Salts of 2-S and 2-R rimantadine for the treatment of cancer

Abstract

Disclosed herein is the use of purified 2-S rimantadine or purified 2-R rimantadine, or pharmaceutically acceptable versions thereof, to treat cancers and precancerous lesions, including cancers and precancerous lesions associated with papillomavirus, in a subject in need of such treatment.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 398,826, filed August 17, 2022, which is incorporated herein by reference in its entirety. INCORPORATION BY REFERENCE

[0002] All publications, patents, and patent applications disclosed herein are incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. In the event of a conflict between a term disclosed herein and a term of an incorporated reference, the term of the present specification shall control. [Technical Field]

[0003] The present disclosure relates to a method of treating or preventing cancer, including cancer caused by papillomavirus, comprising administering an enantiomerically pure salt of the 2-S enantiomer of rimantadine. Background of the Disclosure

[0004] Genital human papillomavirus (HPV) is the most common sexually transmitted disease in the United States. According to the Centers for Disease Control and Prevention (CDC), 90% of HPV infections are asymptomatic and resolve spontaneously within two years. However, in some cases, HPV infection persists, resulting in the development of either warts or precancerous lesions. These lesions increase the risk of cancer of the cervix, vulva, vagina, penis, anus, rectum, and oropharynx, depending on the site of infection. HPV types associated with cervical carcinogenesis are classified into 15 "high-risk types" (HPV 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68, 73, and 82) and three "probable high-risk types" (HPV 26, 53, and 66). Researchers have also shown that HPV 16 and 18 are associated with breast cancer. HPV types (HPV 6, 11, 40, 42, 43, 44, 54, 61, 70, 72, and 81) are classified as "low-risk types" and are known to cause benign low-grade cervical changes, genital warts, and recurrent respiratory papillomatosis. Cutaneous HPV types 5, 8, and 92 are associated with skin cancer.

[0005] Rimantadine hydrochloride (α-methyl-l-adamantane-methalamine hydrochloride) is an oral medication used to treat influenza A, sold under the brand name Flumadine®. Rimantadine inhibits influenza activity by binding to amino acids within the viral M2 transmembrane channel and inhibiting proton transport through the M2 channel. Flumadine® contains a racemic mixture of rimantadine. One study found evidence that the R-enantiomer binds to the M2 channel pore with higher affinity than the S-enantiomer. However, this result contradicts several previous findings that showed no difference between the enantiomers for M2 binding. Subsequent studies confirmed this. Rimantadine has also been suggested to have some anti-Parkinsonian activity. However, it has not been developed or approved for use in this indication.

[0006] Flumadin® has gastrointestinal and central nervous system side effects, including nausea, upset stomach, vomiting, loss of appetite, dry mouth, abdominal pain, asthenia, irritability, fatigue, lightheadedness, dizziness, headache, sleep disturbances, decreased concentration, confusion, and anxiety. Anxiety and insomnia are the toxicities most frequently cited as reasons for discontinuing treatment.

