renal cancer, laryngeal cancer, liver cancer, head and neck cancer, nasopharyngeal cancer, osteosarcoma, oral cancer, ovarian cancer, pancreatic cancer, prostate cancer, rhabdomyosarcoma, salivary gland cancer, gastric cancer, testicular cancer, thyroid cancer, vaginal cancer, lung cancer, lymphoma, myeloma, and neuroendocrine cancer.

[0031] A "chemotherapeutic regimen," as used in the methods described herein, generally refers to one Concomitant or sequential administration of one or two different chemotherapeutic agents, or concomitant or sequential administration of three to ten different chemotherapeutic agents. In the methods described herein, the choice of two or fewer drugs (which may not include chemotherapeutic agents) or the choice of three or more drugs depends on the oxidation level of the blood sample. If the sample from the subject shows high glutathione (GSH) recycle-dependent antioxidant activity (i.e., high oxidative stress), the chemotherapy regimen may be modified to include an increased number of chemotherapeutic agents. If the sample from the subject shows low glutathione (GSH) recycle-dependent antioxidant activity (i.e., low oxidative stress), the dosing regimen may be modified to include a decreased number of chemotherapeutic agents, or the chemotherapy regimen of treatment may be terminated earlier or may even not be used as it is unnecessary. In certain embodiments, the chemotherapy regimen includes a combination of folinic acid (leucovorin, FOL), fluorouracil (5-FU, F), and oxaliplatin (Eloxatin, OX) (i.e., FOLFOX). In certain embodiments, the chemotherapy regimen includes a combination of R-CHOP (rituximab, cyclophosphamide, doxorubicin hydrochloride / hydroxydaunomycin, vincristine sulfate / Oncovin, and prednisone). In certain embodiments, the chemotherapy regimen includes ABVD (doxorubicin hydrochloride / adriamycin, bleomycin, vinblastine sulfate, and dacarbazine). In certain embodiments, the chemotherapy regimen includes R-CVP (rituximab, cyclophosphamide, vincristine sulfate / Oncovin, and prednisone).

[0032] Compounds that are "administered together" or "combined" can be administered as part of the same composition or can be administered separately, at the same time or at different times in the same therapeutic regimen.

[0033] "Glutathione recycling activity" refers to the extent to which RBCs can convert the tripeptide GSH from its oxidized state to its reduced state, thereby neutralizing ROS. A relative increase in GSH recycling activity protects cells from oxidative stress and ROS by recycling the oxidized isoform glutathione disulfide (GSSG) to the reduced isoform glutathione monomer (GSH). Appropriate GSH recycling-dependent antioxidant activity can be measured in biological samples in such a manner, and in one embodiment, can be measured by using the OxPhos™ Cell Survival Kit, Catalog No. KLD-02, Rockland Inc. In this assay, hydroxyethyl disulfide (HEDS) is used as an indirect indicator of glutathione-dependent detoxification, which involves the conversion of GSH→GSSG→GSH, releasing β-mercaptoethanol (ME). In certain embodiments, the method utilizes a measure of GSH recycling activity by quantifying the amount of β-mercaptoethanol (ME) released by a blood sample treated with a reagent, spectrophotometrically measuring the absorbance reading of ME, converting the absorbance reading to ME concentration, normalizing the ME concentration to the total red blood cell count at the time of blood collection, and assessing the GSH recycling-dependent antioxidant activity of intact red blood cells in the sample. In the Ox-Phos™ assay, the conversion of HEDS to ME is determined spectrophotometrically using a premixed dithiobisnitrobenzoic acid (DNTB) reagent, the absorbance reading is converted to ME concentration, and normalized to the total red blood cell count (RBC×10) determined at the time of blood collection. 6In certain embodiments, GSH-dependent antioxidant activity is calculated using the conversion factor provided with the OxPhos™ assay protocol and normalized to total red blood cell count. In certain embodiments, the GSH assay is that described in U.S. Pat. No. 8,697,391, which is incorporated herein by reference. The GSH assay provides a measure of the efficiency of enzymes in six separate pathways that can reliably and repeatedly convert GSH from a reduced to an oxidized state. Measurements obtained using the methods and assays provided herein are sometimes referred to as "Chemotox" scores. In certain embodiments, the Chemotox score ... a single sample. In other embodiments, the Chemotox score is obtained from multiple samples, e.g., represents an average value. Multiple samples may be obtained at different time points during the treatment of the patient.

[0034] Methods and assays for measuring GSH recycling activity are further described in WO2018 / 071323A1, Kutner et al. Support Care Cancer. 2017 Feb;25(2):581-587. doi:10.1007 / s00520-016-3442-5, and McCourt et al. J Unexplored Med Data 2019;4:6. doi:10.20517 / 2572-8180.2019.01, all of which are incorporated herein by reference.

[0035] In one aspect, a method for predicting or assessing a subject's susceptibility to CIPN is provided, the method comprising assaying a biological sample comprising RBCs for a level of oxidative stress, the sample being obtained from a mammalian subject before and / or after administration of a chemotherapeutic agent. By carrying out the method, the risk of developing CIPN in the subject can be assessed and / or the severity of CIPN can be predicted.

[0036] In certain embodiments, the indicator of oxidative stress is GSH recycling capacity. Thus, the subject's blood is obtained prior to treatment with a chemotherapeutic agent. In certain embodiments, a sample (e.g., a sample of whole blood) is obtained from a mammalian subject prior to administration of a chemotherapeutic agent. The sample is contacted with assay components that allow for assessment of the oxidative capacity of RBCs in the sample (such as GSH recycling-dependent antioxidant activity of blood cells). In one embodiment, the blood is tested for GSH recycling activity immediately after collection. In other embodiments, the blood is tested for GSH recycling activity after storage. The GSH recycling activity of blood is expected to remain stable for several hours to several days after collection.

[0037] In certain embodiments, the method includes contacting the sample with HEDS, and further includes the steps described above for GSH assay, namely quantifying the amount of ME released by the HEDS-treated sample, measuring the absorbance reading of ME spectrophotometrically, converting the absorbance reading to ME concentration, normalizing the ME concentration to total RBC number, and evaluating the GSH recycling-dependent antioxidant activity of intact RBCs in the sample. These steps are performed to determine whether the subject's blood sample or biological sample is characterized as having low or reduced (GSH) recycling-dependent antioxidant activity. This method further allows the evaluation of whether the subject is prone to develop CIPN.

[0038] The method is applicable to subjects with cancer who require treatment with a chemotherapeutic agent. The chemotherapeutic agent can be administered in any conventional manner. In certain embodiments, the chemotherapeutic agent is administered intravenously. In certain embodiments, the chemotherapeutic agent is administered orally.

[0039] In one aspect, provided herein is a method of assessing susceptibility to chemotherapy-induced neuropathy (CIPN) in a patient receiving a chemotherapeutic agent, the method comprising: (a) obtaining a measure of glutathione (GSH) recycling activity in one or more biological samples comprising red blood cells (RBCs) obtained from the patient prior to administration of the chemotherapeutic agent; (b) obtaining a measure of GSH recycling activity in one or more biological samples comprising RBCs obtained from the patient after treatment with the chemotherapeutic agent; and (c) comparing the one or more measures of GSH recycling activity in (a) to the one or more measures of GSH recycling activity in (b). In certain embodiments, the reduction in GSH recycling after treatment is indicative of a decrease in CSH recycling activity. Predicts increased susceptibility to IPN. In certain embodiments, decreased GSH recycling following treatment predicts increased severity, frequency, and / or symptoms of CIPN.

[0040] In one aspect, a method is provided for managing a patient's treatment with a chemotherapeutic agent and reducing the patient's susceptibility to CIPN, the method comprising: (a) obtaining a measure of GSH recycle activity in one or more biological samples comprising red blood cells (RBCs) obtained from the patient prior to administration of the chemotherapeutic agent; (b) obtaining a measure of GSH recycle activity in one or more biological samples comprising RBCs obtained from the patient after treatment with the chemotherapeutic agent; (c) comparing the one or more measures of GSH recycle activity in (a) with the one or more measures of GSH recycle activity in (b); and (d) modifying the subject's treatment with the chemotherapeutic agent and / or administering a treatment for CIPN.

