Glutathione trisulfide administration for improving peripheral neuropathy

Abstract

Methods and devices for administering a composition comprising glutathione trisulfide (GSSSG), pantethine trisulfide (PTN-SSS), or lipoic acid trisulfide (LA-SSS) to treat peripheral neuropathy, such as chemotherapy-induced peripheral neuropathy (CIPN), e.g., by oral or nasal administration. The methods can be used, for example, to reduce pain associated with CIPN or to reduce the risk of developing CIPN.

Description

[Technical Field]

[0001] Priority claims This application claims the benefit of U.S. Provisional Application No. 63 / 377,379, filed September 28, 2022, and U.S. Provisional Application No. 63 / 379,298, filed October 13, 2022, the entire contents of which are incorporated herein by reference.

[0002] Sequence Listing This application contains a Sequence Listing that has been submitted electronically as an XML file named "29539-0707WO1_SL_ST26.XML". The XML file was created on September 25, 2023, and is 9,860 bytes in size. The material in the XML file is hereby incorporated by reference in its entirety.

[0003] Methods and devices for administering a composition comprising glutathione trisulfide (GSSSG), pantethine trisulfide (PTN-SSS), or lipoic acid trisulfide (LA-SSS) to treat peripheral neuropathy, such as chemotherapy-induced peripheral neuropathy (CIPN), e.g., by oral or nasal administration. The methods can be used, for example, to reduce pain associated with CIPN or to reduce the risk of developing CIPN. [Background technology]

[0004] As of 2018, 9.8 million cancer patients were treated with chemotherapy, and the number of patients requiring cancer chemotherapy is expected to reach 15 million by 2040 [1]. Paclitaxel (PTX) is one of the most common chemotherapy drugs used to treat breast, ovarian, and prostate cancer. Paclitaxel-induced peripheral neuropathy (PIPN) is a dose-limiting side effect of paclitaxel, affecting 30%–70% of patients treated with paclitaxel [2].

[0005] PIPN manifests as allodynia, hyperalgesia, and spontaneous pain primarily involving the feet and hands

[48] . PIPN typically develops during chemotherapy and often persists even after paclitaxel therapy is discontinued [3]. PIPN manifests as mechanical allodynia and hyperalgesia primarily involving the feet and hands during chemotherapy and often persists even after chemotherapy is discontinued [3]. PIPN can worsen a cancer survivor's quality of life and be severe enough to prompt chemotherapy discontinuation. Unfortunately, other chemotherapy drugs, including cisplatin and vincristine, can also cause peripheral neuropathy with similar clinical symptoms and common pathogenic mechanisms, collectively referred to as chemotherapy-induced peripheral neuropathy (CIPN) [4]. With the increasing number of cancer survivors worldwide [1], the health and economic impact of CIPN has increased significantly. Currently, available treatment options for CIPN, including PIPN, are extremely limited. Summary of the Invention

[0006] Provided herein is a method for treating or reducing the risk of a peripheral neuropathy-related disease in a subject, comprising administering, for example, orally or nasally, to a subject in need thereof a therapeutically or prophylactically effective amount of a composition comprising glutathione trisulfide (GSSSG), pantethine trisulfide (PTN-SSS), or lipoic acid trisulfide (LA-SSS). Also provided herein is a composition for administration, for example, oral or nasal administration, comprising glutathione trisulfide (GSSSG), pantethine trisulfide (PTN-SSS), or lipoic acid trisulfide (LA-SSS) for use in treating or reducing the risk of a peripheral neuropathy-related disease in a subject, wherein the method comprises administering, for example, orally or nasally, a therapeutically or prophylactically effective amount of the composition to a subject in need thereof, and optionally, the composition is formulated for oral or nasal administration.

[0007] In some embodiments, compositions comprising GSSSG are prepared by dissolving a crystalline form of GSSSG in a buffered saline solution of pH 3-6.

[0008] In some embodiments, the disease is chemotherapy-induced peripheral neuropathy (CIPN). In some embodiments, the CIPN is peripheral neuropathy associated with the administration of another platinum-containing chemotherapy, such as paclitaxel, cisplatin, vincristine, carboplatin, oxaliplatin, or nedaplatin, e.g., paclitaxel-induced peripheral neuropathy (PIPN). In some embodiments, the method comprises administering a composition comprising an effective amount of GSSSG, PTN-SSS, or LA-SSS within minutes to hours before or after administration of a dose of chemotherapy. In some embodiments, the method comprises administering a composition comprising an effective amount of GSSSG, PTN-SSS, or LA-SSS daily for the first 1 to 2 weeks of a chemotherapy regimen.

[0009] In some embodiments, the disease is diabetic neuropathy, Guillain-Barré syndrome neuropathy, chronic inflammatory demyelinating polyneuropathy, post-herpetic neuralgia, or peripheral neuropathy caused by hepatitis B, hepatitis C, myeloma, lymphoma, or kidney disease.

[0010] Provided herein are methods for treating peripheral neuropathy in a patient, comprising orally administering to the patient a therapeutically effective amount of a persulfide. In some embodiments, the patient's peripheral neuropathy is diabetic neuropathy, rheumatoid arthritis neuropathy, Guillain-Barré syndrome neuropathy, chronic inflammatory demyelinating polyneuropathy, or peripheral neuropathy caused by Lyme disease, shingles (i.e., post-herpetic neuralgia), hepatitis B, hepatitis C, myeloma, lymphoma, or kidney disease. In some embodiments, the patient's peripheral neuropathy is induced by a chemotherapy drug. In some embodiments, the drug is paclitaxel, cisplatin, or vincristine. In some embodiments, the persulfide is GSSSG. In some embodiments, GSSSG is administered to the patient daily in the range of 25-100 mg per kg for 20-35 days. In some embodiments, GSSSG is administered to a patient in an amount that results in plasma levels of GSSSG in the range of 40-80 pmol per ml for 20-35 days.

[0011] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs. Methods and materials are described herein for use in the present invention, but other suitable methods and materials known in the art can also be used. The materials, methods, and examples are merely intended to aid understanding and are not intended to be limiting. All publications, patent applications, patents, sequences, input databases, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control.

[0012] Other features and advantages of the invention will be apparent from the following detailed description and drawings, and from the claims. [Brief explanation of the drawings]

