Methods for reducing neuropathy and neuropathic symptoms and treating cancer - Patents.com

Abstract

Disclosed herein are methods for reducing neuropathy and neuropathy symptoms and / or promoting neurogenesis, neuritogenesis, neuroprotection, and neuroregeneration using saposin C-phospholipid compositions. Also disclosed are methods for treating cancer by administering saposin C-phospholipid nanovesicle formulations and one or more antitumor agents or immune checkpoint inhibitors, and kits for treating cancer, comprising (a) a saposin C-phospholipid pharmaceutical composition and (b) a pharmaceutical composition containing an antitumor agent or a pharmaceutical composition containing an immune checkpoint inhibitor in separate containers. TIFF2024504066000039.tif106161

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Application No. 63 / 135,579, filed January 9, 2021, U.S. Provisional Application No. 63 / 135,585, filed January 9, 2021, and U.S. Provisional Application No. 63 / 159,809, filed March 11, 2021.

[0002] Sequence Listing This application contains a Sequence Listing that has been submitted electronically as an ASCII text file named "SequenceListing.txt". The size of this ASCII text file, created on January 5, 2022, is 1.10 KB. The data in this ASCII text file is incorporated herein by reference in its entirety.

[0003] Technical Field The present disclosure relates to reducing neurological disorders or neuropathic symptoms; promoting neurogenesis, neuritogenesis, neuroprotection, and neuroregeneration; and treating cancer. [Background technology]

[0004] background Neuropathy is a common disorder of the peripheral nervous system in adults and children, affecting approximately 2-7% of the world's population (Callaghan et al. (2015) JAMA. 314: 2172-2181 (Non-Patent Document 1)). It is associated with acute pain, chronic pain, or phantom limb pain, numbness, and muscle weakness, among many other symptoms. Neuropathy has a variety of etiologies, including underlying conditions such as autoimmune diseases, tumors, trauma, infections, metabolic problems, genetic causes, and exposure to toxins and drugs, such as chemotherapy (Hakim et al. (2019), J. Clin & Exp Pharmacol.; 9(4): 262 (Non-Patent Document 2)) or high doses of many common dietary supplements (e.g., vitamin B6, pyridoxine).

[0005] The prevalence of neuropathy is believed to be even higher in vulnerable cohorts, such as cancer patients undergoing chemotherapy.For example, the overall incidence of chemotherapy-induced peripheral neuropathy (CIPN) in patients treated with multiple drugs is estimated to be approximately 38% (Hershman (2014) J Clin Oncol,;32:1941-1967 (Non-Patent Document 3)).CIPN is a serious side effect that may limit the administration and effectiveness of many of the most advanced drugs used in cancer treatment.

[0006] Neuropathies are often associated with nerve injury. Although the mechanisms involved in the pathophysiology and natural regeneration of peripheral nerve injury are beginning to be understood, finding a treatment that significantly improves neurogenesis, neuritogenesis, neuroprotection, and / or nerve regeneration after injury has been an elusive goal. Furthermore, despite the widespread prevalence of neuropathy, treatment options for this condition generally only provide symptomatic relief, rather than treating the underlying cause. Thus, there is a great need for additional treatment options for neuropathy. Many neurotoxic drugs and treatments are essential for the treatment of many types of cancer, and cancer itself also slightly damages the peripheral nervous system (Housley SN, et al. Cancer Res. 2020 Jul 1; 80(13): 2940-2955 (Non-Patent Document 4)).

[0007] Cancer treatments often include radiation therapy, steroids, and chemotherapy, which are often poorly tolerated by patients. The immune system plays a role in various types of cancer, and cancer immunotherapy involves the attack of cancer cells by the patient's immune system. Often performed as an alternative to poorly tolerated treatments, immunotherapy includes treatment with immune checkpoint inhibitors that target cancer cells expressing specific proteins, such as programmed death ligand 1 (PDL1). Furthermore, although immunotherapy has been used successfully in subjects with certain cancers, there is a great need to fine-tune immunotherapy to develop safe and effective treatment options with minimal side effects and maximum efficacy.

[0008] Gastrointestinal (GI) cancers, such as colorectal, gastric, esophageal, and pancreatic cancers, are prominent global causes of malignant disease and death. Gastric cancer is the fifth most commonly diagnosed cancer and the third most common cause of cancer death worldwide (Leiting, JL, & Grotz, TE (2019). World J. of Gastrointest. Oncol. 11(9), 652-664 (Non-Patent Document 5)). In addition, colorectal cancer is the second most common cause of cancer death in the United States (American Cancer Society, Colorectal Cancer Facts & Figures 2020-2022. Atlanta, Ga (Non-Patent Document 6)).

[0009] GI cancers such as metastatic colorectal cancer (mCRC) have a poor prognosis, with a 5-year survival rate of approximately 14% (Siegel RL, et al (2020). CA Cancer J Clin. 70(3):145-64 (Non-Patent Document 7)). Surgery, radiation therapy, and chemotherapy are key components of colorectal cancer treatment. Although some patients with recurrent and metastatic disease can be salvaged by surgery, chemotherapy remains the mainstay of treatment for advanced colorectal cancer. The main first- and second-line regimens for mCRC are 5-fluorouracil (5-FU) / capecitabine-based combination therapy, such as FOLFOX (5-FU, oxaliplatin, and folinic acid), FOLFIRI (5-FU, folinic acid, and irinotecan), and CAPOX (capecitabine and oxaliplatin) (Sonbol MB, et al. (2019). JAMA Oncol; e194489 (Non-Patent Document 8)). Because oxaliplatin in FOLFOX causes severe CIPN and interferes with the patient's quality of life, oncologists are often forced to reduce or discontinue the dose of oxaliplatin, which can have a negative impact on disease control and progression-free survival. Selvy M, et al. J Clin Med. 2020; 9(8): 2400 (Non-Patent Document 9). Targeted biological treatments such as anti-epidermal growth factor receptor (EGFR) treatments, cetuximab and panitumumab, have also been recommended for first-line treatment of RAS wild-type mCRC (Fornasier G, et al., (2018) Adv Ther. 35(10):1497-509). Several potential postsecond-line agents exist, including EGFR inhibitors (cetuximab / panitumumab), bevacizumab, regorafenib, and trifluridine / tipiracil (Vogel A, et al. Cancer Treat Rev. 2017;59:54-60). Each of the agents used in the treatment of mCRC has a severe side effect profile and offers little hope of a good clinical outcome.

[0010] Thus, there is a great need for safe and effective treatment options for gastrointestinal cancer, including colorectal cancer, that minimize side effects such as CIPN while maximizing treatment efficacy. [Prior art documents] [Non-patent literature]

[0011] [Non-Patent Document 1] Callaghan et al.(2015)JAMA.314:2172-2181 [Non-Patent Document 2] Hakim et al. (2019), J. Clin & Exp Pharmacol.;9(4):262 [Non-Patent Document 3] Hershman(2014)J Clin Oncol,;32:1941-1967 [Non-Patent Document 4] Housley SN,et al.Cancer Res.2020 Jul 1;80(13):2940-2955 [Non-Patent Document 5] Leiting, JL, & Grotz, TE (2019).World J.of Gastrointest.Oncol.11(9),652-664 [Non-Patent Document 6] American Cancer Society,Colorectal Cancer Facts & Figures 2020-2022.Atlanta,Ga [Non-Patent Document 7] Siegel RL,et al (2020).CA Cancer J Clin.70(3):145-64 [Non-Patent Document 8] Sonbol MB,et al(2019).JAMA Oncol;e194489 [Non-Patent Document 9] Selvy M,et al.J Clin Med.2020;9(8):2400 [Non-Patent Document 10] Fornasier G,et al.,(2018)Adv Ther.35(10):1497-509 [Non-Patent Document 11] Vogel A,et al.Cancer Treat Rev.2017;59:54-60 Summary of the Invention

[0012] overview The present disclosure is based, at least in part, on at least the following surprising discoveries.

[0013] (1) Saposin C-phospholipid-containing nanovesicle compositions can, for example, alleviate symptoms of neuropathy in a human subject. Thus, the present disclosure provides a novel treatment for reducing neuropathy or neuropathy symptoms.

[0014] (2) The nanovesicle composition enhances the anticancer effects of two components of standard chemotherapy regimens for gastrointestinal cancer, oxaliplatin and 5-fluorouracil (5-FU). The nanovesicle composition also reduced the symptoms of CIPN in animals. Thus, the present disclosure provides a novel combination therapy for the treatment of gastrointestinal cancer, such as colorectal cancer.

[0015] (3) The nanovesicle composition enhances the anti-cancer effect of immune checkpoint inhibitors. Thus, the present disclosure provides yet another novel combination therapy for the treatment of cancer, e.g., cancers susceptible to treatment with immune checkpoint inhibitors.

[0016] In a first aspect, the disclosure features a method of reducing a neuropathic symptom in a human subject, the method including: identifying a human subject suffering from a neuropathic symptom; administering to the subject a nanovesicle formulation including (i) a saposin C polypeptide comprising SEQ ID NO:1 having 0 to 4 amino acid insertions, substitutions, deletions, or combinations thereof; and (ii) a phospholipid having a net negative charge at neutral pH; and confirming that the subject's neuropathic symptom is reduced.

[0017] In a second aspect, the disclosure features a method of reducing the incidence, intensity, and / or duration of, or delaying the onset of, a neuropathic symptom in a human subject in need thereof, wherein the method includes identifying a human subject at risk for experiencing a neuropathic symptom; and treating the subject with a nanovesicle formulation including (i) a saposin C polypeptide comprising SEQ ID NO:1 having 0-4 amino acid insertions, substitutions, deletions, or combinations thereof, and (ii) a phospholipid having a net negative charge at neutral pH, wherein the treatment is effective in reducing the incidence, intensity, and / or duration of, or delaying the onset of, a neuropathic symptom in the subject.

[0018] In a third aspect, the disclosure features a method of reducing the incidence, intensity, and / or duration of a neuropathic symptom or delaying the onset of a neuropathic symptom in a human subject in need of treatment with a chemotherapeutic agent associated with a neuropathic symptom side effect, where the method includes administering to the subject a dose of a chemotherapeutic agent; and administering to the subject, concurrently with the dose of the chemotherapeutic agent, or immediately before or after administration of the dose of the chemotherapeutic agent, at least one dose of a nanovesicle formulation comprising (i) a saposin C polypeptide comprising SEQ ID NO:1 having 0-4 amino acid insertions, substitutions, deletions, or combinations thereof, and (ii) a phospholipid having a net negative charge at neutral pH.

[0019] In a fourth aspect, the disclosure features a method of treating cancer, the method including co-administering to a human subject in need thereof a chemotherapeutic agent associated with a neuropathic symptom side effect; and a nanovesicle formulation including (i) a saposin C polypeptide comprising SEQ ID NO:1 having 0-4 amino acid insertions, substitutions, deletions, or combinations thereof, and (ii) a phospholipid having a net negative charge at neutral pH, wherein the nanovesicle formulation is administered in an amount that reduces the incidence, intensity, and / or duration of or delays the onset of the neuropathic symptom side effect associated with the chemotherapeutic agent.

[0020] In a fifth aspect, the disclosure features a method of reducing a neuropathic symptom in a human subject in need thereof, where the method includes identifying a subject experiencing a neuropathic symptom; and treating the subject with an effective amount of a nanovesicle formulation to reduce the neuropathic symptom in the subject, the nanovesicle formulation including (i) a saposin C polypeptide comprising SEQ ID NO:1 having 0 to 4 amino acid insertions, substitutions, deletions, or combinations thereof; and (ii) a phospholipid having a net negative charge at neutral pH.

[0021] In a sixth aspect, the disclosure features promoting neurogenesis, neuritogenesis, neuroprotection, and / or neuroregeneration in a subject, the method comprising: identifying a subject in need of one or more of neurogenesis, neuritogenesis, neuroprotection, and neuroregeneration; and administering to the subject a nanovesicle formulation in an amount sufficient to promote neurogenesis, neuritogenesis, neuroprotection, and / or neuroregeneration in the subject, the nanovesicle formulation comprising (i) a saposin C polypeptide comprising SEQ ID NO:1 having 0-4 amino acid insertions, substitutions, deletions, or combinations thereof, and (ii) a phospholipid having a net negative charge at neutral pH. In some embodiments, the subject has one or more of the following conditions: a neurodegenerative disorder, brain injury, brain damage, spinal cord injury, peripheral nerve injury, multiple sclerosis, or disseminated sclerosis. In some embodiments, the subject has Parkinson's disease, Alzheimer's disease, or amyotrophic lateral sclerosis. In some embodiments, the subject is about to undergo anti-cancer chemotherapy. In some embodiments, the method further comprises monitoring neurogenesis, neuritogenesis, neuroprotection, and / or neuroregeneration in the subject using one or more tests selected from the Weinstein Enhanced Sensory Test (WEST), the Semmes-Weinstein Monofilament Test (SWMT), shape-texture identification (STI), magnetic resonance imaging (MRI), computed tomography (CT), intraepidermal nerve fiber density (IENFD), and nerve conduction velocity. In some embodiments, the method further comprises confirming that the subject has experienced neurogenesis, neuritogenesis, neuroprotection, and / or neuroregeneration following administration of the formulation.

[0022] In some embodiments of the above method, the subject has or is at risk of having peripheral neuropathy. In some embodiments, the subject has or is at risk of having peripheral neuropathy. In some embodiments, the subject has or is at risk of having chemotherapy-induced peripheral neuropathy (CIPN) induced by one or more chemotherapeutic agents. In some embodiments, the chemotherapeutic agent is selected from the group consisting of platinum-based agents, taxanes, epothilones, plant alkaloids, immunomodulators, and proteasome inhibitors. In some embodiments, the chemotherapeutic agent is oxaliplatin, cisplatin, carboplatin, paclitaxel, docetaxel, cabazitaxel, ixabepilone, vinblastine, vincristine, vindesine, vinorelbine, vincaminol, vineridine, vinbrunin, etoposide, thalidomide, lenalidomide, pomalidomide, bortezomib, carfilzomib, ixazomib, eribulin, or suramin. In some embodiments, the subject has or is at risk of having oxaliplatin-induced acute pain syndrome.

[0023] In some embodiments of the first, second, or fifth aspect, the subject has or is at risk of having a neuropathy associated with one or more of the following conditions: amyotrophic lateral sclerosis (ALS), spinal muscular atrophy (SMA), rheumatoid arthritis, systemic lupus erythematosus (SLE), post-polio syndrome, Guillain-Barre syndrome, chronic inflammatory demyelinating polyneuropathy (CIDP), multifocal motor neuropathy, muscular dystrophy, peripheral nerve neuropathy, and / or myocardial infarction. Injury, demyelination, acute disseminated leukoencephalitis, progressive multifocal leukoencephalitis (PML), adrenoleukodystrophy, optic neuritis, kidney disease, liver damage, transverse myelitis, Parkinson's disease, stroke, Alzheimer's disease, Lyme disease, carpal tunnel syndrome, lymphoma, neuroma, multiple myeloma, vitamin B12 deficiency, postherpetic neuralgia, leprosy, Charcot-Marie-Tooth disease, Fabry disease, critical illness polyneuropathy, Bell's palsy, ulnar nerve palsy, and peroneal nerve palsy. In some embodiments, the subject has or is at risk of having a neuropathy associated with diabetes or a viral infection.

[0024] In some embodiments of some of the above aspects, the reduction in neuropathy or neuropathy symptoms includes a change in at least one parameter selected from a reduction in the incidence of neuropathy, a reduction in the intensity of neuropathy, a reduction in the duration of neuropathy, a reduction in the duration of neuropathy episodes, a reduction in the frequency of neuropathy episodes, and a delay in the onset of neuropathy.

[0025] In some embodiments of some of the above aspects, reducing neuropathy includes reducing neuropathy symptoms. In some embodiments, reducing the incidence, intensity, and / or duration of neuropathy or delaying the onset of neuropathy includes reducing the incidence, intensity, and / or duration of neuropathy symptoms or delaying the onset of neuropathy symptoms.

[0026] In some embodiments of the second aspect, the method is effective in at least one of (a)-(d): (a) reducing the intensity and / or duration of an episode of neuropathic symptoms in the subject after administration of the nanovesicle formulation compared to the intensity and / or duration of the episode of neuropathic symptoms in the subject before the formulation is administered; (b) reducing the intensity and / or duration of neuropathic symptoms in the subject compared to the intensity and / or duration of the episode of neuropathic symptoms in a control population; (c) reducing the incidence of neuropathy in the subject compared to the incidence of neuropathy in a control population; and (d) delaying the onset of neuropathy in the subject after a triggering event compared to the time to onset of neuropathy in a control population after a similar event.

[0027] In some embodiments of some of the above aspects, one or more additional doses of the nanovesicle formulation and one or more additional doses of the chemotherapeutic agent are administered to the subject. In some embodiments, the nanovesicle formulation does not contain a chemotherapeutic agent.

[0028] In some embodiments of the fifth aspect, the neuropathic condition is (a) one or more of the following conditions: diabetes, amyotrophic lateral sclerosis (ALS), spinal muscular atrophy (SMA), rheumatoid arthritis, systemic lupus erythematosus (SLE), post-polio syndrome, Guillain-Barré syndrome, chronic inflammatory demyelinating polyneuropathy (CIDP), multifocal motor neuropathy, muscular dystrophy, peripheral nerve injury, demyelination, acute disseminated leukoencephalitis, progressive multifocal leukoencephalitis (PML), adrenoleukodystrophy, optic neuritis, kidney disease, liver damage, transverse myelitis, Parkinson's disease, stroke, Alzheimer's disease, Lyme disease, viral infection, carpal tunnel syndrome, lymphoma, neuroma, multiple myeloma. , vitamin B12 deficiency, post-herpetic neuralgia, leprosy, Charcot-Marie-Tooth disease, Fabry disease, critical illness polyneuropathy, Bell's palsy, ulnar nerve palsy, or peroneal nerve palsy; or (b) treatment with a chemotherapeutic agent selected from the group consisting of oxaliplatin, cisplatin, carboplatin, paclitaxel, docetaxel, cabazitaxel, ixabepilone, vinblastine, vincristine, vindesine, vinorelbine, vincaminol, vineridine, vinbrunin, etoposide, thalidomide, lenalidomide, pomalidomide, bortezomib, carfilzomib, ixazomib, eribulin, and suramin. In some embodiments, the neuropathic symptom is pain. In some embodiments, the method further comprises monitoring the neuropathic symptom of the subject. In some embodiments, the method further comprises confirming that the neuropathic symptom of the subject is reduced. In some embodiments, the neuropathic symptom is one or more of the following: pain; hyperalgesia; allodynia; loss of pain sensation; loss of sensation of hot, cold, or physical injury; numbness; hypersensitivity to touch; loss of coordination and proprioception; muscle weakness; muscle wasting; muscle spasms; muscle cramps; or muscle paralysis.

[0029] In some embodiments, some of the above methods further include administering to the subject one or more additional anti-neuropathy treatments selected from the group consisting of transcutaneous electrical nerve stimulation (TENS), therapeutic plasma exchange (TPE), intravenous immunoglobulin (IVIG) treatment, physical therapy, analgesics, antiepileptic drugs, topical treatments, and antidepressants. In some embodiments, some of the above methods further include administering to the subject ibuprofen, gabapentin, pregabalin, capsaicin cream, lidocaine patch, amitriptyline, doxepin, nortriptyline, duloxetine, or venlafaxine.

[0030] In some embodiments of some of the above aspects, a reduction in neuropathy or neuropathy symptoms is confirmed as measured using one or more assessment tools selected from the group consisting of the National Cancer Institute-Common Toxicity Criteria (NCI-CTC), Numeric Rating Scale (NRS), Visual Analog Scale (VAS), European Organization for Research and Treatment of Cancer (EORTC) Quality of Life (QLQ)-CIPN20, QLC-C30, Functional Assessment of Cancer Therapy / Gynecologic Oncology Group-Neurotoxicity (FACT / GOG-Ntx), Total Neuropathy Score (TNS) questionnaire, Chemotherapy-Induced Peripheral Neuropathy Assessment Tool (CIPNAT), and Post-Oxiplatin Symptom Survey.

[0031] In a seventh aspect, the disclosure features a method of treating gastrointestinal cancer in a subject, the method including the steps of: (a) administering to the subject a chemotherapy regimen including one or more anti-tumor agents; and (b) administering to the subject a nanovesicle formulation including (i) a saposin C polypeptide including SEQ ID NO:1 having 0 to 4 amino acid insertions, substitutions, deletions, or combinations thereof; and (ii) a phospholipid having a net negative charge at neutral pH.

[0032] In some embodiments of the seventh aspect, the chemotherapy regimen comprises modified FOLFOX7 (mFOLFOX7). In some embodiments, the antitumor agent comprises oxaliplatin. In some embodiments, the nanovesicle formulation is administered simultaneously with the chemotherapy regimen, or within 48 hours before or after the start of administration of the chemotherapy regimen. In some embodiments, the nanovesicle formulation is administered intravenously. In some embodiments, the chemotherapy regimen is administered intravenously. In some embodiments, the gastrointestinal cancer is esophageal cancer, gastric cancer, anal cancer, colorectal cancer, intestinal cancer, gallbladder cancer, pancreatic cancer, liver cancer, islet cell cancer, rectal cancer, small intestine cancer, gastrointestinal carcinoid tumor, or gastrointestinal stromal tumor. In some embodiments, the method further comprises administering to the subject a biological treatment for the gastrointestinal cancer. In some embodiments, the method further comprises administering to the subject an anti-vascular endothelial growth factor (VEGF) monoclonal antibody. In some embodiments, the method further comprises administering to the subject an anti-epidermal growth factor receptor (EGFR) antibody. In some embodiments, the method further comprises administering to the subject bevacizumab, cetuximab, or panitumumab.

[0033] In some embodiments of the seventh aspect, the method includes the steps of: (a) administering 2.4 mg / kg of the nanovesicle formulation over about 45 minutes to about 120 minutes on day 1 of the first week of treatment; and (b) after completion of step (a), (i) administering 200 mg / m over about 2 hours on day 1 of the first week of treatment. 2 85 mg / m2 infused with leucovorin calcium 2 oxaliplatin; and (ii) 2400 mg / m over approximately 46 hours on days 1 and 2 of the first week of treatment.2 In some embodiments of the seventh aspect, the method includes administering a chemotherapy regimen in the following steps: (a) administering 2.4 mg / kg of the nanovesicle formulation over about 45 minutes to about 120 minutes on day 1 of the first week of treatment; (b) after completion of step (a), (i) administering 200 mg / m over about 2 hours on day 1 of the first week of treatment. 2 85 mg / m2 infused with leucovorin calcium 2 oxaliplatin, and (ii) 2400 mg / m over approximately 46 hours on days 1 and 2 of the first week of treatment. 2 (c) administering about 5 mg / kg to about 15 mg / kg of bevacizumab over about 30 to about 90 minutes on day 1 of the first week of treatment.

[0034] In some embodiments of the seventh aspect, the method further comprises administering an additional dose of the nanovesicle formulation three times per week during the second week of treatment. In some embodiments, the method further comprises administering an additional dose of the nanovesicle formulation once per week (7 (+ / - 3) days) during the third and fourth weeks of treatment. In some embodiments, the method further comprises administering an additional dose of the nanovesicle formulation once per 14 (+ / - 3) days during weeks 5-23 of treatment. In some embodiments, multiple doses of the nanovesicle formulation are administered over a period of at least 8 weeks. In some embodiments, the nanovesicle formulation is administered at least once per week during the first four weeks of treatment. In some embodiments, the nanovesicle formulation is administered at least three times per week during the first two weeks of treatment. In some embodiments, the nanovesicle formulation is administered at least once per 14 (+ / - 3) days after the first four weeks of treatment. In some embodiments, the nanovesicle formulation is administered at least once every 14 (+ / - 3) days in weeks 5-24. In some embodiments, the nanovesicle formulation is administered at least once every 28 (+ / - 3) days after the first 24 weeks of treatment. In some embodiments, the nanovesicle formulation is administered repeatedly to the subject as follows: week 1: one dose on each day of days 1-5; week 2: one dose every other day for a total of three doses; week 3 and week 4: one dose per week (7 (+ / - 3) days); week 5-24: one dose per 14 days (+ / - 3 days). In some embodiments of the seventh aspect, the method further comprises administering additional doses of the chemotherapy regimen in week 3 and once every 14 days (+ / - 3 days) in weeks 5-24. In some embodiments of the seventh aspect, the method further comprises administering additional doses of bevacizumab in week 3 and once every 14 days (+ / - 3 days) from weeks 5 to 24.

[0035] In some embodiments of the seventh aspect, the nanovesicle formulation is not combined with any anti-tumor agent of a chemotherapy regimen at the time the nanovesicle formulation is administered to the subject.

[0036] In some embodiments of the seventh aspect, the treatment increases at least one of the following parameters in the subject compared to a control population treated with a chemotherapy regimen and not treated with the nanovesicle formulation: (a) objective response rate (ORR); (b) overall survival (OS); (c) progression-free survival (PFS); and (d) duration of response (DoR).

[0037] In an eighth aspect, the disclosure features a method of treating cancer in a subject in need thereof, the method comprising administering to the subject: (a) a nanovesicle formulation comprising (i) a saposin C polypeptide comprising SEQ ID NO:1 having 0-4 amino acid insertions, substitutions, deletions, or combinations thereof, and (ii) a phospholipid having a net negative charge at neutral pH; and (b) an immune checkpoint inhibitor. In some embodiments, the immune checkpoint inhibitor is an anti-CTLA-4 antibody. In some embodiments, the anti-CTLA antibody is selected from the group consisting of ipilimumab, tremelimumab, and combinations thereof. In some embodiments, the immune checkpoint inhibitor is an anti-PD-1 antibody. In some embodiments, the anti-PD-1 antibody is selected from the group consisting of nivolumab, pembrolizumab, pidilizumab, MEDI0680, and combinations thereof. In some embodiments, the immune checkpoint inhibitor is an anti-PD-L1 antibody. In some embodiments, the anti-PD-L1 antibody is selected from the group consisting of atezolizumab, BMS-936559, MEDI4736, MSB0010718C, and combinations thereof.