[0041] In certain embodiments, the method comprises modifying the treatment regimen during or after the administration of chemotherapeutic agents if the RBC of the subject shows a decrease in GSH recycle activity.In certain embodiments, if the RBC of the subject shows a decrease in glutathione recycle capacity after treatment with chemotherapeutic agents, the chemotherapeutic regimen is modified to reduce the subsequent dose or dosage of the chemotherapeutic agent delivered to the subject.In certain embodiments, the method comprises discontinuing the regimen comprising one or more chemotherapeutic agents.In certain embodiments, the method comprises starting treatment with a different or additional chemotherapeutic agent.Additional measurements of GSH recycle activity can be obtained after modifying the treatment regimen.

[0042] In certain embodiments, the method comprises modifying the treatment regimen during or after the administration of the chemotherapeutic agent if the RBC of the subject does not show a decrease in GSH recycling activity.In certain embodiments, the chemotherapeutic regimen is modified so that the subsequent dose or dosage of the chemotherapeutic agent delivered to the subject is increased.In certain embodiments, the method comprises continuing the regimen comprising one or more chemotherapeutic agents.In certain embodiments, the method comprises starting treatment with a different or additional chemotherapeutic agent.Additional measurements of GSH recycling activity can be obtained after modifying the treatment regimen.

[0043] In one aspect, provided herein is a method for treating a patient having cancer, the method comprising: (a) obtaining a measure of GSH recycle activity in one or more biological samples comprising red blood cells (RBCs) obtained from the patient prior to administration of a chemotherapeutic agent; (b) administering the chemotherapeutic agent; (c) obtaining a measure of GSH recycle activity in one or more biological samples comprising RBCs obtained from the patient after treatment with the chemotherapeutic agent; and (d) altering treatment of the subject with the chemotherapeutic agent and / or administering a treatment for CIPN.

[0044] In certain embodiments, the method of treating a patient comprises modifying the dose or dosage of the chemotherapeutic agent delivered to the patient when the RBC of the subject shows reduced GSH recycling activity.In certain embodiments, when the RBC of the subject shows reduced glutathione recycling capacity after treatment with a chemotherapeutic agent, the chemotherapeutic regimen is modified so that the subsequent dose or dosage of the chemotherapeutic agent delivered to the subject is reduced.In certain embodiments, the method comprises stopping the treatment of the subject with one or more chemotherapeutic agents.In certain embodiments, the method comprises starting treatment with a different or additional chemotherapeutic agent.

[0045] In certain embodiments, the method of treating a patient comprises modifying the dose or dosage of a chemotherapeutic agent delivered to the patient if the subject's RBCs do not exhibit reduced GSH recycling activity. In certain embodiments, the method comprises increasing the subsequent dose or dosage of a chemotherapeutic agent delivered to the subject. In certain embodiments, the method comprises continuing to treat the subject with one or more chemotherapeutic agents. In certain embodiments, the method comprises , including initiating treatment with a different or additional chemotherapeutic agent.

[0046] In one aspect, provided herein is a method of reducing susceptibility to CIPN following treatment with a chemotherapeutic agent, the method comprising: (a) obtaining a measure of glutathione (GSH) recycle activity in one or more biological samples comprising red blood cells (RBCs) obtained from the patient prior to administration of the chemotherapeutic agent; (b) obtaining a measure of GSH recycle activity in one or more biological samples comprising RBCs obtained from the patient after treatment with the chemotherapeutic agent; (c) comparing the one or more measures of GSH recycle activity in (a) with the one or more measures of GSH recycle activity in (b); and (d) modifying the patient's treatment to reduce susceptibility to CIPN and / or administering a treatment for CIPN in response to the change in GSH recycle activity determined in (c).

[0047] In certain embodiments, the method of reducing susceptibility to CIPN in a subject comprises modifying the dose or dosage of chemotherapeutic agent delivered to the subject when the RBC of the subject shows reduced GSH recycling activity.In certain embodiments, the subsequent dose or dosage of chemotherapeutic agent delivered to the subject is reduced when the RBC of the subject shows reduced glutathione recycling capacity after treatment with chemotherapeutic agent.In certain embodiments, the method of reducing susceptibility to CIPN in a subject comprises ceasing treatment of the subject with one or more chemotherapeutic agents.In certain embodiments, the method comprises starting treatment with a different or additional chemotherapeutic agent.In certain embodiments, the method of reducing susceptibility to CIPN comprises one or treatment for CIPN or its symptoms.

[0048] In one aspect, provided herein is a method for assessing the ability of a treatment to alter susceptibility to CIPN in a subject, the method comprising: (a) obtaining a measure of glutathione (GSH) recycle activity in one or more biological samples comprising red blood cells (RBCs) obtained from the subject prior to administration of a chemotherapeutic agent; (b) obtaining a measure of GSH recycle activity in one or more biological samples comprising RBCs obtained from the subject following treatment with a chemotherapeutic agent; (c) comparing the one or more measures of GSH recycle activity in (a) with the one or more measures of GSH recycle activity in (b); and (d) administering a treatment for CIPN to the subject.

[0049] In certain embodiments, the method includes administering a chemotherapeutic agent in combination with the treatment for CIPN. In certain embodiments, the CIPN treatment is administered prior to administration of the chemotherapeutic agent. In certain embodiments, the CIPN treatment is administered after administration of the chemotherapeutic agent. In certain embodiments, the method includes obtaining a measurement of glutathione (GSH) recycling activity in one or more biological samples comprising red blood cells (RBCs) obtained from the subject prior to administration of the chemotherapeutic agent and treatment for CIPN. In certain embodiments, the method includes obtaining a measurement of glutathione (GSH) recycling activity in one or more biological samples comprising red blood cells (RBCs) obtained from the subject after administration of the chemotherapeutic agent and treatment for CIPN. In certain embodiments, the method includes obtaining a measurement of glutathione (GSH) recycling activity in one or more biological samples comprising red blood cells (RBCs) obtained from the subject after administration of the chemotherapeutic agent and before treatment for CIPN. In certain embodiments, the method includes obtaining a measurement of glutathione (GSH) recycling activity in one or more biological samples comprising red blood cells (RBCs) obtained from the subject after treatment for CIPN and before administration of the chemotherapeutic agent.

[0050] The methods provided herein are intended to be used in a clinical setting, including to evaluate a patient's treatment during a chemotherapy regimen. The regimen may involve multiple chemotherapy treatments or administrations over a period of days, weeks, or years. In certain embodiments, as part of the treatment regimen, treatment with a chemotherapy agent includes at least The subject is administered at least 2 times, at least 3 times, at least 4 times, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, at least 10 times, at least 11 times, at least 12 times, at least 13 times, at least 14 times, at least 15 times, or at least 16 times. In certain embodiments, the measurements are obtained from samples obtained before the start of treatment (including the day of treatment before administration of chemotherapy). In some cases, the measurements are referred to as baseline measurements, pre-treatment measurements, or "pre-1" measurements. Furthermore, one or more measurements can be obtained from samples obtained at such time points as after the first administration of the chemotherapeutic agent but before the second administration of the chemotherapeutic agent (including the day of the second administration), or after the second administration of the chemotherapeutic agent but before the third administration of the chemotherapeutic agent (including the day of the third administration).

[0051] In one aspect, provided herein is a method of assessing susceptibility to or severity of chemotherapy-induced neuropathy (CIPN) in a subject to which a chemotherapeutic agent is administered, the method comprising: (a) obtaining a baseline measurement of glutathione (GSH) recycle activity in one or more biological samples comprising red blood cells (RBCs) obtained from the subject prior to a first administration of the chemotherapeutic agent; (b) obtaining a first measurement of GSH recycle activity in one or more biological samples comprising RBCs obtained from the subject after the first administration of the chemotherapeutic agent, but prior to a second administration of the chemotherapeutic agent; (c) obtaining a second measurement of GSH recycle activity in one or more biological samples comprising RBCs obtained from the subject after the second administration of the chemotherapeutic agent, but prior to a third administration of the chemotherapeutic agent; (d) obtaining a third measurement of GSH recycle activity in one or more biological samples comprising RBCs obtained from the subject after the third administration of the chemotherapeutic agent, but prior to a fourth administration of the chemotherapeutic agent; and (e) obtaining an average of the baseline measurement, the first measurement, the second measurement, and the third measurement. In certain embodiments, a mean value of 1.85 or less indicates susceptibility to or severity of CIPN. In certain embodiments, a mean value of 1.50 or less, 1.55 or less, 1.65 or less, 1.70 or less, 1.75 or less, 1.80 or less, 1.90 or less, 1.95 or less, or 2.00 or less indicates susceptibility to or severity of CIPN.