[0013] [Figure 1-1]Figure 1A-C: Chemical structures of polysulfides. (A) Chemical structures of GSSSG and GSH. Purified GSSSG is a white, odorless solid powder at 20°C. Its molecular weight is 644.7. One molecule of GSSSG consists of one sulfane sulfur (arrow) and two GSH molecules. (B) Chemical structures of PTN-SSS and PTN. One molecule of PTN-SSS consists of one sulfane sulfur (arrow) and one PTN molecule. (C) Chemical structures of LA-SSS and LA. One molecule of LA-SSS consists of one sulfane sulfur and one LA molecule. Abbreviations: GSH, glutathione; GSSSG, glutathione trisulfide; PTN, pantethine; PTN-SSS, pantethine trisulfide. LA-SSS. Lipoic acid trisulfide. [Figure 1-2] (As mentioned above.) [Figure 1-3] (As mentioned above.) [Figure 2] Figure 2A-B: GSSSG prevented mechanical allodynia caused by paclitaxel-induced peripheral neuropathy. (A) Mechanical pain threshold in the von Frey test 4 weeks after paclitaxel treatment with or without concomitant GSSSG treatment. 50 mg / kg GSSSG prevented mechanical allodynia over the experimental period. (B) Relative change in response time compared to baseline in the hot plate test at 4 weeks. PTX induced thermal hyperalgesia. Data were analyzed using a mixed-effects model and adjusted with Bonferroni correction. Data are presented as mean ± SD, n = 6 mice per group. PTX, paclitaxel. [Figure 3-1] Figure 3A-C: (A) Representative immunofluorescence images of intraepidermal nerve fibers (stained with PGP9.5, represented by yellow arrowheads) and basement membrane (stained with collagen IV, represented by white dashed lines) in the hind paws 1 and 4 weeks after paclitaxel treatment. Quantification of unmyelinated fiber density was calculated by dividing the number of intraepidermal nerve fibers by the basement membrane at 1 week (B) and 4 weeks (C). Data were analyzed by one-way ANOVA and Dunnett's multiple comparison test. Data are presented as mean ± SD, n = 6 mice per group. PTX, paclitaxel; G, GSSSG. [Figure 3-2](As mentioned above.) [Figure 4] Figure 4A-D: Neither PTX nor GSSSG altered the number of myelinated axons. (A) Representative microscopic images of sciatic nerves 4 weeks after PTX treatment stained with toluidine blue. (B) Quantification of total myelinated neuron axons in the sciatic nerve. (C) Representative high-magnification images of myelinated neuron axons for measuring myelin thickness. Ten neurons were randomly selected per image. Calculations were achieved using G-ratio software, ImageJ plugin. (D) Quantification of G-ratio. Neither PTX nor GSSSG altered myelin thickness. Data were analyzed by one-way ANOVA and Dunnett's multiple comparison test. Data are presented as mean ± SD, n = 6 mice per group. PTX, paclitaxel; G, GSSSG. [Figure 5] Figure 5A-B: GSSSG prevented the loss of unmyelinated axons in the sciatic nerve. (A) Representative transmission electron microscopy images of sciatic nerves at 4 weeks. The numbers of unmyelinated axons (arrows) and myelinated axons (arrowheads) were counted. (B) The ratio of the number of unmyelinated axons to the total number of axons in the sciatic nerve at 4 weeks. Data were analyzed by one-way ANOVA and Tukey's multiple comparison test. Data are presented as mean ± SD, n = 3 mice per group. PTX, paclitaxel; G, GSSSG. [Figure 6] Figure 6A-B: GSSSG prevented mitochondrial degeneration. (A) Representative transmission electron microscopy images of sciatic nerves 4 weeks after paclitaxel treatment. Asterisks indicate Schwann cells, arrows indicate mitochondria, and arrowheads indicate swollen mitochondria. (B) Quantification of mitochondrial area in unmyelinated neurons. The median mitochondrial area for control, paclitaxel, and paclitaxel + GSSSG was 0.036 μm, 0.057 μm, and 0.041 μm, respectively. Red bars indicate the median for each group. Data were analyzed by Kruskal-Wallis test and Dunn's multiple comparison test. Four high-magnification (×11,000) images from each mouse were examined. n = 3 mice per group. PTX, paclitaxel; G, GSSSG. [Figure 7]Figure 7A-D: 34S-labeled GSSSG and related polysulfides were detected in peripheral tissues 2 h after oral administration of 34S-labeled GSSSG. Quantification of GSSSG and reactive sulfur species by LC-MS / MS. (A) Concentrations of 34S-labeled GSSSG in four tissues obtained 2 h after oral administration. The relative ratios of 34S-labeled reactive sulfur species to endogenous reactive sulfur species (32S-) were calculated. The ratios of 34S-labeled glutathione persulfide (GSSH) (B), cysteine ​​persulfide (CysSSH) (C), and cysteine ​​trisulfide (CysSSSCys) (D) to endogenous in lumbar DRG, lumbar spinal cord, brain, and liver are reported. Data are expressed as mean ± SD, n = 4 per group; DRG, dorsal root ganglion; LSC, lumbar spinal cord. [Figure 8-1] Figure 8A-E: GSSSG prevented axonal degeneration in primary DRG neurons. (A-B) Representative immunofluorescence images of primary DRG neurons stained with NF200. Neurons were cultured for 24 hours and then incubated with paclitaxel for 1 hour with or without GSSSG (A) or PTNSSS (B). (C) Sholl circles of the cultured neurons in (A). The spacing between Sholl circles is 10 μm. (D-E) Analysis of neuronal intersections by Sholl circles after treatment with paclitaxel for 1 hour with or without GSSSG (D) or PTNSSS (E). Data were analyzed by repeated ANOVA and Dunnett's multiple comparison test. P values ​​are shown to indicate the comparison between paclitaxel and paclitaxel + GSSSG. Data are expressed as mean ± SD; n = 3 mice per group, 9-10 neurons per group. PTX, paclitaxel. [Figure 8-2] (As mentioned above.) [Figure 8-3] (As mentioned above.) [Figure 8-4] (As mentioned above.) [Figure 9-1]Figure 9A-C: GSSSG protected mitochondria in the axons of primary DRG neurons. (A) Representative images of nerve axons stained with NF200 and axonal mitochondria stained with Mito Tracker. The number and length of mitochondria (yellow arrowheads) in axons were measured manually. (B) Ratio of total mitochondrial length to total axon length, and (C) number of Mito Tracker puncta per 100 μm of axon. Data were analyzed by one-way ANOVA and Dunnett's multiple comparison test. Data are presented as mean ± SD; n = 3 mice per group, 8 images per group, 450–790 mitochondria per group. PTX, paclitaxel; G, GSSSG. [Figure 9-2] (As mentioned above.) [Figure 10] GSSSG treatment inhibited the increase in superoxide. Relative fluorescence change of GSSSG after paclitaxel exposure with dihydroethidium (DHE) for 30 minutes and 60 minutes. Data were analyzed by one-way ANOVA and Dunnett's multiple comparison test. Data are expressed as mean ± SD, n = 8 mice per group. PTX, paclitaxel; G, GSSSG. [Figure 11] Figure 11A-D: GSSSG enhanced antioxidant signaling in DRG. Gene expression relative values ​​of Nrf2 (A), HO1 (B), NQO1 (C), and GCLC (D) in DRG tissue 2 hours after a single 16 mg / kg paclitaxel dose with or without 50 mg / kg GSSSG treatment. Data were analyzed by one-way ANOVA and Dunnett's multiple comparison test. Data are presented as mean ± SD, n = 8 mice per group. PTX, paclitaxel; G, GSSSG; Nrf2, nuclear factor-erythroid factor 2-related factor 2; HO1, heme oxygenase 1; NQO1, NAD(P)H quinone dehydrogenase 1; GCLC, glutamate cysteine ​​ligase catalytic subunit. [Figure 12]Figure 12A-B: Concomitant administration of GSSSG did not inhibit the antitumor effect of paclitaxel. (A) Cell viability of the human breast cancer cell line MDA-MB-231 measured by LDH assay 24 hours after PTX with or without 10 μM GSSSG. (B) Relative viability of MDA-MB-231 cells 24 hours after 2 μM PTX with or without 10 μM GSSSG. Data were analyzed by two one-tailed t-tests. The limit of equivalence was defined as a 10% difference in cell counts. Data are presented as mean ± SD, n = 5 mice per group; LDH, lactate dehydrogenase, PTX, paclitaxel; G, GSSSG. DETAILED DESCRIPTION OF THE INVENTION

[0014] Although the precise mechanism of PIPN remains elusive, its pathological location is the axons of peripheral sensory neurons [5-7], explaining why the longer axons of sensory neurons in the limbs are primarily affected. Damage to Aβ fibers causes mechanical allodynia, while hyperalgesia arises from damaged Aδ and unmyelinated C fibers, which transmit temperature and cold sensations, respectively [8]. Paclitaxel prevents cancer cell proliferation by stabilizing microtubule polymers that need to be disassembled during mitosis. In addition to this antitumor effect, paclitaxel has been shown to cause mitochondrial dysfunction manifested as mitochondrial swelling [9], reduced mitochondrial membrane potential [10, 11], elevated reactive oxygen species (ROS)

[12] , and impaired oxidative phosphorylation in peripheral neurons

[13] . These off-target effects of paclitaxel have been implicated in the development of PIPN. Nevertheless, no treatments exist that protect mitochondria in peripheral neuron axons.

[0015] Reactive sulfur species, including persulfides and polysulfides, contain reactive sulfur that oxidizes or reduces other molecules. In particular, one type of reactive sulfur is sulfane sulfur (S 0The ) atom has strong nucleophilicity, which promotes the persulfation of protein thiols (cysteine ​​residues). Thiol persulfation competes with ROS-mediated thiol oxidation, thereby protecting proteins from irreversible oxidation. Indeed, endogenous persulfides, such as glutathione persulfide (GSSH) and cysteine ​​persulfide (CysSSH), exert potent antioxidant effects and play an important role in maintaining intracellular reduction equilibrium

[14] . Nevertheless, it is unknown whether systemic administration of polysulfides protects peripheral sensory neurons from the adverse effects of paclitaxel by increasing the local concentration of reactive sulfur.