[0038] In some embodiments, the cancer is B-cell lymphoma, basal cell carcinoma, bladder cancer, blastoma, brain metastasis, breast cancer, Burkitt's lymphoma, cervical cancer, colon cancer, colorectal cancer, endometrial cancer, esophageal cancer, Ewing's sarcoma, fibrosarcoma, follicular lymphoma, gastric cancer, gastroesophageal junction cancer, gastrointestinal cancer, glioblastoma, glioma, head and neck cancer, liver metastasis, Hodgkin's or non-Hodgkin's lymphoma, kidney cancer, laryngeal cancer, leukemia, liver cancer, lung cancer, Selected from lymphoblastic lymphoma, lymphoma, mantle cell lymphoma, metastatic brain tumor, metastatic carcinoma, myeloma, neuroblastoma, ocular melanoma, oropharyngeal carcinoma, osteosarcoma, ovarian cancer, pancreatic cancer, prostate cancer, renal cell carcinoma, salivary gland carcinoma, sarcoma, skin cancer, soft tissue sarcoma, solid tumor, squamous cell carcinoma, synovial sarcoma, testicular cancer, thyroid cancer, transitional cell carcinoma, uveal melanoma, verrucous carcinoma, vulvar carcinoma, and Waldenstrom's hypergammaglobulinemia.

[0039] In some embodiments, the cancer is a gastrointestinal cancer. In some embodiments, one or more cells of the cancer express PD-L1 at an elevated level compared to a reference sample. In some embodiments, one or more cells of the cancer express CTLA-4 at an elevated level compared to a reference sample. In some embodiments, the reference sample is selected from (a) a non-cancerous sample from the subject; or (b) a non-cancerous sample from a different subject. In some embodiments, the one or more cells of the cancer are of the same cell type as the cells of the reference sample. In some embodiments, the method further comprises performing an assay to determine whether treatment with the nanovesicle formulation and an immune checkpoint inhibitor increased the immune response of the subject to the tumor compared to the immune response before treatment. In some embodiments, the assay measures the level of one or more cytokines in the plasma of the subject. In some embodiments, the assay measures the level of mRNA encoding one or more cytokines in the subject. In some embodiments, the assay measures the level of tumor-associated M1 or M2 macrophages of the subject. In some embodiments, the assay detects the presentation of PD-L1 on the surface of macrophages of the subject. In some embodiments, the assay measures the level of activated cytotoxic T cells of the subject. In some embodiments, the nanovesicle formulation does not include an immune checkpoint inhibitor.

[0040] In some embodiments of any of the above aspects, the amino acid sequence of the saposin C polypeptide comprises SEQ ID NO:1 with one or two amino acid insertions, substitutions, deletions, or a combination thereof. In some embodiments, the amino acid sequence of the saposin C polypeptide comprises SEQ ID NO:1. In some embodiments, the amino acid sequence of the saposin C polypeptide consists of SEQ ID NO:1.

[0041] In some embodiments of any of the above aspects, the phospholipid comprises phosphatidylserine. In some embodiments, the phospholipid comprises dioleoylphosphatidylserine (DOPS) or a salt thereof. In some embodiments, the phospholipid comprises a sodium salt of DOPS. In some embodiments, the phospholipid comprises a phosphoglyceride. In some embodiments, the phospholipid comprises one or more of dihexanoylphosphatidylserine lipid, dioctanoylphosphatidylserine lipid, didecanoylphosphatidylserine lipid, dilauroylphosphatidylserine lipid, dimyristoylphosphatidylserine lipid, dipalmitoylphosphatidylserine lipid, palmitoyl-oleoylphosphatidylserine lipid, 1-stearoyl-2-oleoylphosphatidylserine lipid, or diphytanoylphosphatidylserine lipid. In some embodiments, the phosphoglyceride comprises phosphatidic acid.

[0042] In some embodiments of the above aspects, the molar ratio of phospholipid to saposin C polypeptide in the nanovesicle formulation ranges from 8:1 to 20:1. In some embodiments, the molar ratio of phospholipid to saposin C polypeptide in the nanovesicle formulation ranges from 11:1 to 13:1. In some embodiments, the molar ratio of phospholipid to saposin C polypeptide in the nanovesicle formulation is 12:1.

[0043] In some embodiments of the above methods, the subject has cancer. In some cases, the subject has gastrointestinal cancer, pancreatic cancer, colorectal cancer, bone cancer, brain cancer, sarcoma, neuroblastoma, breast cancer, or squamous cell carcinoma.

[0044] In some embodiments of any of the above aspects, the nanovesicle formulation is administered intravenously, intraarterially, intradermally, intramuscularly, intracardially, intracranially, subcutaneously, intraperitoneally, by inhalation, intranasally, orally, or sublingually. In some embodiments, each dose of the nanovesicle formulation administered to the subject contains 0.4 mg / kg to 7 mg / kg of saposin C polypeptide. In some embodiments, the nanovesicle formulation is administered intravenously. In some embodiments, the nanovesicle formulation is administered at least once a day, once every other day, three times a week, approximately once a week (7 (+ / - 3) days), or approximately once every two weeks (14 (+ / - 3) days). In some embodiments, multiple doses of the nanovesicle formulation are administered over a period of at least 8 weeks.

[0045] In a ninth aspect, the disclosure above features a kit for the treatment of cancer, wherein the kit comprises, in separate containers, (a) a first pharmaceutical composition comprising at least one anti-tumor agent; and (b) a second pharmaceutical composition comprising (i) a saposin C polypeptide comprising SEQ ID NO:1 having 0-4 amino acid insertions, substitutions, deletions, or combinations thereof, and (ii) a phospholipid having a net negative charge at neutral pH. In some embodiments, the anti-tumor agent in the kit is oxaliplatin.

[0046] In a tenth aspect, the disclosure above features a kit for the treatment of cancer, wherein the kit comprises, in separate containers, (a) a first pharmaceutical composition comprising at least one immune checkpoint inhibitor; and (b) a second pharmaceutical composition comprising (i) a saposin C polypeptide comprising SEQ ID NO:1 having 0-4 amino acid insertions, substitutions, deletions, or combinations thereof, and (ii) a phospholipid having a net negative charge at neutral pH. In some embodiments, the at least one immune checkpoint inhibitor is an anti-CTLA-4 antibody, an anti-PD-1 antibody, or an anti-PD-L1 antibody.

[0047] In some aspects, the disclosure features the use of a nanovesicle formulation comprising (i) a saposin C polypeptide comprising SEQ ID NO:1 having 0-4 amino acid insertions, substitutions, deletions, or combinations thereof, and (ii) a phospholipid having a net negative charge at neutral pH in any of the above methods of treatment. In some embodiments, the disclosure features the use of a nanovesicle formulation to reduce a neuropathic symptom in a human subject suffering from a neuropathic symptom. In some embodiments, the disclosure features the use of a nanovesicle formulation to prevent or reduce the incidence, intensity, and / or duration of a neuropathic symptom, or to delay the onset of a neuropathic symptom, in a human subject at risk of experiencing a neuropathic symptom. In some embodiments, the disclosure features the use of a nanovesicle formulation to reduce the incidence, intensity, and / or duration of a neuropathic symptom side effect associated with a chemotherapeutic agent, or to delay the onset of a neuropathic symptom side effect associated with a chemotherapeutic agent, in a human subject. In some embodiments, the present disclosure features the use of nanovesicle formulations to treat cancer in human subjects administered chemotherapy agents associated with neuropathic symptoms side effects, where the nanovesicle formulations are administered in an amount that prevents or reduces the incidence, intensity, and / or duration of chemotherapy agent-associated neuropathic symptoms side effects, or delays the onset of chemotherapy agent-associated neuropathic symptoms side effects.In some embodiments, the present disclosure features the use of nanovesicle formulations to prevent or reduce neuropathic symptoms in human subjects identified as experiencing neuropathic symptoms.In some embodiments, the present disclosure features the use of nanovesicle formulations to promote neurogenesis, neuritogenesis, neuroprotection, and / or neuroregeneration in subjects.

[0048] In some embodiments, the disclosure features the use of the nanovesicle formulation described above to treat gastrointestinal cancer in a subject concurrently administered a chemotherapy regimen including one or more anti-tumor agents. In some embodiments, the use includes the steps of: (a) administering to the subject 2.4 mg / kg of the nanovesicle formulation over about 45 minutes to about 120 minutes on day 1 of the first week of treatment; (b) after completion of step (a), (i) administering to the subject 200 mg / m over about 2 hours on day 1 of the first week of treatment. 2 85 mg / m2 infused with leucovorin calcium 2 oxaliplatin, and (ii) 2400 mg / m over approximately 46 hours on days 1 and 2 of the first week of treatment. 2 (c) administering about 5 mg / kg of bevacizumab over a period ranging from about 30 to about 90 minutes on the first or second day of the first week of treatment. In some embodiments, the use comprises administering to the subject the nanovesicle formulation as follows: week 1: one dose each day, days 1-5; week 2: one dose every other day, for a total of three doses; week 3 and week 4: one dose every week (7 (+ / - 3 days); week 5-24: one dose every 14 days (+ / - 3 days). In some embodiments, the use comprises administering an additional dose of the chemotherapy regimen in week 3 and once every 14 days (+ / - 3 days) in weeks 5-24. In some embodiments, the use comprises administering an additional dose of bevacizumab in week 3 and once every 14 days (+ / - 3 days) in weeks 5-24.

[0049] In some aspects, the disclosure features the use of the nanovesicle formulations described above to treat cancer in a subject co-administered with an immune checkpoint inhibitor.

[0050] In some aspects, the disclosure features a nanovesicle formulation comprising (i) a saposin C polypeptide comprising SEQ ID NO:1 having 0-4 amino acid insertions, substitutions, deletions, or combinations thereof, and (ii) a phospholipid having a net negative charge at neutral pH, for use in any of the above methods of treatment. In some embodiments, the disclosure features a nanovesicle formulation for use in reducing a neuropathic symptom in a human subject suffering from a neuropathic symptom. In some embodiments, the disclosure features a nanovesicle formulation for use in preventing, or reducing the incidence, intensity, and / or duration, or delaying the onset of a neuropathic symptom in a human subject at risk of experiencing a neuropathic symptom. In some embodiments, the disclosure features a nanovesicle formulation for use in reducing the incidence, intensity, and / or duration, or delaying the onset of a neuropathic symptom side effect associated with a chemotherapeutic agent in a human subject. In some embodiments, the present disclosure features a nanovesicle formulation for use in the treatment of cancer in a human subject receiving a chemotherapeutic agent associated with neuropathic symptoms side effects, wherein the nanovesicle formulation is administered in an amount that prevents or reduces the incidence, intensity, and / or duration of the chemotherapeutic agent-associated neuropathic symptoms side effects, or delays the onset of the chemotherapeutic agent-associated neuropathic symptoms side effects.In some embodiments, the present disclosure features a nanovesicle formulation for use in preventing or reducing neuropathy symptoms in a human subject identified as experiencing neuropathy symptoms.In some embodiments, the present disclosure features a nanovesicle formulation for use in promoting neurogenesis, neuritogenesis, neuroprotection, and / or neuroregeneration in a subject.

[0051] In some embodiments, the disclosure features the nanovesicle formulation described above for use in treating gastrointestinal cancer in a subject concurrently administered a chemotherapy regimen including one or more anti-tumor agents. In some embodiments, the use comprises the steps of: (a) administering to the subject 2.4 mg / kg of the nanovesicle formulation over about 45 to about 120 minutes on day 1 of the first week of treatment; (b) after completion of step (a), (i) administering to the subject 200 mg / m over about 2 hours on day 1 of the first week of treatment. 2 85 mg / m2 infused with leucovorin calcium 2 oxaliplatin, and (ii) 2400 mg / m over approximately 46 hours on days 1 and 2 of the first week of treatment. 2 (c) administering about 5 mg / kg of bevacizumab over a period ranging from about 30 to about 90 minutes on the first or second day of the first week of treatment. In some embodiments, the use comprises administering to the subject the nanovesicle formulation as follows: week 1: one dose each day, days 1-5; week 2: one dose every other day, for a total of three doses; week 3 and week 4: one dose every week (7 (+ / - 3 days); week 5-24: one dose every 14 days (+ / - 3 days). In some embodiments, the use comprises administering an additional dose of the chemotherapy regimen in week 3 and once every 14 days (+ / - 3 days) in weeks 5-24. In some embodiments, the use comprises administering an additional dose of bevacizumab in week 3 and once every 14 days (+ / - 3 days) in weeks 5-24.

[0052] In some aspects, the disclosure features the nanovesicle formulation described above for use in the treatment of cancer in a subject co-administered with an immune checkpoint inhibitor.

[0053] In any of the above embodiments, the saposin C polypeptide may be a polypeptide that includes or consists of SEQ ID NO:1, or may include SEQ ID NO:1 with only one or two amino acid changes. In any of the above embodiments, the phospholipid may include dioleoylphosphatidylserine (DOPS) or a salt thereof. The phospholipid may include a sodium salt of DOPS. In any of the above embodiments, the molar ratio of phospholipid to saposin C polypeptide in the nanovesicle formulation may range from 8:1 to 20:1. In some embodiments, the molar ratio of phospholipid to saposin C polypeptide in the nanovesicle formulation may range from 11:1 to 13:1. In some embodiments, the molar ratio of phospholipid to saposin C polypeptide in the nanovesicle formulation may be 12:1. [Brief description of the drawings]

[0054] [Figure 1] Figures 1A and 1B show the timeline of administration of various treatments to mice in the oxaliplatin chemotherapy mouse pain model in two individual studies 1 and 2. In Figure 1A, male mice in groups A-E of study 1 were administered 10 mg / kg oxaliplatin or vehicle intraperitoneally on day 0 and test articles as follows: 10 mL / kg vehicle or 20 mg / kg BXQ-350 on days -3, -2, -1, and 1-9, or 30 mg / kg pregabalin on days 3, 5, 7, and 9. n=12 mice per group. vF: von Frey; CP: cold plate. In Figure 1B, male mice in groups A-E of study 2 were administered 10 mg / kg oxaliplatin or vehicle intraperitoneally on day 0 and test articles as follows: 10 mL / kg vehicle, 2 mg / kg BXQ-350 (group C), or 10 mg / kg BXQ-350 (group D) on days -3, -2, -1, and 1-9, or 30 mg / kg pregabalin on days 3, 5, 7, and 9. n=12 mice per group. [Diagram 2]FIG. 2 is a bar graph showing mechanical hypersensitivity via paw withdrawal threshold 50% (g) using von Frey filaments for each of the study groups in Study 1 at baseline (BL), days 3, 5, 7, and 9 (D3, D5, D7, and D9). *, **, ***, and **** indicate statistically significant differences of p<0.05, p<0.01, p<0.001, and p<0.0001, respectively, when compared to the Oxal / vehicle group at the same time points. Values ​​are presented as mean±SEM. Oxal: oxaliplatin; BXQ: BXQ-350. [Diagram 3] FIG. 3 is a line graph showing functional preservation as percent change in paw withdrawal threshold 50% (g) using von Frey filaments for each of Study 1 Groups A-D on days 0, 3, 5, 7, and 9. See Table 2 for Study Groups A-D. * and ** indicate statistically significant differences of p<0.05 and p<0.01, respectively, when compared to the oxaliplatin group at the same time points. Values ​​are presented as mean±SEM. [Figure 4] FIG. 4 is a bar graph showing the latency to withdrawal in the cold plate test for each of the study groups in Study 1 at baseline (BL), days 3, 5, 7, and 9 (D3, D5, D7, and D9). *, **, ***, and **** indicate statistically significant differences of p<0.05, p<0.01, p<0.001, and p<0.0001, respectively, when compared to the Oxal / vehicle group at the same time points. Values ​​are presented as mean±SEM. Oxal: oxaliplatin; BXQ: BXQ-350. [Diagram 5] FIG. 5 is a line graph showing functional preservation as percent change in withdrawal latency in the cold plate test for each of Study 1 Groups A-D on days 0, 3, 5, 7, and 9. See Table 2 for Study Groups A-D. ** and *** indicate statistically significant differences of p<0.01 and p<0.001, respectively, when compared to the oxaliplatin group at the same time points. Values ​​are presented as mean±SEM. [Figure 6]FIG. 6 is a bar graph showing mechanical hypersensitivity via paw withdrawal threshold 50% (g) using von Frey filaments for each of the study groups in Study 2 at baseline (BL) and days 3, 5, 7, and 9 (D3, D5, D7, and D9). Bars refer to Study 2 Groups A-E from left to right for each time point. See Table 8 for Study Groups A-E. *, **, ***, and **** indicate statistically significant differences of p<0.05, p<0.01, p<0.001, and p<0.0001, respectively, when compared to the Veh / Oxal group at the same time points. Values ​​are presented as mean±SEM. Veh: vehicle; Oxal: oxaliplatin; BXQ 2: 10 mg / kg vehicle, 2 mg / kg BXQ-350; BXQ 10: 10 mg / kg vehicle, 10 mg / kg BXQ-350; PGB: 30 mg / kg pregabalin. [Figure 7] Figure 7 is a line graph showing functional preservation as percent change in paw withdrawal threshold 50% (g) using von Frey filaments for Study 2 Groups A-D on days 0, 3, 5, 7, and 9. ** and *** indicate statistically significant differences of p<0.01 and p<0.001, respectively, when compared to the oxaliplatin group at the same time points. Values ​​are presented as mean ± SEM. See Table 8 for Study Groups A-D. [Figure 8]FIG. 8 is a bar graph showing the latency to withdrawal in the cold plate test for each of the study groups in Study 2 at baseline (BL), days 3, 5, 7, and 9 (D3, D5, D7, and D9). Bars refer to Study 2 groups A-E from left to right for each time point. See Table 8 for Study Groups A-E. *, **, ***, and **** indicate statistically significant differences of p<0.05, p<0.01, p<0.001, and p<0.0001, respectively, when compared to the Veh / Oxal group at the same time points. Values ​​are presented as mean±SEM. Veh: vehicle; Oxal: oxaliplatin; BXN 2: 10 mg / kg vehicle, 2 mg / kg BXQ-350; BXQ 10: 10 mg / kg vehicle, 10 mg / kg BXQ-350: BXQ-350; PGB: 30 mg / kg pregabalin. [Figure 9] Figure 9 is a line graph showing functional preservation as percent change in withdrawal latency in the cold plate test for Study 2 Groups A-D on days 0, 3, 5, 7, and 9. * and ** indicate statistically significant differences of p<0.05 and p<0.01, respectively, when compared to the oxaliplatin group at the same time points. Values ​​are presented as mean ± SEM. See Table 8 for Study Groups A-D. [Figure 10] 10A-10D are images (magnification 20x) of neurite outgrowth by NS20Y or PC-12 cells. NS20Y cells were treated with 50 nM BXQ-350 (Fig. 10A) or left untreated (Fig. 10B) in a neurite outgrowth assay. PC-12 cells were treated with 50 nM BXQ-350 (Fig. 10C) or left untreated (Fig. 10D) in a neurite outgrowth assay. [Figure 11]Figures 11A-D are four pairs of images of neurite outgrowth in PC-12 cells. The left image of each pair is at 4x magnification; the right image of each pair is at 20x magnification. BXQ-350 was administered at 50 nM; oxaliplatin was administered at 2 μM. Treatments consisted of BXQ-350 alone (Figure 11A), BXQ-350 and oxaliplatin (Figure 11B), oxaliplatin alone (Figure 11C), and untreated cells (Figure 11D). [Figure 12] 12 is a bar graph showing the percentage of PC-12 cells with neurite outgrowth under various conditions, i.e., untreated or after treatment with BXQ-350 (50 nM), oxaliplatin (2 μM), or a combination of BXQ-350 and oxaliplatin. Oxaliplatin alone resulted in less than 1% cell viability, and therefore neuritogenesis could not be determined. [Figure 13] 13 is a bar graph showing the percentage of viable PC-12 cells compared to the total number of viable cells in the untreated control (100%) under various conditions, i.e., untreated or after treatment with BXQ-350 (50 nM), oxaliplatin (2 μM), or a combination of BXQ-350 and oxaliplatin. Oxaliplatin alone resulted in negligible cell viability. [Figure 14] Figures 14A and 14B are bar graphs showing the percentage of live PC-12 cells (Figure 14A) and NS20Y cells (Figure 14B) with 50 nM BXQ-350 treatment compared to the total number of live cells in untreated controls (normalized to the results for untreated controls shown as 100%). Figures 14C and 14D are two pairs of 20x magnified images of neurite outgrowth in PC-12 cells (Figure 14C) and NS20Y cells (Figure 14D). The left image of each pair shows untreated control cells; the right image of each pair shows cells treated with BXQ-350. Values ​​are shown as mean ± SEM. * and ** indicate statistically significant differences of p<0.05 and p<0.001, respectively. [Figure 15]Figure 15A is a bar graph showing the percentage of neurite outgrowth in live PC-12 cells treated with 2 μM oxaliplatin, 50 nM BXQ-350, a combination of the two, or left untreated for 72 hours. Cells were scored for neurite outgrowth; results are reported in the figure as the percentage of cells found to have outgrowth. Figure 15B is a bar graph showing the number of live PC-12 cells (normalized to the results for the untreated control shown as 100%) after 72 hours of treatment with 2 μM oxaliplatin, 50 nM BXQ-350, or a combination of the two, or left untreated. Each sample was run in triplicate. Values ​​are shown as mean ± SEM. ** and **** indicate statistically significant differences of p<0.01 and p<0.0001, respectively. Figure 15C shows four images of cells and neurite outgrowth in PC-12 cells for each treatment group. Images are at 20x magnification. From left to right, images correspond to untreated cells, cells treated with oxaliplatin, cells treated with BXQ-350, and cells treated with BXQ-350 + oxaliplatin. [Figure 16]Figure 16A is a bar graph showing the percentage of neurite outgrowth in live NS20Y cells treated with 2 μM oxaliplatin, 50 nM BXQ-350, a combination of the two, or left untreated for 72 hours. Cells were scored for neurite outgrowth; results are reported in the figure as the percentage of cells found to have outgrowth. Figure 16B is a bar graph showing the number of live NS20Y cells (normalized to the results for the untreated control shown as 100%) after 72 hours of treatment with 2 μM oxaliplatin, 50 nM BXQ-350, or a combination of the two, or left untreated. Each sample was run in triplicate. Values ​​are shown as mean ± SEM. **, ***, and **** indicate statistically significant differences of p<0.01, p<0.001, and p<0.0001, respectively. Figure 16C shows four images of cells and neurite outgrowth in NS20Y cells for each treatment group.Images are 20x magnification.From left to right, the images correspond to untreated cells, cells treated with oxaliplatin, cells treated with BXQ-350, and cells treated with BXQ-350+oxaliplatin. [Figure 17]Figure 17A is a bar graph showing the percentage of neurite outgrowth in live PC-12 cells treated with 10 μM vincristine, 50 nM BXQ-350, a combination of the two, or left untreated for 72 hours. Cells were scored for neurite outgrowth; results are reported in the figure as the percentage of cells found to have outgrowth. Figure 17B is a bar graph showing the number of viable PC-12 cells (normalized to the results for the untreated control shown as 100%) after 72 hours of treatment with 10 μM vincristine, 50 nM BXQ-350, or a combination of the two, or left untreated. Each sample was run in triplicate. Values ​​are shown as mean ± SEM. *** and **** indicate statistically significant differences of p<0.001 and p<0.0001, respectively. Figure 17C shows four images of cells and neurite outgrowth in PC-12 cells for each treatment group. Images are at 20x magnification. From left to right, images correspond to untreated cells, cells treated with vincristine, cells treated with BXQ-350, and cells treated with BXQ-350 + vincristine. [Figure 18]Figure 18A is a bar graph showing the percentage of neurite outgrowth in live NS20Y cells treated with 10 μM vincristine, 50 nM BXQ-350, a combination of the two, or left untreated for 72 hours. Cells were scored for neurite outgrowth: results are reported in the figure as the percentage of cells found to have outgrowth. Figure 18B is a bar graph showing the number of live NS20Y cells (normalized to the results for the untreated control shown as 100%) after 72 hours of treatment with 10 μM vincristine, 50 nM BXQ-350, or a combination of the two, or left untreated. Each sample was run in triplicate. Values ​​are shown as mean ± SEM. ** and **** indicate statistically significant differences of p<0.01 and p<0.0001, respectively. Figure 18C shows four images of cells and neurite outgrowth in PC-12 cells for each treatment group. Images are at 20x magnification. From left to right, images correspond to untreated cells, cells treated with vincristine, cells treated with BXQ-350, and cells treated with BXQ-350 + vincristine. [Figure 19]Figure 19A is a bar graph showing the percentage of neurite outgrowth in live PC-12 cells treated with 3 μM paclitaxel, 50 nM BXQ-350, a combination of the two, or left untreated for 72 hours. Cells were scored for neurite outgrowth; results are reported in the figure as the percentage of cells found to have outgrowth. Figure 19B is a bar graph showing the number of live PC-12 cells (normalized to the results for the untreated control shown as 100%) after 72 hours of treatment with 3 μM paclitaxel, 50 nM BXQ-350, or a combination of the two, or left untreated. Each sample was run in triplicate. Values ​​are shown as mean ± SEM. *** and **** indicate statistically significant differences of p<0.001 and p<0.0001, respectively. Figure 19C shows four images of cells and neurite outgrowth in PC-12 cells for each treatment group. Images are at 20x magnification. From left to right, images correspond to untreated cells, cells treated with paclitaxel, cells treated with BXQ-350, and cells treated with BXQ-350 + paclitaxel. [Figure 20]Figure 20A is a bar graph showing the percentage of neurite outgrowth in live NS20Y cells treated with 3 μM paclitaxel, 50 nM BXQ-350, a combination of the two, or left untreated for 72 hours. Cells were scored for neurite outgrowth; results are reported in the figure as the percentage of cells found to have outgrowth. Figure 20B is a bar graph showing the number of live NS20Y cells (normalized to the results for the untreated control shown as 100%) after 72 hours of treatment with 3 μM paclitaxel, 50 nM BXQ-350, or a combination of the two, or left untreated. Each sample was run in triplicate. Values ​​are shown as mean ± SEM. *** and **** indicate statistically significant differences of p<0.001 and p<0.0001, respectively. Figure 20C shows four images of cells and neurite outgrowth in NS20Y cells of each treatment group. Images are at 20x magnification. From left to right, images correspond to untreated cells, cells treated with paclitaxel, cells treated with BXQ-350, and cells treated with BXQ-350 + paclitaxel. [Figure 21] Figures 21A and 21B are bar graphs showing the percent cell viability of NS20Y cells (Figure 21A) and PC-12 cells (Figure 21B) that were untreated or pretreated with 50 nM BXQ-350 for 72 hours. Both groups were then treated with medium containing 200 μM H2O2 for 24 hours. Values ​​are shown as mean ± SEM. * and *** indicate statistically significant differences of p<0.05 and p<0.001, respectively. [Figure 22]Figure 22 is a bar graph showing the percent cell viability of human HT-29 colorectal cancer cells in response to treatment with oxaliplatin / 5-FU+BXQ-350 (SapC-DOPS) compared to untreated cells. Combination treatment with BXQ-350+oxaliplatin / 5-FU induces significant synergistic cell death in cultured human HT-29 cells. Cells were treated with BXQ-350 alone (12 μM); BXQ-350+oxaliplatin (0.3 μM); BXQ-350+5-FU (12 μM); oxaliplatin (0.3 μM)+5-FU (12 μM); or BXQ-350+oxaliplatin+5-FU for 72 hours. n=3 per group. Values ​​are shown as mean±SEM. [Figure 23] Figure 23A is a graph showing the toxicity curves (% viable cells) for six colorectal cancer cell lines treated with SapC-DOPS. SW480 and SW620 cells were treated with 3 μM, 6 μM, 9 μM, 12 μM, 15 μM, 20 μM, 25 μM, and 30 μM SapC-DOPS. HT29, HTC116, LoVo, and SW48 cells were treated with 5 μM, 10 μM, 20 μM, 30 μM, 40 μM, and 50 μM. Figure 23B is a bar graph showing the percentage of cells treated with FOLFOX (10 μM oxaliplatin and 10 μM 5-FU) compared to untreated cells for six colorectal cancer cell lines. [Figure 24] 24 is a bar graph showing the percentage of cells treated with 15 μM SapC-DOPS alone, 10 μM FOLFOX alone, or a combination of 10 μM FOLFOX and 15 μM SapC-DOPS (triple combination) compared to untreated cells for six colorectal cancer cell lines. Values ​​are shown as mean ± SEM. [Diagram 25]FIG. 25A and FIG. 25B are line graphs showing TNF-α concentration (left Y-axis, dark shaded line (2)) and percent cell viability (right Y-axis, light shaded line (1)) of human CD14+ macrophages from two healthy subjects (FIG. 25A and FIG. 25B, respectively) in response to in vitro treatment with BXQ-350 (SapC-DOPS). Cells were treated with BXQ-350 at concentrations ranging from 2.5 μM to 9 nM and 1 hour later with lipopolysaccharide. After 72 hours, cells were fixed and analyzed for TNF-α concentration and percent cell viability. Values ​​are shown as mean ± SEM. [Figure 26] Figure 26 is a series of line graphs showing the fluorescence intensity (Incucyte® analyzer) of three-dimensional A549 tumor spheroids as a function of time in the presence of PMBCs stimulated to promote cytotoxic T cells. Line (12): control (no PMBC activation); Line (11): activation with anti-CD3+IL-2; Line (13): activation with anti-CD3+IL-2 and treatment with pembrolizumab; All other lines (lines (1)-(10)): tumor killing curves for BXQ-350 and activation with anti-CD3+IL-2 at concentrations ranging from 1.27 nM to 25 μM. [Figure 27] Figure 27 is a line graph showing the AUC at various BXQ-350 concentrations based on the data from the series of line graphs in Figure 26. The fluorescence intensities from Figure 26 are integrated; as cells die, the fluorescence changes and the total integrated fluorescence also changes, resulting in different AUC values. AUC: Area under the curve. IC50: Half the maximum concentration required to stimulate cell death using BXQ-350. [Figure 28]Figure 28 is a line graph showing tumor volume over time in different treatment groups after administration of sterile PBS (line (1)), m-SapC-DOPS (line (2)), m-anti-PD-1 (line (3)), or both m-SapC-DOPS and m-anti-PD-1 (line (4)) to female BALB / c mice bearing established CT-26 tumors. Animals were sacrificed when tumor volume exceeded 3000 mm3. IV: intravenous injection; IP: intraperitoneal injection; m-SapC-DOPS: mouse SapC-DOPS; D#: days; mpk: mg / kg; BIW: twice weekly. [Figure 29] FIG. 29 is a line graph showing the body weight of the different treatment groups over time following administration of sterile PBS (line (1)), m-SapC-DOPS (line (2)), m-Anti-PD-1 (line (3)), or both m-SapC-DOPS and m-Anti-PD-1 (line (4)) to female BALB / c mice bearing established CT-26 tumors. [Diagram 30] Figure 30 is a line graph showing tumor volumes over time for individual animals in different treatment groups after administration of m-anti-PD-1 (circles, dark shaded lines) or both m-SapC-DOPS and m-anti-PD-1 (diamonds, light shaded lines) to female BALB / c mice bearing established CT-26 tumors. [Figure 31-1] Figures 31A-31H show various bar graphs. Figure 31A is a bar graph showing the increase in IFN-γ concentration in the culture medium of cultured myeloid-derived suppressor cells from healthy donors as a function of BXQ-350 concentration. "T": T cells without stimulation; "T+beads": T cells stimulated to express a biomarker (here interferon-γ). Figures 31B and 31C are bar graphs showing the percentage of expanded CD4+ and CD8+ cells, respectively, as a function of BXQ-350 concentration. [Figure 31-2]Figures 31A-31H show various bar graphs. Figure 31D is a bar graph showing the concentration of IL-10 in the medium from cultured myeloid-derived suppressor cells from healthy donors as a function of BXQ-350 concentration. Figures 31E-31H are bar graphs showing the expression of HLA-DR, CD86, CD11b, and CD80 in cultured myeloid-derived suppressor cells from healthy donors as a function of BXQ-350 concentration, respectively. [Figure 31-3] Figures 31A-31H show various bar graphs, and Figures 31E-31H show bar graphs depicting the expression of HLA-DR, CD86, CD11b, and CD80, respectively, in cultured myeloid-derived suppressor cells from healthy donors as a function of BXQ-350 concentration. [Figure 31-4] See description of Figure 31-3. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0055] Detailed Description The present disclosure relates to a method for reducing neuropathy or neuropathy symptoms, and a method for promoting neurogenesis, neuritogenesis, neuroprotection, and / or neuroregeneration. The present disclosure also relates to a method for treating cancer. In some embodiments, the present disclosure relates to a method for treating gastrointestinal cancer. The composition used in these methods comprises a saposin polypeptide, such as saposin C (SapC), and a phospholipid, such as a phosphatidylserine, such as dioleoylphosphatidylserine (DOPS). These saposin-based formulations, as well as their components and preparation techniques, are described in U.S. Patent No. 10,682,411, the disclosure of which is incorporated herein by reference in its entirety. When used to treat cancer, such as gastrointestinal cancer, the saposin C / phospholipid composition is administered with one or more antitumor agents, such as chemotherapeutic agents or immune checkpoint inhibitors.