[0052] In another aspect, provided herein is a method for assessing susceptibility to chemotherapy-induced neuropathy (CIPN) or severity of CIPN in a subject administered a chemotherapeutic agent, the method comprising: (a) obtaining a first measurement of GSH recycle activity in one or more biological samples comprising RBCs obtained from the subject after a first administration of a chemotherapeutic agent but before a second administration of the chemotherapeutic agent; (b) obtaining a second measurement of GSH recycle activity in one or more biological samples comprising RBCs obtained from the subject after a third administration of a chemotherapeutic agent but before a fourth administration of the chemotherapeutic agent; and (c) obtaining an average value of the baseline measurement, the first measurement, and the second measurement. In certain embodiments, an average value of 1.8 or less indicates susceptibility to CIPN or severity of CIPN. In certain embodiments, an average value of 1.50 or less, 1.55 or less, 1.65 or less, 1.70 or less, 1.75 or less, 1.85 or less, 1.90 or less, 1.95 or less, or 2.00 or less indicates susceptibility to or severity of CIPN.

[0053] In another aspect, provided herein is a method of assessing susceptibility to or predicting severity of chemotherapy-induced neuropathy (CIPN) in a subject to which a chemotherapeutic agent has been administered, the method comprising: (a) obtaining a baseline measure of glutathione (GSH) recycle activity in one or more biological samples comprising red blood cells (RBCs) obtained from the subject prior to a first administration of a chemotherapeutic agent; and (b) obtaining a baseline measure of GSH recycle activity in one or more biological samples comprising RBCs obtained from the subject after (i) the first administration of the chemotherapeutic agent, but prior to a second administration of the chemotherapeutic agent, (ii) the measure of GSH recycle activity in one or more biological samples comprising RBCs obtained from the subject after the second administration of the chemotherapeutic agent, but prior to a third administration of the chemotherapeutic agent. or (iii) obtaining a measure of GSH recycle activity in one or more biological samples comprising RBCs obtained from the subject after a third administration of a chemotherapeutic agent but before a fourth administration of the chemotherapeutic agent, wherein a decrease in GSH recycle activity predicts increased susceptibility to or severity of CIPN.

[0054] In another aspect, provided herein is a method of reducing susceptibility to CIPN following treatment with a chemotherapeutic agent, the method including: (a) obtaining a baseline measure of glutathione (GSH) recycle activity in one or more biological samples comprising red blood cells (RBCs) obtained from the subject prior to a first administration of the chemotherapeutic agent; (b) obtaining a first measure of GSH recycle activity in one or more biological samples comprising RBCs obtained from the subject after the first administration of the chemotherapeutic agent, but prior to a second administration of the chemotherapeutic agent; and (c) obtaining a first measure of GSH recycle activity in one or more biological samples comprising RBCs obtained from the subject after the second administration of the chemotherapeutic agent, but prior to a third administration of the chemotherapeutic agent. (d) obtaining a second measurement of GSH recycling activity in one or more biological samples comprising RBCs obtained from the subject after a third administration of a chemotherapy agent but before a fourth administration of a chemotherapy agent; (e) obtaining an average of the baseline measurement, the first measurement, the second measurement, and the third measurement; and (f) modifying the subject's treatment regimen and / or administering a treatment for chemotherapy-induced neuropathy (CIPN) to reduce susceptibility to or severity of CIPN. In certain embodiments, an average value of 1.85 or less indicates susceptibility to or severity of CIPN. In certain embodiments, an average value of 1.50 or less, 1.55 or less, 1.65 or less, 1.70 or less, 1.75 or less, 1.80 or less, 1.90 or less, 1.95 or less, or 2.00 or less indicates susceptibility to or severity of CIPN.

[0055] In another aspect, provided herein is a method of reducing susceptibility to CIPN after treatment with a chemotherapeutic agent, the method comprising: (a) obtaining a first measurement of GSH recycle activity in one or more biological samples comprising RBCs obtained from the subject after a first administration of the chemotherapeutic agent but before a second administration of the chemotherapeutic agent; (b) obtaining a second measurement of GSH recycle activity in one or more biological samples comprising RBCs obtained from the subject after a third administration of the chemotherapeutic agent but before a fourth administration of the chemotherapeutic agent; (c) obtaining an average value of the first and second measurements; and (d) modifying the subject's treatment regimen to reduce susceptibility to chemotherapy-induced neuropathy (CIPN) or severity of CIPN and / or administering a treatment for CIPN. In certain embodiments, an average value of 1.8 or less indicates susceptibility to CIPN or severity of CIPN. In certain embodiments, an average value of 1.50 or less, 1.55 or less, 1.65 or less, 1.70 or less, 1.75 or less, 1.85 or less, 1.90 or less, 1.95 or less, or 2.00 or less indicates susceptibility to or severity of CIPN.

[0056] In another aspect, provided herein is a method of reducing susceptibility to CIPN following treatment with a chemotherapeutic agent, the method comprising: (a) obtaining a baseline measure of glutathione (GSH) recycle activity in one or more biological samples comprising red blood cells (RBCs) obtained from the subject prior to a first administration of the chemotherapeutic agent; and (b) obtaining (i) a measure of GSH recycle activity in one or more biological samples comprising RBCs obtained from the subject after the first administration of the chemotherapeutic agent, but prior to a second administration of the chemotherapeutic agent, (ii) a measure of GSH recycle activity in one or more biological samples comprising RBCs obtained from the subject after the second administration of the chemotherapeutic agent, but prior to a third administration of the chemotherapeutic agent, or (iii) a measure of GSH recycle activity in one or more biological samples comprising RBCs obtained from the subject after the third administration of the chemotherapeutic agent, but prior to a fourth administration of the chemotherapeutic agent, wherein a decrease in GSH recycle activity indicates a decrease in susceptibility to CIPN or a decrease in the severity of CIPN. (c) predicting or obtaining an increase in severity of chemotherapy-induced neuropathy (CIPN); and (c) modifying the subject's treatment regimen and / or administering a treatment for CIPN to reduce susceptibility to or severity of CIPN.

[0057] In certain embodiments, modifying the subject's treatment comprises reducing the subsequent dose or administration of the chemotherapeutic agent. For example, the patient's planned treatment regimen may be modified based on the measured GSH recycling activity. In certain embodiments, modifying the subject's treatment comprises treatment with a different chemotherapeutic agent. In certain embodiments, modifying the subject's treatment comprises administering an alternative or additional chemotherapeutic agent. In another embodiment, modifying the subject's treatment comprises ceasing administration of the chemotherapeutic agent. In certain embodiments, modifying the subject's treatment comprises treatment with a different chemotherapeutic agent.

[0058] In one aspect, provided herein is a method for assessing susceptibility to chemotherapy-induced neuropathy (CIPN) or the ability of a treatment to alter the severity of CIPN in a subject, the method comprising: (a) obtaining a baseline measure of glutathione (GSH) recycle activity in one or more biological samples comprising red blood cells (RBCs) obtained from the subject prior to a first administration of a chemotherapeutic agent; and (b) obtaining a first measure of GSH recycle activity in one or more biological samples comprising RBCs obtained from the subject after the first administration of the chemotherapeutic agent but prior to a second administration of the chemotherapeutic agent. (c) obtaining a second measurement of GSH recycling activity in one or more biological samples comprising RBCs obtained from the subject after a second administration of a chemotherapeutic agent but before a third administration of the chemotherapeutic agent; (d) obtaining a third measurement of GSH recycling activity in one or more biological samples comprising RBCs obtained from the subject after a third administration of a chemotherapeutic agent but before a fourth administration of the chemotherapeutic agent; (e) obtaining an average of the baseline measurement, the first measurement, the second measurement, and the third measurement; and (f) administering a treatment for CIPN to the subject. In certain embodiments, an average value of 1.85 or less indicates susceptibility to CIPN or severity of CIPN. In certain embodiments, an average value of 1.50 or less, 1.55 or less, 1.65 or less, 1.70 or less, 1.75 or less, 1.80 or less, 1.90 or less, 1.95 or less, or 2.00 or less indicates susceptibility to CIPN or severity of CIPN.