[0016] This study was designed to address this knowledge gap by examining the effects of a systemically administered stable formulation of the endogenous polysulfide glutathione trisulfide (GSSSG) in a mouse model of PIPN. GSSSG contains a sulfane sulfur in its structure (Figure 1A, arrow) and is in equilibrium with GSSH in the following formula

[15] :

[0017]

number

[0018] Without wishing to be bound by theory, it is hypothesized that systemic administration of GSSSG ameliorates PIPN by increasing the local concentration of reactive sulfur species in peripheral sensory neurons and protecting mitochondria in nerve axons. As shown herein, GSSSG attenuates PIPN without reducing the antitumor effects of paclitaxel.

[0019] Previous studies have demonstrated that polysulfides, such as GSSSG, have the potential to alleviate paclitaxel-induced peripheral neuropathy by protecting mitochondria in the axons of peripheral neurons. This conclusion is based on the following findings: 1) daily oral administration of GSSSG attenuated mechanical allodynia in the hind paws of paclitaxel-treated mice; 2) orally administered GSSSG was absorbed and increased reactive sulfur levels in the lumbar DRG and spinal cord (both regions containing primary sensory neurons innervating the hind paws); 3) GSSSG inhibited axonal degeneration and prevented mitochondrial swelling in paclitaxel-treated mice, maintaining the number of axonal mitochondria in cultured primary DRG neurons exposed to paclitaxel; and 4) GSSSG attenuated the increase in superoxide levels in primary cortical neurons incubated with paclitaxel. Collectively, these results suggest that GSSSG improves PIPN. The beneficial effects of GSSSG were associated with preserving mitochondrial integrity in peripheral nervous system axons.

[0020] As shown herein, oral administration of GSSSG at 50 mg / kg / day for 4 weeks improved paclitaxel-induced mechanical allodynia, 34 S-labeled GSSSG was detected in the lumbar DRG and lumbar spinal cord 2 hours after a single oral administration. The results indicated that GSSSG was well absorbed from the gastrointestinal tract and readily incorporated into central and peripheral nervous tissues

[33] . 34 The concentrations of relevant polysulfides and persulfides, including S, are 34 Two hours after administration of S-labeled GSSSG, the levels of polysulfides and persulfides were more than 10-fold higher than the respective endogenous polysulfides and persulfides. These observations suggest that GSSSG is in dynamic equilibrium with other reactive sulfur species. This study demonstrated a link between neuroprotection and increased polysulfide levels in the peripheral nervous system after systemic administration of a polysulfide donor.

[0021] Mitochondrial dysfunction and increased oxidative stress play a crucial role in PIPN

[34] . Mitochondria are the main source of intracellular ROS but are also important targets of ROS. Normal mitochondria emit low levels of ROS because a small amount of electrons leak from complexes I, III, and IV of the electron transport chain (ETC) and combine with molecular oxygen to generate superoxide. When electron transport is impaired, more electrons leak from ETC complexes, generating more ROS. Increased ROS production by dysfunctional mitochondria further impairs mitochondrial function and ultimately leads to the destruction of the mitochondrial membrane potential. Mitochondria that malfunction are degraded and replaced by quality control mechanisms, such as mitophagy. Because neurons primarily rely on oxidative phosphorylation in mitochondria to generate ATP, mitochondrial dysfunction can lead to the degeneration of neuronal axons

[35] .

[0022] Reactive sulfur species are thought to protect mitochondria through several mechanisms: 1) supporting bioenergetics

[36] ; 2) scavenging ROS

[14] ; 3) activating superoxide dismutase (SOD)

[37] ; 3) preventing mitochondrial fission by inhibiting Drp1 activity

[36] ; and 4) enhancing the Nrf2 / Keap1 pathway by oversulfating Keap1

[38] . Previous studies have shown that administration of GSSSG attenuated paclitaxel-induced ROS generation and prevented mitochondrial swelling and loss in peripheral nerve axons. These observations suggest that GSSSG has antioxidant properties and may support bioenergetics. Our results in primary DRG neurons suggest that GSSSG prevents not only paclitaxel-induced mitochondrial fragmentation (or fission) but also mitochondrial loss in nerve axons. As previously reported, redox balance and glutathione oxidation in neuronal axons partially regulate mitochondrial transport from the cell body to the axon

[39] , and loss of axonal mitochondria leads to axonal degeneration

[40] and neuronal damage

[41] . This observation suggests that GSSSG attenuates PIPN through mitochondrial protection by regulating redox balance in peripheral neurons. Interestingly, paclitaxel with or without GSSSG enhanced Nrf2, a potent regulator of cellular homeostasis, redox balance, and inflammation

[42] , suggesting that paclitaxel triggers an antioxidant defense mechanism. However, we observed that GSSSG and paclitaxel, but not paclitaxel alone, enhanced the expression of HO1, NQO1, and GCLC, downstream genes of Nrf2. Without wishing to be bound by theory, one possible reason why GSSSG upregulates genes downstream of Nrf2 is that it induces the oversulfation of Keap1, an Nrf2-binding protein. Oversulfation of Keap1 promotes the activation of Nrf2, thereby upregulating downstream genes and exerting antioxidant effects

[38] . In addition to the Nrf2 pathway, GSSSG may exert protective effects on peripheral neurons through multiple antioxidant mechanisms.

[0023] Treatment methods As shown herein, systemic administration of GSSSG attenuated paclitaxel-induced mechanical allodynia in a model of CIPN. After oral administration, GSSSG reached the DRG and prevented mitochondrial impairment and axonal degeneration in peripheral neurons. GSSSG enhanced Nrf2-dependent antioxidant signaling in vivo and reduced ROS levels in vitro. Because mitochondrial impairment is a common feature of various forms of peripheral neuropathy

[42] , these results suggest the potential therapeutic effect of GSSSG not only for CIPN but also for other forms of peripheral neuropathy caused by mitochondrial dysfunction, such as diabetic neuropathy.

[0024] Thus, provided herein are methods for treating or reducing the risk of a disease associated with peripheral neuropathy in a subject, e.g., a mammalian subject, e.g., a human or non-human veterinary subject. In some embodiments, the disease is CIPN, e.g., PIPN. In some embodiments, the disease is diabetic neuropathy, which involves sensory nerves and usually causes axonal damage in peripheral neurons and is pathologically similar to CIPN. In some embodiments, the disease is Guillain-Barré syndrome neuropathy, which involves sensory nerves and usually causes axonal damage in peripheral neurons and is pathologically similar to CIPN. In some embodiments, the disease is chronic inflammatory demyelinating polyneuropathy with demyelination similar to CIPN. In some embodiments, the disease is neuropathy caused by herpes zoster (i.e., post-herpetic neuralgia), which affects peripheral neurons. In some embodiments, the disease is peripheral neuropathy caused by hepatitis B, hepatitis C, myeloma, lymphoma, or kidney disease.

[0025] Typically, the methods involve systemic (e.g., oral or nasal) administration to a subject in need of, or determined to be in need of, such treatment of, a composition comprising a therapeutically effective amount of a crystalline form of GSSSG, PTN-SSS, or LA-SSS described herein.

[0026] As used in this context, "treating" means improving at least one symptom of a disease associated with neurodegeneration. The conditions that can be treated using the methods described herein can be related to peripheral neuropathy and pain. Administration of a therapeutically effective amount of the compounds described herein can result in the relief of peripheral neuropathy and pain.

[0027] Furthermore, the methods can result in a reduced risk of developing peripheral neuropathy and pain. Subjects at risk of developing peripheral neuropathy and pain can include those with cancer who are being treated with a chemotherapy regimen containing an agent that causes or can cause peripheral neuropathy and pain, such as a platinum-containing agent, e.g., cisplatin, paclitaxel, vincristine, carboplatin, oxaliplatin, or nedaplatin. These methods can include oral or intranasal administration of an effective amount of a GSSSG, PTN-SSS, or LA-SSS composition described herein within minutes to hours or days before and / or after chemotherapy administration. Subjects at risk of developing neuropathy and pain can include those who have (or have been diagnosed with) diabetic neuropathy, Guillain-Barré syndrome neuropathy, chronic inflammatory demyelinating polyneuropathy, shingles, hepatitis B, hepatitis C, myeloma, lymphoma, or kidney disease.