[0007] In some embodiments, the present disclosure relates to various salts and solid forms of 2-S-limantidine. In some embodiments, the composition comprises a salt comprising 2-S-limantidine and fumaric acid. In some embodiments, the salt is anhydrous. In some embodiments, the salt is crystalline. In some embodiments, the salt has an X-ray powder diffraction pattern comprising a peak at about 6.09 2θ. In some embodiments, the salt has an X-ray powder diffraction pattern further comprising peaks at about 14.36 2θ, about 17.56 2θ, about 18.45 2θ, about 18.81 2θ, and / or about 27.17 2θ. In some embodiments, the composition comprises a salt comprising 2-S-limantidine and tartaric acid. In some embodiments, the salt is anhydrous. In some embodiments, the salt is crystalline. In some embodiments, the salt has an X-ray powder diffraction pattern comprising a peak at about 7.50 2θ. In some embodiments, the salt has an X-ray powder diffraction pattern further comprising peaks at about 17.64 2θ, about 18.68 2θ, about 15.43 2θ, about 19.37 2θ, and / or about 22.48 2θ. In some embodiments, the composition comprises a salt comprising 2-S-limantidine and galactaric acid. In some embodiments, the salt is anhydrous. In some embodiments, the salt is crystalline. In some embodiments, the salt has an X-ray powder diffraction pattern further comprising peaks at about 5.71 2θ. In some embodiments, the salt has an X-ray powder diffraction pattern further comprising peaks at about 15.85 2θ, about 16.96 2θ, about 19.76 2θ, and / or about 19.43 2θ. In some embodiments, the salt has an X-ray powder diffraction pattern further comprising peaks at 5.83 2θ. In some embodiments, the salt has an X-ray powder diffraction pattern further comprising peaks at about 14.89 2θ, about 16.87 2θ, about 17.62 2θ, about 30.87 2θ, and / or about 19.72 2θ. In some embodiments, the composition comprises a salt comprising 2-S-limantidine and benzoic acid. In some embodiments, the salt is anhydrous. In some embodiments, the salt is crystalline. In some embodiments, the salt has an X-ray powder diffraction pattern comprising a peak at 7.85 2θ.In some embodiments, the salt has an X-ray powder diffraction pattern further comprising peaks at about 9.72 2θ, about 11.49 2θ, about 12.29 2θ, about 15.66 2θ, about 19.06 2θ, about 20.38 2θ, and / or about 29.91 2θ. In some embodiments, the composition comprises a salt comprising 2-S-limantidine and benzenesulfonate. In some embodiments, the salt is anhydrous. In some embodiments, the salt is crystalline. In some embodiments, the salt has an X-ray diffraction powder pattern further comprising peaks at about 8.02 2θ, about 8.44 2θ, about 14.11 2θ, about 15.39 2θ, about 16.01 2θ, and / or about 19.08 2θ. In some embodiments, the salt has an X-ray diffraction powder pattern comprising a peak at 8.04 2θ. In some embodiments, the salt has an X-ray diffraction powder pattern further comprising peaks at about 8.45 2θ, about 14.13 2θ, about 15.37 2θ, about 16.86 2θ, and / or about 19.10 2θ. In some embodiments, the composition comprises a crystalline form of 2-S-rimantadine, with XRPD peaks at about 14.36 2θ, about 17.56 2θ, about 18.45 2θ, about 18.81 2θ, and / or about 27.17 2θ. In some embodiments, the composition comprises a salt of 2-S-rimantadine. In some embodiments, the composition comprises a fumarate salt, a tartrate salt, a galactarate salt, a benzoate salt, a benzenesulfonate salt, or a combination thereof. In some embodiments, the composition comprises a fumarate salt. In some embodiments, the fumarate salt is fumarate type A. In some embodiments, the composition is characterized by comprising at least one or more of the following peaks in an X-ray powder diffraction pattern ("XRPD") diffractogram: a peak in an XRPD diffractogram at about 6.09°2θ, a peak in an XRPD diffractogram at about 14.36°2θ, a peak in an XRPD diffractogram at about 17.56°2θ, a peak in an XRPD diffractogram at about 18.45°2θ, a peak in an XRPD diffractogram at about 18.81°2θ, or a peak in an XRPD diffractogram at about 27.17°2θ. In some embodiments, the salt is a tartrate salt.In some embodiments, the tartrate salt is tartrate Type A. In some embodiments, the composition is characterized by comprising at least one or more of the following peaks in an X-ray powder diffraction pattern ("XRPD") diffractogram: a peak at about 6.09°2θ, a peak at about 7.50°2θ, a peak at about 17.64°2θ, a peak at about 18.68°2θ, a peak at about 15.43°2θ, a peak at about 19.37°2θ, or a peak at about 22.48°2θ. In some embodiments, the salt is a galactarate salt. In some embodiments, the galactarate salt is galactarate Type A. In some embodiments, the salt is characterized by at least one or more of the following peaks in an XRPD diffractogram: 5.71°2θ, about 15.85°2θ, about 16.96°2θ, about 19.76°2θ, or about 19.43°2θ. In some embodiments, the galactarate salt is galactarate Type B. In some embodiments, the composition is characterized by at least one or more of the following peaks in an XRPD diffractogram: 5.83°2θ, about 14.89°2θ, about 16.87°2θ, about 17.62°2θ, about 30.87°2θ, or about 19.72°2θ. In some embodiments, the salt is a benzoate salt. In some embodiments, the benzoate salt is benzoate Type A. In some embodiments, the composition is characterized by comprising at least one or more peaks in an XRPD diffractogram at 7.85°2θ, 9.72°2θ, 11.49°2θ, about 12.29°2θ, 15.66°2θ, about 19.06°2θ, about 20.38°2θ, or about 29.91°2θ. In some embodiments, the salt is a benzenesulfonate salt.In some embodiments, the benzenesulfonate salt is benzenesulfonate Type A. In some embodiments, the salt is characterized by comprising at least one or more of the following peaks in an XRPD diffractogram: a peak in an XRPD diffractogram at about 6.21°2θ, a peak in an XRPD diffractogram at about 8.02°2θ, a peak in an XRPD diffractogram at about 8.44°2θ, a peak in an XRPD diffractogram at about 14.11°2θ, a peak in an XRPD diffractogram at about 15.39°2θ, a peak in an XRPD diffractogram at about 16.01°2θ, or a peak in an XRPD diffractogram at about 19.08°2θ. In some embodiments, the benzenesulfonate salt is benzenesulfonate Type B. In some embodiments, the salt is characterized by at least one or more of the following peaks in an XRPD diffractogram: a peak in an XRPD diffractogram at about 8.04°2θ, a peak in an XRPD diffractogram at about 8.45°2θ, a peak in an XRPD diffractogram at about 14.13°2θ, a peak in an XRPD diffractogram at about 15.37°2θ, a peak in an XRPD diffractogram at about 16.86°2θ, or a peak in an XRPD diffractogram at about 19.10°2θ. In some embodiments, the composition is crystalline. In some embodiments, the differential scanning calorimetry is substantially as shown in FIG. 11. In some embodiments, the differential scanning calorimetry is substantially as shown in FIG. 12. In some embodiments, the differential scanning