[0059] In one aspect, provided herein is a method for assessing susceptibility to chemotherapy-induced neuropathy (CIPN) or the ability of a treatment to alter the severity of CIPN in a subject, the method comprising: (a) obtaining a first measurement of GSH recycle activity in one or more biological samples comprising RBCs obtained from the subject after a first administration of a chemotherapeutic agent but before a second administration of the chemotherapeutic agent; (b) obtaining a second measurement of GSH recycle activity in one or more biological samples comprising RBCs obtained from the subject after a third administration of a chemotherapeutic agent but before a fourth administration of the chemotherapeutic agent; (c) obtaining an average value of the baseline measurement, the first measurement, and the second administration; and (d) administering a treatment for CIPN to the subject. In certain embodiments, an average value of 1.8 or less indicates susceptibility to CIPN or severity of CIPN. In certain embodiments, an average value of 1.50 or less, 1.55 or less, 1.65 or less, 1.70 or less, 1.75 or less, 1.85 or less, 1.90 or less, 1.95 or less, or 2.00 or less indicates susceptibility to or severity of CIPN.

[0060] In one aspect, provided herein is a method for assessing susceptibility to chemotherapy-induced neuropathy (CIPN) or the ability of a treatment to alter the severity of CIPN in a subject, the method comprising: (a) obtaining a baseline measure of glutathione (GSH) recycling activity in one or more biological samples comprising red blood cells (RBCs) obtained from the subject prior to a first administration of a chemotherapeutic agent; and (b) (i) obtaining a baseline measure of glutathione (GSH) recycling activity in one or more biological samples comprising red blood cells (RBCs) obtained from the subject prior to a first administration of a chemotherapeutic agent, but not prior to a first administration of a chemotherapeutic agent. (ii) a measure of GSH recycle activity in one or more biological samples comprising RBCs obtained from the subject prior to a second administration of a therapeutic agent, or (iii) a measure of GSH recycle activity in one or more biological samples comprising RBCs obtained from the subject after a second administration of a chemotherapeutic agent but prior to a third administration of the chemotherapeutic agent, or (iii) a measure of GSH recycle activity in one or more biological samples comprising RBCs obtained from the subject after a third administration of a chemotherapeutic agent but prior to a fourth administration of the chemotherapeutic agent, where a decrease in GSH recycle activity is predictive of increased susceptibility to CIPN or severity of CIPN; and (c) administering a treatment for CIPN to the subject.

[0061] In certain embodiments, the provided method predicts the increased susceptibility to grade 0 CIPN, grade 1 CIPN, grade 2 CIPN, or grade 3 CIPN. The method can predict CIPN occurring during or after a treatment regimen. In certain embodiments, the method predicts the onset of CIPN at least 1 week, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 5 weeks, at least 6 weeks, at least 7 weeks, at least 8 weeks, at least 9 weeks, at least 10 weeks, at least 11 weeks, at least 12 weeks, at least 13 weeks, at least 14 weeks, at least 15 weeks, at least 16 weeks, at least 17 weeks, at least 18 weeks, at least 19 weeks, at least 20 weeks, at least 3 months, at least 6 months, or at least 1 year before the onset of CIPN or severe CIPN symptoms.

[0062] Measurements taken before and during treatment, or measurements taken during treatment (e.g., after various times of treatment) can be used to measure changes in GSH recycling activity for an individual. In certain embodiments, the decrease in GSH recycling activity is a percentage decrease calculated as (measurement taken at one time during treatment - baseline) / baseline). A decrease in GSH recycling that predicts increased susceptibility to CIPN or severity of CIPN can be at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, or at least 50%.

[0063] In certain embodiments, the methods provided are directed to patients of a certain age or gender. In certain embodiments, the patient is male. In certain embodiments, the patient is female. In certain embodiments, the patient is 25-100 years old, 25-50 years old, 25-75 years old, 50-75 years old, or 50-100 years old.

[0064] As used herein, the term "treatment" or "treating" refers to a composition(s) and / or method(s) aimed at improving one or more symptoms of a disease or condition. For example, treating CIPN may include administering a composition to alleviate neuropathy or hyperalgesia, including preventing or reducing the frequency, severity, and / or duration of symptoms (such as pain in the limbs). Thus, treatment may include one or more of: reducing the onset or progression of CIPN, preventing CIPN, reducing the frequency and / or severity of CIPN symptoms (e.g., pain in the limbs), slowing the progression of CIPN, and / or slowing the progression of CIP. Treatments for CIPN include, but are not limited to, oral analgesics, antidepressants, lidocaine patches, menthol creams, serotonin-norepinephrine reuptake inhibitors (SNRIs) (e.g., duloxetine), tricyclic antidepressants (TCAs), anticonvulsants, combination topicals, nonsteroidal anti-inflammatory drugs (NSAIDs), and opioid therapy, physical therapy, and occupational therapy. In certain embodiments, a treatment reduces the frequency and / or severity of symptoms in a treated subject compared to an untreated control subject. As used herein, a treatment for CIPN can include a composition (i.e., a test compound) whose potential as a treatment for CIPN is under investigation.

[0065] In certain embodiments, the treatment of CIPN includes administering a "sodium channel blocker", which is a compound that selectively binds to a sodium channel, thereby inactivating the sodium channel. Specifically, sodium channel blockers include compounds that bind to the SS1 or SS2 extracellular domain of the alpha subunit of the sodium channel. Sodium channel blocker compounds that bind to the SS1 or SS2 subunit of the sodium channel, specifically tetrodotoxin and saxitoxin, have been shown to have similar pharmaceutical activity (see, e.g., US6,407,088, which is incorporated herein by reference).

[0066] In certain embodiments, the treatment of CIPN comprises administering calcium / magnesium supplementation to the subject. In certain embodiments, the treatment of CIPN comprises a calpain inhibitor.

[0067] In certain embodiments, the treatment of CIPN comprises administering a composition comprising one or more phytocannabinoids or synthetic derivatives thereof. In certain embodiments, the treatment of CIPN comprises administering a composition comprising cannabidiol (CBD).

[0068] As used herein, the phrase low glutathione (GSH) recycle-dependent antioxidant activity (i.e. high oxidative stress) in biological samples is used to indicate predisposition to CIPN. In certain embodiments, low GSH recycle-dependent antioxidant activity score is ≦1.0. As used herein, the phrase high glutathione recycle-dependent antioxidant activity (i.e. low oxidative stress) in biological samples is used to indicate increased risk of CIPN. In certain embodiments, high GSH recycle-dependent antioxidant activity score is >1.0.

[0069] As described herein, the inventors have disclosed methods that utilize, for example, one or more measurements of GSH recycling activity in a patient sample obtained before administration of a chemotherapeutic agent compared to one or more measurements of GSH recycling activity in a patient sample obtained after administration of a chemotherapeutic agent. In certain embodiments, the methods include obtaining measurements over multiple days before and / or after administration of a chemotherapeutic agent, or over the course of weeks or months. In certain embodiments, the methods include comparing average values ​​of measurements of GSH recycling activity obtained on a single day before and / or after administration of a chemotherapeutic agent. In certain embodiments, the methods include obtaining an average value of GSH recycling activity based on measurements obtained on the same day before administration of a chemotherapeutic agent to a subject, or over the course of days or weeks. In certain embodiments, the methods include obtaining an average value of GSH recycling activity based on measurements obtained on the same day after administration of a chemotherapeutic agent to a subject, or over the course of days or weeks. Thus, a "measurement" of GSH recycling activity may refer to an average value of measurements obtained from a series of samples obtained (e.g., before treatment with a chemotherapeutic agent or after treatment with a chemotherapeutic agent).

[0070] In certain embodiments, the method includes obtaining one or more measurements of GSH recycling activity in one or more biological samples obtained from the subject less than 12 hours, less than 24 hours, or less than 36 hours prior to treatment with a chemotherapeutic agent. In certain embodiments, the method includes obtaining one or more measurements of GSH recycling activity in one or more biological samples obtained from the subject less than 12 hours, less than 24 hours, or less than 36 hours after treatment with a chemotherapeutic agent.