[0028] An "effective amount" is an amount sufficient to produce a beneficial or desired result. For example, a therapeutic amount is one that achieves a desired therapeutic effect. This amount may be the same as or different from a prophylactically effective amount, which is the amount needed to prevent the onset of a disease or disease symptoms. An effective amount can be administered in one or more administrations, applications, or dosages. The compositions can be administered one or more times per day to one or more times per week, including every other day. In some embodiments, the GSSSG, PTN-SSS, or LA-SSS is administered daily for at least 2, 3, 4, 5, 6, or 7 days before or after a dose of chemotherapy is administered, or daily for the first one or two weeks of a chemotherapy regimen that includes the administration of chemotherapy that can cause peripheral neuropathy. One of skill in the art will appreciate that certain factors can affect the dosage and timing required to effectively treat a subject, including, but not limited to, the severity of the disease or disorder, previous treatments, the overall health and / or age of the subject, and other illnesses present. Furthermore, treatment of a subject with a therapeutically effective amount of a therapeutic compound described herein can include a single treatment or a series of treatments.

[0029] The dosage, toxicity, and therapeutic effect of therapeutic compounds can be determined by standard pharmaceutical procedures in cell culture or animal studies, for example, to determine the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index, which can be expressed as the ratio LD50 / ED50. Compounds that exhibit high therapeutic indices are preferred. Compounds that exhibit toxic side effects may also be used, but care should be taken to design a delivery system that targets such compounds to the site of affected tissue in order to minimize potential damage to non-infected cells and thereby reduce side effects.

[0030] Data obtained from cell culture assays and animal studies can be used to formulate a range of dosages for use in humans. The dosage of such compounds preferably lies within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage may vary within this range depending on the dosage form used and the route of administration utilized. For any compound used in the methods of the invention, the therapeutically effective dose can be initially estimated from cell culture assays. A dose may be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (i.e., the concentration of the test compound that achieves a half-maximal inhibition of symptoms) determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Plasma levels may be measured, for example, by high-performance liquid chromatography.

[0031] Pharmaceutical Compositions and Methods of Administration The methods described herein involve the use of pharmaceutical compositions containing GSSSG, PTN-SSS, or LA-SSS as an active ingredient. In some embodiments, compositions are prepared using crystalline forms of GSSSG by dissolving crystalline GSSSG in a buffer solution, such as saline, at a pH of 3 to 6, e.g., 4.8 to 5, using the methods described in EP 3560947 A1. The pH can be adjusted with an acid or base, e.g., hydrochloric acid or sodium hydroxide. Compositions containing PTN-SSS or LA-SSS can be prepared by dissolving them in a buffer solution, such as saline or water, at a pH of 4 to 9, e.g., 5 to 8.

[0032] An exemplary method for producing a crystalline form of glutathione trisulfide dehydrate can include precipitating glutathione trisulfide dihydrate crystals in an aqueous solution containing dissolved glutathione trisulfide, and recovering the precipitated glutathione trisulfide dihydrate crystals. PTN-SSS or LA-SSS can be prepared as described in WO 2022 / 045212 (LA-SSS) and WO 2022 / 045052 (PTN-SSS).

[0033] Pharmaceutical compositions typically contain pharmaceutically acceptable carriers. As used herein, the term "pharmaceutically acceptable carriers" includes physiological saline, solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, etc., that are compatible with pharmaceutical administration. Supplementary active compounds can also be incorporated into the composition.

[0034] The pharmaceutical compositions used in the present methods are formulated to be suitable for nasal or oral administration, or for parenteral, eg, intravenous, administration.

[0035] Methods for formulating suitable pharmaceutical compositions are known in the art; see, e.g., Remington: The Science and Practice of Pharmacy, 21st ed., 2005; and Drugs and the Pharmaceutical Sciences: a Series of Textbooks and Monographs (Dekker, NY).

[0036] Oral compositions usually contain an inert diluent or an edible carrier. For the purpose of oral therapeutic administration, the active agent can be incorporated with an excipient or used in the form of tablets, troches, or capsules, such as gelatin capsules. Oral compositions can also be prepared using a liquid carrier for use as a mouthwash. Pharmaceutically compatible binders and / or adjuvant materials can be included as part of the composition. Tablets, pills, capsules, troches, etc. can contain any of the following ingredients, or substances of similar nature: binders, such as microcrystalline cellulose, tragacanth gum, or gelatin; excipients, such as starch or lactose; disintegrants, such as alginic acid, Primogel, or corn starch; lubricants, such as magnesium stearate or sterotes; glidants, such as colloidal silicon dioxide; sweeteners, such as sucrose or saccharin; or flavorings, such as peppermint, methyl salicylate, or fruit (e.g., orange or cherry) flavorings.

[0037] For purposes of mucosal therapeutic administration, the active compound (e.g., GSSSG, PTN-SSS, or LA-SSS) can be incorporated with excipients or carriers suitable for administration by inhalation or absorption, for example, by nasal spray or nasal drops. For nasal administration, the formulation can be, for example, an aerosol or dry powder in a sealed vial or other suitable container.

[0038] Pharmaceutical compositions and dosage forms can further include one or more compounds that reduce the rate at which the active ingredient decomposes. Thus, the oral or nasal dosage forms described herein can be converted into immediate-release or sustained-release dosage forms. Immediate-release dosage forms may release GSSSG, PTN-SSS, or LA-SSS in a very short time, for example, within minutes to a few hours. Sustained-release dosage forms may, if desired, release GSSSG, PTN-SSS, or LA-SSS over several hours, for example, up to 24 hours or longer. In either case, delivery can be controlled to be substantially at a predetermined rate over the delivery period.

[0039] Nasal delivery is considered an attractive route for needle-free systemic drug delivery, especially when rapid absorption and efficacy are desired. Furthermore, nasal delivery can help address issues related to poor bioavailability, slow absorption, drug degradation, and gastrointestinal adverse events (AEs), and avoids first-pass metabolism in the liver.

[0040] Liquid nasal formulations are primarily aqueous solutions, although suspensions and emulsions can also be delivered. In traditional spray pump systems, antimicrobial preservatives are typically required to maintain microbial stability in liquid formulations.

[0041] Metering spray pumps can be used. Pumps typically deliver approximately 25-200 μL per spray, providing high reproducibility of emitted dose and plume shape. Particle size and plume shape can vary within certain limits and depend on the characteristics of the pump, formulation, actuator opening, and applied force. Traditional spray pumps displace the emitted liquid with air, and therefore require preservatives to prevent contamination.

[0042] Alternative spray systems or devices that avoid the need for preservatives can also be used. These systems use collapsible bags, movable pistons, or compressed gas to compensate for the volume of the dispensed liquid. Solutions with collapsible bags and movable pistons that compensate for the volume of the dispensed liquid offer the added benefit of being able to dispense them upside down without the risk of drawing air into the dip tube and complicating subsequent spraying. This can be useful for some products where head-down application is recommended for bedridden patients. Another method used to avoid preservatives is to pass the air replacing the dispensed liquid through a sterile air filter. Additionally, some systems have a ball valve at the tip to prevent contamination of the liquid inside the applicator tip.

[0043] For administration by inhalation, the GSSSG, PTN-SSS, or LA-SSS compounds can be delivered in the form of a dry powder or aerosol spray from a pressured container or dispenser containing a suitable propellant, e.g., a gas such as carbon dioxide, or an inhaler. Such methods include those described in U.S. Patent No. 6,468,798.

[0044] Also provided herein are devices for nasal administration, eg, as described herein, comprising a GSSSG, a PTN-SSS, or a LA-SSS.