[0056] The term "deletion," as used herein, refers to a change in amino acid or nucleotide sequence that results in the absence of one or more amino acid residues or nucleotides.

[0057] The words "insertion" or "addition" as used herein refer to a change in amino acid or nucleotide sequence that results in the addition of one or more amino acid residues or nucleotides, respectively, to the sequence found in the naturally occurring molecule.

[0058] Saposin C-DOPS Nanovesicles containing saposin C ("SapC") and the phospholipid dioleoylphosphatidylserine (DOPS) have high affinity for phosphatidylserine-rich membranes and can induce apoptosis and / or necrosis in target cells in vitro and in vivo (Qi et al. (2009), Clin Cancer Res., 15:5840-5851). The mechanism by which SapC-DOPS nanovesicles induce apoptosis is proposed to be via ceramide elevation by activation of β-glucosidase and acid sphingomyelinase (and subsequent degradation of glucosylceramide and sphingomyelin, respectively), which leads to activation of caspases. Nanovesicle preparations have been found to be effective against a wide variety of tumor types in vitro and in orthotopic mouse tumor models (Qi X, et al. (2009) Clin Cancer Res., 15(18):5840-51; Wojton et al. (2013), Mol Ther, 21:1517-1525; Abu-Baker et al. (2012), J Cancer Ther, 3:321-326; Chu et al. (2013), PLoS One; 8:e75507; U.S. Patent No. 7,834,147).

[0059] Active Agent The "active agent" used in the methods of the invention is a combination of saposin C ("SapC") polypeptide and phospholipids, which together form lipid vesicles (also called "liposomes" or "nanovesicles") when suspended in aqueous solution. Such suspensions are also called "nanovesicle formulations." The amino acid sequence of naturally occurring human SapC is: TIFF2024504066000002.tif34158. The SapC polypeptide used in the methods of the invention consists of or comprises the amino acid sequence of SEQ ID NO:1, or comprises SEQ ID NO:1 with one to four amino acid modifications (a total of four or less insertions, substitutions, deletions, or combinations thereof): for example, an insertion of one to four amino acid residues into SEQ ID NO:1; or a substitution of one, two, three, or four residues in SEQ ID NO:1; or a deletion of one, two, three, or four residues from the amino terminus, or from the carboxy terminus, or at an internal site of SEQ ID NO:1. SEQ ID NO:1 was disclosed as SEQ ID NO:1 in issued U.S. Patent No. 10,682,411, which is incorporated by reference in its entirety.

[0060] Phospholipids that can be incorporated into active agents include phospholipids that have a net negative charge at neutral pH, such as phosphoglycerides, such as phosphatidic acid (diacylglycerol 3-phosphate) and phosphatidylserine. The two fatty acids attached to the phosphoglyceride backbone can be the same or different; for example, they can have 0, 1, or 2 carbon-carbon double bonds in each carbon chain; and they can have carbon chains from C6 to C20, such as oleic acid, hexanoic acid, octanoic acid, decanoic acid, lauric acid, myristic acid, stearic acid, palmitic acid, linoleic acid, and phytanic acid, and any combination of two of these. Thus, when the phospholipid is phosphatidylserine, the following are representative: dioleoyl phosphatidylserine (DOPS), dihexanoyl phosphatidylserine lipid, dioctanoyl phosphatidylserine lipid, didecanoyl phosphatidylserine lipid, dilauroyl phosphatidylserine lipid, dimyristoyl phosphatidylserine lipid, dipalmitoyl phosphatidylserine lipid, palmitoyl-oleoyl phosphatidylserine lipid, 1-stearoyl-2-oleoyl phosphatidylserine lipid, and diphytanoyl phosphatidylserine lipid. The active agent can include any combination of two or more (e.g., two or three) different phospholipids, e.g., two or three different phosphatidylserine lipids. (The terms "phosphatidylserine" and "phosphatidylserine lipid" are used interchangeably.)

[0061] According to the present disclosure, the active agent may include SapC (SEQ ID NO:1) and DOPS. Alternatively, an anionic phospholipid or a phospholipid with an overall negative charge may be used in place of DOPS. SapC-DOPS is described in U.S. Pat. No. 7,834,147, which is incorporated by reference in its entirety. The combination of SapC and DOPS may form nanovesicles. The nanovesicles may be 10 nm to 800 nm, e.g., 40 nm to 200 nm. The formulation including the active agent may have a pH of 5 to 8, e.g., 7 to 7.4.

[0062] In aqueous compositions at neutral pH, phospholipids are typically present in the form of salts with cations, and therefore, reference to DOPS and other phospholipids used in the compositions of the present invention includes both salt and non-salt forms of phospholipids.Suitable cations include any pharma- ceutically acceptable cations that form salts with phospholipids, such as any of the following: ammonium ion; L-arginine ion; benzathine ion; deanol ion; diethanolamine (2,2'-iminodiethanol) ion; hydrabamine ion; lysine ion; potassium ion; sodium ion; triethanolamine (2,2',2''-nitrilotri(ethane-1-ol)) ion; and tromethamine (2-amino-2-(hydroxymethyl)propane-1,3-diol) ion. Sodium, potassium, and ammonium salts are typical.

[0063] The molar ratio of SapC polypeptide to phospholipid in the composition of the present invention may range from 1:2 to 1:50, for example, from 1:5 to 1:30, or from 1:8 to 1:20, or from 1:11 to 1:13. Suitable molar ratios include, but are not limited to, 1:10, 1:11, 1:12, 1:13, 1:14, and 1:15. The mass ratio of polypeptide to phosphatidylserine lipid ranges from about 1:0.11 to 1:4.8, or from about 1:0.18 to about 1:4.5, or from about 1:0.45 to about 1:2.7, or from about 1:0.72 to about 1:1.81, or from about 1:1 to about 1:1.2.

[0064] The active agent may be supplied in the form of a solid (e.g., a lyophilized powder), with or without pharma- ceutically acceptable buffers and other inactive ingredients. The solid is typically reconstituted with sterile water or saline prior to administration to form a suspension of liposomes in aqueous solution.

[0065] When the active agent is in the form of liposomes suspended in an aqueous solution, the solution may also contain a pharma- ceutically acceptable buffer and other inactive ingredients. Suitable formulations (and methods for preparing them) include those described in U.S. Patent No. 10,682,411, which is incorporated by reference in its entirety. One example of such a suspension of an active agent in an aqueous solution is designated "BXQ-350". It contains a saposin C polypeptide consisting of SEQ ID NO:1 and a phospholipid consisting of the sodium salt of DOPS in a molar ratio (SapC to DOPS) of approximately 1:12 (i.e., in the range of 1:11 to 1:13).

[0066] Administration of active agent is typically via injection (e.g., infusion) of a suspension, and can be by any suitable injectable route, for example, intravenous, intraarterial, intradermal, intramuscular, intracardiac, intracranial, transdermal, subcutaneous, intraocular, or intraperitoneal. In appropriate circumstances, dry particles of active agent or a solution containing active agent can be aerosolized or nebulized and delivered via inhalation through nose or mouth, or the solution can be delivered as a liquid, for example, eye drops, nose drops, or spray. Oral delivery, for example, as a mouthwash or gargle or sublingual formulation, or swallowed as a liquid, capsule, or tablet, is also contemplated. Liquids can be delivered as lavage fluids or enemas. Further details regarding routes of administration can be found, for example, in U.S. Pat. No. 7,834,147.

[0067] Administration may be at least once a day for several consecutive days, for example, 3, 4, 5, 6, 7, 8, 9 or more consecutive days, or may be, for example, every other day, or 3 times a week, or once every 7±3 days, or once every 14±3 days, or once every 28±3 days. The timing of administration may be started on one of these schedules, and after a suitable treatment period, may be changed to other schedules, more or less frequently. The entire treatment period may be completed in, for example, 8 weeks, 12 weeks, 16 weeks, or up to 6 months, but may continue as long as the patient is considered to be benefiting from the treatment.

[0068] The SapC-DOPS formulation for use in the method of the present invention may be supplied in lyophilized form and then reconstituted with water prior to use. An example of a formulation for use in the method is shown in Table 1. The molar ratio of DOPS to SapC in the formulation in Table 1 is 12:1. The target pH is 7.2±0.4.

[0069] Table 1. Reconstituted SapC-DOPS formulations TIFF2024504066000003.tif29128

[0070] Formulations for use in the disclosed methods can include compositions disclosed in US Pat. No. 10,682,411, the teachings of which are incorporated by reference in their entirety.

[0071] SapC and polypeptides derived therefrom can be produced by any useful method, for example, chemically, enzymatically, or recombinantly. Methods for producing polypeptides and fragments thereof are known in the art and include, but are not limited to, chemical peptide synthesis, in vitro translation systems, and expression (and purification) in recombinant host organisms.

[0072] Useful background information and technical details can be found in US Pat. Nos. 7,834,147 and 9,271,932, which are incorporated herein by reference in their entireties.

[0073] "Lipid vesicles," "liposomes," and "nanospores" are used interchangeably to refer to clusters or aggregates of amphiphilic lipids that are generally spherical and typically in the form of one or more concentric layers, e.g., bilayers.

[0074] As used herein, the term "SapC-DOPS" refers to a combination of SapC and DOPS.

[0075] As used herein, when a dosage of SapC-DOPS is reported, the dosage refers to the dose of SapC. For example, a dosage of 2.4 mg / kg of SapC-DOPS refers to 2.4 mg / kg of SapC.

[0076] A "patient," "subject," or "individual" refers to an animal, e.g., a mammal, preferably a human, monkey, chimpanzee, horse, cow, sheep, goat, pig, cat, dog, mouse, rat, or rabbit.

[0077] With respect to the treatment of neuropathy, a "therapeutically effective dose" or "therapeutically effective amount" of an active agent is an amount useful for treating a neuropathy in a patient. In general, a therapeutically effective dose of the composition of the present invention will contain an amount of SapC (or a derivative thereof) ranging from about 0.01 to 30 mg / kg body weight, preferably about 0.05 to 20 mg / kg body weight, more preferably about 0.1 to 15 mg / kg body weight, and even more preferably about 0.5 to 10 mg / kg. For example, the amount of SapC in an intravenous dose can be about 0.4 mg / kg, 0.7 mg / kg, 1.1 mg / kg, 1.4 mg / kg, 1.8 mg / kg, 2.4 mg / kg, 2.8 mg / kg, 3.0 mg / kg, 3.2 mg / kg, 3.6 mg / kg, 7 mg / kg, or more. A given patient may receive a given dose level for one or more initial administrations and different (lower or higher) levels for further administrations.

[0078] Neuropathy "Peripheral neuropathy" is a collection of diverse clinical symptoms, natural histories, and pathologies that affect the peripheral nervous system (PNS). The PNS consists of sensory neurons that run from stimulatory receptors, which transmit stimuli to the central nervous system (CNS), and motor neurons that run from the spinal cord to effector organs, which produce actions. The term "neuropathy" includes peripheral symmetric neuropathies, autonomic neuropathies, proximal neuropathies, focal neuropathies, and mononeuropathy.

[0079] Patients with peripheral neuropathy may present with motor dysfunction (weakness), paresthesia (numbness, paralysis, hyperalgesia / allodynia, pain), autonomic symptoms, or a combination of all, often depending on the specific disease (Cashman, CR, & Hoke, A. (2015) Neuroscience letters, 596, 33-50). Most neuropathies are chronic, slowly progressive conditions, but some have a more acute onset and slow recovery (Kuwabara S, Yuki N. (2013) Lancet Neurol. 2013; 12: 1180-1188; Winer JB. (2014) Autoimmune Dis.; 2014: 1-6; Yuki N, and Hartung HP. (2012) N. Engl. J. Med. 2012; 366: 2294-2304). Isolated neuropathy is rare and often secondary to other systemic diseases, such as diabetes and infections with human immunodeficiency virus and hepatitis C virus. In addition, peripheral neuropathy can be iatrogenic, resulting from the toxicity of drugs given as part of antiretroviral or chemotherapy regimens. In chemotherapy-induced peripheral neuropathy (CIPN), anticancer drugs can damage both sensory and motor functions.

[0080] Symptoms of neuropathy ("Neuropathy Symptoms") include pain, hyperalgesia, allodynia, analgesia, loss of sensation of warmth, cold, or physical injury, hypersensitivity to touch, loss of coordination and proprioception, muscle weakness, muscle wasting, muscle spasms, muscle paralysis, tingling in fingers or hands, tingling in toes or feet, numbness in fingers or hands, numbness in toes or feet, electric shock or burning pain in fingers or hands, electric shock or burning pain in toes or feet, muscle cramps in hands and / or feet, difficulty feeling the ground under the soles of the feet which causes problems standing or walking, difficulty distinguishing between hot and cold water, These include, but are not limited to, difficulty holding a pen, problems holding a pen and difficulty writing, difficulty manipulating small items with the fingers (e.g., fastening a small button), difficulty opening jars or bottles due to hand weakness, difficulty walking due to a falling sensation, difficulty climbing stairs or rising from a chair due to leg weakness, dizziness when rising from a sitting or lying position, blurred vision, difficulty hearing, difficulty using the pedals of a car, and difficulty achieving or maintaining an erection. "Neuropathy side effects" are neuropathic symptoms that occur as a side effect of (i.e., associated with) the use of chemotherapeutic agents, antivirals, toxic agents, e.g., pesticides, and the like.

[0081] Neuropathy is the main cause of chronic pain, which is pain that lasts for more than 3 months. It is estimated that 10% of the population has neuropathic pain. "Neuropathic pain" is defined by the International Association For The Study Of Pain (IASP) as "pain that is initiated or caused by a primary lesion or dysfunction in the nervous system" (IASP, Classification of chronic pain, 2nd Edition, IASP Press (2002), p.210). In some embodiments of the method of the present disclosure, the neuropathic pain is caused by chemotherapy, i.e., caused by the administration of chemotherapeutic agents in chemotherapy. Chemotherapeutic agents can cause neurotoxicity, especially peripheral neurotoxicity, which leads to peripheral neuropathy. Allodynia (pain with stimuli that normally do not provoke pain) and hyperalgesia or hypersensitivity (increased pain with stimuli that normally provoke lesser degrees of pain), along with dysesthesias (unpleasant sensations that are not actually painful in themselves) and dysesthesias (abnormal sensations, e.g., pins and needles, or a burning sensation), are prominent symptoms in patients with neuropathic pain (Jensen TS, The Lancet (2014); 13 (9), P924-935).

[0082] Peripheral neuropathy is associated with a variety of conditions, including but not limited to: diabetes, amyotrophic lateral sclerosis (ALS), spinal muscular atrophy (SMA), rheumatoid arthritis, systemic lupus erythematosus (SLE), post-polio syndrome, Guillain-Barré syndrome, chronic inflammatory demyelinating polyneuropathy (CIDP), multifocal motor neuropathy, primary muscular dystrophy (PML), adrenoleukodystrophy, optic neuritis, kidney disease, liver disease, transverse myelitis, Parkinson's disease, stroke, and Alzheimer's disease. Marr's disease, Lyme disease, viral infections (e.g., infection with varicella-zoster virus, West Nile virus, cytomegalovirus, herpes simplex virus, or human immunodeficiency virus), carpal tunnel syndrome, peripheral nerve injury, demyelination, acute disseminated leukoencephalitis, progressive multifocal leukoencephalitis lymphoma, neuroma, multiple myeloma, vitamin B12 deficiency, postherpetic neuralgia, leprosy, Charcot-Marie-Tooth disease, Fabry disease, critical illness polyneuropathy, Bell's palsy, ulnar nerve palsy, and peroneal nerve palsy.

[0083] Peripheral neuropathy can also be induced by a wide range of medications, including, but not limited to, chemotherapeutic agents (e.g., as described in detail elsewhere in this application), biologic drugs (e.g., efalizumab, infliximab, etanercept, and adalimumab), anti-alcohol drugs (e.g., disulfiram), anticonvulsants (e.g., phenytoin), heart or blood pressure medications (e.g., amiodarone), antibiotics (e.g., metronidazole and fluoroquinolones (e.g., levofloxacin and ciprofloxacin)), skin medications (e.g., dapsone), and radiation.

[0084] In the context of the present disclosure, the term "reducing neuropathy" means alleviating, ameliorating, or eliminating one or more symptoms associated with neuropathy (e.g., peripheral neuropathy), including, but not limited to, neuropathic pain, hyperalgesia, and / or allodynia. Reduction of neuropathy symptoms may be achieved by administering to a patient a SapC-phospholipid nanovesicle formulation of the present disclosure.

[0085] The administration protocol for treating neuropathy may vary depending on the type of neuropathy that the subject experiences and how quickly symptoms resolve upon treatment.In some embodiments, a relatively long-term administration protocol may be required to treat subjects with chronic neuropathy.Thus, in some embodiments, the nanovesicle formulation is administered by any acceptable route disclosed elsewhere in this application for at least 24 hours, or individual doses of the nanovesicle formulation are administered periodically, for example, over a period of days, weeks, or months to years, as needed, to reduce neuropathy and / or treat or reduce symptoms associated with neuropathy.

[0086] In some embodiments, treatment with the disclosed SapC-phospholipid nanovesicle formulations reduces the incidence or occurrence of neuropathic symptoms in a subject compared to the incidence or occurrence of neuropathic symptoms in a control population (i.e., a group of subjects not receiving SapC-phospholipid treatment).

[0087] Neuropathic symptoms can be experienced by patients as a single, constant event, or as a series of separate occurrences or episodes.For example, in some embodiments, a subject who receives chemotherapy associated with neuropathic side effects can experience the onset of one or more neuropathic episodes during or after treatment with the chemotherapy.

[0088] The SapC-phospholipid nanovesicle formulations may reduce the frequency, duration, and / or intensity of neuropathy symptoms experienced by a subject and / or delay the onset of neuropathy in a subject.

[0089] In some embodiments, treatment with the disclosed SapC-phospholipid nanovesicle formulations can reduce the intensity of neuropathy symptoms in a subject compared to the intensity of neuropathy symptoms experienced by the subject prior to treatment. The intensity of neuropathy symptoms can be measured subjectively or objectively using one or more assessment tools known to those skilled in the art (e.g., patient self-report via the CIPN20 questionnaire). In one example, the change in the intensity of a neuropathy symptom (e.g., a CIPN symptom or symptom cluster) is determined by continuously measuring a total neuropathy "score" derived from one or more individual questions (e.g., one or more questions regarding numbness, tingling, and pain in the EORTC QLQ-CIPN20 questionnaire) in a patient survey that quantifies the intensity of the symptom as a score. The resulting total neuropathy score is then converted to a scale of 0 to 100. The change in neuropathy score (e.g., improvement or worsening of the score) can then be determined by comparing the scores obtained at various time points during the course of treatment (e.g., before, during, or after treatment with the SapC-phospholipid formulation). Depending on the wording of the question, the reduction (i.e., improvement) of the intensity of neuropathy symptoms can be reflected as either an increase or a decrease in score. Other methods of evaluating the improvement of neuropathy scores are known in the art and described elsewhere in this application. In some embodiments, treatment with SapC-phospholipid formulations can improve neuropathy symptom scores (e.g., reduce the intensity of episodes of neuropathy symptoms) by at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 97%, or at least about 99%.

[0090] In some embodiments, treatment with the disclosed SapC-phospholipid nanovesicle formulations can reduce the duration of neuropathic symptoms in a subject compared to the duration of neuropathic symptoms experienced in a suitable control population.For example, a patient who is administered a SapC-phospholipid formulation simultaneously with a chemotherapy agent (i.e., a drug associated with neuropathic symptoms side effects) may experience neuropathic symptoms for a shorter period of time after starting chemotherapy treatment when compared to the average duration of neuropathic symptoms experienced in a control population (i.e., a population of patients who are administered a chemotherapy agent but not a SapC-phospholipid nanovesicle formulation).

[0091] In some embodiments, treatment with the disclosed SapC-phospholipid nanovesicle formulation can reduce the duration or frequency of neuropathic episodes in a subject, compared with the duration or frequency of similar neuropathic episodes that the subject experiences before starting treatment with SapC-phospholipid nanovesicle formulation.For example, before treatment, a patient may regularly experience episodes of neuropathic pain, each lasting several weeks.After treatment, the duration of each episode that the patient experiences may be reduced to 1 week or less, or the frequency of episodes may be reduced, and the symptom-free period between episodes may be longer.