[0071] Decreased GSH recycling activity or decreased GSH recycle activity following treatment of a subject with a chemotherapeutic agent. Cycle activity is associated with an increased risk of CIPN. In certain embodiments, the method comprises identifying a decrease in GSH recycling activity that is greater than about 10%, greater than about 20%, greater than about 25%, greater than about 30%, greater than about 35%, greater than about 40%, greater than about 45%, greater than about 50%, greater than about 55%, greater than about 60%, greater than about 65%, greater than about 70%, greater than about 75%, greater than about 85%, greater than about 90%, or at least about 95% or greater, or any amount between these specific percentages.

[0072] In certain embodiments, the method includes identifying an absence of a decrease in GSH recycle activity or a relative unchanged GSH recycle activity after treatment compared to measurements obtained from a pre-treatment sample. In certain embodiments, the method includes identifying an absence of a decrease in GSH recycle activity (or an unchanged GSH recycle activity) in which measurements obtained after treatment deviate from the pre-treatment measurement(s) by less than about 5%, less than about 10%, less than about 15%, less than about 20%, less than about 25%, less than about 30%, less than about 35%, less than about 40%, less than about 45%, less than about 50%, or any amount of decrease between these specifically recited percentages.

[0073] In certain embodiments, the method includes diagnosing CIPN or assessing the severity of CIPN in a subject. The diagnosis of CIPN can be based on the results of both subjective and / or objective assessment methods. In certain embodiments, the method includes subjective assessments such as patient considerations, previous and current treatments (both chemotherapeutic and non-chemotherapeutic), and social history. In certain embodiments, the method includes objective assessment strategies such as physical examination, laboratory tests, standardized questionnaires, electrodiagnostic tests, and nerve biopsy. The National Comprehensive Cancer Network (NCCN) details the most common physician-based grading scales used to assess the severity of CIPN. These grading scales include the Ajani Sensory, Eastern Cooperative Oncology Group (ECOG), National Cancer Institute-Common Terminology Criteria for Adverse Events (NCI-CTCAE), and the World Health Organization (WHO) system, which grades CIPN severity on a scale of 0 (normal) to 5 (death).

[0074] In yet another aspect, a method is provided for determining a personalized chemotherapy regimen for an individual human subject having cancer, the method comprising performing an assay to assess glutathione recycle dependent antioxidant activity and free radical scavenging efficiency of a blood sample from the subject prior to initiating chemotherapy.

[0075] As shown in the following examples, a redox assay capable of measuring GSH recycling in blood was used to compare the patient's inherent GSH recycling capacity before chemotherapy with the patient's subsequent susceptibility to CINV after treatment. The examples focus on platinum-based therapy. However, the accompanying data shown in the following examples can be extended to a broader patient population. The examples describe preliminary results showing that a decrease in GSH recycling capacity in blood is an objective indicator of the development of CIPN.

[0076] Assay As mentioned above, methods for assessing the antioxidant glutathione recycling capacity of a sample are known in the art. The present inventors have developed an improved method over previous assays including the OxPhos™ Cell Survival Kit (Cat. No. KLD-02, Rockland Inc.) that utilizes the conversion of hydroxyethyl disulfide (HEDS) to mercaptoethanol (ME) via a bioreduction mechanism (see also U.S. Pat. No. 8,697,391, which is incorporated herein by reference). The improved method involves the use of multiple cell cultures (e.g., in a 96-well plate format) to measure the amount of glutathione in the sample. These methods are easy to process samples of and provide accurate readings when used with automated plate readers typically found in CLIA certified clinical laboratories. Of note, certain methods do not require sample treatment with a sulfosalicylic acid (SSA) buffer.

[0077] In one aspect, provided herein is an assay for measuring glutathione (GSH) recycling capacity in a sample comprising red blood cells (RBCs), the assay comprising: (a) combining the sample with a solution comprising hydroxy-ethyl-disulfide (HEDS) to obtain a first volume, incubating the first volume to sediment a substantial RBC fraction from the sample; and (b) combining an aliquot of the first volume substantially free of RBCs with a solution comprising magnetic nanobeads to obtain a first volume of the sample. (c) exposing the second volume, or an aliquot thereof, to a magnetic field; (d) obtaining a supernatant from the second volume and mixing the supernatant, or an aliquot thereof, with a 5,5'-disulfanediylbis(2-nitrobenzoic acid) (DNTB)-containing solution to obtain a third volume; and (e) subjecting the third volume, or an aliquot thereof, to spectrophotometric analysis to obtain an absorbance reading, wherein the absorbance measured in (e) is indicative of GSH recycling capacity in the sample comprising RBCs.

[0078] In another aspect, provided herein is an assay for measuring GSH recycling capacity in a series of samples comprising RBCs, the assay comprising: (a) mixing each of the samples with a HEDS-containing solution to obtain a series of first volumes, and incubating the series of first volumes to sediment a substantial RBC fraction from the samples; (b) obtaining aliquots of each of the first volumes substantially free of RBCs, and mixing each aliquot with a magnetic nanobeads-containing solution to obtain a series of second volumes; (c) exposing the series of second volumes, or aliquots thereof, to a magnetic field; (d) obtaining a series of supernatants from the second volumes, and mixing each of the supernatants, or aliquots thereof, with a 5,5'-disulfanediylbis(2-nitrobenzoic acid) (DNTB)-containing solution to obtain a series of third volumes; and (e) subjecting the series of third volumes, or aliquots thereof, to spectrophotometric analysis to obtain a series of absorbance readings, wherein the absorbance measured in (e) is indicative of GSH recycling capacity in the samples comprising RBCs.

[0079] In another aspect, provided herein is an assay for measuring GSH recycling capacity in a series of samples comprising RBCs, comprising: (a) mixing each of the samples with a HEDS-containing solution to obtain a series of first volumes and incubating the series of first volumes; (b) centrifuging the samples to remove RBCs and debris from the suspension; (c) obtaining aliquots of each of the first volumes substantially free of RBCs and debris and mixing each aliquot with trichloroacetic acid (TCA) to obtain a series of second volumes; (d) subjecting the series of second volumes or aliquots thereof to centrifugation; (e) obtaining a series of supernatants from the second volumes and mixing each of the supernatants or aliquots thereof with a 5,5'-disulfanediylbis(2-nitrobenzoic acid) (DNTB)-containing solution to obtain a series of third volumes; and (f) subjecting the series of third volumes or aliquots thereof to spectrophotometric analysis to obtain a series of absorbance readings, wherein the absorbance readings obtained in (e) are indicative of GSH recycling capacity in the samples comprising RBCs.

[0080] As described in Example 3, the inventors have developed an assay that can be performed using one 8-well strip or a series of 8-well strips. In one particular embodiment of this assay, each 8-well strip is assigned to each sample. The design of the assay facilitates sample processing and improves the accuracy of measurements compared to available assays. 8-well strips are readily available and are designed to fit into frames that hold up to 12 strips (e.g., JG Finneran and Porvair Sciences, Model No. 208107, 96-well plate, 12 (See design of 8-well strips on x8 frame.) 8-well strips are available with V-, U-, and flat bottoms depending on the application.

[0081] For example, as shown in Figure 12, the wells of the 8-well strip allow for dilutions of the RBC-containing sample, as well as one or more of a blank, negative control, and positive control. In certain embodiments, the wells are duplicated to control for precision. The arrangement of the sample volumes in the 8-well strip facilitates the movement and ordering of the sample at various steps of the assay, including the movement of the supernatant after a clarification step (e.g., centrifugation).

[0082] As described herein, the inventors have identified L-cysteine ​​hydrochloride monohydrate (LCHM) as a useful positive control that can be included in assays to measure GSH recycling activity. L-cysteine ​​hydrochloride monohydrate (LCHM) is readily available, for example, in lyophilized form or dissolved in buffer, through various suppliers (e.g., Alfa Aesar, CAS7048-04-6). The positive control can be included in the assay at one or more concentrations. In certain embodiments, LCHM is included in solution at a concentration of about 25 μMol, about 50 μMol, and / or about 25 μMol to about 50 μMol. LCHM can be dissolved, for example, in low pH ultrapure water (Cayman Chemicals).