[0045] For example, kits are described herein that can include compositions containing GSSSG, PTN-SSS, or LA-SSS as a pre-prepared dry powder or liquid nasal or oral form ready for administration, or alternatively, as a solid pharmaceutical composition that can be reconstituted with a solvent to provide a liquid nasal or oral dosage form. When the kit includes a GSSSG composition as a solid pharmaceutical composition that can be reconstituted with a solvent to provide a liquid dosage form (e.g., for oral or nasal administration), the kit may optionally include a reconstitution solvent having a pH of 3-6, e.g., pH 4.8-5.0. When the kit includes a PTN-SSS or LA-SSS composition as a solid pharmaceutical composition that can be reconstituted with a solvent to provide a liquid dosage form (e.g., for oral or nasal administration), the kit may optionally include a reconstitution solvent having a pH of 4-9, e.g., pH 5-8. In this case, the reconstitution or reconstitution solvent is combined with the active ingredient to provide a liquid oral dosage form of the active ingredient. Typically, the active ingredient is soluble in the solvent to form a solution. The solvent can be, for example, water, a non-aqueous liquid, or a combination of a non-aqueous component and an aqueous component.Suitable non-aqueous components include, but are not limited to, oil; alcohol, such as ethanol; glycerin; and glycol, such as polyethylene glycol and propylene glycol.In some embodiments, the solvent is phosphate buffered saline (PBS).

[0046] The pharmaceutical composition can be included in a container, pack, or dispenser along with instructions for administration. For example, GSSSG can be provided in a kit in crystalline form with a sterile buffer solution (e.g., saline) of pH 3-6 used to dissolve the crystals and prepare a solution for nasal or oral administration. [Example]

[0047] The invention is further described in the following examples, which do not limit the scope of the invention described in the claims.

[0048] material and method The following materials and methods were used in the examples below.

[0049] animal All animal protocols were approved by the Massachusetts General Hospital Animal Care and Use Committee. Animal care was in accordance with guidelines established by the NIH and the International Association for the Study of Pain

[16] . Male C57BL / 6J mice (6–7 weeks old) were purchased from the Jackson Laboratory (Bar Harbor, ME, USA). Until the time of experiment, mice were housed in our animal facility in a light-controlled environment with a 12-hour light shift schedule (7:00 AM to 7:00 PM) with free access to food and water.

[0050] Drugs and Animal Models Paclitaxel (Sigma-Aldrich) was dissolved in ethanol and cremophor (1:1) and diluted with normal saline (1:4). Peripheral neuropathy was induced in mice by intraperitoneal (ip) administration of 4 mg / kg paclitaxel every other day for a total of four injections (cumulative dose of 16 mg / kg) according to a previously described protocol

[17] . A stable crystalline form of GSSSG was manufactured and supplied by Kyowa Hakko Bio Co., Ltd. GSSSG was ground and mixed in 0.5% methylcellulose. To evaluate the therapeutic effect of GSSSG in the PIPN model, mice were randomly divided into three groups of six. Mice were treated with 1) paclitaxel alone, 2) paclitaxel and GSSSG, or 3) vehicle alone (control). Mice in groups 1 and 2 received paclitaxel as described above, and mice in the control group received the same volume of vehicle. The first dose of GSSSG was given within 1 hour after the first PTX injection. Treatment with 50 mg / kg / day of GSSSG (Group 2) or 0.5% methylcellulose (Groups 1 and 3) was administered by oral gavage for 28 days. The dose of GSSSG was determined based on a pilot study. To determine the effect of GSSSG on the development of PIPN, behavioral tests to assess allodynia and hyperalgesia were performed on all mice on days -1 (baseline), 7, 14, 21, and 28 after paclitaxel administration. Behavioral tests were performed by an investigator blinded to the treatment groups as described in the following section.

[0051] PTN-SSS was synthesized and supplied by Kyowa Hakko Bio Co., Ltd. The purity of PTN-SSS was 96.3%, and PTN-SSS is highly water-soluble (>50 g / L). PTN-SSS and PTN (Toronto Research Chemicals, Toronto, ON, Canada) were suspended in distilled water. PTN-SSS is stable in solutions with pH 4.0–9.0 at room temperature for at least 4 days. In studies of PTN-SSS, distilled water was used as the only vehicle.

[0052] To determine the tissue distribution of orally administered GSSSG, 34 S-labeled GSSSG was administered by gavage to each of four mice. 34 S-labeled GSSSG was synthesized and supplied by Kyowa Hakko Bio CO., LTD. 34 Two hours after a single dose of S-GSSSG, DRG, lumbar spinal cord, brain, liver, and plasma were obtained and immediately frozen for subsequent analysis.

[0053] Behavioral testing 1) Measurement of mechanical allodynia To assess mechanical allodynia, mechanical pain thresholds were measured using a manual von Frey filament test. Prior to testing, mice were acclimatized for 3–4 days in our animal facility and then acclimatized for 30 minutes in plastic cages on a wire mesh floor for three consecutive days. For testing, mice were placed in the same cages as the acclimatized mice. An experimenter blinded to the mouse's treatment stimulated the mid-plantar surface of the hind paw with von Frey filaments (Ugo Basile, catalog no. 37450-275) at a force ranging from 0.04 grams (g) to 2.0 g, starting at 0.6 g. The filaments were applied at a constant rate until they bent. A behavioral response of paw withdrawal and licking was considered a response. If the mouse did not respond, stimulation with the same filament size was repeated up to two times. The filament was changed according to the previous filament response (we applied a smaller filament after a positive response and a larger filament after a negative response). The total number of filament applications was limited to nine. The 50% threshold for paw withdrawal was calculated using the Up-down Reader

[18] based on the up-and-down method

[19] . This behavioral test was performed weekly for 4 weeks.

[0054] 2) Hot plate test To assess hyperalgesia, thermal thresholds were determined using the hot plate test

[20] . Mice were placed on a hot plate (Hot / Cold Plate NG, Ugo Basile, Cat. No. 35150) heated to 52°C. The latency to exhibit nociceptive behaviors, including hind paw withdrawal or licking, stamping, and jumping, was measured. The test was repeated twice by a blinded examiner after a 5-minute interval, and the mean time was calculated. The ratio of change from baseline was calculated as the difference in latency from baseline divided by the baseline time. This test was performed weekly for 4 weeks in the same group of mice after the von Frey test.

[0055] Immunohistochemical staining of intraepidermal nerve fibers (IENF) To assess paclitaxel-induced peripheral nerve fiber damage, the density of intraepidermal nerve fibers (fibers / mm) in the hind paws was calculated. Mice were deeply anesthetized with isoflurane (4%) and euthanized by exsanguination at 1 or 4 weeks after paclitaxel treatment for tissue collection. They were perfused with cold 4% paraformaldehyde in PBS via the left ventricle. Skin from the hind paws was harvested and fixed overnight in 4% paraformaldehyde. Then, the tissue was cryoprotected in 20% sucrose solution at 4°C until infiltrated, and then in 30% sucrose solution at 4°C overnight. The tissue blocks were then submerged in optimal cutting temperature medium frozen at -80°C and thinly sliced ​​(25 μm) on a cryostat. Sections were then incubated with 0.3% hydrogen peroxide for 10 minutes and blocking solution (5% donkey serum; Sigma-Aldrich, catalog number D9663, 0.3% Triton-114) for 1 hour. Then, sections were incubated with anti-protein gene product (PGP) 9.5 antibody (1:100, rabbit; Abcam, catalog number ab108986) and anti-collagen IV antibody (1:400, goat; SouthernBiotech, catalog number 1340-01) overnight at 4°C. They were then incubated with secondary antibodies (1:300, donkey anti-rabbit; Abcam, catalog number ab150073; donkey anti-goat; Abcam, catalog number ab150132) for 1 hour at room temperature and covered with fluoroshield mounting medium containing DAPI (Sigma-Aldrich, catalog number F6057). Ten images per mouse (six mice per group) were obtained by fluorescence microscopy (Nikon Eclipse 80i, Nikon Instruments, Inc., Melville, NY), and four images were randomly selected from the ten images. Density (fibers / mm basement membrane) was calculated as the number of intraepidermal nerve fibers divided by the length of the basement membrane according to the guidelines

[21] .