[0092] In some embodiments, treatment with a SapC-phospholipid formulation can reduce the duration and / or frequency of episodes of neuropathic symptoms by at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 97%, or at least about 99% as determined by patient self-report as described elsewhere in this disclosure.

[0093] In some embodiments, treatment with the SapC-phospholipid formulation reduces the intensity of neuropathy (e.g., CIPN) in the NCI-CTC tool (Kaplow, R (2017) Nursing: 47 (2)-p67-68) to a toxicity grade of 1 or less.

[0094] cancer The term "cancer" refers to a mammalian physiological condition typically characterized by uncontrolled cell growth / proliferation, and includes all types of cancer, neoplasm, or malignant tumors found in mammals (e.g., humans), such as leukemia (e.g., acute or chronic leukemia), carcinoma, and sarcoma. In some embodiments, cancers treatable in the methods described herein include, for example, any solid tumor or neurological cancer, such as prostate cancer, liver cancer, lung cancer, pancreatic cancer, renal cell carcinoma, breast cancer, bladder cancer, ovarian cancer, testicular cancer, ependymoma, brain cancer, such as high-grade glioma (HGG), such as glioblastoma multiforme (GBM), neuroblastoma, and gastrointestinal (GI) cancer, such as appendix cancer and colorectal cancer. The compositions are useful for treating neurological disorders associated with chemotherapy treatment of metastatic tumors, regardless of the type of primary tumor or the organ to which it metastasizes. Metastatic tumors can arise from a number of primary tumor types, including, but not limited to, those originating from the prostate, colon, lung, breast, bone, and liver. "Cancer" includes malignancies of various organ systems, such as those affecting the lung, breast, thyroid, lymphatic system, gastrointestinal, or genitourinary tract, and also includes adenocarcinomas, including most colon cancers, renal cell carcinoma, prostate cancer, and / or testicular cancer, non-small cell lung cancer, small intestine cancer, and esophageal cancer. Other examples of cancers that can be treated include Hodgkin's and non-Hodgkin's lymphomas, rhabdomyosarcoma, Ewing's sarcoma, Wilms' tumor, and multiple myeloma. The term "tumor" is recognized in the art and refers to malignancies of epithelial or endocrine tissues, such as respiratory system cancer, gastrointestinal system cancer, genitourinary system cancer, testicular cancer, breast cancer, prostate cancer, endocrine system cancer, squamous cell carcinoma, and melanoma. Exemplary cell tumors include those formed from tissues of the cervix, lung, prostate, breast, head and neck, colon, and ovary. The term also includes carcinosarcomas, which include malignant tumors composed of carcinomatous and sarcomatous tissues. "Adenocarcinoma" refers to cell tumors derived from glandular tissue or in which the tumor cells form recognizable glandular structures. Further details can be found, for example, in U.S. Patent No. 7,834,147, which is incorporated herein by reference in its entirety.

[0095] In some embodiments, the term "cancer" includes all types of cancer, neoplasms, or malignant tumors found in the gastrointestinal system of a subject. In some embodiments, the cancers treatable in the methods described herein (e.g., saposin C-DOPS nanovesicle formulations and chemotherapy regimens of one or more antitumor agents) include any gastrointestinal cancer, including, but not limited to, esophageal cancer, gastric cancer, anal cancer, colorectal cancer, intestinal cancer, gallbladder cancer, pancreatic cancer, liver cancer, pancreatic islet cell cancer, rectal cancer, small intestine cancer, gastrointestinal carcinoid tumor, or gastrointestinal stromal tumor. The compositions are useful for treating metastatic tumors regardless of the type of primary tumor or the organ to which it metastasizes. Metastatic gastrointestinal tumors can arise from a number of primary tumor types, including, but not limited to, those originating from the esophagus, stomach, small intestine, large intestine, rectum, anus, liver, gallbladder, and pancreas.

[0096] In other embodiments, cancers treatable in the methods described herein (e.g., saposin C-DOPS nanovesicle formulations and one or more immune checkpoint inhibitors) include, for example, B cell lymphoma (e.g., B cell chronic lymphocytic leukemia, B cell non-Hodgkin's lymphoma, cutaneous B cell lymphoma, diffuse large B cell lymphoma, primary mediastinal large B cell lymphoma (PBMCL)), basal cell carcinoma, bladder cancer, blastoma, brain metastases, breast cancer, Burkitt's lymphoma, carcinomas (e.g., phagocytosis, leukemia, and lymphomas (e.g., leukemia, bronchitis, and bronchial asthma)). adenocarcinoma of the gastroesophageal junction), cervical cancer, colon cancer, colorectal cancer (colon and rectal cancer; microsatellite instability-high (MSI-H)), endometrial cancer, esophageal cancer (e.g., esophageal squamous cell carcinoma (ESCC)), Ewing's sarcoma, fibrosarcoma, follicular lymphoma, gastric cancer, esophagogastric junction cancer, gastrointestinal cancer, glioblastoma (e.g., glioblastoma multiforme), glioma, head and neck cancer (e.g., squamous cell carcinoma of the head and neck (SCCHN or HNSSC)), Hodgkin's lymphoma (classical Hodgkin's lymphoma (cHL)), non-Hodgkin's lymphoma , kidney cancer (e.g., renal cell carcinoma (RCC) and Wilms' tumor), laryngeal cancer, leukemia (e.g., chronic myelocytic leukemia, hairy cell leukemia), liver cancer (e.g., hepatocellular carcinoma (HCC); hepatocellular carcinoma), lung cancer (e.g., non-small cell lung cancer and small cell lung cancer), mesothelioma (malignant pleural mesothelioma), lymphoblastic lymphoma, lymphoma, mantle cell lymphoma, metastatic brain tumor, metastatic carcinoma, myeloma (e.g., multiple myeloma), neuroblastoma, ocular melanoma, oropharyngeal cancer, osteosarcoma, ovarian cancer, pancreatic cancer (e.g., pancreatic ductal adenocarcinoma), prostate cancer, Adenocarcinoma (e.g., hormone refractory (e.g., castration resistant), metastatic, metastatic hormone refractory (e.g., castration resistant, androgen independent)), salivary gland carcinoma, sarcoma (e.g., rhabdomyosarcoma), skin cancer (e.g., melanoma (e.g., metastatic melanoma); Merkel cell carcinoma (MCC)), soft tissue sarcoma, squamous cell carcinoma, synovial sarcoma, testicular cancer, thyroid cancer, transitional cell carcinoma (urothelial cell carcinoma; urothelial carcinoma), uveal melanoma (e.g., metastatic), verrucous carcinoma, vulvar carcinoma, and Waldenstrom's hypergammaglobulinemia. In some cases, the cancer includes gastrointestinal cancer, including, but not limited to, esophageal cancer, gastric cancer, anal cancer, colorectal cancer, intestinal cancer, gallbladder cancer, pancreatic cancer, liver cancer, islet cell carcinoma, rectal cancer, small intestine cancer, gastrointestinal carcinoid tumor, or gastrointestinal stromal tumor.The composition is useful for treating metastatic tumors regardless of the type of primary tumor or the organ to which it metastasizes.Metastatic gastrointestinal tumors can arise from a number of primary tumor types, including but not limited to those originating from the esophagus, stomach, small intestine, large intestine, rectum, anus, liver, gallbladder, and pancreas.In some cases, the cancer comprises cells that express one or more immune checkpoint biomarkers, such as PD-L1 and CTLA-4.In some cases, the abundance of genomic tumor abnormalities in additional biomarkers (e.g., EGFR or ALK) is determined in the cells.In some cases, the detection of immune checkpoint biomarkers or genomic tumor abnormalities is performed using a test approved by the FDA.

[0097] As used herein, the terms "treat", "treating", "treatment" and variations thereof refer to partial or complete alleviation, inhibition, remission, or remission of a disease or condition (e.g., gastrointestinal cancer, head and neck cancer, or melanoma) suffered by a subject. "Treating cancer" means using the treatment methods described herein to cause a partial or complete reduction in the rate of tumor growth, the size of a tumor, the rate of local or distant tumor metastasis, the total tumor burden in a subject, and / or a reduction in tumor survival rate. In certain embodiments, "treat" and variations thereof refer to delaying or reversing the progression of cancer (e.g., gastrointestinal cancer) compared to untreated controls. Response to treatment using the disclosed methods is evaluated by a number of parameters known in the art, including:

[0098] (1) "Objective response rate" or "ORR" is an important parameter for proving the efficacy of treatment in oncology (Aykan NF, Ozatli T. (2020) World J Clin Oncol. Feb 24; 11(2): 53-73). It determines the tumor burden (TB) after a given treatment in patients with solid tumors by determining the proportion of patients with a planned amount of tumor size reduction in a minimum period. Tumors are evaluated using the World Health Organization (WHO) criteria and Response Evaluation Criteria in Solid Tumors (RECIST), which are anatomical response criteria developed primarily for cytotoxic chemotherapy and are based on anatomical imaging. ORR is measured as the total number of subjects with a best overall response (BOR) of either complete response (CR) or partial response (PR) divided by the total number of subjects in the population of interest.

[0099] (2) "Overall survival" or "OS" is the survival time of a patient diagnosed with a disease from the date of diagnosis of the disease, e.g., cancer, or from the date of initiation of treatment. For the purposes of this disclosure, it is defined as the time from the date of randomization (the date on which a clinical trial subject is assigned to a particular treatment group) to the date of death from any cause.

[0100] (3) "Progression-free survival" or "PFS" is the length of time that a patient survives with disease but without progression during and after treatment for a disease, e.g., cancer. For the purposes of this disclosure, it is defined as the time from the date of randomization (the date that a clinical trial subject is assigned to a particular treatment group) to the date of disease progression or death.

[0101] (4) "Duration of response" or "DoR" is the length of time that a tumor continues to respond to treatment without the cancer growing or spreading. For the purposes of this disclosure, it is defined as the time from demonstration of a positive response to treatment in a patient to disease progression.

[0102] (5) Disease control rate (DCR) is the percentage of patients with cancer who achieved complete response (CR), partial response (PR), or stable disease (SD). In the context of the present disclosure, it is defined as the proportion of participants whose best overall response (BOR) was complete response (CR), partial response (PR), and stable disease (SD) according to RECIST v1.1 or a later updated version of RECIST (e.g., as described in Eisenhauer et al., Eur J Cancer, (2009) 45(2):228-47, the disclosure of which is incorporated herein by reference).

[0103] Thus, in some embodiments, treatment with the disclosed methods results in an increase in at least one of the following parameters in a subject compared to a control population: (a) objective response rate (ORR); (b) overall survival (OS); (c) progression-free survival (PFS); and (d) duration of response (DoR). For example, treatment with the disclosed methods can increase one or more of ORR, OS, PFR, and / or DoR by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 97%, or at least about 99%. Furthermore, treatment with the disclosed methods may increase the DCR in a treated population (i.e., a group of subjects treated with the disclosed methods) by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 97%, or at least about 99% compared to a control population.

[0104] Chemotherapy-induced peripheral neuropathy Chemotherapy-induced peripheral neuropathy (CIPN) is defined as a physical or autonomic sign or symptom resulting from injury to the peripheral nervous system (PNS) or autonomic nervous system (ANS) caused by chemotherapy agents (Cooper C; Chemotherapy-Induced Peripheral Neuropathy, in Cooper's Fundamentals of Hand Therapy, 2020). In CIPN, chemotherapy agents (anticancer drugs) can impair both sensory and motor function. CIPN is a common and serious side effect of cancer treatment that can lead to dose reduction or early cessation of chemotherapy, thereby reducing the effectiveness of cancer treatment. It can cause debilitating symptoms and also has a significant impact on the patient's quality of life. Approximately 30 to 40 percent of cancer patients treated with chemotherapy experience CIPN.

[0105] Platinum-based anticancer drugs are commonly used to treat lung cancer, colorectal cancer, ovarian cancer, breast cancer, head and neck cancer, and urogenital cancer. However, peripheral neuropathy, which mainly affects dorsal root ganglion (DRG) neurons, is a common side effect of their chemotherapy. The mechanism underlying peripheral neuropathy has not been fully elucidated.

[0106] Oxaliplatin, a platinum-based chemotherapy drug, is commonly used for the treatment of various types of cancer. Oxaliplatin induces two distinct types of neuropathy: a transient, very common acute pain syndrome that appears during or shortly after exposure to oxaliplatin, and a dose-limiting chronic sensory neurotoxicity that is cumulative in nature and resembles the characteristics of those caused by other types of chemotherapy-related neurotoxicity, e.g., cisplatin and paclitaxel. CIPN is a well-described clinical problem (Grothey A,et al(2011)Journal of Clinical Oncology.29(4):421-7; Loprinzi CL,et al(2014)J Clin Oncol.32(10):997-1005; Pachman DR,et al(2015).Journal of Clinical Oncology 33(30):3416-22; and Pachman DR,et al(2016)Supportive Care in Cancer.24(12):5059-68).

[0107] Chemotherapeutic agents known to induce neuropathy include, but are not limited to, platinum-based agents (e.g., oxaliplatin, cisplatin, carboplatin), taxanes (e.g., paclitaxel, docetaxel, cabazitaxel), epothilones (e.g., ixabepilone), plant alkaloids (e.g., vinblastine, vincristine, vindesine, vinorelbine, vincaminol, vineridin, vinbrunin, etoposide), immunomodulators (e.g., thalidomide, lenalidomide, pomalidomide), proteasome inhibitors (e.g., bortezomib, carfilzomib, ixazomib), eribulin, and suramin.

[0108] Patients with CIPN often experience symmetric symptoms, typically beginning in the hands and / or feet and gradually moving to the arms and legs. Patients may also or alternatively experience symptoms such as pain (shooting, sharp, stabbing, and / or burning), numbness, temperature sensitivity, difficulty walking, and difficulties with fine motor skills such as writing, undoing buttons, and packing small items. CIPN symptoms may also include hearing loss, blurred vision, and taste changes. In addition, motor neuron dysfunction may manifest as muscle spasms, impaired gait, paralysis, muscle cramps, tremors, and weakness. CIPN may be characterized by paresthesia, dysesthesias, loss of joint and vibration sensation, and loss of deep tendon reflexes. The development of symptomatic CIPN typically results in a reduction in chemotherapy dose and / or interruption of treatment, which may adversely affect cancer-related outcomes.

[0109] In some embodiments, the nanovesicle formulation utilized in the disclosed method is administered simultaneously (at the same time as the start or within 48 hours before or after the start) with the chemotherapy treatment associated with the neuropathic symptom side effect, thereby reducing the incidence, intensity, and / or duration of neuropathic symptoms or delaying the onset of neuropathic symptoms. In some embodiments, the nanovesicle formulation is administered immediately before or after the administration of the dose of chemotherapy, so that the period from the end of administration of one of these agents to the start of administration of the other is 2 hours or less. For example, the period can be 90 minutes or less, 80 minutes or less, 70 minutes or less, 1 hour or less, 50 minutes or less, 40 minutes or less, 30 minutes or less, 20 minutes or less, 10 minutes or less, 5 minutes or less, or within 1 minute. Furthermore, one or more additional doses of the nanovesicle formulation can be administered over the weeks, months, or years that the patient continues to receive chemotherapy. The administration of one or more additional doses of the nanovesicle formulation can each be administered simultaneously with the administration of some or all of the chemotherapy doses, or can be administered on a different schedule.

[0110] In other embodiments, the nanovesicle formulations utilized in the disclosed methods are administered before and / or after treatment with a chemotherapeutic agent associated with a neuropathic symptom side effect, thereby reducing the incidence, intensity, and / or duration of neuropathy or delaying the onset of neuropathy. Additionally, the nanovesicle formulations may be administered before, during, and after administration of the chemotherapeutic agent.

[0111] Neuropathy assessment tool Neuropathic symptoms are evaluated by several methods known in the art, including, but not limited to, physical examination (assessment of sensation (e.g., evoked pain), motor function, and autonomic changes), sensory testing to determine sensory deficits to various stimuli (e.g., touch, puncture, temperature, vibration), blood tests, imaging studies, electrodiagnostic tests, clinical tests of nerve function (e.g., electromyography (EMG)), nerve conduction velocity testing (nerve conduction studies), autonomic reflex screening, and patient self-report.

[0112] Methods have been developed to distinguish the various clinical components of peripheral neuropathy, to separate pain, numbness, and tingling, and to measure them on a scale of 0 to 10. The National Cancer Institute's Common Toxicity Criteria score is one of the protocols for the assessment of peripheral neuropathy. Visual analog scale (VAS) scores provide a complete assessment of the patient's symptoms, signs, abilities, and electrophysiology. Nerve conduction studies and needle electromyography help identify the severity of nerve structures, axonal degeneration or demyelination, and axonal injury and confirm the diagnosis of peripheral neuropathy. Neurofilament light chain (NFL) is a reliable and easily accessible biomarker of CIPN progression and severity. Monitoring neurofilament levels (e.g., serum neurofilament light chain (sNFL) levels) during chemotherapy can indicate ongoing nerve axonal damage and the severity of CIPN (Kim, SH., et al. (2020) Sci Rep 10, 7995). Other biomarkers include NGF, BDNF, ceramide, etc. (Meregalli C, et al Neurosci Lett.2021 Apr 1;749:135739). The European Organization for Research and Treatment of Cancer has developed a questionnaire to determine the severity of neuropathy by patient self-assessment.

[0113] Due to the subjective nature of neuropathic symptoms, measurement of these symptoms relies, in part, on patient self-report. In some cases, neuropathic symptoms (e.g., neuropathic side effects of CIPN) can be assessed based on assessment tools and inventories, such as patient self-report questionnaires (e.g., the CIPN20, which complements the European Organization for Research and Treatment of Cancer (EORTC) quality of life questionnaire (Postma TJ, et al. (2005). Eur J Cancer. 2005 May; 41(8): 1135-9; Beijers AJ, et al. (2016). Support Care Cancer. 2016 Jun; 24(6): 2411-20). Other assessment tools include the Chemotherapy-Induced Peripheral Neuropathy Assessment Tool (CIPNAT), Post-Oxaliplatin Symptom Survey, National Cancer Institute-Common Toxicity Criteria (NCI-CTC), Numeric Rating Scale (NRS), Visual Analog Scale (VAS), Functional Assessment of Cancer Therapy / Gynecologic Oncology (FANCA)). Group-Neurotoxicity (FACT / GOG-Ntx), Total Neuropathy Score (TNS) questionnaire, Patient-Reported Outcome Measurement Information System (PROMIS), Cold Hypersensitivity Survey, Thermal Sensitivity Test (Tofthagen CS,et al.(2011)Cancer Nurs.2011 Jul-Aug;34(4):E10-20;Leonard,GD,et al.(2005)BMC Cancer 5,116;Dy SM,et al.(2017)Agency for Healthcare Research and Quality(US);Mar.(Comparative Effectiveness Reviews,No.187.)Table 3;Calhoun EA,et al.(2003) Int J Gynecol Cancer. Nov-Dec; 13(6): 741-8; Cheng, HL, et al. (2020) Health Qual Life Outcomes 18, 246; Cavaletti G, et al. (2007) J Peripher Nerv Syst. Sep; 12(3): 210-5; Askew, RL, et al. (2016). Value Health, 19(5), 623-630, Bae, KH, et al. (2018). Integrative medicine research, 7(1), 61-67; Ceynowa M. et al, (2015) BioMed Research International, Article ID 528356).

[0114] These assessments are based on the patient's responses and are administered through abbreviated surveys, customized paper or electronic surveys, computer adaptive tests, etc., intended to capture a comprehensive assessment of the patient's pain experience. The questionnaire may include a scoring system (Yang Z, et al. (2018) Cochrane Database Syst Rev. 2018 Jul 30; 2018(7):CD010974) to further characterize the level of neuropathy the subject is experiencing. Such scoring systems include the Clinical Neurological Examination (CNE), Michigan Neuropathy Screening Instrument (MNSI), Neuropathy Disability Score (NDS), Neuropathy Impairment Score (NIS), Neuropathy Impairment Score in the Lower Limbs (NIS-LL), Neuropathy Symptom Profile (NSP), Neuropathy Symptom Score (NSS), Toronto Clinical Scoring System (TCSS), Neuropathy Total Symptom Score-6 (NTSS-6), Neuropathy Symptom Change Score (NSC), Total Neuropathy Symptom Score (TCSS), Neuropathy Disability Score (NDS), Neuropathy Impairment Score (NIS), Neuropathy Impairment Score in the Lower Limbs (NIS-LL), Neuropathy Symptom Profile (NSP), Neuropathy Symptom Score (NSS), Toronto Clinical Scoring System (TCSS), Neuropathy Total Symptom Score-6 (NTSS-6), Neuropathy Symptom Change Score (NSC), Total Neuropathy Symptom Score (NSS), Neuropathy Disability Score (NDS), Neuropathy Impairment Score in the Lower Limbs (NIS-LL), Neuropathy Disability Score (NDS), Neuropathy Impairment Score in the Lower Limbs (NIS-LL), Neuropathy Disability Score (NSS ... Scoring systems for diabetic neuropathy include, but are not limited to, the Total Symptom Score (TNS), and the Total Symptom Score (TSS). Scoring systems for diabetic neuropathy include the Diabetic Neuropathy Examination (DNE) and the Diabetic Neuropathy Symptom Score (DNSS).

[0115] In some cases, neuropathic pain can be measured as a score based on the patient's response to questions about pain intensity on the neuropathy pain scale (NPS), which was developed to monitor response to treatment (May S, Serpell M. (2009). F1000 Med Rep. 2009 Oct 14;1:76). On this scale, 0 indicates no pain and 10 indicates the most intense pain imaginable.

[0116] Screening tools to identify and assess neuropathic pain experienced by a subject include, but are not limited to, the Leeds Assessment of Neuropathic Symptoms and Signs (LANSS), the Neuropathic Pain Diagnostic Questionnaire (DN4), the Neuropathic Pain Scale (NPS), the Neuropathic Pain Symptom Inventory (NPSI), and the Neuropathic Pain Questionnaire (NPQ).

[0117] In other cases, the neuropathic symptoms of chemotherapy-induced peripheral neuropathy (CIPN) can be characterized using a toxicity grading system that typically uses a combination of clinical and paraclinical parameters and relies on the judgment of a physician and / or nurse (Postma TJ, et al. (2005) Eur J Cancer. May; 41(8):1135-9).

[0118] Several preclinical animal models of peripheral neuropathy have been used in the art to evaluate the efficacy of treatments (see, e.g., Gadgil S et al., 2019). These models include, for example, rodent neuropathic pain models induced by paclitaxel, cisplatin, and oxaliplatin. Such models can be used to evaluate the efficacy of proposed neuropathy treatments.

[0119] Neurogenesis, neuroregeneration, neuritogenesis, and neuroprotection Neurogenesis is the generation of pre-neuronal cells. Neural regeneration is the regrowth or repair of neural tissue by the generation of new neurons, axons, and / or synapses after injury that causes nervous system injury and / or degeneration. Neuroprotection refers to the preservation of neuronal structure and / or function, and the prevention of neuronal loss. Neuritogenesis is the extension of neurites from neuronal cell bodies via neurotrophic factors, such as nerve growth factors. In some embodiments, the present disclosure provides methods for promoting neurogenesis, neurite outgrowth, neuroprotection (e.g., prevention of neuronal loss), and neural regeneration (e.g., axonal regeneration) in neurons of the central nervous system (CNS) and peripheral nervous system (PNS).

[0120] Neurodegeneration is a feature of many acute and chronic neurological disorders. One mode of axonal degeneration, called Wallerian degeneration (WD), is a highly destructive process in which segments of axons distal to the injury die. Initial abnormalities are seen as early as a few hours after injury, with more pronounced WD becoming evident one or two days later (Ballin RH and Thomas PK (1969) Acta Neuropathol (Berl) 14:237). For example, the myelin sheath breaks down and is phagocytosed by scavenger cells (Leonhard et al. (2002) Eur. J. Neurosci. 16:1654).

[0121] The disclosed method can be used to prevent or treat acute or chronic nerve injury, or to reduce symptoms associated with acute or chronic nerve injury. Whether acute or chronic, conditions that require neurogenesis, neuritogenesis, neuroprotection, and nerve regeneration are disclosed, for example, in WO2007 / 044928 and the references cited therein. Acute trauma to peripheral nerves includes not only blunt trauma, but also trauma from penetrating missiles, such as bullets or other objects. Needle puncture or foreign body (e.g., glass, sheet metal) injuries that result in clean rupture of nerves are known, as are nerve injuries caused by fractures and fracture-dislocations, such as ulnar nerve neurapraxia and radial nerve lesions and paralysis. In general, acute nerve injury often results in long-term neuropathic pain that manifests as allodynia, decreased pain threshold, and hyperplasia, and increased pain in response to noxious stimuli. See Colohan AR, et al. (1996) Injury to the peripheral nerves. In: Feliciano DV, Moore EE, Mattox K L. Trauma. 3rd ed. Stamford, Conn.: Appleton & Lange; 1996: 853. Other types of acute nerve injuries within the scope of this disclosure include traumatic brain injury (TBI) and acute injuries to the spinal cord and peripheral / sensory nerves, such as sports injuries with nerve injury.

[0122] In some embodiments, administration of nanovesicle formulations in response to acute nerve injury promotes neurogenesis, neuritogenesis, neuroprotection, and nerve regeneration when the formulation is administered as soon as possible, for example, within about 24 hours after injury (for example, within 12 hours, within 6 hours, within 3 hours, within 2 hours, or within 1 hour after injury).In addition, nanovesicle formulations can be administered prophylactically (as a preventative measure) before medical interventions (e.g., surgery) associated with the risk of nerve injury.This allows for advantageous enhancement of nerve regeneration and shortened recovery time.