[0083] In certain embodiments, the first volume (each volume in the series of first volumes) is about 250 μl or less, about 200 μl or less, about 150 μl or less, about 100 μl or less, about 75 μl or less, about 50 μl or less, or about 25 μl or less. In certain embodiments, the second volume (or each volume in the series of second volumes) is about 250 μl or less, about 200 μl or less, about 150 μl or less, about 100 μl or less, about 75 μl or less, about 50 μl or less, or about 25 μl or less. In certain embodiments, the third volume (or each volume in the series of third volumes) is about 350 μl or less, about 300 μl or less, about 250 μl or less, about 200 μl or less, about 150 μl or less, about 100 μl or less, about 75 μl or less, about 50 μl or less, or about 25 μl or less. In certain embodiments, the method includes at least about 5, at least about 10, at least about 20, at least about 50, at least about 100, at least about 200, at least about 250, at least about 300, or at least about 350 first volumes, second volumes, and / or third volumes.

[0084] In certain embodiments, step (a) of the described method comprises incubating the sample(s) for at least about 15 minutes, at least about 30 minutes, at least about 1 hour, at least about 1.5 hours, or at least about 2 hours. The incubation time allows for conversion of hydroxyethyl disulfide (HEDS) to mercaptoethanol (ME) and sedimentation of RBCs. In certain embodiments, step (a) comprises shaking the sample, for example at 700 rpm, during all or a portion of the incubation time. In certain embodiments, the first volume is incubated in a V-bottom microplate. In certain embodiments, the plate is a 96-well microplate. In certain embodiments, the plate is a 384-well microplate. In certain cases, step (a) comprises incubating the sample volume at or about 21° C. If the incubation is not performed at 21° C., the following formula can be utilized to obtain an adjusted final GSH activity measurement:

number

[0085] In certain embodiments, the magnetic nanobeads of the described methods are glutathione magnetic agarose beads (see, e.g., Pierce™ Glutathione Magnetic Agarose Beads; Catalog Number: 78601). In certain embodiments, the nanobeads are composed of silica (SiO2) and maghemite (γ-Fe2O3). Suitable nanobeads composed of silica (SiO2) and maghemite (γ-Fe2O3) or the like can be produced according to the methods described in Stoeber, Werner; Fink, Arthur; Bohn, Ernst (January 1968). “Controlled growth of monodisperse silica spheres in the micron size range”. Journal of Colloid and Interface Science., which is incorporated herein by reference.

[0086] Magnetic separation can be accomplished using conventional means available to those skilled in the art. In certain embodiments, the method includes contacting the plate containing the sample or the sample-containing well with a magnetic separation device (e.g., Dexter LifeSep® biomagnetic separator tray; Dexter Magentic Technologies). Other suitable magnetic separation devices are known in the art.

[0087] As previously reported, extracellular ME in biological samples can be measured by the 5,5-dithiobis-2-nitrobenzoic acid (DTNB) assay (e.g., Ayene et al. (See, e.g., J. Biol. Chem., 277:9929-35, 2002). For example, the extracellular medium can be mixed with DTNB and then the OD measured at 412 nm. The concentration of ME is determined by the extinction coefficient of reduced DTNB, 1.36×10 4The absorbance can be calculated using the following formula: In certain embodiments, the absorbance measurements in the described methods are obtained using an automated plate reader. In certain embodiments, the step of measuring absorbance is performed on samples in a flat-bottom plate. In certain embodiments, the plate is a 96-well microplate. In certain embodiments, the plate is a 384-well microplate. In certain embodiments, the plate is an optical bottom microplate. In certain embodiments, the absorbance measurements are performed on one or more 8-well strips in a frame capable of holding up to 12 8-well strips. In certain embodiments, the method includes comparing the amount of absorbance from the test sample to at least one standard. For example, the standard can be an amount from a biological sample (e.g., subject). In certain embodiments, the standard is a solution comprising hydroxy-ethyl-disulfide (HEDS) and L-cysteine ​​hydrochloride monohydrate (LCHM).

[0088] kit Kits for carrying out the methods described herein are also provided.

[0089] In certain embodiments, the kit comprises HEDS and nanobeads. In other embodiments, the kit comprises HEDS, nanobeads, and DTNB. In certain embodiments, one or more of HEDS, nanobeads, and DTNB are included in a composition that includes a carrier. In certain embodiments, the kit further comprises one or more microtiter plates. In certain embodiments, the kit comprises a V-bottom microplate.

[0090] In another embodiment, the kit includes hydroxy-ethyl-disulfide (HEDS) and L-cysteine ​​hydrochloride monohydrate (LCHM) as a positive control. In certain embodiments, the LCHM is lyophilized. In other embodiments, the LCHM is in solution at a concentration of about 25 μMol, about 50 μMol, and / or about 25 μMol to about 50 μMol. In certain embodiments, the kit includes DTNB (Ellman's Reagent), V-bottom 8-well strips, flat-bottom 8-well strips, up to twelve 8-well strips. Also included is one or more of a frame capable of holding, trichloroacetic acid (TCA), and glutathione buffer.

[0091] The invention will now be described with reference to the following examples, which are provided for illustrative purposes only and should not be construed as limiting the invention in any manner to these examples, but rather as embracing any and all variations that become evident as a result of the teachings provided herein. EXAMPLES

[0092] Example 1: Measurement of GSH recycling activity to assess susceptibility to CIPN and predict the severity of CIPN Vinca alkaloids (e.g., vincristine) are important anticancer drugs that are primarily used to treat hematological cancers. Their primary mechanism of antitumor action is microtubule disruption. However, these drugs also cause mitochondrial damage, which leads to oxidative stress and the generation of reactive oxygen species (ROS). Other chemotherapeutic agents suggested to cause peripheral neuropathy by this mechanism, i.e., increased oxidative stress, include taxanes and platinum compounds. Oxidative damage to peripheral neurons can cause damage to the myelin sheath, mitochondrial proteins, and other antioxidant enzymes, resulting in hyperexcitability of peripheral neurons. This nerve damage results in chemotherapy-induced peripheral neuropathy (CIPN), a commonly observed dose-limiting neurological side effect.

[0093] The inventors hypothesized that chemotherapeutic agents with similar actions on mitochondria (vinca alkaloids, taxanes, and platinum compounds) would result in specific glutathione recycling patterns in patients predisposed to CIPN.

[0094] METHODS: Patients who consented to participate in this exploratory, single-center, prospective, IRB-approved study provided blood samples before each treatment cycle. Baseline / pretreatment samples were obtained before the start of the treatment regimen. Additional samples were obtained on the day of each treatment before administration of any chemotherapy. The Rotterdam Symptom Checklist (RSCL) was completed at each visit and reported symptoms were confirmed by comparison with those in the medical record.

[0095] Whole blood was analyzed for glutathione recycling capacity using the bioactive probe hydroxyethyl disulfide (HEDS) and incubated for 2 hours at room temperature with gentle mixing. Blood cells and proteinaceous thiols were removed from the samples by acid precipitation and centrifugation prior to spectrophotometric determination. The OxPho assay kit (Rockland Final spectrophotometric readings were converted to ME using conversion factors provided by NIH (Inc.) to obtain a "chemotox" score. Recycling ability (Chemotox score) was compared to self-reported grade of CIPN. "Severe CIPN" refers to patients who self-reported grade 3 CIPN on three or more consecutive treatment visits. Figure 1 provides a table showing guidelines for grading nervous system disorders, including CIPN.