calorimetry is substantially as shown in FIG. 13. In some embodiments, the differential scanning calorimetry is substantially as shown in FIG. 14. In some embodiments, the differential scanning calorimetry is substantially as shown in FIG. 15. In some embodiments, the differential scanning calorimetry is substantially as shown in Figure 16. In some embodiments, the differential scanning calorimetry is substantially as shown in Figure 17. In some embodiments, the differential scanning calorimetry is substantially as shown in Figure 18. In some embodiments, the differential scanning calorimetry is substantially as shown in Figure 19. In some embodiments, the differential scanning calorimetry is substantially as shown in Figure 20. In some embodiments, the differential scanning calorimetry is substantially as shown in Figure 21.In some embodiments, the composition comprises a salt of 2-S-limantidine characterized by a sample weight loss of about 0.01% w / w in a thermogravimetric analysis over a temperature range of 0° C. to 150° C. In some embodiments, the salt is the tartrate salt. In some embodiments, the composition comprises a salt of 2-S-limantidine characterized by a sample weight loss of about 6.72% w / w in a thermogravimetric analysis over a temperature range of 0° C. to 150° C. In some embodiments, the salt is the maleate salt. In some embodiments, the composition comprises a salt of 2-S-limantidine characterized by a sample weight loss of about 10.30% w / w in a thermogravimetric analysis over a temperature range of 0° C. to 85° C. In some embodiments, the salt is the hippurate salt. In some embodiments, the composition comprises a salt of 2-S-limantidine characterized by a sample weight loss of about 0.15% w / w in a thermogravimetric analysis over a temperature range of 0° C. to 150° C. In some embodiments, the salt is the galactarate salt. In some embodiments, the composition comprises a salt of 2-S-limantidine characterized by a sample weight loss of about 0.00% w / w in a thermogravimetric analysis over a temperature range of 0° C. to 150° C. In some embodiments, the salt is a galactarate salt. In some embodiments, the composition comprises a salt of 2-S-limantidine characterized by a sample weight loss of about 0.26% w / w in a thermogravimetric analysis over a temperature range of 0° C. to 100° C. In some embodiments, the salt is a benzoate salt. In some embodiments, the composition comprises a salt of 2-S-limantidine characterized by a sample weight loss of about 14.77% w / w in a thermogravimetric analysis over a temperature range of 0° C. to 110° C. In some embodiments, the salt is a benzoate salt. In some embodiments, the composition comprises a salt of 2-S-limantidine characterized by a sample weight loss of about 1.68% w / w in a thermogravimetric analysis over a temperature range of 0° C. to 150° C. In some embodiments, the salt is a gentisate salt. In some embodiments, the composition comprises a salt of 2-S-limantidine characterized by a sample weight loss of about 3.55 w / w% in thermogravimetric analysis over a temperature range of 0°C to 200°C.In some embodiments, the salt is a toluenesulfonate salt. In some embodiments, the composition comprises a salt of 2-S-limantidine characterized by a sample weight loss of about 0.02 w / w% in thermogravimetric analysis over a temperature range of 0°C to 200°C. In some embodiments, the salt is a benzenesulfonate salt. In some embodiments, the composition comprises a salt of 2-S-limantidine characterized by a sample weight loss of about 0.09 w / w% in thermogravimetric analysis over a temperature range of 0°C to 200°C. In some embodiments, the salt is a benzenesulfonate salt. In some embodiments, the composition comprises a salt of 2-S-limantidine that exhibits a differential scanning calorimetry measurement comprising an endothermic peak at about 221°C. In some embodiments, the salt is a tartrate salt. In some embodiments, the composition comprises a salt of 2-S-limantidine that exhibits a differential scanning calorimetry measurement comprising endothermic peaks at about 63°C, 74°C, and 121°C. In some embodiments, the salt is a maleate salt. In some embodiments, the composition comprises a salt of 2-S-limantidine, which exhibits an endothermic peak at about 62°C in differential scanning calorimetry. In some embodiments, the salt is hippurate. In some embodiments, the composition comprises a salt of 2-S-limantidine, which exhibits an endothermic peak at about 193°C in differential scanning calorimetry. In some embodiments, the salt is galactarate. In some embodiments, the composition comprises a salt of 2-S-limantidine, which exhibits an endothermic peak at about 195°C in differential scanning calorimetry. In some embodiments, the salt is galactarate. In some embodiments, the composition comprises a salt of 2-S-limantidine, which exhibits an endothermic peak at about 199°C in differential scanning calorimetry. In some embodiments, the salt is benzoate. In some embodiments, the composition comprises a salt of 2-S-limantidine, which exhibits an endothermic peak at about 198°C in differential scanning calorimetry. In some embodiments, the salt is a benzoate salt. In some embodiments, the composition comprises a salt of 2-S-limantidine, wherein the salt exhibits a differential scanning calorimetry (DSC) with an endothermic peak at about 206°C. In some embodiments, the salt is a gentisate salt. In some embodiments, the composition comprises a salt of 2-S-limantidine, wherein the salt exhibits a DSC with an endothermic peak at about 214°C. In some embodiments, the salt is a toluenesulfonate salt. In some embodiments, the composition comprises a salt of 2-S-limantidine, wherein the salt exhibits a DSC with an endothermic peak at about 223°C. In some embodiments, the salt is a benzenesulfonate salt. In some embodiments, the composition comprises a salt of 2-S-limantidine, wherein the salt exhibits a DSC with an endothermic peak at about 232°C. In some embodiments, the salt is a benzenesulfonate salt. In some embodiments, the composition comprises a salt of 2-S-limantidine, wherein the salt exhibits a crystalline particle size of 1 to 50 μm. In some embodiments, the composition comprises a salt of 2-S-limantidine and is characterized by an XRPD diffractogram substantially as shown in Figure 4. In some embodiments, the composition comprises a salt of 2-S-limantidine and is characterized by an XRPD diffractogram substantially as shown in Figure 5. In some embodiments, the composition comprises a salt of 2-S-limantidine and is characterized by an XRPD diffractogram substantially as shown in Figure 6. In some embodiments, the composition comprises a salt of 2-S-limantidine and is characterized by an XRPD diffractogram substantially as shown in Figure 7. In some embodiments, the composition comprises a salt of 