[0123] In some embodiments, the nanovesicle formulations treat, prevent, or reduce symptoms of conditions associated with chronic damage to the nervous system by promoting neurogenesis, neuritogenesis, neuroprotection, and neuroregeneration, including neurodegenerative disorders (e.g., Parkinson's disease, Alzheimer's disease, or amyotrophic lateral sclerosis), brain injury, peripheral nerve injury, multiple sclerosis, disseminated sclerosis, chronic demyelinating neuropathy (CMT type 1), HMSN (CMT) disease types 1A and 1B, hereditary neuropathy with liability to pressure palsies (HNPP) and other pressure palsies, Bethlem myopathy, limb-girdle muscular dystrophy, Miyoshi myopathy, radicular chondrodysplasia punctata, HMSN-Lom, PXE (pseudoxanthoma elasticum), CCFDN (congenital cataract, facial dysmorphism, and neuropathy), Huntington's disease, Charcot-Marie's disease, and other conditions associated with chronic damage to the nervous system ... chronic damage to the These include, but are not limited to, Myers-Tooth disease, Guillain-Barre syndrome (GBS), leukodystrophies, motor neuron diseases, diabetic neuropathy, distal axonopathies, such as those resulting from metabolic or toxic neuronal derangements (e.g., those associated with diabetes, renal failure, exposure to drugs or toxins (e.g., anticancer drugs), malnutrition, or alcoholism), mononeuropathies, radiculopathies (e.g., of cranial nerve VII; facial nerve), leprosy, plexopathies, such as brachial neuritis, and focal entrapment neuropathy (e.g., carpal tunnel syndrome).

[0124] In embodiments where the goal of treatment is to treat chronic neuronal injury, a relatively long-term administration protocol will generally be preferred. Thus, in one embodiment, the nanovesicle formulation is administered by any acceptable route disclosed elsewhere herein for at least 24 hours, or individual doses of the nanovesicle formulation are administered periodically over a period of days, weeks, or months to years, as needed to promote neurogenesis, neuritogenesis, neuroprotection, and / or neuroregeneration, and / or to treat or reduce symptoms associated with a particular indication.

[0125] Determining neurogenesis, neuritogenesis, neuroprotection, and neuroregeneration Methods for monitoring improvements in neurogenesis, neuritogenesis, neuroprotection, and neuroregeneration have been described and generally include various functional tests that can be performed in human patients. Such tests typically monitor the recovery of sensory and / or motor function. Examples include, but are not limited to, the Weinstein Enhanced Sensory Test (WEST), the Semmes-Weinstein Monofilament Test (SWMT), and the like. For methods of detecting and monitoring neurogenesis, neuritogenesis, neuroprotection, and neuroregeneration, as well as methods of classifying various neuronal injuries, see WO2008 / 044928, Ristic S,et al.(2000)Clin Orthop Relat Res.370:138, Sierra,A.,et al(2011).Frontiers in neuroscience,5,47; and references cited therein. Further methods for determining neuronal regeneration and neuronal protection include, but are not limited to, cell-based, culture-based, and tissue-based assays; neurite outgrowth assay; neurite retraction assay; stripe assay; spot assay; scratch assay; radial assay; neuronal explant; and brain and spinal cord slice culture.See, for example, Al-Ali H, et al., Exp Neurol.2017;287(Pt 3):423-434) for further description of these assays.

[0126] Other methods for evaluating neuroregeneration and neuroprotection include shape texture identification (STI). Imaging techniques such as magnetic resonance imaging (MRI), MRI spectroscopy, PET imaging, and computed tomography (CT) can be used to evaluate neurogenesis, neuritogenesis, neuroprotection, and neuroregeneration. In addition, neurogenesis and neuroprotection can be evaluated by techniques known in the art, such as obtaining tissue (e.g., via surgery) and staining it with appropriate neuromarkers using immunohistochemistry, or using tissue for single-cell RNA sequencing.

[0127] In one example, a suitable dose of the nanovesicle formulation can be shown to promote nerve regeneration and neuronal preservation as measured by intraepidermal nerve fiber density (IENFD) using a skin biopsy (see, e.g., Myers, MI, & Peltier, AC (2013). Current neurology and neuroscience reports, 13(1), 323; and Collongues N, et al., (2018). PLoS One. Jan 25; 13(1): e0191614) and / or by taking cross-section nerve fibers and counting the different types of nerves (small unmyelinated, medium, large, large myelinated). See also Chattopadhyay M, et al (2004) Brain. Apr; 127(Pt 4): 929-39. Appropriate dosing can be determined by methods known in the art.

[0128] Methods of Treating Cancer Sections A through E below relate to disclosed methods of treating cancer.

[0129] A. Antitumor Agents The active agent of the chemotherapy regimen of the disclosed method is an antitumor agent. The term "antitumor agent" refers to a drug that prevents, inhibits, or delays the development of a neoplasm (tumor) and is used in the treatment of gastrointestinal cancer. An antitumor agent may also be referred to as an anticancer agent, a chemotherapeutic agent, a chemotherapy drug, or a cytotoxic agent. The antitumor agents of the present disclosure include, but are not limited to, alkylating agents, antimetabolites, plant alkaloids, and various other agents, as well as checkpoint inhibitors. Alkylating agents include, but are not limited to, platinum-containing compounds (e.g., oxaliplatin, cisplatin, carboplatin, etc.), cyclophosphamide, ifosfamide, chlorambucil, busulfan and melphalan, and carmustine. Antimetabolites include, but are not limited to, methotrexate, 5-fluorourocil, capecitabine, and cytosine arabinoside. Plant alkaloids include, but are not limited to, vinca alkaloids (e.g., vincristine and vinblastine), epipodophyllotoxins (e.g., etoposide and teniposide), and taxanes (e.g., paclitaxel and docetaxel). Miscellaneous other agents include, but are not limited to, topoisomerase I inhibitors (e.g., irinotecan). Checkpoint inhibitors include, but are not limited to, anti-PD1 and anti-PDL1 agents. Most antitumor agents are delivered intravenously, but many agents can be administered orally (e.g., melphalan, busulfan, capecitabine). In some embodiments, the disclosed methods include the use of the antitumor agents, oxaliplatin and 5-fluorouracil. For purposes of this disclosure and claims, the above formulations containing both SapC polypeptide and phospholipids are not encompassed by the terms "chemotherapeutic agent," "chemotherapy," and "antitumor agent."

[0130] The combination of a chemotherapy regimen and a saposin C polypeptide / phospholipid formulation (e.g., BXQ-350) can enhance or prolong the anti-tumor response of a subject.Furthermore, the administration of a saposin C polypeptide / phospholipid formulation (e.g., BXQ-350) and a chemotherapy regimen can enhance or prolong the therapeutic effect of the chemotherapy regimen, allow subjects who would otherwise not respond or would not respond as well to respond to the chemotherapy regimen, reduce the toxicity of the chemotherapy regimen, or allow the dose of the chemotherapy regimen required to achieve the desired therapeutic effect in the patient to be reduced.

[0131] In some embodiments, the combination of the chemotherapy regimen and the saposin C polypeptide / phospholipid formulation of the present disclosure can be used in combination with biological treatments, such as antibodies, including but not limited to, bevacizumab, cetuximab, and panitumumab. Bevacizumab is an anti-vascular endothelial growth factor (VEGF) monoclonal antibody. Bevacizumab has been approved by the FDA for the first-line treatment of mCRC in combination with 5-FU-based chemotherapy (Strickler JH, Hurwitz HI. Oncologist. 2012; 17(4): 513-524). Cetuximab is an anti-epidermal growth factor receptor (EGFR) chimeric antibody, and panitumumab is an anti-epidermal growth factor receptor (EGFR) monoclonal antibody. Both cetuximab and panitumumab have shown clinical efficacy in patients with chemotherapy-refractory wild-type KRAS exon 2 mCRC (Taniguchi H, et al. Cancers (Basel). 2020; 12(7): 1715).

[0132] B. Immune Checkpoint Inhibitors The present disclosure also provides methods of using immune checkpoint inhibitors in combination with saposin C polypeptide / phospholipid formulations (e.g., BXQ-350) to treat cancer. As used herein, an "immune checkpoint inhibitor" is a molecule (e.g., a protein, a small molecule, or a molecule that includes a protein) that blocks checkpoint protein function in immune cells, e.g., T cells, and some cancer cells. When the checkpoint is blocked using an inhibitor disclosed herein, T cells can more effectively kill cancer cells. Examples of checkpoint proteins found in T cells or cancer cells include PD-1 / PD-L1 and CTLA-4 / B7-1 / B7-2. In some cases, the immune checkpoint inhibitor does not include a nanovesicle formulation that includes (i) a saposin C polypeptide comprising SEQ ID NO:1 having 0-4 amino acid insertions, substitutions, deletions, or combinations thereof, and (ii) a phospholipid having a net negative charge at neutral pH.

[0133] The combination of immune checkpoint inhibitors and saposin C polypeptide / phospholipid formulations (e.g., BXQ-350) can enhance or prolong the antitumor response of a subject. Furthermore, administration of saposin C polypeptide / phospholipid formulations (e.g., BXQ-350) and immune checkpoint inhibitors can enhance or prolong the therapeutic effect of immune checkpoint inhibitors, allow subjects who would otherwise not respond or would not respond as well to respond to immune checkpoint inhibitors, reduce the toxicity of immune checkpoint inhibitors, or allow the dose of immune checkpoint inhibitors required to achieve the desired therapeutic effect in patients to be reduced.

[0134] In some cases, the immune checkpoint inhibitor is a biotherapeutic or small molecule. In some cases, the immune checkpoint inhibitor is a monoclonal antibody, a humanized antibody, a fully human antibody, a single chain Fv (scFv), a fusion protein, or a combination thereof. In some cases, the immune checkpoint inhibitor is an inhibitor of programmed death receptor-1 (PD-1). The PD-1 inhibitor can be selected from the group consisting of nivolumab, pembrolizumab, pidilizumab, MEDI0680, and combinations thereof. In some cases, the immune checkpoint inhibitor is an inhibitor of programmed death ligand 1 (PD-L1). The PD-L1 inhibitor can be selected from the group consisting of atezolizumab, BMS-936559, MEDI4736, MSB0010718C, and combinations thereof. In some cases, the immune checkpoint inhibitor is an inhibitor of cytotoxic T-lymphocyte-associated protein 4 (CTLA-4). The CTLA-4 inhibitor may be selected from the group consisting of ipilimumab, tremelimumab, and combinations thereof.

[0135] In some cases, the patient has unresectable and / or metastatic tumors or cancer. In other cases, the combination treatment is performed after complete or partial resection, and may be performed in combination with steroids. In some cases, the combination treatment described herein is a first-line treatment, a second-line treatment, or a third-line treatment. The combination treatment can be given after detection of certain genomic tumor abnormalities, such as EGFR or ALK genomic tumor abnormalities, or in their absence. The subject who receives the treatment can have a tumor PD-L1 positivity rate (Tumor Proportion Score) (TPS) of 1% or more, or a combined PD-L1 positivity rate (Combined Positive Score) (CPS) of 10 or more, as determined by a test approved by the FDA.

[0136] C. Chemotherapy regimens The term "chemotherapeutic regimen" refers to a standardized plan or protocol for cancer treatment using one or more antitumor agents. Current chemotherapy regimens apply drug treatments in cycles, but the frequency and duration of treatment is limited by toxicity. A commonly used combination chemotherapy regimen called FOLFOX is used to treat colorectal cancer. It contains the components leucovorin calcium (folinic acid) and the antitumor agents 5-fluorouracil (5FU) and oxaliplatin. There are several different FOLFOX regimens, including FOLFOX-4, FOLFOX-6, modified FOLFOX-6 (mFOLFOX-6), FOLFOX-7, and modified FOLFOX-7 (mFOLFOX-7). The FOLFOX regimens differ in the doses and manner in which the three drugs are given. Examples of chemotherapy regimens used in the treatment of gastrointestinal cancer (e.g., colorectal cancer) include, but are not limited to, 5-fluorouracil (5-FU) / capecitabine-based combination therapy, such as FOLFOX, FOLFIRI (5-FU, folinic acid, and irinotecan), and CAPOX (capecitabine and oxaliplatin) (Sonbol MB, et al (2019). JAMA Oncol; e194489). Any of these chemotherapy regimens may be used in the methods of the disclosure. In some embodiments, the methods of the disclosure include administering the chemotherapy regimen mFOLFOX7. The mFOLFOX7 regimen consists of (i) about 200 mg / m2 over about 2 hours on the first day of treatment. 2 85 mg / m2 infused with leucovorin calcium 2 oxaliplatin; and (ii) 2400 mg / m2 over approximately 46-48 hours on days 1 and 2 of the first week of treatment. 2 This involves the sequential administration of 5-fluorouracil (5-FU).

[0137] C.1. Oxaliplatin The antitumor agent of the chemotherapy regimen of the present disclosure may include oxaliplatin, a well-known chemotherapy drug with significant antitumor activity. It is sold under the trade name Eloxatin® and is an anticancer drug used to treat colorectal cancer. Oxaliplatin is a platinum-based antitumor drug that exerts its cytotoxic effect primarily through DNA damage (Alcindor T, et al (2011). Curr Oncol. 2011; 18(1): 18-25). Apoptosis of cancer cells can be caused by the formation of DNA damage, the cessation of DNA synthesis, the inhibition of RNA synthesis, and the induction of an immune response. Oxaliplatin is a cytotoxic agent that inhibits ... 14 It has the chemical formula NOPt and the chemical name cis-[(1R,2R)-1,2-cyclohexanediamine-N,N'][oxalate(2)-O,O']platinum. It is described in U.S. Patent No. 4,169,846, which is incorporated herein by reference.

[0138] C.2. 5-Fluorouracil The antitumor agent of the chemotherapy regimen of the present disclosure may include 5-fluorouracil (5-FU), an antimetabolite that is a fluoropyrimidine analog of nucleoside pyrimidines with antitumor activity. It is sold under the trade names Adrucil®, Carac®, Efudex®, Efudix®, etc., and is an anticancer drug that has been used to treat several cancers, such as anal cancer, colorectal cancer, colon cancer, esophageal cancer, gastric cancer, pancreatic cancer, breast cancer, and cervical cancer. It exerts its cytotoxic effect by inhibition of thymidylate synthase (TS) and incorporation of its metabolites into RNA and DNA (Longley, D., et al. (2003) Nat Rev Cancer 3, 330-338). 5-FU is a cytotoxic agent that is synthesized by the method of the present invention and is synthesized by the method of the present invention. 2 and is described in U.S. Pat. No. 4,336,381 A, which is incorporated herein by reference.

[0139] C.3. Leucovorin Leucovorin (also called folinic acid, Wellcovorin®) or leucovorin calcium is not a chemotherapy drug, but is given in conjunction with chemotherapy. It acts as a biochemical cofactor for one-carbon transfer reactions in the synthesis of purines and pyrimidines. Leucovorin does not require the enzyme dihydrofolate reductase (DHFR) for conversion to tetrahydrofolate. The effects of methotrexate and other DHFR antagonists are inhibited by leucovorin. Leucovorin can potentiate the cytotoxic effects of fluorinated pyrimidines (i.e., fluorouracil and floxuridine). After being activated in cells, 5-FU, along with the folate cofactor, inhibits the enzyme thymidylate synthase, thus inhibiting pyrimidine synthesis. Leucovorin increases the folate pool, thereby increasing the binding of the folate cofactor and active 5-FU to thymidylate synthase. Leucovorin inhibits C 20 H 23 It has the chemical formula N7O7 and is described in U.S. Pat. No. 4,500,711 A, which is incorporated herein by reference.

[0140] In some embodiments, the method of the present disclosure includes administering chemotherapy regimen mFOLFOX7 in combination with bevacizumab. Bevacizumab, cetuximab, and panitumumab are included in the National Comprehensive Cancer Network guidelines for first-line therapy in combination with FOLFOX to treat cancer (NCCN.org [Internet]. Guidelines for Patients®-Colon Cancer. Plymouth Meeting, PA: National Comprehensive Cancer Network; July 28, 2021). Each dose of bevacizumab is administered over about 30 to about 90 minutes, for example, 45 minutes or 60 minutes.

[0141] D. Treatment Cycle The SapC-phospholipid nanovesicle formulations and chemotherapy regimens of the present disclosure may be administered to a subject over multiple treatment cycles (e.g., 1, 2, 3, 4, 5, or 6 cycles). The term "cycle" refers to a period of treatment followed by a rest (treatment-free) period that is repeated on a regular schedule.

[0142] The term "day 1 of the first week of treatment" refers to the first day that treatment with a SapC-phospholipid nanovesicle formulation is initiated in a subject.

[0143] In one example, cycle 1 of treatment includes the first 4 weeks of treatment. The SapC-phospholipid nanovesicle formulation is administered once a day for 5 consecutive days during the first week of treatment, once every 2 days (or 3 times a week) during the second week, and approximately once a week (i.e., once every 7 (+ / - 3) days) during the third and fourth weeks of treatment. Cycles 2-6 include weeks 5-24 of treatment. In cycles 2-6, the SapC-phospholipid nanovesicle formulation is administered once every 14 (+ / - 3) days. Meanwhile, the chemotherapy regimen is administered once every 14 (+ / - 3) days in all cycles (1-6), starting from day 1 of week 1. Thus, a total of 12 infusions of the chemotherapy regimen are administered to the subject with cancer during the course of 6 cycles of treatment.

[0144] In another example, cycle 1 of treatment includes the first 4 weeks of treatment. The SapC-phospholipid nanovesicle formulation is administered once a day for 5 consecutive days during the first week of treatment, once every 2 days (or 3 times a week) during the second week, and approximately once a week (i.e., 7 (+ / - 3) days) during the third and fourth weeks of treatment. Cycles 2-6 include weeks 5-24 of treatment. In cycles 2-6, the SapC-phospholipid nanovesicle formulation is administered once every 14 (+ / - 3) days. Meanwhile, the chemotherapy regimen and bevacizumab are administered once every 14 (+ / - 3) days in all cycles (1-6), starting from day 1 of week 1. Thus, a total of 12 infusions of the chemotherapy regimen are administered to the subject with cancer during the course of 6 cycles of treatment.

[0145] In some embodiments, treatment can be extended for a period of time. After completing cycles 1-6, subjects can receive additional treatment. Cycles 7-26 include weeks 25-104 of treatment. In cycles 7-26, SapC-phospholipid nanovesicle formulation is administered once every 28 (+ / - 3) days. Meanwhile, chemotherapy regimen and / or bevacizumab treatment are continued as maintenance treatment at the discretion of the attending physician and / or according to standard of care.

[0146] As used herein, the term "concomitant administration" refers to the administration of a first treatment, e.g., a first pharmaceutical composition (e.g., a nanovesicle formulation comprising SapC-phospholipid), a second treatment, e.g., a second pharmaceutical composition (e.g., an anti-tumor agent or an immune checkpoint inhibitor of a chemotherapy regimen), and a third treatment, e.g., a third pharmaceutical composition (e.g., a biologic, e.g., bevacizumab), wherein the first, second, and third treatments are separate and administered at about the same time, i.e., the first, second, and third pharmaceutical compositions are administered within 48 hours of each other.

[0147] In one example, the second pharmaceutical composition (i.e., at least one anti-tumor agent or at least one immune checkpoint inhibitor of a chemotherapy regimen) is administered immediately after the SapC-phospholipid nanovesicle formulation is administered, for example, administration of oxaliplatin or administration of an immune checkpoint inhibitor in an mFOLFOX7 chemotherapy regimen can be initiated at least 1 hour after completion of administration of the SapC-phospholipid nanovesicle formulation. In another example, administration of a dose of an anti-tumor agent (e.g., oxaliplatin) or an immune checkpoint inhibitor of a chemotherapy regimen is initiated within 48 hours (e.g., within 24 hours or within 12 hours or within 6 hours or within 2 hours or within 1 hour) after completion of administration of a dose of a SapC-phospholipid nanovesicle formulation. In another example, administration of a dose of a SapC-phospholipid nanovesicle formulation is initiated within 48 hours (e.g., within 24 hours, or within 12 hours, or within 6 hours, or within 2 hours, or within 1 hour) after completion of administration of a dose of an anti-tumor agent or immune checkpoint inhibitor.

[0148] In some cases, the second pharmaceutical composition (i.e., at least one anti-tumor agent of a chemotherapy regimen) and the third pharmaceutical composition (e.g., bevacizumab) are administered simultaneously such that their administration overlaps. In one example, at least one anti-tumor agent (e.g., 5-FU) is administered intravenously over a period of minutes, and the administration is continued via a pump (e.g., an ambulatory infusion pump) that delivers 5-FU continuously for 46-48 hours. During the course of the 5-FU infusion, bevacizumab is administered over 30-90 minutes. Alternatively, bevacizumab is administered before (e.g., 1 hour before) the 5-FU infusion begins or after (e.g., 1 hour after) the 5-FU infusion is completed.

[0149] In yet another example, the SapC-phospholipid nanovesicle formulation is initially administered at a given dose on multiple consecutive days over a period of time (e.g., once a day for 3, 4, 5, or 6 consecutive days in the first week). Then, in weeks 2-4, the frequency of administration of the SapC-phospholipid formulation is reduced (e.g., 3 times a week or once a week). From week 5 to 6 months, the SapC-phospholipid formulation is administered once every 14 (+ / - 3) days. From 6 months to 2 years, the SapC-phospholipid formulation is administered once every 28 (+ / - 3) days. Meanwhile, the chemotherapy regimen or immune checkpoint inhibitor, and optional bevacizumab, may be administered according to standard protocols, for example, once every 14 (+ / - 3) days, starting within 48 hours (e.g., within 24 hours or within 12 hours or within 6 hours or within 2 hours or within 1 hour) after the completion of the last administration of the consecutive dose of SapC-phospholipid nanovesicle formulation, and continued for up to 6 months. The chemotherapy regimen or immune checkpoint inhibitor, and optional bevacizumab treatment may be continued as a maintenance treatment for up to 2 years, at the discretion of the attending physician or according to standard treatment. In a further example, the chemotherapy regimen or immune checkpoint inhibitor regimen, and optional bevacizumab treatment may be initiated long after the initiation of the SapC-phospholipid regimen, for example, at week 2, week 3, week 4, week 5, or week 6.

[0150] As an example, the SapC-phospholipid nanovesicle formulation is administered once a day for 5 consecutive days during the first week of treatment, once every 2 days (or 3 times a week) during the second week, and approximately once a week (i.e., once every 7 (+ / - 3) days) during the third and fourth weeks of treatment. Thereafter, the SapC-phospholipid nanovesicle formulation is administered once every 14 (+ / - 3) days, or once every 7 (+ / - 3 days), or once every 21 (+ / - 3 days), or once every 28 (+ / - 3 days). The chemotherapy regimen or immune checkpoint inhibitor regimen is administered once every 14 (+ / - 3) days. Bevacizumab is administered once every 14 (+ / - 3) days, or once every 21 (+ / - 3) days.

[0151] In another example, the SapC-phospholipid nanovesicle formulation is administered as in the above example after completing a standard chemotherapy regimen (optionally including a biologic, e.g., bevacizumab) or immune checkpoint inhibitor regimen. In yet another example, administration of the SapC-phospholipid nanovesicle formulation is initiated midway through a chemotherapy regimen (optionally including a biologic, e.g., bevacizumab) or immune checkpoint inhibitor regimen.

[0152] Each dose of the SapC-phospholipid nanovesicle formulation is administered over about 45 minutes to about 120 minutes (eg, about 45 minutes, about 60 minutes, or about 90 minutes).

[0153] The active ingredients of the first, second, and third pharmaceutical compositions (i.e., the nanovesicle formulation comprising SapC-phospholipid, the one or more anti-tumor agents of the chemotherapy regimen, or the one or more immune checkpoint inhibitors, and / or the biologics) are typically not present in the same composition.

[0154] In another example, treatment with SapC-phospholipid nanovesicle formulations and immune checkpoint inhibitors is indicated for subjects who have not responded to other treatments, such as steroids, radiation therapy, or chemotherapy. In some cases, treatment includes front-loading with SapC-phospholipid nanovesicle formulations prior to treatment with immune checkpoint inhibitors.

[0155] E. Therapeutically Effective Doses for Treating Cancer As described above, the methods of treating cancer (e.g., gastrointestinal cancer) provided herein include administering to a subject in need thereof a nanovesicle formulation having SapC-DOPS as an active agent and a chemotherapy regimen having one or more antitumor agents, each agent being administered in an amount and for such time as is necessary to achieve the desired result (i.e., a "therapeutically effective amount").

[0156] With respect to the treatment of cancer, a "therapeutically effective amount" or "therapeutically effective dose" of a nanovesicle formulation or anti-tumor agent is an amount useful to treat cancer in a patient, i.e., an amount of agent that modulates or ameliorates a symptom or condition of a cancer, tumor, or neoplastic disease, e.g., prevents or reduces survival of cancer cells. It may include a single treatment or a series of treatments.

[0157] The therapeutic dose of the composition of the present disclosure (i.e., nanovesicle formulations containing SapC-phospholipids and / or antitumor agents of chemotherapy regimens) can be determined by the attending physician within the scope of sound medical judgment and experience. For active agents, therapeutically effective doses can be estimated initially in cell culture assays or in animal models, such as mice, rats, rabbits, dogs, pigs, or monkeys. Animal models can be used to achieve or determine desirable concentrations and full dosage ranges and administration routes, which can be used to determine useful dosage ranges and administration routes for administration to humans. In addition, clinical trials and individual patient response can determine recommended therapeutic doses.

[0158] In general, a therapeutically effective dose of a nanovesicle formulation will contain an amount of SapC (or a derivative thereof) ranging from about 0.01 to 30 mg / kg body weight, preferably about 0.05 to 20 mg / kg body weight, more preferably about 0.1 to 15 mg / kg body weight, and even more preferably about 0.5 to 10 mg / kg. For example, the amount of SapC in an intravenous dose can be about 0.4 mg / kg, 0.7 mg / kg, 1.1 mg / kg, 1.4 mg / kg, 1.8 mg / kg, 2.4 mg / kg, 2.8 mg / kg, 3.0 mg / kg, 3.2 mg / kg, 3.6 mg / kg, 7 mg / kg, or more. A given patient may receive a given dose level for one or more initial administrations and different (lower or higher) levels for further administrations.

[0159] The therapeutic dose of the antitumor drug oxaliplatin is 50-150 mg / m 2 In the range of, for example, 85 mg / m2 The therapeutic dose of the antitumor agent 5-fluorouracil is 500-2400 mg / m 2 In the range of, for example, 2400 mg / m 2 The therapeutic dose of the antitumor agent paclitaxel is 90 mg / m 2 ~250mg / m 2 , e.g., 125 mg / m 2 ~175mg / m 2 The therapeutic dose of bevacizumab is in the range of 5-15 mg / kg, for example, 5 mg / kg, 7.5 mg / kg, 10 mg / kg, or 15 mg / kg. The 5 mg / kg, 7.5 mg / kg, and 10 mg / kg doses are generally administered every 2 weeks. The 15 mg / kg dose is generally administered every 3 weeks.