[0096] Results: There were 428 enrolled patients, with a mean age of 63.84 years (12.85 STDEV ± 13.00; range; 25-92). Patients were predominantly of Caucasian ethnic background (82%), with additional African American (16.59%) and Asian (0.43%) ethnic backgrounds, and Latino (0.48%) ethnic backgrounds. Women constituted 57.94% of the cohort. To date, there have been 306 patients with a follow-up period of more than 24 months, of which 19.28% reported grade 3 CIPN, 24.51% reported grade 2 symptoms, while 32.35% reported no CIPN symptoms. Figure 2 shows that patient age is a significant CIPN risk factor. Analysis of Chemotox score based on gender and time of year showed that men generally have lower Chemotox scores (i.e., lower levels of glutathione recycling) than women, and that levels may vary depending on the time of year (Figure 3). An initial analysis of a series of four patient Chemotox scores revealed that combining time points may be useful in determining patient susceptibility to CIPN (Figures 4 and 8). Prediction of CIPN was obtained 5-12 weeks before the onset of grade 2 or 3 CIPN symptoms. Using this method, 80.77% of patients with persistent CIPN (defined as persisting beyond three treatment cycles or follow-up visits) were correctly identified (AUC 0.869281) (Figure 8). Using baseline / pre-treatment measurements alone was not useful for predicting severe CIPN, as evidenced by an AUC of 0.47 (Figure 5). Alternatively, using measurements obtained after the first treatment (and before the second treatment) yielded an AUC of 0.74 (Figure 6). We next evaluated whether severe CIPN could be predicted by using a combination of Chemotox scores obtained from patients at different time points and additional factors (such as patient age and sex). Using the mean of four measurements (1=pretreatment / baseline, 2=after first treatment / before second treatment, 2=after second treatment / before third treatment, and 4=after third treatment / before fourth treatment) as well as additional criteria improved the AUC of the assessment to 0.81 (Figure 7). Further analysis showed that the mean Chemotox score could be used to predict severe CIPN in patients undergoing chemotherapy regimens. Using the mean of the four Chemotox scores revealed a cutoff value of 1.85 for the prediction of severe CIPN (Figure 9A). Surprisingly, simply utilizing the average of the Chemotox scores obtained after the first treatment / before the second treatment, the second score obtained, and the Chemotox scores obtained after the third treatment / before the fourth treatment was highly predictive of severe CIPN with a cutoff value of 1.8 (Figure 9B).Figures 11A and 11B show that analysis using the mean Chemotox score obtained at four time points is useful for predicting CIPN in cohorts receiving taxol-containing or oxaliplatin / FOLFOX combination therapy. Additional risk factors / variables can be included and used in combination with the Chemotox score to improve CIPN prediction. Factors include patient age, race, sex, specific treatment regimen, cancer type, comorbidities, cancer history and treatment history, body mass index, genetic factors, and red blood cell count.

[0097] Example 2: Improved assay for measuring glutathione recycling capacity Determining the level of oxidative stress and adverse effects induced by reactive oxygen species uses an existing tube-based assay (Oxphos™ Cell Survival Assay Kit; Rockland, Inc.) to measure the recycling capacity of the antioxidant glutathione. The inventors adapted this previous microcentrifuge tube-based assay into a 96-well high-throughput assay, replacing some of the older chemistry to ensure accurate final measurements are obtained using automated plate readers typically found in CLIA-certified clinical laboratories.

[0098] The first modification to the assay involves the use of V-shaped 96-well microplates for incubation of blood samples with the bioactive probe hydroxy-ethyl-disulfide. After a standard incubation time, blood cells are allowed to settle to the bottom of the wells and the supernatant can be transferred to a new plate. A slurry of nanobeads is added to remove any interfering substances that may have been released from damaged cells. The nanobeads are composed of silica (SiO2) and maghemite (γ-Fe2O3) or similar, created according to the Stober process (Stoeber, Werner; Fink, Arthur; Bohn, Ernst (January 1968). “Controlled growth of monodisperse silica spheres in the micron size range”.Journal of Coll oid and Interface Science. 26(1):62-69. Bibcode:1968JCIS26-62-S. doi:10.1016 / 0021-9797(68)90272-5. The beads have surface ligands that bind to molecules that may interfere with the final optical measurement. By placing the plate on a magnetic separator unit, the beads containing the ligand-bound material are allowed to settle to the bottom of the wells, after which the clarified supernatant can be transferred to an optical bottom plate for measurement in a plate reader at 412 nm using dithiobisnitrobenzoic acid as done in previous methodologies (Oxphos™ Cell Survival Assay Kit; Rockland, Inc.).

[0099] These improvements would facilitate large-volume testing in clinical laboratories using automated high-throughput equipment, allowing for rapid determination of glutathione recycling capacity in patients.

[0100] Protocol for multi-well assays Non-kit materials: - Two V-bottom 96-well plates -One flat-bottom 96-well plate -Equilibration / wash buffer: 125mM Tris-HCL, 150mM NaCl, 1mM EDTA, pH 7.4 -Pierce Glutathione Magnetic Agarose Beads. Catalog Number 78601 -Dexter LifeSep 96F Magnetic Plate Separator -0.9% sodium chloride

[0101] Preparation of Magnabeads 1. For each patient, add 100 uL of bead slurry (25 uL of stable sedimenting beads) to a 1.5 mL microcentrifuge tube. NOTE: For multiple patients use larger tubes and increase the volume of beads and equilibration / wash buffer, respectively. 2. Add 400 uL of Equilibration / Wash Buffer to the beads and vortex for 10 seconds. 3. Place the tube on the magnetic stand to collect the beads on the wall of the tube. Remove and discard the supernatant. 4. Add 500 uL of Equilibration / Wash Buffer to the tube. Vortex the beads for 10 seconds and collect the beads using the magnetic stand. Remove and discard the supernatant. 5. Add 400 uL of Equilibration / Wash Buffer to the tube and vortex for 10 seconds. Note: Do not allow the beads to dry. Store the beads in equilibration / wash buffer if necessary.

[0102] reagent Reagent #1: (HEDS) 2-Hydroxyethyl disulfide (CAS1892-29-1, MW154.25g / mol) Reagent #2: (PBS) Phosphate buffered saline, pH 7.4 Dissolve 2.0 g KCL, 2.40 g KH2PO4, 80.0 g NaCl, and 14.4 g Na2HPO4 x 7H2O in distilled water, adjust the pH, and adjust the volume to 1 L. Reagent #3: (SS acid) [100mM 5-sulfosalicylic acid dihydrate, BioXtra≧99%] [Sigma S7422-100G] CAS5965-83-3,C7H6O6S·2H2O,MW254.21g / mol To prepare 500 ml, dissolve 12.71 grams in distilled H2O. Upon dissolution, add more water to adjust volume to 500 ml. Reagent #4: (GSH Buffer) [0.1M NaH2PO4·0.5mM EDTA, pH7.5] [Sodium dihydrogen phosphate anhydrous USP, Fisher S397-500] CAS7558-80-7, MW119.96g / mol NaH2PO4; [Ethylenediaminetetraacetic acid (EDTA), disodium salt, anhydrous USP, Fisher S312-500] CAS6381-92-6, MW372.24, C 10 H 14 To prepare 1 L of N2Na2H8·2H2O, dissolve 11.996 grams of sodium dihydrogen phosphate. Add 0.1862 grams of EDTA and continue stirring the solution. A pH adjustment may be necessary to dissolve all of the EDTA. Upon complete dissolution, the pH is adjusted to 7.5. Reagent #5: (DTNB) [10 mM 5,5'-dithiobis(2-nitro-benzoic acid)] [Sigma D8130-10G] CAS69-78-3;MW396.35;C 13 H8N2O8S2 To prepare 250 ml, dissolve 0.9908 grams of DNTB in Reagent 4.

[0103] Multiwell Assay Step 1 Prepare a V-bottom 96-well plate containing saline and reagent 6 as shown in Table 1 to load 5 uL and 10 uL blood samples in duplicate for a total of 5 wells per patient. After blood is dispensed into each well and mixed gently, cover the plate with microplate sealing film and then incubate on a rocker for 2 hours. [Table 1]

[0104] Step 2 After the 2 hour incubation, transfer 75 uL of supernatant from each well from step 1 to a new V-bottom 96-well plate containing 75 uL of washed magnetic bead solution in each well and incubate for 10 minutes.

[0105] Step 3 After incubation, place the V-bottom 96-well plate from step 2 on the Dexter magnetic plate for 30-60 seconds. The magnetic beads will be attracted to the bottom of each well. Transfer 10 uL of supernatant from each well to a flat-bottom 96-well plate containing reagents 4 and 5 as shown in Table 2 for a total of 5 wells per patient, plus one blank. [Table 2] Place the lid on the flat-bottom 96-well plate and read the absorbance at 412 nm. Subtract the blank from the experimental samples.