2-S-limantidine and is characterized by an XRPD diffractogram substantially as shown in Figure 8. In some embodiments, the composition comprises a salt of 2-S-limantidine and is characterized by an XRPD diffractogram substantially as shown in Figure 9. In some embodiments, the composition comprises a salt of 2-S-limantidine and is characterized by an XRPD diffractogram substantially as shown in Figure 10. In some embodiments, the salt of 2-S-rimantadine is the tartrate salt of 2-S-rimantadine.In some embodiments, the salt of 2-S-rimantadine is the fumarate salt of 2-S-rimantadine.In some embodiments, the salt of 2-S-rimantadine is a galactarate salt of 2-S-rimantadine. In some embodiments, the salt of 2-S-rimantadine is a benzoate salt of 2-S-rimantadine. In some embodiments, the salt of 2-S-rimantadine is a benzenesulfonate salt of 2-S-rimantadine. In some embodiments, the methods of the present disclosure include methods comprising administering to a subject a therapeutically effective amount of a salt or composition of any one of the preceding claims. In some embodiments, the cancer is selected from one or more of melanoma, head and neck cancer, lung cancer, colon cancer, breast cancer, esophageal cancer, pancreatic cancer, prostate cancer, cervical cancer, and gastric cancer. In some embodiments, the cancer is a sarcoma, carcinoma, lymphoma, or leukemia. In some embodiments, the carcinoma is squamous cell carcinoma. In some embodiments, the squamous cell carcinoma is head and neck squamous cell carcinoma. In some embodiments, the cancer is selected from the group consisting of head and neck cancer, breast cancer, and melanoma. In some embodiments, the cancer is an HPV-associated cancer. In some embodiments, the HPV-associated cancer is associated with the alpha genus of HPV. In some embodiments, one or more cancer cells from the subject express a human papillomavirus (HPV) protein. In some embodiments, the HPV protein is an E5 HPV protein. In some embodiments, the HPV E5 protein is from one or more HPV subtypes selected from the group consisting of HPV 6, HPV 11, HPV 16, HPV 18, HPV 31, HPV 33, HPV 35, HPV 39, HPV 45, HPV 51, HPV 52, HPV 56, HPV 58, HPV 66, and HPV 69. In some embodiments, the HPV protein is E5 from HPV 16. In some embodiments, the HPV protein is E5 from HPV 18. In some embodiments, the described methods include a method of treating cancer in a subject, the method comprising detecting cancer cells that express a human papillomavirus (HPV) protein in a sample from the subject, and administering to the subject a therapeutically effective amount of a salt or composition of any one of the preceding claims. In some embodiments, the cancer is associated with the alpha genus of HPV.In some embodiments, the HPV protein is one or more of E5, E6, or E7 HPV proteins. In some embodiments, the HPV E5, E6, or E7 proteins are from one or more HPV subtypes selected from the group consisting of HPV 6, HPV 11, HPV 16, HPV 18, HPV 31, HPV 33, HPV 35, HPV 39, HPV 45, HPV 51, HPV 52, HPV 56, HPV 58, HPV 66, and HPV 69. In some embodiments, the cancer is selected from the group consisting of head and neck cancer, mucosal squamous cell carcinoma, cutaneous squamous cell carcinoma, liver cancer, cervical cancer, vaginal cancer, vulvar cancer, penile cancer, and anal cancer. In some embodiments, the method further comprises administering an additional anticancer agent. In some embodiments, the additional anticancer agent is selected from the group consisting of carboplatin, cisplatin, gemcitabine, methotrexate, paclitaxel, pemetrexed, lomustine, temozolomide, dacarbazine, and combinations thereof. In some embodiments, the additional anticancer agent is an immunotherapy. In some embodiments, the additional anticancer agent is an immune checkpoint inhibitor. In some embodiments, the immune checkpoint inhibitor targets one or more of CTLA-4, PD-1, PD-L1, BTLA, LAG-3, A2AR, TIM-3, B7-H3, VISTA, and IDO. In some embodiments, the immune checkpoint inhibitor is selected from the group consisting of ipilimumab, nivolumab, pembrolizumab, atezolizumab, avelumab, durvalumab, tremelimumab, cemiplimab, and combinations thereof. In some embodiments, the method further comprises exposing the subject to radiation therapy, surgery, or a combination thereof. In some embodiments, the subject is human. In some embodiments, the methods described herein include methods for treating precancerous HPV lesions in a subject in need thereof, comprising administering a therapeutically effective amount of a salt or composition of 2-S-rimantidine. In some embodiments, the HPV lesion is associated with the alpha genus of HPV. In some embodiments, the precancerous HPV lesion is a lesion of the cervix, skin, urethra, nasal cavity, paranasal sinuses, larynx, tracheobronchial mucosa, or oral cavity.In some embodiments, the HPV precancerous lesions express one or more HPV proteins selected from one or more of the E5, E6, or E7 HPV proteins. In some embodiments, the HPV E5, E6, or E7 proteins are from one or more HPV subtypes selected from the group consisting of one or more of HPV 6, HPV 11, HPV 16, HPV 18, HPV 31, HPV 33, HPV 35, HPV 39, HPV 45, HPV 51, HPV 52, HPV 56, HPV 58, HPV 66, and HPV 69. In some embodiments, rimantadine is administered topically, orally, subcutaneously, or parenterally. In some embodiments, the methods described herein include methods for treating or preventing avian influenza in poultry, comprising administering a therapeutically effective amount of a salt or composition of any one of the preceding claims. In some embodiments, the avian influenza is H5N1. In some embodiments, side effects associated with administration of a salt or composition of 2-S rimantadine are reduced compared to side effects associated with racemic rimantadine or enantiomerically pure 2-R rimantadine. In some embodiments, the composition comprises a salt or composition of 2-S-rimantadine and one or more immune checkpoint inhibitors. In some embodiments, the one or more immune checkpoint inhibitors include CTLA-4, PD-1, PD-L1, BTLA, LAG-3, A2AR, TIM-3, B7-H3, VISTA, IDO, or any combination thereof. In some embodiments, the salt or composition comprises enantiomerically pure 2-S-rimantadine. In some embodiments, the salt or composition does not include R-rimantadine. In some embodiments, the salt or composition is formulated for injection. In some embodiments, the method disclosed herein is a method for preparing a salt of 2-S-rimantadine, comprising dissolving 2-S-rimantadine in a first solvent to form a first solution, adding a second solvent to the solution to form a second solution, cooling the second solution, and evaporating the second solution. In some embodiments, the first solvent is selected from the group consisting of methanol, ethanol, chloroform, and water. [Brief explanation of the drawings]