[0160] The results of treatment with the methods of the present disclosure may be assessed or determined by any method known to one of skill in the art, including, but not limited to, imaging, ultrasound, physical examination, or blood tests.

[0161] As used herein, a "pharmaceutical composition" refers to any chemical or biological composition, material, agent, etc., that is capable of inducing a therapeutic effect when properly administered to a subject, including, for example, the inactive form of the composition, material, agent, etc., and any active metabolites thereof that may be formed in vivo.

[0162] The results of treatment with the methods of the present disclosure may be assessed or determined by any method known to one of skill in the art, including, but not limited to, imaging, ultrasound, physical examination, blood tests, and radioimmunoassays.

[0163] kit The pharmaceutical compositions described herein may be included in a kit, pack, or dispenser, optionally together with instructions for administration of such pharmaceutical compositions, which are collectively referred to herein as "kits". In some embodiments, the kit may include a first pharmaceutical composition comprising a saposin C-phospholipid formulation (e.g., in a solid form, e.g., in the form of a lyophilized powder) and a second pharmaceutical composition comprising at least one immune checkpoint inhibitor (e.g., a PD-1 inhibitor selected from nivolumab, pembrolizumab, pidilizumab, or MEDI0680; a PD-L1 inhibitor selected from atezolizumab, BMS-936559, MEDI4736, or MSB0010718C; or a CTLA-4 inhibitor selected from ipilimumab or tremelimumab).

[0164] In some embodiments, the kit may include a first pharmaceutical composition, i.e., a nanovesicle formulation comprising SapC-phospholipid, and a second pharmaceutical composition comprising at least one anti-tumor agent (e.g., oxaliplatin or 5-FU) in separate containers, and may further include a third container containing a third composition, which may be, for example, another anti-tumor agent, e.g., oxaliplatin or 5-FU. The kit may also include a container containing a composition comprising a biologic, e.g., bevacizumab.

[0165] Optionally, additional pharmaceutical compositions may be included in the kit. The kit may also include one or more containers of pharma- ceutically acceptable carriers (e.g., sterile water or saline) suitable for reconstituting or diluting each pharmaceutical composition included in the kit. The compositions and carriers may be contained in vials or other suitable containers, such as syringes. Typically, the first and second pharmaceutical compositions will be in separate containers in the kit. Each composition may be in the form of a dry powder, liquid, suspension in liquid, frozen, or any other suitable form.

[0166] It should be understood that the disclosed methods and compositions may assume various alternative variations and sequences of steps unless expressly specified to the contrary. Moreover, except in the examples or where otherwise indicated, all numerical values, e.g., values, amounts, percentages, ranges, subranges, and fractions, may be read as if preceded by the word "about", even if the term "about" is not expressly stated. Thus, unless otherwise indicated, the numerical parameters set forth in this specification and the appended claims are approximations that may vary depending on the results desired to be obtained by the disclosed method. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should be construed by applying ordinary rounding techniques, at least in light of the number of reported significant digits. When a closed-ended or open-ended numerical range is set forth herein, all numbers, values, amounts, percentages, subranges, and fractions within or subsumed within that numerical range are to be considered as specifically included and belonging to the initial disclosure of this application as if those numbers, values, amounts, percentages, subranges, and fractions were fully and explicitly written out.

[0167] Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values ​​set forth in the specific examples are reported as precisely as possible, however, any numerical value inherently contains certain errors necessarily resulting from the standard variation found in their respective testing measurements.

[0168] As used herein, unless otherwise indicated, plural terms may include their corresponding singular forms and vice versa. For example, although a "composition" is referred to herein, a combination of those components (i.e., a plurality of those components) may be used. Additionally, in this application, the use of "or" means "and / or" unless otherwise indicated, although in certain instances "and / or" may be explicitly used.

[0169] As used herein, the terms "including," "containing," and the like are understood to be synonymous with "comprising" with respect to this application and, therefore, are open-ended and do not exclude the presence of additional, unrecited and / or unrecited elements, materials, components, and / or method steps.

[0170] As used herein, "consisting of" is understood in the context of the present application to exclude the presence of any unspecified element, component, and / or method step.

[0171] As used herein, "consisting essentially of" is understood in the context of this application to include specified elements, materials, components, and / or method steps, as well as unspecified others that "do not materially affect the basic and novel characteristics" of what is described.

[0172] The compositions and methods are further supported by the information provided in the following examples. It should be understood that the embodiments described in the examples are illustrative only and are not intended to limit the scope of the present disclosure, which is limited only by the appended claims. EXAMPLES

[0173] Example 1. SapC-DOPS (BXQ-350) ameliorates neuropathy in subjects diagnosed with cancer and / or diabetes A Phase 1 study to determine the anti-cancer efficacy of BXQ-350 was conducted in adult patients with advanced solid tumors who had previously been treated with chemotherapy (Clinical Trials.gov Identifier NCT02859857). Each participant received multiple doses of BXQ-350 at a dose level of 2.4 mg / kg. Unexpectedly, some patients in the study experienced relief of ongoing neuropathic symptoms. These patients are described below.

[0174] Patient 1: This patient is a 70-year-old female with a history of metastatic pancreatic cancer. The patient completed 5 cycles of FOLFIRINOX (folinic acid / fluorouracil / irinotecan / oxaliplatin) with a good response. Oxaliplatin was discontinued due to grade 2 peripheral sensory neuropathy. One additional cycle of FOLFIRI was completed followed by 3 weeks of gemcitabine and radiation therapy. Two years later, the patient developed metastases to the lungs from a known primary pancreatic cancer. The patient was treated with symptomatic gemcitabine and nab-paclitaxel for approximately 2 more years (22 months) with persistent grade 1 peripheral sensory neuropathy before disease progression. The patient was enrolled in the BXQ-350 clinical trial. After two cycles of 2.4 mg / kg intravenous BXQ-350, the patient reported complete resolution of grade 1 peripheral sensory neuropathy, which had been present since treatment with oxaliplatin 5 years earlier, along with redness of the feet accompanied by neuropathic pain. The patient's neuropathy improved to the point that FOLFOX could be resumed following disease progression.

[0175] Patient 2: A metastatic colorectal cancer patient with persistent neuropathy associated with many years of previous treatment reported resolution of neuropathy symptoms while being treated with 2.4 mg / kg intravenous BXQ-350.

[0176] Patient 3: A patient with metastatic parotid / salivary gland carcinoma with persistent residual peripheral sensory neuropathy in the hands and feet from previous treatment with cisplatin / navelbine and paclitaxel, currently being treated with gabapentin, noted resolution of numbness and tingling in the hands and some improvement in the feet where numbness persisted after treatment with 2.4 mg / kg intravenous BXQ-350.

[0177] Patient 4: A patient with a history of esophageal cancer and peripheral neuropathy in both feet was treated with 2.4 mg / kg BXQ-350 intravenously. The patient reported mild improvement on the CIPN questionnaire: tingling in toes / feet improved from 3 on a scale of 1 to 4 to 2 on day 113 from the start of BXQ-350 treatment (1 indicating no tingling and 4 indicating severe tingling); numbness in toes / feet improved from 3 on day 1 to 2 on the remaining visits; plantar ground sensation improved from 2 on day 1 to 1 on the remaining visits; and walking difficulty due to foot drop improved from 3 on day 1 to 2 on days 57 and 85 and to 1 on day 113.

[0178] Patient 5: A pancreatic cancer patient with persistent grade 1 platinum-induced peripheral neuropathy saw improvement during treatment with BXQ-350.

[0179] Patient 6: A pancreatic cancer patient with neuropathy due to FOLFOX treatment (last dose in November 2018) saw improvement in his neuropathy after starting treatment with BXQ-350 in March 2019.

[0180] Patient 7: A patient with metastatic colon cancer with grade 1 peripheral neuropathy associated with previous treatment with FOLFOX (treatment ended October 2018) began treatment with BXQ-350 on February 18, 2019. The patient subjectively noted improvement in his neuropathy symptoms while receiving BXQ-350.

[0181] Of the 10 patients on the study who had chemotherapy-induced peripheral neuropathy (CIPN) symptoms and were questioned and evaluated for their neuropathy symptoms: 4 patients with long-term peripheral neuropathy (patients 1-4) noted resolution or improvement of symptoms; 3 patients (patients 5-7) noted resolution of symptoms, but due to the timing between chemotherapy treatment and BXQ-350 treatment, it was unclear whether this was due to cessation of chemotherapy, e.g., drug holidays, or due to a beneficial effect of BXQ-350; 3 patients reported no change in their pre-existing neuropathy symptoms while being treated with BXQ-350. Persistent CIPN typically does not improve after several years, so it was surprising and unexpected that 4 out of 10 patients clearly reported resolution / improvement of persistent peripheral neuropathy several years after chemotherapy completion.

[0182] Additionally, the study included a patient with gastrointestinal stromal tumor (GIST). He also had long-standing diabetic neuropathy. After starting 2.4 mg / kg of intravenous BXQ-350, the patient noted that his symptoms of intermittent tingling in his fingers became subjectively much less frequent.

[0183] Example 2. SapC-DOPS (BXQ-350) prevents neuropathic symptoms in a mouse model of chemotherapy (oxaliplatin)-induced peripheral neuropathy (CIPN) BXQ-350 was tested in the mouse oxaliplatin model of chemotherapy-induced neuropathy. All experiments were performed in accordance with the ethical guidelines of the International Association for the Study of Pain (Zimmerman, 1983). All behavioral tests were approved by the United Kingdom Home Office Animals (Scientific Procedures) Act 1986.

[0184] background Chemotherapy-induced neuropathy (CIN) is a significant side effect of various chemotherapy drugs. Oxaliplatin, a platinum-based chemotherapy drug, is commonly used for the treatment of various types of cancer and is known to cause acute and chronic peripheral neuropathy in rodents (Ling et al., 2007). Cold hypersensitivity, which is considered a hallmark of oxaliplatin-induced neuropathy, can be characterized as abnormal cold-induced sensations localized to the hands and feet (Attal et al., 2009). This cold hypersensitivity can also be seen in rodents after a single intraperitoneal injection of oxaliplatin (e.g., Ling et al., 2007; Zhao et al., 2012).

[0185] Research 1 animal Sixty 6-7 week-old adult male C57 / BL6 mice (Charles River) were housed in groups of five in Perspex cages in a controlled environment with constant temperature and humidity (temperature: 21 ± 1 °C, 12:12 h light: dark cycle) and had free access to food and water. The animals were allowed to recover from transport for at least 1 week before the start of the experiment. The mice were assigned to the various groups shown in Table 2.

[0186] (Table 2) Animal group TIFF2024504066000004.tif57160CIN:Chemotherapy-induced neuropathy

[0187] Experimental design As shown in Table 2, mice in groups A-E (n=12 per group) were administered 10 mg / kg oxaliplatin or vehicle intraperitoneally on day 0 and then administered test articles as follows: 10 mg / kg vehicle or 20 mg / kg BXQ-350 on days -3, -2, -1, and days 1-9; or 30 mg / kg pregabalin on days 3, 5, 7, and 9. Figure 1A shows the timeline of administration of various treatments to mice in groups A-E.

[0188] Study 1 Instrumentation and Evaluation Static mechanical tactile allodynia: Static mechanical (tactile) allodynia was determined by measurement of withdrawal thresholds using calibrated (force; g) von Frey monofilaments (Touch-Test Sensory Evaluator; Scientific Marketing Associates) applied to the plantar surface of the left hind paw. Animals were placed on an elevated metal mesh in individual Perspex boxes for 30-40 min for habituation before testing. A series of progressive von Frey hairs (0.07 g, 0.16 g, 0.4 g, 0.6 g, and 1 g) were applied in sequence in a 1 sec on, 1 sec off protocol repeated 10 times. Each hair was applied perpendicular to the center of the ventral surface of the paw until it was slightly bent. The force applied to the animal's hind paw to elicit a response on 5 out of 10 trials was recorded as paw withdrawal threshold (PWT) 50%. The results of the study are shown in Table 3 (raw data), Table 4 (summary), Figure 2, and Figure 3.

[0189] Table 3. Individual paw withdrawal threshold 50% (von Frey filament) values ​​(g) TIFF2024504066000005.tif189170 Table 3: Individual paw withdrawal threshold 50% (von Frey filament) values ​​(g). ID numbers indicate mouse ID. BL: baseline; D3, D5, D7, and D9 refer to days 3, 5, 7, and 9, respectively.

[0190] Table 4. Summary of results: Mechanical allodynia TIFF2024504066000006.tif75170Table 4: Paw withdrawal threshold 50% (g) for the left hind paw. Two-way ANOVA showed a main effect of time (F(3.931,211.3)=5.518; p<0.001), a main effect of treatment (F(4,54)=5.518; p<0.0001), and a significant interaction between the time and treatment factors (F(16,2.2926)=2.926). *, **, ***, and **** indicate statistically significant differences of p<0.05, p<0.01, p<0.001, and p<0.0001, respectively, when compared to the oxaliplatin / vehicle group (group B) at the same time points. Values ​​are presented as mean ± SEM.

[0191] Thermal hypersensitivity: Thermal hypersensitivity was determined using a dynamic cold plate (Bioseb, France) set at a fixed temperature of 15°C. This method allows for the analysis of reflex behaviors, such as the tail immersion test, more integrated than the behavioral analysis. In animal studies, thermal sensitivity is primarily evaluated based on the latency of the nociceptive reaction in response to an applied thermal aversive stimulus. In this classical test, the temperature of the plate is fixed and remains constant: the latency to first lick, jump or withdraw the paw is taken as an index of the nociceptive threshold. Normal animals typically show withdrawal behavior at approximately 5°C, while animals treated with oxaliplatin show the same withdrawal behavior at higher temperatures (approximately 15°C). The results of the study are shown in Table 5 below (raw data), Table 6 below (summary), Figures 4 and 5.

[0192] Table 5. Individual withdrawal latencies in the cold plate test TIFF2024504066000007.tif188170 Table 5: Individual latencies to withdrawal behavior in the cold plate test measured in seconds (s). ID numbers indicate mouse ID. BL: baseline; D3, D5, D7, and D9 refer to days 3, 5, 7, and 9, respectively.

[0193] Table 6. Summary of results: Heat sensitivity TIFF2024504066000008.tif58157Table 6: Latency to withdrawal in the cold plate test (15°C) measured in seconds (s). Summary of data from individual groups and time points. Values ​​are presented as mean ± SEM.

[0194] In the oxaliplatin model of chemotherapy-induced neuropathy (CIN), no iatrogenic effects were demonstrated. Nevertheless, von Frey mechanical allodynia was always tested first, followed by cold-plate thermal allodynia.

[0195] Body weight: Mice were weighed daily starting 1 day before treatment through day 9. Individual body weights are shown in Table 7 below.

[0196] (Table 7) Individual body weight (g) TIFF2024504066000009.tif140170TIFF2024504066000010.tif209170

[0197] BQX-350 and pregabalin formulations A warm vial of BQX-350 was brought to room temperature and reconstituted with 4 mL of water for injection via syringe. The vial was gently agitated and / or vortexed for 15-20 seconds and left at room temperature for 5 minutes. The rehydration solution contained 2.2 mg / mL SapC (8.8 mg SapC per vial). Each mouse was injected with approximately 10 ml of the formulation to achieve a target dose of 20 mg / kg. Pregabalin was reconstituted with water and injected into each mouse to achieve a target dose of 30 mg / kg.

[0198] statistical analysis A two-way repeated measures analysis of variance with “treatment” as a between-subject effect and “day” as a within-subject effect (using GraphPad Prism version 9.0 for Windows, GraphPad Software, San Diego California USA) was used, followed by Dunnett's multiple comparison test. Data are presented as means ± SEM.

[0199] Results of Study 1 Oxaliplatin administration induced a consistent and sustained mechanical allodynia response and increased heat (cold) sensitivity as measured from day 3 after oxaliplatin administration through day 9, the final day of behavioral assessment.

[0200] In oxaliplatin-treated animals, administration of pregabalin at a dose of 30 mg / kg 2 hours prior to behavioral assessment reduced mechanical allodynia and reduced heat sensitivity. Pregabalin was used as a standard positive control in the assay.

[0201] BXQ-350 showed a statistically significant protective effect in the cold plate test, but not in the mechanical allodynia assessment, in animals treated with oxaliplatin. Administration of BXQ-350 alone led to a trend towards increased mechanical allodynia. This trend was not evident in the cold hypersensitivity test.

[0202] Research 2 animal Sixty 6-7 week old adult male C57 / BL6 mice (Charles River) were housed in groups of five in Perspex cages in a controlled environment with constant temperature and humidity (Temperature: 21 ± 1 °C, Light: Dark cycle 12:12 h) and had free access to food and water. The animals were allowed to recover from transport for at least one week before the start of the experiment. The mice were assigned to the various groups shown in Table 8.

[0203] (Table 8) Animal group TIFF2024504066000011.tif57160CIN:Chemotherapy-induced neuropathy

[0204] Experimental design As shown in Table 8, mice in groups A-E (n=12 per group) were administered 10 mg / kg oxaliplatin or vehicle intraperitoneally on day 0 and test articles as follows: 10 mg / kg vehicle, 2 mg / kg BXQ-350, or 10 mg / kg BXQ-350 on days -3, -2, -1, and 1-9; or 30 mg / kg pregabalin on days 3, 5, 7, and 9. Figure 1B shows the timeline of administration of various treatments to mice in groups A-E.

[0205] Study 2: Instrumentation and Evaluation Static mechanical tactile allodynia: The same apparatus and protocol were used as in Study 1. The results of Study 2 are shown in Table 9 (raw data), Table 10 (summary), Figure 6, and Figure 7.

[0206] Table 9. Individual paw withdrawal threshold 50% (von Frey filament) values ​​(g) TIFF2024504066000012.tif104170TIFF2024504066000013.tif158170Table 9: Individual paw withdrawal threshold 50% (von Frey filament) values ​​(g). ID numbers indicate mouse ID. BL: baseline; D3, D5, D7, and D9 refer to days 3, 5, 7, and 9, respectively.

[0207] Table 10. Summary of Results: Mechanical Allodynia TIFF2024504066000014.tif81170Table 10: Paw withdrawal threshold 50% (g) for the left hind paw. Two-way ANOVA showed a main effect of time (F(4.834,265.9)=4.829; p<0.001), a main effect of treatment (F(4,55)=17.50; p<0.0001), and a significant interaction between the time and treatment factors (F(24,330)=6.502. *, **, ***, and **** indicate statistically significant differences of p<0.05, p<0.01, p<0.001, and p<0.0001, respectively, when compared to the oxaliplatin / vehicle group at the same time points. Values ​​are presented as mean ± SEM.

[0208] Heat Hypersensitivity: The same equipment and protocol was used as in Study 1. The results of Study 2 are shown in Table 11 (raw data), Table 12 (summary), Figures 8 and 9.

[0209] Table 11. Individual withdrawal latencies (s) in the cold plate test TIFF2024504066000015.tif107170 Table 11: Individual latencies to withdrawal behavior in the cold plate test measured in seconds (s). ID numbers indicate mouse ID. BL: baseline; D3, D5, D7, and D9 refer to days 3, 5, 7, and 9, respectively.

[0210] Table 12. Summary of Results: Heat Sensitivity TIFF2024504066000016.tif62170Table 12: Latency to withdrawal in the cold plate test (15°C). Two-way ANOVA showed a main effect of time (F(6,330) = 8.849; p < 0.0001), a main effect of treatment (F(4,55) = 2.775; p = 0.0358), and a significant interaction between time and treatment factors (F(24,330) = 8.849. *, **, and *** indicate statistically significant differences of p < 0.05, p < 0.01, and p < 0.001, respectively, when compared to the oxaliplatin / vehicle group at the same time point. Values ​​are presented as mean ± SEM.

[0211] Body weight: Mice were weighed daily starting 1 day before treatment through day 9. Individual body weights are shown in Table 13 below.

[0212] Table 13. Individual body weight (g) TIFF2024504066000017.tif169170TIFF2024504066000018.tif168170TIFF2024504066000019.tif86170

[0213] Results of Study 2 Administration of oxaliplatin induced a consistent and sustained mechanical allodynia response on the days assessed. Treatment with BXQ-350 significantly prevented the reduction of PWT (paw withdrawal threshold level) in a dose-dependent manner. On all days assessed, the group treated with BXQ-350 showed significantly higher PWT levels compared to the Veh / Oxal group, but the group treated with BXQ-350 at a dose of 2 mg / kg was not significantly different from the Veh / Oxal group on days 3 and 9.

[0214] BXQ-350 showed a consistent trend in protective effects in animals treated with oxaliplatin in the cold plate test. On days 7 and 10, there was lower intragroup variability and some statistically significant differences. On day 7, the group treated with BXQ-350 10mg / kg was significantly different from the Veh / Oxal group.

[0215] conclusion These results indicate that BXQ-350 can protect mice from both oxaliplatin-induced neuropathic pain and oxaliplatin-induced cold hypersensitivity in a mouse model of mechanical allodynia.

[0216] Example 3. Efficacy and safety of BXQ-350 (SapC-DOPS) for reducing oxaliplatin-induced sensory neurotoxicity and treating newly diagnosed metastatic colorectal cancer A placebo-controlled, double-blind, Phase 2 efficacy and safety study to determine (a) the efficacy of BXQ-350 + mFOLFOX7 (modified FOLFOX7: 5-fluorouracil (5-FU) / leucovorin (LV) / oxaliplatin) and bevacizumab (Avastin) in treating patients with metastatic colorectal cancer (mCRC); and (b) whether BXQ-350 reduces the onset, intensity, and / or duration of oxaliplatin-induced neurosensory toxicity in subjects with mCRC receiving mFOLFOX7 may be configured as follows: Leucovorin calcium (200 mg / m 2 ) and oxaliplatin (85 mg / m 2 ) was administered to patients over 2 hours on day 1, followed by 5-fluorouracil (5FU) infused over 46 hours on days 1 and 2 (2,400 grams [g] / m 2), a chemotherapy regimen mFOLFOX7, infused every 2 weeks for 6 months for a total of 12 infusions. Eligible subjects will be randomized in a 1:1 ratio to receive either 2.4 milligrams (mg) / kilogram (kg) BXQ-350 or placebo administered intravenously concomitantly with mFOLFOX7 (days 1-5, 8, 10, 12, 15, and 22 in cycle 1; every 14 days starting on day 29 in cycles 2-6). Eligible subjects are aged 18 years or older, have newly diagnosed metastatic colon / rectal adenocarcinoma, are receiving mFOLFOX7, have an Eastern Cooperative Oncology Group (ECOG) Performance Status of 0 or 1, and have a life expectancy of greater than 3 months.

[0217] Subjects will receive either BXQ-350 or placebo concurrently with mFOLFOX7 and bevacizumab treatment according to the dosing schedule in Table 14.

[0218] Table 14: BXQ-350 / placebo will be administered by IV infusion over 60 minutes ± 5 minutes according to the following schedule: TIFF2024504066000020.tif83160* 2-day mFOLFOX7 regimen: Leucovorin calcium (200 mg / m 2 ) along with oxaliplatin (85 mg / m 2 ) was infused over 2 hours, followed by 5-FU (2,400 milligrams [mg] / m2) infused over 46 hours. 2 ) infused every 2 weeks for 6 months (total of 12 infusions). mFOLFOX7 = infused according to institutional policy and procedure. Bevacizumab = 5 mg / kg IV. Infused according to institutional policy and procedure.

[0219] Study Duration: Treatment with BXQ-350 or placebo in combination with mFOLFOX7 and bevacizumab may begin after 6 cycles of trial entry (primary treatment period). After 6 cycles (extension treatment period), all participants without progression or other discontinuation criteria may continue maintenance mFOLFOX7 and / or bevacizumab according to the discretion of the attending physician. After 6 cycles, BXQ-350 / placebo treatment transitions to a once every 28 days dosing schedule and continues for up to 720 days (approximately 26 cycles) from study day 1 until disease progression or other discontinuation rules are met, with an end-of-treatment visit occurring 2 weeks after discontinuation of BXQ-350 treatment. Participants will be followed every 28 days from study day 1 to study day 548 for neuropathy assessment, and every 12 weeks for survival from study day 1 to study day 1,825, death, withdrawal of consent, or completion of the study, whichever occurs first.

[0220] Research objectives: Treatment of mCRC One objective may include determining the efficacy of BXQ-350 plus modified FOLFOX7 (mFOLFOX7) treatment and bevacizumab in patients with mCRC, as measured by objective response rate (ORR), which will be determined according to Response Evaluation Criteria in Solid Tumors (RECIST) v1.1, as assessed by the investigator.

[0221] Secondary objectives included: (a) Overall survival (OS), defined as the time from the date of randomization to the date of death from any cause; (b) progression-free survival (PFS), defined as the time from the date of randomization to the date of disease progression or death; (c) duration of response (DoR), defined as the time from demonstration of disease response to disease progression; and (d) Disease control rate (DCR), defined as the proportion of participants with best overall response (BOR) complete response (CR), partial response (PR), and stable disease (SD) The efficacy of BXQ-350 + modified FOLFOX7 (mFOLFOX7) treatment and bevacizumab in patients with mCRC may be assessed, as measured by:

[0222] In addition, the pharmacokinetics (PK) of BXQ-350 combined with mFOLFOX7 and bevacizumab can be characterized. Furthermore, the overall safety and tolerability of BXQ-350 combined with mFOLFOX7 and bevacizumab can be determined in all participants compared to mFOLFOX7 and bevacizumab alone. Adverse events for this purpose will be graded by the Common Terminology Criteria for Adverse Events [CTCAE] v5. In addition, clinical chemistry, hematology, coagulation and urinalysis, vital signs, physical examination, and weight will be determined. In addition, 12-lead electrocardiograms (ECGs) will be performed and Eastern Cooperative Oncology Group (ECOG) Performance Status will be determined.

[0223] Treatment of Neuropathy One objective may include determining whether BXQ-350 reduces the onset, intensity, and / or duration of chronic oxaliplatin-induced sensory neurotoxicity in subjects receiving modified FOLFOX7 (mFOLFOX7), as measured using the European Organization for Research and Treatment of Cancer (EORTC) Quality of Life (QLQ) questionnaires (QLC-C30 and CIPN 20).

[0224] Other purposes include: (a) To determine whether BXQ-350 administered with mFOLFOX7 provides greater exposure to oxaliplatin in terms of number of cycles received and total dose administered; (b) to determine the overall safety and tolerability of BXQ-350 in subjects diagnosed with mCRC receiving treatment with mFOLFOX7, with respect to adverse events; and (c) To determine whether BXQ-350 reduces symptoms of oxaliplatin-associated acute pain syndrome in subjects receiving mFOLFOX7 may be included.