[0056] Histological evaluation of the sciatic nerve 1) Toluidine blue staining Four weeks after paclitaxel treatment, sciatic nerves were evaluated by toluidine blue staining as previously described

[22] . Briefly, mice were deeply anesthetized, and the sciatic nerve was exposed in the prone position. The sciatic nerve was covered with Trump's fixative (Quimigen, Cat. No. 11750) for 10 minutes. It was then harvested and fixed in the same fixative for 1 week, with the fixative changed every other day. The specimens were immersed in 2% osmium tetroxide (TGI, Cat. No. O0308) for 2 hours and embedded in Resin-Epoxy medium (Sigma-Aldrich, Cat. No. 45359-4) overnight at 60°C according to the manufacturer's protocol. The embedded nerve blocks were sectioned at 1 μm using an ultramicrotome (Reichert-Jung, Ultracut E, Australia) and stained in 1% toluidine blue (Sigma-Aldrich, Cat. No. T3260). Two images per mouse (six mice per group) were acquired using a light microscope (Nikon Eclipse 80i, Nikon Instruments, Inc., Melville, NY) and analyzed using ImageJ. Myelin thickness was assessed by the G-ratio, which is the ratio of the inner radius to the outer radius. The G-ratio was calculated using GRatio software, an ImageJ plugin available online (http: / / gratio.efil.de / ), which converts the inner and outer circumferences of myelin to radii. Neuron counts and size measurements were performed by an examiner blinded to the treatment of the mice.

[0057] 2) Transmission electron microscopy To evaluate unmyelinated neurons and mitochondria, we examined neurons in the sciatic nerve by transmission electron microscopy. Using the same method as above, resin-embedded nerve blocks were sectioned at 50 nm using an ultramicrotome. Sections were examined with an FEI Morgagni transmission electron microscope. Low-magnification (×2200) and high-magnification (×11000) images were captured with an AMT 2K charge-coupled device camera (Advanced Microscopy Techniques, Woburn, MA, USA). Unmyelinated neurons were quantified in nine low-magnification images per group (three mice per group). The percentage of unmyelinated neurons was calculated by dividing the unmyelinated neurons by the total number of neurons. The cross-sectional area of ​​mitochondria in unmyelinated neurons (μm 2 ) were quantified in 18–20 high-magnification images per group (3 mice per group). A total of 80–100 mitochondria were evaluated per group. Quantification was performed by a blinded examiner, and images were evaluated by a pathologist (ASR) blinded to sample identity.

[0058] Real-time quantitative polymerase chain reaction (qPCR) Paclitaxel- and GSSSG-induced changes in gene expression in lumbar dorsal root ganglia (DRG) were examined by real-time qPCR after a single dose of paclitaxel with or without GSSSG. We simultaneously administered 16 mg / kg paclitaxel via intraperitoneal injection and 50 mg / kg GSSSG via oral gavage, and examined mRNA expression 2 hours later. Lumbar DRG were isolated from mice as previously described

[23] . Harvested DRG were immersed in RNA later (Invitrogen, Cat. No. AM7020) overnight at 4°C. After recovery from RNA later, they were stored at -80°C. DRG were homogenized in TRIzol reagent (ThermoFisher Scientific, Cat. No. 15596026) and mixed with chloroform. After centrifugation at 14,000 × g for 15 minutes at 4°C, the clear top layer was transferred to a new tube. The sample was mixed with 400 μl of isopropanol and incubated at -20°C for 20 minutes. After centrifugation at 14,000×g for 15 minutes at 4°C, the pellet was collected, mixed with 70% ethanol, and centrifuged at 14,000×g for 10 minutes at 4°C. The pellet was dried for 10 minutes and incubated with 50 μl of nuclease-free water. Complementary DNA was synthesized using a cDNA Reverse Transcription Kit (Applied Biosystems, Cat. No. 4368814), and quantitative PCR was performed using SYBR green (Applied Biosystems, Cat. No. A46109). Primers are listed in Table 1. Relative quantification of gene expression was performed using 2 -ΔΔCT This was carried out in accordance with the law.

[0059] [Table 1]

[0060] Mass spectrometry for the detection of GSSSG administered by oral gavage We used liquid chromatography tandem mass spectrometry (LC-MS / MS) to investigate whether GSSSG administered by oral gavage reaches the peripheral nervous system. 34 S-labeled GSSSG (the central sulfur is a sulfane sulfur) 34 Substituted with S;G- 32 S- 34 S- 32 Mice were orally administered SG. Two hours after administration, plasma and tissues from the liver, brain, lumbar spinal cord, and lumbar DRG (L1–L6) were collected and immediately frozen at −80°C. Tissues were homogenized with 5 mM β-(4-hydroxyphenyl)ethyl iodoacetamide (HPE-IAM) (Santa Cruz, catalog number SC-473766) and incubated at 37°C for 20 minutes to promote the HPE-IAM reaction, which stabilizes persulfide residues

[24] . Proteins were removed by centrifugation at 15,000 g for 10 minutes at 4°C, and total protein concentration was measured by BCA assay. The supernatant was diluted with 0.1% formic acid for LC-MS / MS analysis. 34 The amount of S-labeled GSSSG was quantified by selected reaction monitoring (SRM) using the precursor ion (647.14 m / z), product ion (389.1 m / z), and HCD (21v) and normalized to the protein concentration. 32 S) reactivity to sulfur species (GSSH, CysSSH, and CysSSSCys) 34The ratios of S-labeled reactive sulfur species were also calculated from their peak areas measured using a Dionex UltiMate 3000 RS UPLC-Orbitrap Exploris 480 mass spectrometer (Thermo Scientific, Waltham, MA, USA). Briefly, samples were subjected to a UPLC system equipped with a Hypersil Gold C-18 (100 × 2.1 mm, 3.0 μm, Thermo Fisher Scientific) column and eluted with a linear methanol gradient (0–90% in 15 min) at a flow rate of 0.2 ml / min in the presence of 0.1% formic acid at 40 °C. Raw data were acquired using Compound Discoverer software 3.3. The molecular weights of the reactive sulfur species combined with HPE-IAM were reported in previous studies [25, 26].

[0061] In vitro studies 1) Isolation and histological evaluation of primary DRG neurons Primary DRG neurons were prepared from 8- to 10-week-old mice as previously described

[27] . Briefly, lumbar DRG (L1-L6) were isolated from mice and centrifuged at 176 × g (1000 rpm) for 3 minutes. After aspirating the solution, DRG were incubated with Dispase-II Solution (Sigma-Aldrich, Catalog No. SCM133) and collagenase type II (Worthington, Catalog No. LS004176) for 70 minutes and 0.25% trypsin for 5 minutes. Then, they were triturated with a flame-polished glass pipette, and the neurons were seeded onto 12-well plates with 15 mm coverslips. Cells were incubated for 24 hours in Neurobasal A medium containing 2% B27 supplement (Gibco, Catalog No. 10889038), 1% penicillin / streptomycin, 1% Glutamax (Gibco, Catalog No. 35050061), and nerve growth factor (Sigma-Aldrich, Catalog No. N6009). After 24 hours, cells were exposed to 100 nM paclitaxel with or without 500 nM GSSSG or PTNSSS for 1 hour and 50 nM MitoTracker (Invitrogen, Catalog No. M22426) for 30 minutes. After fixation with 4% paraformaldehyde for 10 minutes at room temperature, cells were immersed in 0.2% Triton X-100 in PBS for 7 minutes and washed with PBS. They were incubated overnight at 4°C with anti-NF200 antibody (1:400, mouse, Sigma-Aldrich, catalog no. N0142) and then with secondary antibody (1:1000, donkey anti-mouse, Abcam, catalog no. ab150105) for 1 hour at room temperature. After covering with fluoroshield mounting medium containing DAPI (Sigma-Aldrich, catalog no. F6057), 9–10 cells per group (3 mice per group) were imaged using a confocal microscope (ZEISS LSM 800, Carl Zeiss, Thornwood, NY) with a 63× oil immersion objective with an image size of 1,024 × 1,024 pixels.

[0062] 2) Isolation of primary cortical neurons and ROS assay Primary cortical neurons were prepared from mouse cortices at embryonic day 15 as previously described

[28] . Briefly, embryonic cortices were isolated in Hanks' balanced salt solution and centrifuged at 176 × g (1000 rpm) for 3 minutes. After aspirating the solution, cells were incubated with 0.25% trypsin for 15 minutes and seeded at 20,000 cells / well onto 96-well plates coated with poly-D-lysine (Gibco, catalog no. A3890401). They were incubated in Neurobasal medium (Gibco, catalog no. 21103049) containing 2% B27 supplement (Gibco, catalog no. 10889038), 1% penicillin / streptomycin, and 1% Glutamax (Gibco, catalog no. 35050061) until day 11, when they were used for experiments.