[0008] Figure 1A-B shows peak current amplitude and steady-state current measurements of 2-S rimantadine (TGN-S15) and 2-R rimantadine (TGN-S16) on NR2A. Figure 1C-D shows peak current amplitude and steady-state current measurements of 2-S rimantadine (TGN-S15) and 2-R rimantadine (TGN-S16) on NR2B.

[0009] Figure 2 shows the proliferation of CAL-27 cells at various concentrations of RS-rimantadine (TGN-S11), S-rimantadine (TGN-S15), R-rimantadine (TGN-S16), and memantine (TGN-S13).

[0010] FIG. 3 shows the interaction map of 2-S-limantidine.

[0011] FIG. 4 shows the X-ray powder diffraction ("XRPD") diffractogram of 2-S-rimantidine fumarate salt Type A.

[0012] FIG. 5 shows the XRPD diffractogram of 2-S-rimantidine tartrate salt Type A.

[0013] FIG. 6 shows the XRPD diffractogram of 2-S-rimantidine galactarate type A.

[0014] FIG. 7 shows the XRPD diffractogram of 2-S-rimantidine galactarate type B.

[0015] FIG. 8 shows the XRPD diffractogram of 2-S-rimantidine benzoate type A.

[0016] FIG. 9 shows the XRPD diffractogram of 2-S-rimantidine benzenesulfonate salt Type A.

[0017] FIG. 10 shows the XRPD diffractogram of 2-S-rimantidine benzenesulfonate salt Type B.

[0018] FIG. 11 shows the thermogravimetric analysis and differential scanning calorimetry of 2-S-rimantidine tartrate salt Type A.

[0019] FIG. 12 shows the thermogravimetric analysis and differential scanning calorimetry of 2-S-rimantidine maleate salt Type A.

[0020] FIG. 13 shows the thermogravimetric analysis and differential scanning calorimetry of 2-S-rimantidine hippurate salt Type A.

[0021] FIG. 14 shows the thermogravimetric analysis and differential scanning calorimetry of 2-S-rimantidine galactarate type A.

[0022] FIG. 15 shows the thermogravimetric analysis and differential scanning calorimetry of 2-S-rimantidine galactarate type B.

[0023] FIG. 16 shows the thermogravimetric analysis and differential scanning calorimetry of 2-S-rimantidine benzoate type A.

[0024] FIG. 17 shows the thermogravimetric analysis and differential scanning calorimetry of 2-S-rimantidine benzoate type B.

[0025] FIG. 18 shows the thermogravimetric analysis and differential scanning calorimetry of 2-S-rimantidine gentisate type A.

[0026] FIG. 19 shows the thermogravimetric analysis and differential scanning calorimetry of 2-S-rimantidine toluenesulfonate salt Type A.

[0027] FIG. 20 shows the thermogravimetric analysis and differential scanning calorimetry of 2-S-rimantidine benzenesulfonate salt Type A.

[0028] FIG. 21 shows the thermogravimetric analysis and differential scanning calorimetry of 2-S-rimantidine benzenesulfonate salt Type B.

[0029] FIG. 22 shows a polarized light microscopy analysis of 2-S-rimantidine fumarate salt Type A.

[0030] FIG. 23 shows a polarized light microscopy analysis of 2-S-rimantidine tartrate salt Type A.

[0031] FIG. 24 shows a polarized light microscopy analysis of 2-S-rimantidine maleate salt Type A.

[0032] FIG. 25 shows a polarized light microscopy analysis of 2-S-rimantidine hippurate salt Type A.

[0033] FIG. 26 shows a polarized light microscopy analysis of 2-S-rimantidine galactarate type A.

[0034] FIG. 27 shows a polarized light microscopy analysis of 2-S-rimantidine galactarate type B.

[0035] FIG. 28 shows a polarized light microscopy analysis of 2-S-rimantidine benzoate salt Type A.

[0036] FIG. 29 shows a polarized light microscopy analysis of 2-S-rimantidine benzoate type B.

[0037] FIG. 30 shows a polarized light microscopy analysis of 2-S-rimantidine gentisate type A.

[0038] FIG. 31 shows a polarized light microscopy analysis of 2-S-rimantidine toluenesulfonate salt Type A.

[0039] FIG. 32 shows a polarized light microscopy analysis of 2-S-rimantidine benzenesulfonate salt Type A.

[0040] Figure 33 shows a polarized light microscopy analysis of 2-S-rimantidine benzenesulfonate salt Type B. DETAILED DESCRIPTION OF EMBODIMENTS

[0041] One aspect of the present disclosure is the use of enantiomerically pure 2-S rimantadine (e.g., an enantiomerically pure salt of 2-S rimantadine (e.g., fumarate, tartrate, galactarate, benzoate, benzenesulfonate, or any combination thereof)) or enantiomerically pure 2-R rimantadine to treat cancer. In some embodiments disclosed herein, the use of 2-S rimantadine (e.g., a salt of 2-S rimantadine) (also referred to as "S-rimantadine") or enantiomerically pure 2-R rimantadine to treat cancer associated with papillomaviruses, such as human papillomavirus (HPV), is disclosed. In some embodiments, the HPV is an alpha genus HPV.

[0042] Another aspect of the present disclosure is the use of enantiomerically pure 2-S rimantadine (e.g., a salt of 2-S rimantadine) or enantiomerically pure 2-R rimantadine in the treatment of precancerous lesions associated with papillomaviruses, such as human papillomaviruses.

[0043] Racemic rimantadine is associated with side effects at currently prescribed doses. These side effects include central nervous system (CNS), drowsiness, gastrointestinal, and atropine-related side effects, including, but not limited to, lightheadedness, dizziness, depression, confusion, difficulty concentrating, anxiety (e.g., nervousness), irritability, hallucinations, headache, insomnia, excessive fatigue, loss of appetite, nausea, vomiting, constipation, dry mouth, blurred vision, difficulty urinating, and difficulty swallowing. Anxiety and insomnia are the most commonly cited racemic rimantadine toxicities leading to treatment discontinuation.