[0225] Secondary objectives may be measured, for example, using the CIPN Assessment Tool (Tofthagen CS, et al. (2011) Cancer Nurs. 2011 Jul-Aug;34(4):E10-20) and the Post-Oxaliplatin Symptoms Questionnaire (Pachman DR, et al (2016) Supportive Care in Cancer. 24(12):5059-68).

[0226] Efficacy Determination for Neuropathy: One efficacy outcome for this pilot study may be a continuously measured Total Sensory Neuropathy Score derived from six individual EORTC QLQ-CIPN20 questions quantifying numbness, tingling, and pain in the fingers (or hands) and toes (or feet); the Total Sensory Neuropathy Score is converted to a scale of 0 to 100 (higher numbers are better). In one example, participants complete a questionnaire according to Table 15 below.

[0227] Table 15: Questionnaire schedule TIFF2024504066000021.tif83166

[0228] Questionnaires used for endpoint purposes may include the EORTC questionnaire (Table 16), the CIPN Assessment Tool questionnaire (Table 17), and the Post-Oxaliplatin Symptoms Questionnaire (Table 18), as shown below. The questionnaire in Table 17 was developed and used with permission from Dr. Cindy Tofthagen (Cancer Nursing, Vol. 00, No. 0, 2011). Table 18 was developed and used with permission from Dr. Charles Loprinzi.

[0229] Table 16: EORTC QLQ - CIPN20 questionnaire TIFF2024504066000022.tif100170TIFF2024504066000023.tif154170

[0230] (Table 17) CIPN Assessment Tool Questionnaire (Quantitative Items) If you have had any neuropathy symptoms in the past week (for example, numbness, tingling, or pain in your hands and / or feet), how much did these symptoms prevent you from: TIFF2024504066000024.tif140169

[0231] (Table 18) Post-Oxaliplatin Symptom Questionnaire TIFF2024504066000025.tif151170

[0232] Example 4 SapC-DOPS (BXQ-350) protects neurons from oxaliplatin and promotes neurite formation The neurostimulatory, neuroprotective, and neurotrophic properties of a SapC-phospholipid formulation (BXQ-350) were tested in vitro in two cell lines, PC-12 cells (derived from a rat adrenal medulla pheochromocytoma) and NS20Y cells (a cholinergic cell line derived from a mouse neuroblastoma). BXQ-350 alone and in combination with oxaliplatin were administered to the cells.

[0233] PC-12 cells (ATCC, Cat. No. CRL-1721) and NS20Y cells (Millipore Sigma, Cat. No. 08062517) were cultured at 1 × 10 per well in 1 mL of DMEM (Gibco, Cat. No. 11965-092) supplemented with 10% FBS and PenStrep. 4 Cells were seeded overnight in 24-well plates. Each drug tested was made into a solution at the appropriate concentration in dosing medium consisting of high glucose, glutamine-free, calcium-free DMEM (Gibco, catalog number 21068-028) supplemented with 4 mM glutamine, 1 mM calcium gluconate, and PenStrep. BXQ-350 was tested at SapC concentrations ranging from 5 nM to 1 μM, and oxaliplatin was tested at concentrations ranging from 100 nM to 4 μM. The seeding medium was completely removed from the cells, and then dosing medium containing the test drug was added.

[0234] Neurostimulation: BXQ-350 has both neurogenesis and neurotrophic activity in both cell lines tested. NS20Y cells (FIG. 10A) and PC-12 cells (FIG. 10C) treated with 50 nM BXQ-350 produced more cells and a higher percentage of cells with neuronal morphology compared to NS20Y and PC-12 cells grown in control medium (FIGS. 10B and 10D, respectively).

[0235] Neuroprotection: BXQ-350 has neuroprotective activity in PC-12 cells when challenged with oxaliplatin, a known cytotoxic drug with neuropathic effects. Although a 2 μM dose of oxaliplatin can significantly reduce cell viability (FIG. 13) and inhibit neurite outgrowth (FIG. 11C), too few viable cells remain to allow accurate calculation of the percentage of cells with neurites. Cells exposed to a combination of 2 μM oxaliplatin and 50 nM BXQ-350 showed both preserved cell viability (FIG. 13) and increased neurite outgrowth or neuritogenesis (FIG. 11B) compared to cells treated with oxaliplatin alone (FIG. 13 and FIG. 11C). The left image of each pair in FIG. 11 is at 4x magnification, and the right image of each pair in FIG. 11 is at 20x magnification.

[0236] Neurite generation: BXQ-350 was shown to stimulate neurite outgrowth in PC-12 cells. Images of cells were acquired on a Biotek Cytation™ 5. Images at 4x magnification were analyzed for neurite outgrowth by determining the percentage of cells with neurite growth at least 1 mm longer than the diameter of the cell body using Biotek Gen5 (ver. 3.05) software. This analysis of cell neurite outgrowth (Figure 12) shows that cells treated with the combination of oxaliplatin / BXQ-350 had a significantly higher percentage of cells with neurite outgrowth than untreated control cells. Indeed, the percentage of cells with neurite outgrowth in cells treated with oxaliplatin / BXQ-350 (Figure 11B) was closer to the percentage with neurite outgrowth in cells treated with BXQ-350 alone (Figure 11A) than the percentage with neurite outgrowth in untreated cells (Figure 11D).

[0237] Taken together, these results provide evidence that BXQ-350 has both neurodevelopmental and neurotrophic effects and can provide both neuroprotection and neurite outgrowth stimulation to neuronal cells under neuropathic stress, e.g., due to chemotherapy.

[0238] Example 5. SapC-DOPS (BXQ-350) protects neurons from cytotoxic agents and has neurostimulatory, neurotrophic, and neuroprotective properties The neurostimulatory, neuroprotective, and neurotrophic properties of the SapC-phospholipid formulation (BXQ-350) were further tested in vitro in two PC-12 and NS20Y cells. BXQ-350 alone and in combination with oxaliplatin, vincristine, paclitaxel, or hydrogen peroxide (H2O2) were administered to the cells.

[0239] PC-12 cells (ATCC, Cat. No. CRL-1721) and NS20Y cells (Millipore Sigma, Cat. No. 08062517) were cultured at 1 × 10 cells per well in 1 mL of DMEM (Gibco, Cat. No. 11965-092) supplemented with 10% FBS and PenStrep. 4 Cells were seeded overnight in 24-well plates. Each drug tested was brought to an appropriate concentration in dosing medium consisting of high glucose, glutamine-free, calcium-free DMEM (Gibco, catalog number 21068-028) supplemented with 4 mM glutamine, 1 mM calcium gluconate, and PenStrep. BXQ-350 was tested at concentrations ranging from 5 nM to 1 μM SapC concentration. The seeding medium was completely removed from the cells, followed by addition of dosing medium containing the test drug.

[0240] PC-12 and NS20Y cells were treated with BXQ-350 alone or in combination with oxaliplatin, vincristine, or paclitaxel. Oxaliplatin was tested at concentrations from 100 nM to 4 μM; vincristine was tested at 100 nM to 100 μM; and paclitaxel was tested at 100 nM to 20 μM. For combination studies, 50 nM BXQ-350 was co-administered with 100 nM to 4 μM oxaliplatin or 100 nM to 10 μM vincristine or 1 μM to 10 μM paclitaxel for 72 hours. Images were acquired on a Biotek Cytation™ 5 cell imaging system after full exposure to the compounds. Biotek Gen5 (ver. 3.05) software was used to determine neurite outgrowth, defined as tendril outgrowth >1 mm longer than the cell body. Cell numbers were determined from these images, with untreated cells serving as a baseline.

[0241] PC-12 and NS20Y cells were treated with 50 nM BXQ-350 alone or left untreated for 72 hours. After 72 hours, cells were washed with ice-cold PBS and treated with 200 μM H2O2 for 24 hours. Images were acquired on a Biotek Cytation™ 5 cell imaging system after full compound exposure. Biotek Gen5 (ver. 3.05) software was used to determine neurite outgrowth, defined as tendril growth >1 mm longer than the cell body. Cell numbers were determined from these images, with untreated cells as the baseline.

[0242] BXQ-350 as a single agent treatment exhibited neurotrophic properties, induced neurogenesis, neuritogenesis, and was neuroprotective.

[0243] Neurostimulation: BXQ-350 has both neurogenesis and neurotrophic activity in both cell lines tested. NS20Y cells (FIG. 14B) and PC-12 cells (FIG. 14A) treated with 50 nM BXQ-350 produced approximately 60% more cells than untreated controls. Furthermore, BXQ-350 caused significantly more neurite outgrowth in NS20Y neuronal progenitor cells (FIG. 14D) and PC-12 cells (FIG. 14C), indicating that BXQ-350 stimulates both division and differentiation of neuronal progenitor cell lines.

[0244] Neuroprotection: BXQ-350 has neuroprotective activity in PC-12 cells when challenged with oxaliplatin, vincristine, or paclitaxel, which are known cytotoxic drugs with neuropathic effects. PC-12 and NS20Y cells were co-treated with 50 nM BXQ-350 and either 2 μM oxaliplatin, 10 μM vincristine, or 3 μM paclitaxel for 72 hours. Untreated cells and cells treated with single agent treatments of 50 nM BXQ-350, 2 μM oxaliplatin, 10 μM vincristine, and 3 μM paclitaxel were included. In cells treated with oxaliplatin alone (Figures 15B and 16B), vincristine (Figures 17B and 18B), and paclitaxel (Figures 19B and 20B), massive cytotoxicity was observed, and the viability was far below 10%, close to zero. When BXQ-350 was added to the cytotoxic drug, the cell viability was significantly increased. The viability of the cytotoxic drug + BXQ-350 combination group was about 50% of that of the BXQ-350 alone group.

[0245] Neurite generation: BXQ-350 was shown to stimulate neurite outgrowth in PC-12 and NS20Y cells. Images of cells were obtained with a Biotek Cytation™ 5 cell imaging system. Images at 4x magnification were analyzed for neurite outgrowth by determining the percentage of cells with neurite growth at least 1 mm longer than the cell body using Biotek Gen5 (ver.3.05) software. This analysis of cell neurite outgrowth showed that cells treated with the combinations of oxaliplatin / BXQ-350 (Figures 15A and 16A), vincristine / BXQ-350 (Figures 17A and 18A), paclitaxel / BXQ-350 (Figures 19A and 20A) had a significantly higher percentage of cells with neurite outgrowth than control cells. 75-80% of cells treated with BXQ-350 alone had neurite outgrowth, compared with 35-50% of untreated cells and 35-60% of cells treated with the cytotoxic agent-BXQ-350 combination. Oxaliplatin, vincristine, and paclitaxel alone groups could not be analyzed for neurite outgrowth because cytotoxicity was too high to obtain acceptable sample sizes.

[0246] Next, BXQ-350 was administered in combination with a neurotoxic drug, hydrogen peroxide (H2O2). PC-12 cells and NS20Y cells were pretreated with 50 nM BXQ-350 for 72 hours or left untreated. Then, the medium (with or without drug) was removed, and medium containing 200 μM H2O2 was added to all groups for 24 hours. For PC-12 cells, in the untreated group, only about 35% of the cells were viable, while the group pretreated with BXQ-350 had a viability of about 74% (Figure 21B). For NS20Y cells, about 40% of the cells were viable, while the cells pretreated with BXQ-350 had a viable rate of about 81% (Figure 21A).

[0247] Taken together, these data suggest that BXQ-350 can simultaneously stimulate neurogenic cell division, promote neurite outgrowth, and exhibit neuroprotection when exposed to chemotherapeutic agents. Furthermore, these data indicate that BXQ-350 has the potential to protect against a variety of neurotoxic agents. These data also demonstrate that BXQ-350 has the ability to promote neuronal growth and neurite development in vitro. Furthermore, when combined with general or chemotherapeutic neurotoxic agents, BXQ-350 can exhibit significant neurotrophic activity.

[0248] Example 6. SapC-DOPS enhances the cytotoxic effects of oxaliplatin and 5-fluoracil in HT-29 colorectal cancer cell lines The ability of a SapC-phospholipid formulation (BXQ-350) to enhance the cytotoxic effects of the standard of care drugs oxaliplatin and 5-fluorouracil (5-FU) in FOLFOX was tested in in vitro cancer cell lines.

[0249] 1 × 10 cells per well in 100 μL of DMEM (Gibco, Cat. No. 11965-092) supplemented with 10% FBS and PenStrep. 5 HT-29 cells were seeded overnight in 96-well plates. Blank wells containing DMEM only were included. The next day, BXQ-350 lyophilized powder was resuspended in special dosing medium consisting of high glucose, glutamine-free, calcium-free DMEM (Gibco, catalog number 21068-028) supplemented with 10% FBS, 4 mM glutamine, 1 mM calcium gluconate, and PenStrep. Oxaliplatin and 5-FU were made into pH-controlled solutions. The desired test concentrations, either single agent or combination dosing, were made by diluting aliquots of the stock solutions into dosing medium.

[0250] Growth medium was removed from the culture plates and cells were treated with one of the following: single agents; combinations of BXQ-350 and oxaliplatin, BXQ-350 and 5-FU, or 5-FU and oxaliplatin; and a combination of all three agents.

[0251] To determine IC50, BXQ-350 was dosed at 8 μM-40 μM, with optimal BXQ-350 at 12 μM-16 μM. Oxaliplatin was dosed at 0.2 μM-1 μM, and 5-FU was dosed at 12 μM-24 μM. Each plate contained untreated cells as a control for comparison with treated cells. Plates were incubated at 37°C, 5% CO2 for 72 hours. After 72 hours, 10 μL of MTT salt solution was added to each well and the plates were returned to the incubator for 2 hours. Media was then removed from each well and replaced with 100 μL of DMSO. Plates were then incubated overnight as above. For data analysis, plates were read on a BioTek Synergy plate reader to measure absorbance at 570 nm. Cell viability was determined by first subtracting blank OD570 values ​​from all wells, then dividing the OD570 values ​​of treated cells by the OD570 values ​​of untreated cells and multiplying by 100. This provides the percentage of viable cells in treated samples, with the untreated control used as a baseline of 100% viability. BXQ-350 as a single agent was lethal at 16 μM, but surprisingly, a lower sublethal concentration of 12 μM BXQ-350 at 72 hours enhanced the cytotoxic effects of oxaliplatin and / or 5-FU.

[0252] Optimized dosing and MTT cell viability assay (Roche) were used to determine the percent cell viability in treated and untreated HT29 cells. BXQ-350, 5-FU, and oxaliplatin were used at concentrations of 12 μM, 12 μM, and 0.3 μM, respectively. All combinations were treated simultaneously for 72 h, while BXQ-350 was administered alone for 72 h. All treatments were performed in triplicate.

[0253] The triple treatment combination (BXQ-350 + oxaliplatin + 5-FU) shows significantly more cytotoxicity (reduction in % cell viability) compared to any of the doublet combinations (Figure 22). BXQ-350 and oxaliplatin show little cytotoxicity compared to the triplet combination which shows excellent lethality. There is a doubling of cytotoxicity in the triplet combination compared to the standard of care oxaliplatin and 5-FU or BXQ-350 and 5-FU.

[0254] conclusion These results indicate that BXQ-350 can significantly enhance the cytotoxic effects of oxaliplatin and / or 5-FU in cancer cell lines.

[0255] Example 7. SapC-DOPS and FOLFOX are differentially cytotoxic in various colorectal cancer cell lines The combined effect of SapC-DOPS with 5-fluorouracil (5-FU) or oxaliplatin or FOLFOX (5-FU + oxaliplatin) on the cytotoxicity of cancer cell lines in vitro was tested. Human colorectal adenocarcinoma cell lines, including SW480 (Cat. No.: CCL-228), SW620 (Cat. No.: CCL-227), HT29 (Cat. No.: HTB-38), HCT116 (Cat. No.: CCL-247), LoVo (Cat. No.: CCL229), and SW48 (Cat. No.: CCL231), were obtained from the American Type Culture Collection (ATCC). HT29, HCT116, LoVo, and SW48 cells were cultured in high glucose DMEM (Thermofisher-Gibco, Catalog No.: 11965-092) supplemented with 9% fetal bovine serum and 1% penicillin-streptomycin. SW480 and SW620 cells were cultured in either L-15 (ATCC, Catalog No.: 30-2008) or Leibovitz L-15 (Alphabioregen, Catalog No.: PG058) supplemented with 9% fetal bovine serum and 1% penicillin-streptomycin. Cells were cultured at 37°C in an atmosphere containing 5% CO2. At approximately 80-90% confluency, cells were detached using trypsin (Thermofisher-Gibco, Catalog No.: 25200-072) for use in assays or for passaging.

[0256] MTT Viability Assay: On day 1, 1.5 x 10 cells per well 4 MTT Assay of Cells Cells were seeded in high glucose DMEM medium in 96-well absorbance plates (Falcon, Cat. No.: 353072) and incubated at 37° C., 5% CO 2 for 24 hours to allow attachment.

[0257] On day 2, SapC-DOPS lyophilized powder (Lot No.: 130061) was resuspended and diluted in low calcium medium (calcium-free DMEM (Gibco, Catalog No.: 21068-028) supplemented with 9% FBS, 1% Pen-Strep, 4 mM L-glutamine, and 0.6 mM calcium chloride). Prior to the study, oxaliplatin lyophilizates were suspended in WIFI (Gibco, Lot No.: 2085277) and frozen in single-use aliquots. 5-FU lyophilizates were suspended in ammonium hydroxide (NH4OH; Sigma, Lot No.: BCCF1716) and frozen in single-use aliquots. Reconstituted 5-FU and oxaliplatin were diluted to 500 μM in low calcium medium for further use. Final concentrations and dilution volumes are shown in Table 19 below.

[0258] Table 19 Compound Dilution TIFF2024504066000026.tif67167

[0259] The medium was removed from the overnight cultures and the plates were treated simultaneously with the above compounds alone, in binary combinations, or in triple combination as SapC-DOPS+FOLFOX (without leucovorin). All plates were incubated at 37° C., 5% CO2 for 72 hours.

[0260] On day 4, 10 uL of MTT labeling reagent (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) (Sigma-Aldrich, Catalog Number: 11465007001) was added to each well. The plate was incubated for 2 hours at 37° C., 5% CO2. After incubation, 100 μL of the solution in each well was removed and replaced with 100 μL DMSO. The plate was incubated for 1 hour at room temperature and the absorbance of each well was measured at 570 nm using a Biotek Synergy™ H1 Hybrid microplate reader.

[0261] Data Processing: Percent cell viability is calculated as the absorbance from blank-subtracted treated wells versus the absorbance from blank-subtracted untreated wells. TIFF2024504066000027.tif11145

[0262] DMSO MTT experiment: SapC-DOPS up to 50 μM and FOLFOX up to 50 μM were individually tested for cytotoxicity in all colorectal cancer (CRC) cell lines by DMSO Stop MTT (FIGS. 23A and 23B). 50 μM FOLFOX is equivalent to 50 μM 5-FU and 50 μM oxaliplatin.

[0263] FIG. 23A shows toxicity curves for each of the six CRC cell lines when treated with SapC-DOPS. SW480 and SW620 cells were treated with 3 μM, 6 μM, 9 μM, 12 μM, 15 μM, 20 μM, 25 μM, and 30 μM SapC-DOPS. HT29, HTC116, LoVo, and SW48 cells were treated with 5 μM, 10 μM, 20 μM, 30 μM, 40 μM, and 50 μM SapC-DOPS. A wide range of responses was observed across cell lines. SW480 and SW620 cell lines show increased sensitivity to SapC-DOPS compared to previously tested cell lines. IC50s of 15-20 μM were observed in other cell lines, including melanoma, pancreatic, and brain / CNS cancer cell lines. In contrast, SW480 and SW620 had IC50s of 9-11 μM (data not shown). LoVo and SW48 were weakly sensitive to SapC-DOPS, and HT29 and HTC116 appeared to be at least partially resistant.

[0264] Figure 23B shows the cytotoxic response of each cell line to a single FOLFOX treatment (10 μM oxaliplatin and 10 μM 5-FU). Each cell line responded differently to this concentration of FOLFOX, with two cell lines (LoVo and SW48) being relatively more sensitive to FOLFOX treatment than the other lines tested. Because four of the six cell lines had 50-70% viable cells at 10 μM FOLFOX, this concentration was chosen as the starting point for testing synergy with SapC-DOPS.

[0265] SapC-DOPS was tested in combination with FOLFOX treatment. Several combinations and administration methods were examined, with maximum administration concentrations of 25 μM SapC-DOPS and 15 μM FOLFOX. Figure 24 shows the response of cell lines to one combination of 10 μM FOLFOX and 15 μM SAPC-DOPS administered simultaneously. Sequential administration was also performed (data not shown), but no differences were observed compared to simultaneous administration. In all cell lines, the combination of SapC-DOPS and FOLFOX resulted in lower cell viability than SapC-DOPS or FOLFOX alone.

[0266] SapC-DOPS and FOLFOX appear to function in a complementary manner, which is clinically relevant. SW480 and SW620 cell lines, which showed higher resistance to FOLFOX, were extremely sensitive to SapC-DOPS (see Figures 23A and 23B). LoVo and SW48 cell lines, which showed weak cytotoxicity to SapC-DOPS, were extremely sensitive to FOLFOX treatment (see Figures 23A and 23B). This suggests that the combination of SapC-DOPS and FOLFOX can be used to successfully treat a wider range of CRC tumors than either agent alone. Furthermore, HT29 and HCT116 cells, which showed relatively high resistance to single-agent treatment with FOLFOX and SapC-DOPS, had lower cell viability when treated with the combination (see Figures 23A, 23B, and 24). In such cases, the pro-neurotropic potential of SapC-DOPS may allow patients to tolerate FOLFOX treatment longer until a tumor response can be achieved. Taken together, these data indicate that the combination of SapC-DOPS and FOLFOX is more beneficial than FOLFOX alone.

[0267] Example 8. Repolarization of macrophages to the M1 phenotype The ability of a SapC-phospholipid formulation (BXQ-350) to repolarize M2 macrophages towards an M1 phenotype was examined in an ex vivo immune cell model using TNF-α as a marker of the M2 phenotype.

[0268] Peripheral mononuclear blood cells and macrophages were collected from two healthy human donors ("Donor A" and "Donor B"). + Macrophages were isolated from peripheral mononuclear blood cells and checked for purity and viability. Then, on day 0, CD14 +Macrophages were seeded in 384-well plates at a density of 5,000 cells / well with seeding medium. On day 5, the growth medium was removed from the culture plates and the cells were treated with BXQ-350 ranging from 2.5 μM to 9 nM in two-fold serial dilutions starting from 2.5 μM. Two reference compounds, prednisolone and dexamethasone, were used as positive controls in separate wells. Aliquots of 10 μM prednisolone and 10 μM dexamethasone were made by serially diluting the compounds 8 times in each well. One hour after adding BXQ-350 or the positive control compound, lipopolysaccharide (LPS) at a concentration of 10 ng / ml was added to the cells. As negative controls, LPS alone and LPS + 0.1% DMSO were added to separate wells. On day 8, cells were fixed with 4% formaldehyde, cells were counted using CD80 staining + DAPI counterstain, and TNF-α levels were quantified.

[0269] Figures 25A and 25B show that addition of BXQ-350 ranging from 2.5 μM to 9 nM results in a concentration-dependent decrease in TNF-α concentration in macrophages obtained from donor A (Figure 25A) and donor B (Figure 25B). The number of viable cells was not altered or even increased by the addition of BXQ-350, indicating that BXQ-350 is not cytotoxic. The BXQ-350 IC for repolarization to the M1 phenotype 50 were 0.77 μM and 0.75 μM for cells derived from donor A and donor B, respectively.

[0270] conclusion These results demonstrate that BXQ-350 effectively repolarizes macrophages toward an M1 antitumor phenotype ex vivo.

[0271] Example 9. T cell activation and associated killing effects As demonstrated in Example 1, BXQ-350 functions to repolarize M2 macrophages to an M1 phenotype. Whether BXQ-350 also promotes T cell-mediated antitumor immune responses was examined in an ex vivo 3D tumor spheroid model.

[0272] A549 human lung cancer cells transfected by lentivirus were seeded in 96-well ultra-low attachment plates at a concentration of 750 cells / well. The plates were then centrifuged at 1200 rpm for 2 minutes to promote cell aggregation and spheroid formation. The plates were then incubated at 37°C for 3 days. After 3 days, freshly isolated human peripheral blood mononuclear cells (PBMCs) were added to the spheroids at a concentration of 10,000 PBMCs / well. Various wells were added with one or more of the following: an activation cocktail containing anti-CD3 antibody at a final concentration of 1 ng / ml and IL-12 at a final concentration of 10 ng / ml; serial dilutions of BXQ-350 from 25 μM to 1.27 nM; and a positive control of pembrolizumab at 50 μg / ml. The negative control group had no addition to the spheroids and PBMCs. The final volume of each well was then adjusted to 200 μL per well.

[0273] The plates were then incubated for an additional 30 minutes before being placed in an Incucyte® live cell analyzer; fluorescence was analyzed every 4 hours for 6 days. Images were then analyzed using Incucyte® software to determine IC50. An increase in fluorescence intensity within the A549 tumor cell spheroids correlates with an increase in the number of live Luc-A549 cells within the spheroids, while a lack of fluorescence indicates cell death.

[0274] The results of the fluorescence intensity of each group of wells measured over a 6-day period are shown in FIG. 26. The no activation control group (line (12)) shown in FIG. 26 showed an increase in intensity over time in the 6-day study that correlated with an increase in cell number. Similarly, spheroids treated with 8.33 μM and 25 μM BXQ-350 (lines (1) and (2)) also showed an increase in fluorescence intensity, suggesting that no T cell-mediated anti-tumor immune response was observed in these experimental conditions. On the other hand, treatment with pembrolizumab (line (13)) or BXQ-350 at concentrations between 1.3 nM and 2.8 μM (lines (3)-(10)) showed an increase in cell death by day 6, demonstrating that BXQ-350 was more effective at killing cancer cells in this ex vivo 3D tumor spheroid cytotoxic T cell model than T cell-mediated killing alone. Finally, the data in Figure 26 was used to calculate the area under the curve to determine the concentration of BXQ-350 required for enhanced T cell stimulation and cytotoxicity. As shown in Figure 27, the concentration of BXQ-350 required for T cell stimulation is 0.104 μM.

[0275] conclusion These results demonstrate that BXQ-350 effectively stimulates T cell killing effects when combined with activated cytotoxic T cells.