[0106] Example 3: Chemotox high throughput assay Improved assays have been developed to allow efficient analysis of large numbers of patient samples.

[0107] Materials required for each sample - 3 empty 96-well plate frames - Two V-bottom 8-well strips per sample -One flat-bottom 8-well strip per sample -Four 8-well strip caps per sample - Reagent #1: Hydroxyethyl disulfide (HEDS) (CAS 1892-29-1, MW 154.25 g / mol) - Reagent #2: Trichloroacetic acid (TCA) - Reagent #3: Colorimetric reagent, Ellman's Reagent (DTNB) [10mM 5,5'-Dithiobis(2-nitro-benzoic acid)] [Sigma D8130-10G] CAS69-78-3; MW396.35; C13H8N2O8S2 - Reagent #4 (Glutathione Buffer) -Lyophilized L-cysteine ​​hydrochloride monohydrate (LCHM) [Alfa Aesar: CAS7048-04-6

[0108] Listed below is the ready-to-use layout of 8-well strips (each of the three strips contains wells A-H as shown in FIG. 12) containing the specific reagents for the assay. One of the strips is required for each patient sample, and a single plate frame can hold up to 12 strips in parallel (8 x 12) to perform assays on 12 patient samples at a time. [Table 3] [Table 4] [Table 5]

[0109] Performing the assay: 1.2 Add 10 μL of Reagent #4 to wells B and C of Plate #1. 2. Add 20 μL of duplicate whole blood to wells E and F (containing 180 μL saline) and 60 μL of blood to G and H (containing 140 μL saline). 3. Seal the plate with cap strips and incubate at room temperature on a 700 RPM shaker for 2 hours. NOTE: Optimal performance is achieved at 21°C. If the environmental temperature is different from 21°C, use the following formula to obtain the final Chemotox score:

number

[0110] Parallel analysis of patient samples comparing results from a commercially available tube-based method with results from a high-throughput plate-based format. The assay was found to be at least as accurate or more accurate (Figure 13). The addition of a positive control (LCHM) further increases the reliability of the assay.

[0111] Each and every one of the patents, patent applications, and publications is incorporated herein by reference. U.S. Provisional Patent Application No. 63 / 275,271, filed November 3, 2021, and U.S. Provisional Patent Application No. 63 / 275,279, filed November 3, 2021, are incorporated herein by reference. Although the present invention has been described with reference to certain embodiments, it will be understood that modifications can be made without departing from the spirit of the invention. Such modifications are intended to be within the scope of the appended claims.

Claims

1. 1. A method for assessing susceptibility to or severity of chemotherapy-induced neuropathy (CIPN) in a subject receiving a chemotherapeutic agent, the method comprising: (a) obtaining a baseline measurement of glutathione (GSH) recycling activity in one or more biological samples comprising red blood cells (RBCs) obtained from the subject prior to a first administration of a chemotherapeutic agent; (b) obtaining a first measure of GSH recycling activity in one or more biological samples comprising RBCs obtained from the subject after a first administration of the chemotherapeutic agent but before a second administration of the chemotherapeutic agent; (c) obtaining a second measure of GSH recycling activity in one or more biological samples comprising RBCs obtained from the subject after a second administration of the chemotherapeutic agent but before a third administration of the chemotherapeutic agent; (d) obtaining a third measure of GSH recycling activity in one or more biological samples comprising RBCs obtained from the subject after a third administration of the chemotherapeutic agent but before a fourth administration of the chemotherapeutic agent; (e) obtaining an average value of the baseline measurement, the first measurement, the second measurement, and the third measurement; Including, The method, wherein a mean value of 1.85 or less indicates susceptibility to or severity of CIPN.

2. 1. A method for assessing susceptibility to or severity of chemotherapy-induced neuropathy (CIPN) in a subject receiving a chemotherapeutic agent, the method comprising: (a) obtaining a first measure of GSH recycling activity in one or more biological samples comprising RBCs obtained from the subject after a first administration of the chemotherapeutic agent but before a second administration of the chemotherapeutic agent; (b) obtaining a second measure of GSH recycling activity in one or more biological samples comprising RBCs obtained from the subject after a third administration of the chemotherapeutic agent but before a fourth administration of the chemotherapeutic agent; (c) obtaining an average value of the baseline measurement, the first measurement, and the second measurement; Including, The method, wherein a mean value of 1.8 or less indicates susceptibility to or severity of CIPN.

3. 1. A method for assessing susceptibility to chemotherapy-induced neuropathy (CIPN) or predicting the severity of CIPN in a subject receiving a chemotherapeutic agent, the method comprising: (a) obtaining a baseline measurement of glutathione (GSH) recycling activity in one or more biological samples comprising red blood cells (RBCs) obtained from the subject prior to a first administration of a chemotherapeutic agent; (b)(i) a measure of GSH recycling activity in one or more biological samples comprising RBCs obtained from the subject after a first administration of the chemotherapeutic agent but before a second administration of the chemotherapeutic agent; (ii) a measure of GSH recycling activity in one or more biological samples comprising RBCs obtained from the subject after a second administration of the chemotherapeutic agent but before a third administration of the chemotherapeutic agent; or (iii) obtaining a measure of GSH recycling activity in one or more biological samples comprising RBCs obtained from the subject after a third administration of the chemotherapeutic agent but before a fourth administration of the chemotherapeutic agent; Including, The method, wherein decreased GSH recycling activity predicts increased susceptibility to or severity of CIPN.

4. 4. The method of any one of claims 1 to 3, wherein the method further comprises modifying the subject's planned treatment with the chemotherapeutic agent and / or assessing the ability of treatment to alter susceptibility to or severity of CIPN in the subject.

5. modifying the subject's planned treatment includes reducing a subsequent dose or administration of the chemotherapeutic agent, and / or optionally Altering the subject's treatment regimen includes treatment with a different chemotherapeutic agent, and / or optionally, modifying the subject's treatment regimen includes administering an alternative or additional chemotherapeutic agent, and / or optionally modifying the subject's treatment regimen includes discontinuing administration of the chemotherapeutic agent, and / or optionally modifying the subject's treatment regimen comprises treatment with a different chemotherapeutic agent; The method according to any one of claims 1 to 4.

6. 4. The method of claim 3, wherein the decrease in GSH recycling activity is a percent decrease calculated as (measurement obtained from (i), (ii), or (iii)−baseline) / baseline).

7. 7. The method of claim 6, wherein the decrease in GSH recycling activity is at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, or at least 50%.

8. The method of any one of claims 1 to 7, wherein the planned treatment comprises at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, at least 12, at least 13, at least 14, at least 15, or at least 16 treatments with the chemotherapeutic agent.

9. 9. The method of any one of claims 1 to 8, wherein the method predicts increased susceptibility to grade 0 CIPN, grade 1 CIPN, grade 2 CIPN, or grade 3 CIPN.

10. 10. The method of any one of claims 1-9, wherein the subject's treatment regimen comprises a taxane, a platinum compound, optionally oxaliplatin or cisplatin, a vinca alkaloid, thalidomide, an epothilone, eribulin, ipilimumab, pembrolizumab, nivolumab, or bortezomib, FOLFOX, or a combination of oxiplatin and FOLFOX.

11. The method of any one of claims 1 to 10, wherein the one or more biological samples comprise whole blood.

12. (i) the one or more blood samples obtained from the subject prior to treatment are obtained on one or more days; and / or (ii) The method of any one of claims 1 to 11, wherein the one or more blood samples obtained from the subject after treatment are obtained on one or more days.

13. 13. The method of any one of claims 1 to 12, wherein the one or more measurements of GSH recycling activity comprise measuring the enzymatic conversion of hydroxyethyl disulfide (HEDS).

14. 14. The method of any one of claims 1 to 13, comprising contacting a biological sample with hydroxyethyl disulfide (HEDS), quantifying the amount of mercaptoethanol (ME) released by the HEDS-treated sample, measuring an absorbance reading of ME spectrophotometrically, converting the absorbance reading to an ME concentration, normalizing the ME concentration to the total RBC number at the time the sample was obtained, and assessing the GSH recycling-dependent antioxidant activity of intact RBCs in the sample.

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