[0044] As disclosed herein, 2-S rimantadine inhibits the N-methyl-D-aspartate subtype glutamate receptor (NMDA) subunit NR2B subunit to a lesser extent compared to 2-R rimantadine and racemic rimantadine (see Table 2 in Example 2 below).

[0045] In some embodiments, disclosed herein is the use of 2-S rimantadine (e.g., a salt (e.g., an enantiomerically pure salt) of 2-S rimantadine) to treat cancer, HPV-associated cancer, HPV-associated precancerous lesions, and / or influenza A. 2-S rimantadine has a reduced ability to inhibit NR2B compared to racemic rimantadine, which may result in fewer side effects than treatment of these conditions with racemic or 2-R rimantadine. 2-R rimantadine has a greater ability to inhibit NR2B compared to 2-S rimantadine, which may result in fewer side effects than treatment of these conditions with racemic or 2-S rimantadine.

[0046] In some embodiments, disclosed herein is the use of 2-R rimantadine for the treatment of cancer, HPV-associated cancer, HPV-associated precancerous lesions, and / or influenza A. Due to its increased ability to inhibit NR2B compared to racemic rimantadine, 2-R rimantadine may have fewer side effects compared to treating these conditions with racemic or 2-S rimantadine.

[0047] In some embodiments, the 2-S rimantadine disclosed herein (e.g., a salt (e.g., an enantiomerically pure salt) of 2-S rimantadine) may have fewer side effects compared to treating these conditions with racemic rimantadine or 2-R rimantadine due to the reduced ability of 2-S rimantadine to antagonize NMDA receptors and / or inhibit NMDA-mediated biological pathways. In some embodiments, the degree of NMDA receptor inhibition caused by 2-S rimantadine is about 10% less to about 100% less than that caused by 2-R rimantadine or racemic rimantadine.In some embodiments, the degree of NMDA receptor inhibition caused by 2-S rimantadine is about 10% less to about 20% less, about 10% less to about 30% less, about 10% less to about 40% less, about 10% less to about 50% less, about 10% less to about 60% less, about 10% less to about 70% less, about 10% less to about 80% less, or about 10% less to about 90% less than 2-R rimantadine or racemic rimantadine. , about 10% less to about 100% less, about 20% less to about 30% less, about 20% less to about 40% less, about 20% less to about 50% less, about 20% less to about 60% less, about 20% less to about 70% less, about 20% less to about 80% less, about 20% less to about 90% less, about 20% less to about 100% less, about 30% less to about 40% less, about 30% less to about 50% less, about 30% less to about 60% less, about 30% less ~ about 70% less, about 30% less to about 80% less, about 30% less to about 90% less, about 30% less to about 100% less, about 40% less to about 50% less, about 40% less to about 60% less, about 40% less to about 70% less, about 40% less to about 80% less, about 40% less to about 90% less, about 40% less to about 100% less, about 50% less to about 60% less, about 50% less to about 70% less, about 50% less to about 80% less about 50% less to about 90% less, about 50% less to about 100% less, about 60% less to about 70% less, about 60% less to about 80% less, about 60% less to about 90% less, about 60% less to about 100% less, about 70% less to about 80% less, about 70% less to about 90% less, about 70% less to about 100% less, about 80% less to about 90% less, about 80% less to about 100% less, or about 90% less to about 100% less. In some embodiments, the degree of NMDA receptor inhibition caused by 2-S rimantadine is about 10% less, about 20% less, about 30% less, about 40% less, about 50% less, about 60% less, about 70% less, about 80% less, about 90% less, or about 100% less compared to 2-R rimantadine or racemic rimantadine.In some embodiments, the degree of NMDA receptor inhibition caused by 2-S rimantadine is at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, or about 90% less than that caused by 2-R rimantadine or racemic rimantadine. In some embodiments, the degree of NMDA receptor inhibition caused by 2-S rimantadine is at most about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 100% less than that caused by 2-R rimantadine or racemic rimantadine. In some embodiments, the NMDA receptor is NR2A. In some embodiments, the NMDA receptor is NR2B.

[0048] In some embodiments, the 2-R rimantadine disclosed herein may have fewer side effects compared to treating these conditions with racemic or 2-S rimantadine due to the reduced ability of 2-R rimantadine to antagonize NMDA receptors and / or inhibit NMDA-mediated biological pathways. In some embodiments, the degree of NMDA receptor inhibition caused by 2-R rimantadine is about 10% less to about 100% less than that caused by 2-S rimantadine or racemic rimantadine.In some embodiments, the degree of NMDA receptor inhibition caused by 2-R rimantadine is about 10% less to about 20% less, about 10% less to about 30% less, about 10% less to about 40% less, about 10% less to about 50% less, about 10% less to about 60% less, about 10% less to about 70% less, about 10% less to about 80% less, or about 10% less to about 90% less than 2-S rimantadine or racemic rimantadine. , about 10% less to about 100% less, about 20% less to about 30% less, about 20% less to about 40% less, about 20% less to about 50% less, about 20% less to about 60% less, about 20% less to about 70% less, about 20% less to about 80% less, about 20% less to about 90% less, about 20% less to about 100% less, about 30% less to about 40% less, about 30% less to about 50% less, about 30% less to about 60% less, about 30% less ~ about 70% less, about 30% less to about 80% less, about 30% less to about 90% less, about 30% less to about 100% less, about 40% less to about 50% less, about 40% less to about 60% less, about 40% less to about 70% less, about 40% less to about 80% less, about 40% less to about 90% less, about 40% less to about 100% less, about 50% less to about 60% less, about 50% less to about 70% less, about 50% less to about 80% less about 50% less to about 90% less, about 50% less to about 100% less, about 60% less to about 70% less, about 60% less to about 80% less, about 60% less to about 90% less, about 60% less to about 100% less, about 70% less to about 80% less, about 70% less to about 90% less, about 70% less to about 100% less, about 80% less to about 90% less, about 80% less to about 100% less, or about 90% less to about 100% less. In some embodiments, the degree of NMDA receptor inhibition caused by 2-R rimantadine is about 10% less, about 20% less, about 30% less, about 40% less, about 50% less, about 60% less, about 70% less, about 80% less, about 90% less, or about 100% less compared to 2-S rimantadine or racemic rimantadine.In some embodiments, the degree of NMDA receptor inhibition caused by 2-R rimantadine is at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, or about 90% less than that caused by 2-S rimantadine or racemic rimantadine. In some embodiments, the degree of NMDA receptor inhibition caused by 2-R rimantadine is at most about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 100% less than that caused by 2-S rimantadine or racemic rimantadine. In some embodiments, the NMDA receptor is NR2A. In some embodiments, the NMDA receptor is NR2B.