[0276] Example 10. In vivo activity of BXQ-350 in a syngeneic mouse tumor model The activity of BXQ-350 was examined in the syngeneic CT26 model, a murine colorectal cancer model classified as a resistant model that does not respond adequately to immune checkpoint inhibitors, e.g., anti-PD1 antibodies, anti-CTLA4 antibodies, or anti-PD-L1 antibodies.

[0277] For this study, 7-9 week old female BALB / c mice were used. The study protocol was approved by the site's Institutional Animal Care and Use Committee (IACUC). Animals were maintained in individual cages with housing conditions required by the animal welfare guidelines. Diet also complied with the animal welfare guidelines. As shown in Table 20, this study included four animal groups treated with (1) vehicle control; (2) mouse SapC-DOPS (m-SapC-DOPS); (3) mouse anti-PD1 antibody (m-anti-PD-1) (n=10); and (4) m-SapC-DOPS + m-anti-PD1 antibody (n=10), as shown in the table below. The m-anti-PD-1 antibody was purchased from BioXcell (Lot No. 780120J2). m-SapC-DOPS was prepared using the procedure described by Qi et al., J Biol Chem, (1996) 22;271(12):6874-80, which discloses a procedure for replacing human SapC with mouse SapC.

[0278] (Table 20) Animal group TIFF2024504066000028.tif74147

[0279] CT26 cells were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum, 1% antibiotic-antimycotic solution at 37°C in an atmosphere of 5% CO in air. Exponentially growing cells were harvested and counted for tumor inoculation. For tumor development, CT26 tumor cells (0.3 × 10 6 Each mouse was inoculated subcutaneously into the right lower flank with 0.1 mL of serum-free RPMI-1640 containing 100% PBS (100% PBS / mouse). 3 After reaching a tumor volume of 1000 mg / kg, animals were randomized into four groups using Excel-based randomization software that performed stratified randomization based on tumor volume. Animals with intradermal (ID) or intramuscular (IM) growing tumors were excluded from the study.

[0280] Animals were checked daily for normal behavior, e.g., movement, food and water consumption, weight gain or loss (body weight was measured three times per week), effects of tumor growth and treatment on eye / hair matting, and any other abnormal effects noted in the protocol. Mortality and observed clinical signs were recorded.

[0281] Tumor size was measured three times a week in two dimensions using calipers and volume was calculated according to the formula: V = 0.5a × b 2 (a and b are the long and short diameters of the tumor, respectively) in mm 3 Tumor size was then used to calculate T / C and TGI values. T and C are the mean volumes of the treatment group and control group on a given day, respectively. T / C value (percentage) is an index of antitumor efficacy. TGI was calculated for each group using the formula: TGI(%)=[1-(Ti-T0) / (Vi-V0)]×100 (Ti is the mean tumor volume of the treatment group on a given day, T0 is the mean tumor volume of the treatment group on the first day of treatment, Vi is the mean tumor volume of the vehicle control group on the same day as Ti, and V0 is the mean tumor volume of the vehicle group on the first day of treatment).

[0282] As shown in Table 21 and Figures 28 and 29, the combination of mSapC-DOPS and m-anti-PD1 (line (4) in Figures 28 and 29) results in greater tumor growth inhibition and a significantly smaller standard error of the mean (SEM) than that observed in the groups receiving m-anti-PD1 alone (line (3) in Figures 28 and 29) or m-SapC-DOPS alone (line (2) in Figures 28 and 29). The lower SEM observed in the combination treatment indicates that more tumors responded in the combination treatment group than in the single treatment group. These results and the advantage of combining m-SapC-DOPS and m-anti-PD1 can also be further seen by looking at the tumor growth inhibition plots of individual animals shown in Figure 30. In these plots, it can be seen that the inhibition of tumor growth is significantly better in the combination group (light shaded line) than in the group treated with m-anti-PD1 alone (dark shaded line). Additionally, the results shown in Table 22 show that on day 17, in the combination arm, 3 There were no larger tumors; in contrast, the two single-agent treatment arms both had tumors over 1600 mm on day 17. 3 had larger tumors: 3 of 9 tumors in the m-SapC-DOPS arm and 2 of 10 tumors in the m-anti-PD1 arm, both larger than 1600 mm 3 It shows that it was larger.

[0283] Table 21. Tumor size on day 17 in treatment groups TIFF2024504066000029.tif49161a. Mean ± SEM bT / C%=T 17 / C 17 × 100% (T: treatment group; C: control group). 17 is the mean tumor volume on day 17. c. TGI(%) = [1-(T 17 -T0) / (C 17 -C0)] × 100 NOTE: One mouse in group 1 and one mouse in group 2 were sacrificed on day 14, so data from those two mice were not included in the calculation of tumor size, T / C, or TGI values.

[0284] Table 22. Tumor size on day 17 TIFF2024504066000030.tif34162

[0285] These results indicate that the saposin C / phospholipid formulation SapC-DOPS plus immune checkpoint inhibitor is more effective at reducing tumor growth than either agent alone.

[0286] Example 11: Effect of SapC-DOPS on myeloid-derived suppressor cells Myeloid-derived suppressor cells (MDSCs) are a heterogeneous population of cells that expand during cancer progression. MDSCs have a remarkable ability to suppress T cell responses, thereby minimizing the immune response to cancer in patients. Therefore, inhibiting the expansion of MDSCs and inhibiting their suppressive function is a promising therapeutic approach.

[0287] To determine the effect of SapC-DOPS on MDSCs, on day 0, frozen PBMCs from healthy donors were thawed in a 37°C water bath and washed once with MACS buffer. CD14 microbeads were used to isolate CD14 positive monocytes from the preparation of PBMCs. CD14 positive cells were then diluted to 2 million cells / ml in RPMI1640 culture medium and 50ul / well (100,000 cells / well) were placed in a flat-bottom 96-well plate. Different concentrations of solutions of BXQ-350 were added to the cells, and then rhGM-CSF and rhIL-6 were added to the mixture to stimulate monocyte differentiation. The plates were then incubated at 37°C, 5% CO2 for 7 days.

[0288] On day 3, the medium was replaced with a fresh solution of rhGM-CSF, rhIL-6+IL-2, 1640 medium, and BXQ-350.

[0289] On day 7, approximately 150 μL of supernatant was taken from the wells and analyzed for IL-10 by ELISA. Also on day 7, the plates were centrifuged at 350 g for 5 minutes, the medium was removed with a pipette, the cells were washed once with 100 μL of medium, and a solution of test compound was added to the wells. T cells isolated from thawed PBMCs using a total T cell isolation kit were labeled with cell trace violet dye, diluted to 2 million / ml, and added to the wells at 50 ul / well (100,000 T cells / well). CD3 / 28 Dynabead™ magnetic beads were prepared according to the manufacturer's instructions. The plates were then incubated at 37° C., 5% CO2 for 4 days.

[0290] On day 11, cells were processed for MDSC suppression assay. Plates were centrifuged at 300g for 5 min, and approximately 150 μL of supernatant was collected from each well for determination of interferon gamma (IFN-γ) levels by ELISA. 100 μL of Dulbecco's Phosphate Buffered Saline (DPBS) was then added to each well of the plate, the mixture was mixed well by multiple gentle pipetting, and the cell / bead suspension was transferred to a new 96-well V-bottom plate. The plate containing the T cell / bead suspension was placed on a magnet plate for 2 min, after which the T cell suspension was removed from the plate and added to a new 96-well plate. T cells in the latter plate were then stained with Live / Dead™ near infrared (IR) dye diluted 1:1000 in DPBS for 20 min at 100 μL / well to determine cell viability. The mixture was washed once with staining buffer. Cells in different wells were then labeled with antibodies against CD4+ T cells, CD8+ T cells, IL-10, HLA-DR, CD86, CD80, or CD11b and detected via ELISA (e.g., IL-10) or FACS analysis using a BD FortessaX20™ cell analyzer according to the manufacturer's instructions.

[0291] As shown in Figure 31A, BXQ-350 at concentrations of 0.3-1.5 μM promoted the expression of IFN-γ in this assay. Figures 31B-31C show that proliferation of both CD4+ and CD8+ T cells was increased in a dose-dependent manner in the presence of BXQ-350. Furthermore, BXQ-350 significantly reduced the expression of IL-10 (Figure 31D), HLA-DR (Figure 31E), CD86 (Figure 31F), and CD80 (Figure 31H), and moderately reduced the expression of CD11b (Figure 31G).

[0292] conclusion Overall, the data indicate that BXQ-350 has the ability to reverse the suppressive properties of MDSCs on T cells at relatively low concentrations.

[0293] Example 12: Effect of BXQ-350 on systemic cytokine concentrations in subjects with cancer Cytokines are secreted by specific cells of the immune system and have effects on (1) other cells, including cells of the immune system, (2) expression of complexes of differentiation on immune cells, (3) induction of specific immune responses, and (4) activation and coordination of the overall immune response in various diseases. See Dranoff, Nat Rev Cancer, (2004) 4(1):11-22, which is incorporated by reference in its entirety. Systemic monitoring of concentrations of specific cytokines is necessary to understand the relative changes in specific cytokines over time, which are indicative of the immune system response.

[0294] Monitoring the concentration of certain cytokines over time in cancer patients following therapeutic intervention (e.g., administration of a pharmaceutical compound; e.g., BXQ-350) can be an indication of the stimulation of the immune system of these patients toward an immune response that will inhibit cancer progression and even support cancer regression. Furthermore, as markers of cancer regression or progression, cytokines (by systemic expression) can be identified as having anti-cancer or cancer-promoting effects. For example, systemic cytokines indicative of anti-cancer efficacy include, but are not limited to, interferon gamma (IFN-γ) (see Ikeda, H. et al., Cytokine Growth Factor Rev, (2002) 13(2): 95-109); tumor necrosis factor alpha (TNF-α) (see van Horssen, R. et al., Oncologist (2006); 11(4): 397-408); interleukin 2 (IL-2) (Wrangle, JMet al., J Interferon Cytokine Res, (2018) 38(2): 45-68); interleukin 12 (IL-12); and interleukin 15 (IL-15). Systemic cytokines indicative of cancer progression include, but are not limited to, interleukin 1 (IL-1); interleukin 6 (IL-6) (Kumari, N. et al., Tumour Biol (2016) 37(9):11553-11572); interleukin 8 (IL-8) (Waugh D. et al. Clin Cancer Res (2008) 14(21):6735-41); and interleukin 10 (IL-10) (Wang X. et al. Cold Spring Harb Perspect Biol. (2019) 11(2):a028548). Each of the references cited in this paragraph is incorporated by reference in its entirety.

[0295] A Phase 1 study to determine the anti-cancer efficacy of BXQ-350 was conducted in adult patients with advanced solid tumors who had been previously treated with chemotherapy (Clinical Trials.gov Identifier: NCT02859857). Plasma samples from seven enrolled patients (patients A-G) were collected over time, pre-administration (day 1) and 29 days after BXQ-350 administration. Tables 23-25 ​​show that concentrations of cytokines associated with anti-cancer progression properties (i.e., IFN-γ in patients A-D; TNF-α in patients A, C, E, F, and G; IL-2 in patients B, D, and F) increased after administration of BXQ-350. Tables 26-28 show that concentrations of cytokines associated with cancer progression (i.e., IL-6 in patients A, B, D, and G; IL-8 in patients A, B, C, D, and G; IL-10 in patients A, B, E, and G) were decreased following administration of BXQ-350. These results indicate that BXQ-350 positively impacts the immune system and rebalances the immune system toward an anti-tumor state.

[0296] Furthermore, as shown in Table 29, the changes in various cytokine concentrations between days 1 and 29 for patients A and B indicate that the immune system is switching to an anti-cancer state. The anti-cancer state correlated with clinical benefit: Patient A, a patient with glioblastoma multiforme, survived more than 5 years after initiating BXQ-350 treatment, and Patient B, a patient with appendiceal cancer, survived more than 2 years after initiating BXQ-350 treatment.

[0297] conclusion These results indicate that BXQ-350 positively impacts the immune system and rebalances it toward an antitumor state.

[0298] Table 23. Interferon gamma expression on days 1 and 29 TIFF2024504066000031.tif36128* = IFN-γ concentration (pg / ml) on day 1 or day 29 BLQ: below limit of quantification N / A: Change not calculated as baseline expression was BLQ

[0299] Table 24. TNF-α expression on days 1 and 29 TIFF2024504066000032.tif36129* = TNF-α concentration (pg / ml) on day 1 or day 29 BLQ: below limit of quantification N / A: Change not calculated as baseline expression was BLQ

[0300] Table 25. IL-2 expression on days 1 and 29 TIFF2024504066000033.tif36128* = IL-2 concentration (pg / ml) on day 1 or day 29 BLQ: below limit of quantification N / A: Change not calculated as baseline expression was BLQ

[0301] Table 26. IL-6 expression on days 1 and 29 TIFF2024504066000034.tif36128* = IL-6 concentration (pg / ml) on day 1 or day 29

[0302] Table 27. IL-8 expression on days 1 and 29 TIFF2024504066000035.tif36130* = IL-8 concentration (pg / ml) on day 1 or day 29

[0303] Table 28. IL-10 expression on days 1 and 29 TIFF2024504066000036.tif36128* = IL-10 concentration (pg / ml) on day 1 or day 29

[0304] Table 29. Percentage change in cytokine expression from day 1 to day 29 TIFF2024504066000037.tif26152

[0305] Although specific features of the invention have been described above for purposes of illustration, it will be apparent to those skilled in the art that many variations in the details of the methods disclosed herein may be made without departing from the scope of the appended claims.

[0306] TIFF2024504066000038.tif98128

Claims

1. 1. A nanovesicle formulation for use in a method of reducing or delaying the onset of a neuropathic symptom in a human subject, comprising: (i) a saposin C polypeptide comprising SEQ ID NO:1 having 0-4 amino acid insertions, substitutions, deletions, or combinations thereof; and (ii) a phospholipid having a net negative charge at neutral pH, The method comprises identifying a human subject experiencing or at risk of experiencing a neuropathic symptom, and administering the nanovesicle formulation to the subject.

2. (a) the method further comprises the step of confirming that the subject's neurological symptoms are reduced; and / or (b) the method is effective to reduce the incidence, intensity, and / or duration of, or delay the onset of, a neuropathic symptom in the subject; and / or (c) the subject has cancer.

2. The nanovesicle formulation of claim 1.

3. The human subject is in need of treatment with a chemotherapeutic agent associated with a neuropathic symptom side effect, and the method further comprises administering to the subject a dose of the chemotherapeutic agent; and / or At least one dose of a nanovesicle formulation is administered simultaneously with the dose of chemotherapeutic agent, or immediately before or after administration of the dose of chemotherapeutic agent.

2. The nanovesicle formulation of claim 1.

4. A nanovesicle formulation for use in a method for treating cancer, comprising: (i) a saposin C polypeptide comprising SEQ ID NO:1 having 0-4 amino acid insertions, substitutions, deletions, or combinations thereof; and (ii) a phospholipid having a net negative charge at neutral pH, wherein the nanovesicle formulation is co-administered to a human subject in need thereof with a chemotherapeutic agent associated with a neuropathic symptom side effect; Optionally, the nanovesicle formulation is administered in an amount that reduces the incidence, intensity, and / or duration of chemotherapy-associated neuropathic symptom side effects or delays the onset of chemotherapy-associated neuropathic symptom side effects.

5. 1. A nanovesicle formulation for use in a method of treating gastrointestinal cancer in a subject, comprising: (i) a saposin C polypeptide comprising SEQ ID NO:1 having 0-4 amino acid insertions, substitutions, deletions, or combinations thereof; and (ii) a phospholipid having a net negative charge at neutral pH, wherein the nanovesicle formulation is used in combination with a chemotherapy regimen comprising one or more anti-tumor agents, Optionally, the nanovesicle formulation is administered in an amount that reduces the incidence, intensity, and / or duration of neuropathic symptom side effects associated with a chemotherapy regimen, or delays the onset of neuropathic symptom side effects associated with a chemotherapy regimen. (a) the amino acid sequence of the saposin C polypeptide comprises, comprises, or consists of SEQ ID NO:1 with one or two amino acid insertions, substitutions, deletions, or combinations thereof; and / or (b) the phospholipid comprises phosphatidylserine, dioleoylphosphatidylserine (DOPS) or a salt thereof, optionally a sodium salt, a phosphoglyceride optionally comprising phosphatidic acid, or one or more of dihexanoylphosphatidylserine lipid, dioctanoylphosphatidylserine lipid, didecanoylphosphatidylserine lipid, dilauroylphosphatidylserine lipid, dimyristoylphosphatidylserine lipid, dipalmitoylphosphatidylserine lipid, palmitoyl-oleoylphosphatidylserine lipid, 1-stearoyl-2-oleoylphosphatidylserine lipid, or diphytanoylphosphatidylserine lipid; A nanovesicle formulation according to any one of claims 1 to 5. (a) the molar ratio of phospholipid to saposin C polypeptide in the nanovesicle formulation is in the range of 8:1 to 20:1; (b) the molar ratio of phospholipid to saposin C polypeptide in the nanovesicle formulation is in the range of 11:1 to 13:1; or (c) the molar ratio of phospholipid to saposin C polypeptide in the nanovesicle formulation is 12:1; A nanovesicle formulation according to any one of claims 1 to 6.

8. The nanovesicle formulation of any one of claims 2-4, 6 or 7, wherein the subject has gastrointestinal cancer, pancreatic cancer, colorectal cancer, bone cancer, brain cancer, sarcoma, neuroblastoma, breast cancer, or squamous cell carcinoma.

9. The method of claim 1, wherein the chemotherapeutic agent is selected from the group consisting of platinum-based agents, taxanes, epothilones, plant alkaloids, immunomodulators, and proteasome inhibitors; or (b) the chemotherapeutic agent is oxaliplatin, cisplatin, carboplatin, paclitaxel, docetaxel, cabazitaxel, ixabepilone, vinblastine, vincristine, vindesine, vinorelbine, vincaminol, vineridine, vinbrunin, etoposide, thalidomide, lenalidomide, pomalidomide, bortezomib, carfilzomib, ixazomib, eribulin, or suramin; or (c) the chemotherapeutic agent is oxaliplatin and the subject has or is at risk of having oxaliplatin-induced acute pain syndrome; The nanovesicle formulation of any one of claims 3, 4, and 6-8. (a) each dose of the nanovesicle formulation administered to the subject contains 0.4 mg / kg to 7 mg / kg of saposin C polypeptide; or (b) each dose of the nanovesicle formulation administered to the subject contains between 0.4 mg / kg and 7 mg / kg of saposin C polypeptide, and the nanovesicle formulation is administered intravenously; Optionally, the nanovesicle formulation is administered at least once a day, once every other day, three times a week, approximately once a week (7 (+ / - 3) days), or approximately once every two weeks (14 (+ / - 3) days), and optionally multiple doses of the nanovesicle formulation are administered for at least 8 weeks. A nanovesicle formulation according to any one of claims 1 to 9.

11. The nanovesicle formulation of claim 2, wherein the method is effective in at least one of the following (a) to (d): (a) a reduction in the intensity and / or duration of episodes of neuropathic symptoms in said subject after administration of a nanovesicle formulation compared to the intensity and / or duration of episodes of neuropathic symptoms in said subject before the nanovesicle formulation was administered; (b) a reduction in the intensity and / or duration of neuropathic symptoms in the subject compared to the intensity and / or duration of neuropathic symptoms in a control population; (c) a reduction in the incidence of neuropathy in the subject compared to the incidence of neuropathy in a control population; and (d) A delay in the onset of neuropathy in said subject after a triggering event compared to the time to onset of neuropathy in a control population after a similar event.

12. The nanovesicle formulation of claim 3, wherein one or more additional doses of the nanovesicle formulation and one or more additional doses of a chemotherapeutic agent are administered to the subject.

13. 5. The nanovesicle formulation of claim 3 or 4, which does not contain a chemotherapeutic agent.

14. (a) The neuropathic symptom is one or more of the following, optionally pain: pain; hyperalgesia; allodynia; analgesia; loss of sensation of heat, cold, or physical injury; numbness; hypersensitivity to touch; loss of coordination and proprioception; muscle weakness; muscle wasting; muscle spasms; muscle cramps; or muscle paralysis; and / or (b) the method further comprises monitoring the subject for neuropathic symptoms; and / or (c) the method further comprises confirming that the subject's neuropathic symptoms are reduced; and / or (d) the method further comprises administering to the subject one or more additional anti-neuropathy treatments selected from the group consisting of transcutaneous electrical nerve stimulation (TENS), therapeutic plasma exchange (TPE), intravenous immunoglobulin (IVIG) treatment, physical therapy, analgesics, antiepileptic drugs, topical treatments, and antidepressants, or administering to the subject ibuprofen, gabapentin, pregabalin, capsaicin cream, lidocaine patch, amitriptyline, doxepin, nortriptyline, duloxetine, or venlafaxine; A nanovesicle formulation according to any one of claims 1 to 4 or 6 to 13.

15. (a) The chemotherapy regimen comprises modified FOLFOX7 (mFOLFOX7), (b) the chemotherapy regimen includes oxaliplatin; (c) the nanovesicle formulation is administered simultaneously with the chemotherapy regimen, or within 48 hours before or after the initiation of administration of the chemotherapy regimen; (d) the nanovesicle formulation is administered intravenously; (e) the chemotherapy regimen is administered intravenously; (f) the gastrointestinal cancer is esophageal cancer, gastric cancer, anal cancer, colorectal cancer, intestinal cancer, gallbladder cancer, pancreatic cancer, liver cancer, pancreatic islet cell carcinoma, rectal cancer, small intestine cancer, gastrointestinal carcinoid tumor, or gastrointestinal stromal tumor; (g) used in combination with biological treatments for gastrointestinal cancer; (h) used in combination with an anti-vascular endothelial growth factor (VEGF) monoclonal antibody; (i) in combination with an anti-epidermal growth factor receptor (EGFR) antibody; or (j) used in combination with bevacizumab, cetuximab, or panitumumab; 6. The nanovesicle formulation of claim 5.

16. (A) The method comprises: (a) administering 2.4 mg / kg of the nanovesicle formulation over about 45 minutes to about 120 minutes on day 1 of the first week of treatment; and (b) after completion of step (a), sequentially administering a chemotherapy regimen in the following steps: (i) 200 mg / m over approximately 2 hours on day 1 of the first week of treatment 2 85 mg / m2 infused with leucovorin calcium 2 oxaliplatin; and (ii) 2400 mg / m over approximately 46 hours on days 1 and 2 of the first week of treatment. 2 5-Fluorouracil (5-FU) Contains or (B) The method comprises: (a) administering 2.4 mg / kg of the nanovesicle formulation over about 45 minutes to about 120 minutes on day 1 of the first week of treatment; (b) after completion of step (a), sequentially administering a chemotherapy regimen in the following steps: (i) 85 mg / m2 oxaliplatin infused with 200 mg / m2 leucovorin calcium over approximately 2 hours on day 1 of the first week of treatment; and (ii) 2400 mg / m2 5-FU over approximately 46 hours on days 1 and 2 of the first week of treatment; and (c) administering about 5 mg / kg to about 15 mg / kg of bevacizumab over about 30 to about 90 minutes on day 1 of the first week of treatment. Including, Optionally, method (A) or (B) is (a) administering an additional dose of the nanovesicle formulation three times per week during the second week of treatment; (b) administering an additional dose of the nanovesicle formulation three times per week during the second week of treatment and an additional dose of the nanovesicle formulation once per week (7 (+ / - 3) days) during the third and fourth weeks of treatment; or (c) administering an additional dose of the nanovesicle formulation three times per week during the second week of treatment, administering an additional dose of the nanovesicle formulation once per week (7 (+ / - 3) days) during the third and fourth weeks of treatment, and administering an additional dose of the nanovesicle formulation every 14 (+ / - 3) days during weeks 5-23 of treatment, optionally wherein multiple doses of the nanovesicle formulation are administered for at least 8 weeks.

6. The nanovesicle formulation of claim 5, further comprising:

17. 6. The nanovesicle formulation of claim 5, administered at least once a week for the first four weeks of treatment, optionally comprising: (a) administered at least three times per week during the first 2 weeks of treatment; (b) administered at least three times per week during the first 2 weeks of treatment, and after the first 4 weeks of treatment, administered at least once every 14 (+ / - 3) days, and optionally, after the first 24 weeks of treatment, administered at least once every 28 (+ / - 3) days; (c) administered at least once every 14 (+ / - 3) days after the first 4 weeks of treatment, and optionally administered at least once every 28 (+ / - 3) days after the first 24 weeks of treatment; or (d) administered at least three times per week during the first 2 weeks of treatment, and once every 14 (+ / - 3) days during weeks 5-24, and optionally once every 28 (+ / - 3) days after the first 24 weeks of treatment; The nanovesicle formulation.

18. (a) The nanovesicle formulation is repeatedly administered to the subject as follows: Week 1: 1 dose each day, days 1-5; Week 2: 1 dose every other day for a total of 3 doses; Weeks 3 and 4: 1 dose per week (7 (+ / - 3) days); and Weeks 5-24: 1 dose every 14 days (+ / - 3 days) Optionally, additional doses of the chemotherapy regimen are administered in week 3 and once every 14 days (+ / - 3 days) from weeks 5 to 24; or (b) The method of (B), wherein additional doses of bevacizumab are administered in week 3 and once every 14 days (+ / - 3 days) from weeks 5 to 24; 17. The nanovesicle formulation of claim 16.

19. The nanovesicle formulation of any one of claims 15 to 18, which is administered intravenously, intraarterially, intradermally, intramuscularly, intracardially, intracranially, subcutaneously, intraperitoneally, by inhalation, intranasally, orally, rectally, or sublingually.

20. 6. The nanovesicle formulation of claim 5, wherein each dose of the nanovesicle formulation administered to the subject contains 0.4 mg / kg to 7 mg / kg of saposin C polypeptide, and optionally, said treatment increases at least one of the following parameters in the subject compared to a control population treated with a chemotherapy regimen and not treated with the nanovesicle formulation: (a) Objective response rate (ORR); (b) overall survival (OS); (c) progression-free survival (PFS); and (d) Duration of response (DoR).

21. (a) a first pharmaceutical composition comprising at least one anti-tumor agent; and (b) a second pharmaceutical composition comprising (i) a saposin C polypeptide comprising SEQ ID NO:1 having 0 to 4 amino acid insertions, substitutions, deletions, or combinations thereof; and (ii) a phospholipid having a net negative charge at neutral pH. in separate containers, and optionally the anti-tumor agent is oxaliplatin.

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