[0063] To investigate whether GSSSG reduces ROS generated by paclitaxel, we performed a superoxide-sensitive dihydroethidium (DHE) assay (Abcam, catalog no. ab236206). Experiments were performed according to the manufacturer's protocol. Briefly, primary cortical neurons were incubated in DHE reagent with or without 10 and 30 μM GSSSG for 30 min. After incubation, 100 nM paclitaxel was administered, and the cells were incubated for 1 h. DHE fluorescence was measured at an excitation wavelength of 490 nm and an emission wavelength of 585 nm.

[0064] 3) Cancer cell lines and survival rates To investigate whether coadministration of GSSSG with paclitaxel affected the antitumor effect of paclitaxel, we performed experiments using MDA-MB-231, a human breast cancer cell line (HTB-26, ATCC). Cells were cultured in a medium consisting of 90% RPMI 1640 (Corning, catalog no. 10-040-CV), 10% FBS, and 1% penicillin / streptomycin. After seeding at a density of 20,000 cells / well in a 96-well plate and culturing overnight, they were exposed to different concentrations of paclitaxel (0.125 μM, 0.25 μM, 0.5 μM, 1 μM, and 2 μM) and incubated for 24 hours. Cell viability was assessed using an LDH Cytotoxicity Detection Kit (Roche, catalog no. 11644793001). The plates were immediately centrifuged at 250 × g for 10 minutes. Cells were washed with PBS and incubated with 100 μl of 1% Triton X-100 at 37°C for 30 minutes. After mixing with the assay enzyme for 30 minutes at 25°C, cell viability was determined by measuring absorbance at 492 nm. The inhibitory effect of paclitaxel was calculated using the half-maximal inhibitory concentration (IC50). Using the IC50 of paclitaxel obtained from the LDH assay, we investigated the antitumor effect of combined administration of paclitaxel and GSSSG using a trypan blue dye exclusion assay. We tested the effect of 10 μM GSSSG, as this concentration prevented axonal degeneration in primary cortical neurons. MDA-MB-231 cells were seeded at 5.6 × 10,000 cells / well in 6-well plates and incubated overnight. The IC50-based concentration of paclitaxel and 10 μM GSSSG were applied and incubated for 24 hours. After detachment with 0.25% trypsin, viable cells were counted under 0.04% trypan blue. Five wells per group were evaluated.

[0065] statistical analysis The sample size for behavioral tests was selected based on previous research

[29] . All values ​​are expressed as mean ± standard deviation (SD). Because behavior was measured five times repeatedly from the same mice, the results of behavioral tests were analyzed using a mixed-effects model. Individual differences from baseline were confirmed in a preliminary study. The Bonferroni correction was applied to correct for multiple comparisons in the mixed-effects model. Parametric data were analyzed using one-way analysis of variance (ANOVA) and Dunnett's multiple comparison test. Nonparametric data were analyzed using the Kruskal-Wallis test and Dunn's multiple comparison test. The morphology of primary DRG neurons was analyzed using two-way repeated-measures ANOVA and Dunnett's multiple comparison test. The antitumor effects of paclitaxel and GSSSG were analyzed by equivalence testing using two one-tailed t-tests. The limit of equivalence was defined as a 10% difference in cell number. A probability (p) value of less than 0.05 was considered significant. Statistical analyses were performed using GraphPad Prism 9.1 (GraphPad Software Inc., La Jolla, CA, USA).

[0066] [Example 1] GSSSG prevented paclitaxel-induced mechanical allodynia. Male adult mice were treated with 4 mg / kg paclitaxel every other day for four injections (days 0, 2, 4, and 6). From week 1 to week 4 after initiation of paclitaxel, paclitaxel-treated mice showed signs of mechanical allodynia by exhibiting significantly lower pain thresholds than control mice measured by the von Frey test (Figure 2A, closed circles, p = 0.0014). Paclitaxel also induced thermal hyperalgesia measured by the hot plate test (Figure 2B, closed circles, p < 0.0001). Oral administration of GSSSG at 50 mg / kg / day attenuated mechanical allodynia over the experimental period (Figure 2A, black squares, p = 0.003). In contrast, the thermal hyperalgesia threshold remained unchanged by GSSSG administration (Figure 2B, black squares).

[0067] [Example 2] GSSSG prevented paclitaxel-induced loss of intraepidermal nerve fibers We evaluated the degeneration of unmyelinated nerve endings by measuring the density of intraepidermal nerve fibers, a widely used pathological marker of peripheral neuropathy. The density of intraepidermal nerve fibers in the plantar skin of mouse hind paws was calculated by dividing the number of intraepidermal nerve fibers (Figure 3A, yellow arrowheads) by the length of the epidermal basement membrane (Figure 3A, white dashed line). Paclitaxel reduced the density of intraepidermal nerve fibers 4 weeks after initiation of paclitaxel, but not 1 week after initiation (Figures 3B, C). Daily oral administration of 50 mg / kg GSSSG prevented paclitaxel-induced loss of intraepidermal nerve fibers 4 weeks after initiation of paclitaxel (p = 0.0024).

[0068] [Example 3] GSSSG prevented the loss of unmyelinated axons in the sciatic nerve induced by paclitaxel To evaluate the effects of paclitaxel on myelinated and unmyelinated neurons, we counted the number of neurons in the sciatic nerve 4 weeks after starting paclitaxel. The number of myelinated neurons was similar among the control, paclitaxel, and paclitaxel with GSSSG groups (Figures 4A and 4B). Myelin thickness, calculated by the G ratio (ratio of inner radius to outer radius), was not affected by paclitaxel with or without GSSSG (Figures 4C and 4D). We also analyzed transmission electron microscopy images (low magnification, ×2200) to examine the number of unmyelinated neurons in the sciatic nerve. Unmyelinated (Figure 5A, arrows) and myelinated neurons (Figure 5A, arrowheads) were counted and divided by the total number of neurons. Paclitaxel tended to reduce unmyelinated neurons compared to control mice (p=0.0695) and caused neurons to become disaligned (Figure 5A, double arrows), indicating axonal degeneration. Compared with mice receiving paclitaxel alone, mice receiving paclitaxel and GSSSG had a greater ratio of unmyelinated neurons in the sciatic nerve, similar to that observed in control mice (Figure 5A, B, p = 0.0153). These results suggest that GSSSG prevents axonal loss of unmyelinated neurons after paclitaxel treatment.

[0069] [Example 4] GSSSG prevented mitochondrial swelling in axons of unmyelinated neurons To investigate the underlying mechanism of paclitaxel-induced axonal degeneration in unmyelinated neurons, we examined mitochondrial morphology in unmyelinated sciatic neurons 4 weeks after initiation of paclitaxel. Mitochondria appeared larger and more swollen in unmyelinated sciatic neurons from paclitaxel-treated mice (Figure 6A, arrowheads) compared with control mice and mice treated with paclitaxel and GSSSG. The cross-sectional area of ​​mitochondria in unmyelinated sciatic neurons from paclitaxel-treated mice was significantly larger than that of control mice (Figure 6B, p<0.0001) and mice treated with paclitaxel and GSSSG (Figure 6B, p=0.001). These results suggest that the beneficial effect of GSSSG on paclitaxel-induced axonal degeneration is mediated by protecting mitochondrial integrity.

[0070] [Example 5] 34 S-labeled GSSSG was detected in the DRG, spinal cord, brain, and liver after oral administration. To study the pharmacokinetics of orally administered GSSSG, we 34 The distribution of GSSSG and its metabolites in the central and peripheral nervous system was investigated using S-labeled GSSSG. The lumbar DRG, lumbar spinal cord, brain, liver, and plasma were examined at 50 mg / kg. 34 S-labeled GSSSG was orally administered, and the tissues were collected 2 hours later. 34 The levels of S-labeled GSSSG were determined by liquid chromatography tandem mass spectrometry (LC-MS / MS). Endogenous GSSSG was not detected, whereas the administered 34The mean concentrations of S-labeled GSSSG were 415, 518, 142, and 158 pmol / mg protein in the lumbar DRG, lumbar spinal cord, brain, and liver, respectively (Fig. 7A). The plasma level of GSSSG was 58 pmol / ml. We also investigated the endogenous ( 32 Exogenous to (including S) 34 The ratios of reactive sulfur species (including S): GSSH, CysSSH, and CysSSSCys were determined (Figure 7B, C, and D, respectively). 34 The concentrations of S-labeled GSSH, CysSSH, and CysSSSCys were more than 10-fold higher than the endogenous levels of GSSH, CysSSH, and CysSSSCys in all four tissues. These observations indicate that orally administered GSSSG was absorbed and taken up into the central and peripheral nervous systems and was partially metabolized to other reactive sulfur species.