[0049] In some embodiments, 2-S rimantadine (e.g., a salt (e.g., an enantiomerically pure salt) of 2-S rimantadine) may have fewer side effects compared to treating these conditions with racemic rimantadine or 2-R rimantadine due to the reduced ability of 2-S rimantadine to antagonize GABA receptors and / or inhibit GABA-mediated biological pathways. In some embodiments, the degree of GABA receptor inhibition and / or GABA-mediated biological pathway inhibition caused by 2-S rimantadine is about 10% less to about 100% less than that caused by 2-R rimantadine or racemic rimantadine.In some embodiments, the degree of GABA receptor and / or GABA-mediated biological pathway inhibition caused by 2-S rimantadine (e.g., a salt of 2-S rimantadine) is about 10% less to about 20% less, about 10% less to about 30% less, about 10% less to about 40% less, about 10% less to about 50% less, about 10% less to about 60% less, about 10% less to about 70% less, about 10% less to about 80% less, about 10% less to about 90% less, about 10% less to about 110% less, about 10% less to about 210% less, about 10% less to about 220% less, about 10% less to about 230% less, about 10% less to about 240% less, about 10% less to about 250% less, about 10% less to about 260% less, about 10% less to about 270% less, about 10% less to about 280% less, about 10% less to about 290% less, about 10% less to about 300% less, about 10% less to about 310% less, about 10% less to about 320% less, about 10% less to about 330% less, about 10% less to about 340% less, about 10% less to about 350% less, about 10% less to about 360% less, about 10% less to about 370% less, about 10% less to about 380% less, about 10% less to about 390% less, about 10% less to about 400% less, about 10% less to about 410% less, about 10% less to about 420% less, about Less to about 80% less, about 10% less to about 90% less, about 10% less to about 100% less, about 20% less to about 30% less, about 20% less to about 40% less, about 20% less to about 50% less, about 20% less to about 60% less, about 20% less to about 70% less, about 20% less to about 80% less, about 20% less to about 90% less, about 20% less to about 100% less, about 30% less to about 40% less, about 30% less to about 50% less, about 30% Less to about 60% less, about 30% less to about 70% less, about 30% less to about 80% less, about 30% less to about 90% less, about 30% less to about 100% less, about 40% less to about 50% less, about 40% less to about 60% less, about 40% less to about 70% less, about 40% less to about 80% less, about 40% less to about 90% less, about 40% less to about 100% less, about 50% less to about 60% less, about 50% less to about 70% less, about 50% Less to about 80% less, about 50% less to about 90% less, about 50% less to about 10% less, about 60% less to about 70% less, about 60% less to about 80% less, about 60% less to about 90% less, about 60% less to about 100% less, about 70% less to about 80% less, about 70% less to about 90% less, about 70% less to about 100% less, about 80% less to about 90% less, about 80% less to about 100% less, or about 90% less to about 100% less.In some embodiments, the degree of inhibition of GABA receptors and / or GABA-mediated biological pathways caused by 2-S rimantadine is about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 100% less than that caused by 2-R rimantadine or racemic rimantadine. In some embodiments, the degree of inhibition of GABA receptors and / or GABA-mediated biological pathways caused by 2-S rimantadine is at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, or about 90% less than that caused by 2-R rimantadine or racemic rimantadine. In some embodiments, the degree of GABA receptor and / or GABA-mediated biological pathway inhibition caused by 2-S rimantadine is up to about 20% less, about 30% less, about 40% less, about 50% less, about 60% less, about 70% less, about 80% less, about 90% less, or about 100% less compared to 2-R rimantadine or racemic rimantadine.