[0071] [Example 6] GSSSG and PTNSSS prevented paclitaxel-induced axonal degeneration and mitochondrial fragmentation in cultured primary DRG neurons The effects of PTNSSS and GSSSG on axonal integrity were examined in cultured mouse primary DRG neurons incubated with paclitaxel. Neurons treated with 100 nM paclitaxel for 1 hour displayed bulbous axon terminals compared to the control group (Figure 8A-B). Neuronal morphology, neurite number, length, and branching were analyzed by Sholl analysis performed in ImageJ

[30] . Sholl analysis quantifies neuronal morphology by drawing equally spaced circles from the soma and counting the number of intersections between the neurites and the circles (Figure 8C). Incubation with paclitaxel for 1 hour inhibited axonal outgrowth (Figures 8A, 8B, 8D, and 8E). Combined treatment with GSSSG restored axonal elongation (Figure 8D, >160 μm from the soma, p<0.05, paclitaxel vs. paclitaxel + GSSSG), as did combined treatment with PTNSSS (Figure 8E); while paclitaxel tended to shorten axon length, PTNSSS prevented paclitaxel-induced axonal shortening. Incubation with paclitaxel also reduced the ratio of total mitochondrial length to axon length and the number of mitochondria in axons, a sign of increased mitochondrial fragmentation (Figure 9A). GSSSG prevented the paclitaxel-induced decrease in the ratio of total mitochondrial length to axon length (Figure 9B, p<0.0001) and the number of mitochondria (Figure 9C, p=0.0017). These results indicate that the beneficial effects of GSSSG on PIPN are mediated by the prevention of axonal degeneration through the maintenance of mitochondrial integrity in peripheral nerve axons.

[0072] [Example 7] GSSSG attenuated paclitaxel-induced increase in superoxide levels in primary cortical neurons Since another possible effect of GSSSG is its antioxidant activity

[14] , we investigated the changes in intracellular ROS in primary cortical neurons. After incubating primary cortical neurons with 100 nM paclitaxel for 1 hour, the intracellular level of superoxide was measured using dihydroethidium (DHE). The level of superoxide increased after incubation with paclitaxel (Figure 10, p = 0.01, paclitaxel vs. control). Incubation with 10 μM GSSSG prevented the paclitaxel-induced increase in intracellular superoxide (p = 0.0114, paclitaxel vs. paclitaxel + GSSSG). These results suggest that GSSSG attenuates paclitaxel-induced ROS generation.

[0073] [Example 8] GSSSG mildly enhanced antioxidant signaling in DRG To further characterize the beneficial effects of GSSSG on PIPN, we measured the mRNA levels of antioxidant proteins in the lumbar DRG by real-time qPCR 2 hours after a single paclitaxel injection (16 mg / kg) with or without 50 mg / kg GSSSG administration. Both paclitaxel and paclitaxel plus GSSSG treatment increased the mRNA levels of Nrf2, whereas the levels of NAD(P)H quinone dehydrogenase 1 (NQO1) were only moderately increased in mice given paclitaxel plus GSSSG treatment (Figure 11, p = 0.0472). These results suggest that GSSSG moderately enhances Nrf-2-dependent antioxidant signaling in the DRG after paclitaxel treatment.

[0074] [Example 9] GSSSG did not affect the antitumor effect of paclitaxel in human breast cancer cell lines Because several studies have shown that enhanced antioxidant activity contributes to cancer cell resistance to chemotherapy [31, 32], we evaluated whether GSSSG affects the antitumor effect of paclitaxel. We investigated the antitumor effect of paclitaxel using MDA-MB-231 human breast cancer cells. After 24 h of incubation with paclitaxel, the viability of MDA-MB-231 cells was analyzed by LDH cytotoxicity detection assay (Figure 12A). We found that the IC50 of paclitaxel against MDA-MB-231 cells was 1.66 μM. Based on these results, we treated MDA-MB-231 cells with 2 μM paclitaxel and 10 μM GSSSG for 24 h to investigate whether co-administration of GSSSG altered the cytotoxic effect of paclitaxel on MDA-MB-231 cells. After 24 hours of incubation, the number of surviving MDA-MB-231 cells was reduced by approximately half with or without GSSSG (PTX vs. PTX + GSSSG: 44.9% vs. 48.3% of control cells) (Figure 12B). Statistical testing showed no difference beyond the specified 10% limit (mean difference: 0.034, 90% confidence interval: -0.280 to 0.357). These results indicate that GSSSG does not affect the antitumor effect of paclitaxel.

[0075] References

[0076] [Table 2-1]

[0077] [Table 2-2]

[0078] [Table 2-3]

[0079] [Table 2-4]

[0080] [Table 2-5]

[0081] Other embodiments Although the present invention has been described in conjunction with its detailed description, it will be understood that the above description is intended to illustrate, but not to limit, the scope of the invention as defined by the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

1. 1. A method for treating or reducing the risk of a peripheral neuropathy-related disease in a subject, comprising administering to a subject in need thereof a therapeutically or prophylactically effective amount of a composition comprising glutathione trisulfide (GSSSG), pantethine trisulfide (PTN-SSS), or lipoic acid trisulfide (LA-SSS), and optionally administering the GSSSG orally or intranasally.

2. 10. The method of claim 1, further comprising: preparing the composition comprising GSSSG by dissolving a crystalline form of GSSSG in a buffered saline solution having a pH of 3-6.

3. 2. The method of claim 1, wherein the disease is chemotherapy-induced peripheral neuropathy (CIPN).

4. 4. The method of claim 3, wherein the CIPN is paclitaxel-induced peripheral neuropathy (PIPN).

5. 5. The method of claim 3 or 4, comprising administering a composition comprising an effective amount of GSSSG, PTN-SSS, or LA-SSS within minutes to hours before or after administration of a dose of chemotherapy.

6. 7. The method of any one of claims 3 to 6, comprising administering a composition comprising an effective amount of GSSSG, PTN-SSS, or LA-SSS daily for the first 1 to 2 weeks of a chemotherapy regimen.

7. 2. The method of claim 1, wherein the disease is diabetic neuropathy, Guillain-Barré syndrome neuropathy, chronic inflammatory demyelinating polyneuropathy, post-herpetic neuralgia, or peripheral neuropathy caused by hepatitis B, hepatitis C, myeloma, lymphoma, or kidney disease.

8. 1. A composition for administration comprising glutathione trisulfide (GSSSG), pantethine trisulfide (PTN-SSS), or lipoic acid trisulfide (LA-SSS) for use in treating or reducing the risk of a disease associated with peripheral neuropathy in a subject, the method comprising administering a therapeutically or prophylactically effective amount to a subject in need thereof, and optionally, the composition is formulated for oral or nasal administration.

9. 9. The composition for use according to claim 8, prepared by a method comprising dissolving a crystalline form of GSSSG in a buffered saline solution of pH 3-6.

10. 9. The composition for use according to claim 8, wherein the disease is chemotherapy-induced peripheral neuropathy (CIPN).

11. The composition for use according to claim 10, wherein the CIPN is paclitaxel-induced peripheral neuropathy (PIPN).

12. 12. The composition for use of claim 10 or 11, comprising administering a composition comprising an effective amount of GSSSG, PTN-SSS, or LA-SSS within minutes to hours before or after administration of a dose of chemotherapy.

13. 13. The composition for use according to any one of claims 10 to 12, comprising administering a composition comprising an effective amount of GSSSG, PTN-SSS, or LA-SSS daily for the first 1 to 2 weeks of a chemotherapy regimen.

14. 9. The composition for use according to claim 8, wherein the disease is diabetic neuropathy, Guillain-Barré syndrome neuropathy, chronic inflammatory demyelinating polyneuropathy, post-herpetic neuralgia, or peripheral neuropathy caused by hepatitis B, hepatitis C, myeloma, lymphoma, or kidney disease.

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