[0050] In some embodiments, the 2-R rimantadine disclosed herein may have fewer side effects than treatment of these conditions with racemic or 2-S rimantadine due to its reduced ability to antagonize GABA receptors and / or inhibit GABA-mediated biological pathways. In some embodiments, the degree of inhibition of GABA receptors and / or GABA-mediated biological pathways caused by 2-R rimantadine is about 10% less to about 100% less than that caused by 2-S or racemic rimantadine.In some embodiments, the degree of GABA receptor and / or GABA-mediated biological pathway inhibition caused by 2-R rimantadine is about 10% less to about 20% less, about 10% less to about 30% less, about 10% less to about 40% less, about 10% less to about 50% less, about 10% less to about 60% less, about 10% less to about 70% less, about 10% less to about 80% less, about 10% less to about 90% less, about 10% less to about 100% less, about 20% less to about 30% less, about 20% less to about 40% less, about 20% less to about 50% less, about 20% less to about 60% less, about 20% less to about 70% less, about 20% less to about 80% less, about 20% less to about 90% less, about 20% less to about 100% less, about 30% less to about 40% less, about 30% less to about 50% less, about 30% less to about 60% less None, about 30% less to about 70% less, about 30% less to about 80% less, about 30% less to about 90% less, about 30% less to about 100% less, about 40% less to about 50% less, about 40% less to about 60% less, about 40% less to about 70% less, about 40% less to about 80% less, about 40% less to about 90% less, about 40% less to about 100% less, about 50% less to about 60% less, about 50% less to about 70% less, about 50% less to about 80% less, about 50% less to about 90% less, about 50% less to about 100% less, about 60% less to about 70% less, about 60% less to about 80% less, about 60% less to about 90% less, about 60% less to about 100% less, about 70% less to about 80% less, about 70% less to about 90% less, about 70% less to about 100% less, about 80% less to about 90% less, about 80% less to about 100% less, or about 90% less to about 100% less.In some embodiments, the degree of GABA receptor and / or GABA-mediated biological pathway inhibition caused by 2-R rimantadine is about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 100% less than that caused by 2-S or racemic rimantadine. In some embodiments, the degree of GABA receptor and / or GABA-mediated biological pathway inhibition caused by 2-R rimantadine is at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, or about 90% less than that caused by 2-S or racemic rimantadine. In some embodiments, the degree of GABA receptor and / or GABA-mediated biological pathway inhibition caused by 2-R rimantadine is up to about 20% less, about 30% less, about 40% less, about 50% less, about 60% less, about 70% less, about 80% less, about 90% less, or about 100% less compared to 2-S rimantadine or racemic rimantadine.

[0051] In some embodiments, the 2-S rimantadine disclosed herein (e.g., a salt (e.g., an enantiomerically pure salt) of 2-S rimantadine) may have fewer side effects compared to treating these conditions with racemic rimantadine or 2-R rimantadine due to the reduced dopamine receptor antagonism and / or dopamine-mediated biological pathway inhibition of 2-S rimantadine. In some embodiments, the degree of dopamine receptor inhibition and / or dopamine-mediated biological pathway inhibition caused by 2-S rimantadine is about 10% less to about 100% less than that caused by 2-R rimantadine or racemic rimantadine.In some embodiments, the degree of dopamine receptor and / or dopamine-mediated biological pathway inhibition caused by 2-S rimantadine is about 10% less to about 20% less, about 10% less to about 30% less, about 10% less to about 40% less, about 10% less to about 50% less, about 10% less to about 60% less, about 10% less to about 70% less, or about 10% less to about 80% less than 2-R rimantadine or racemic rimantadine. , about 10% less to about 90% less, about 10% less to about 100% less, about 20% less to about 30% less, about 20% less to about 40% less, about 20% less to about 50% less, about 20% less to about 60% less, about 20% less to about 70% less, about 20% less to about 80% less, about 20% less to about 90% less, about 20% less to about 100% less, about 30% less to about 40% less, about 30% less to about 50% less, about 30% less to about 60% Less, about 30% less to about 70% less, about 30% less to about 80% less, about 30% less to about 90% less, about 30% less to about 100% less, about 40% less to about 50% less, about 40% less to about 60% less, about 40% less to about 70% less, about 40% less to about 80% less, about 40% less to about 90% less, about 40% less to about 100% less, about 50% less to about 60% less, about 50% less to about 70% less, about 50% less to about 80% less, about 50% less to about 90% less, about 50% less to about 100% less, about 60% less to about 70% less, about 60% less to about 80% less, about 60% less to about 90% less, about 60% less to about 100% less, about 70% less to about 80% less, about 70% less to about 90% less, about 70% less to about 100% less, about 80% less to about 90% less, about 80% less to about 100% less, or about 90% less to about 100% less.In some embodiments, the degree of dopamine receptor and / or dopamine-mediated biological pathway inhibition caused by 2-S rimantadine is about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 100% less than that caused by 2-R rimantadine or racemic rimantadine. In some embodiments, the degree of dopamine receptor and / or dopamine-mediated biological pathway inhibition caused by 2-S rimantadine is at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, or about 90% less than that caused by 2-R rimantadine or racemic rimantadine. In some embodiments, the degree of dopamine receptor and / or dopamine-mediated biological pathway inhibition caused by 2-S rimantadine is up to about 20% less, about 30% less, about 40% less, about 50% less, about 60% less, about 70% less, about 80% less, about 90% less, or about 100% less compared to 2-R rimantadine or racemic rimantadine. In some embodiments, the dopamine receptor is D. 2 / 3 It is a receptor.

[0052] In some embodiments, 2-R rimantadine may have fewer side effects than treatment of these conditions with racemic or 2-S rimantadine due to its reduced ability to antagonize dopamine receptors and / or inhibit dopamine-mediated biological pathways. In some embodiments, the degree of inhibition of dopamine receptors and / or dopamine-mediated biological pathways caused by 2-R rimantadine is about 10% less to about 100% less than that caused by 2-S or racemic rimantadine.In some embodiments, the degree of dopamine receptor and / or dopamine-mediated biological pathway inhibition caused by 2-R rimantadine is about 10% less to about 20% less, about 10% less to about 30% less, about 10% less to about 40% less, about 10% less to about 50% less, about 10% less to about 60% less, about 10% less to about 70% less, or about 10% less to about 80% less than 2-S rimantadine or racemic rimantadine. , about 10% less to about 90% less, about 10% less to about 100% less, about 20% less to about 30% less, about 20% less to about 40% less, about 20% less to about 50% less, about 20% less to about 60% less, about 20% less to about 70% less, about 20% less to about 80% less, about 20% less to about 90% less, about 20% less to about 100% less, about 30% less to about 40% less, about 30% less to about 50% less, about 30% less to about 60% Less, about 30% less to about 70% less, about 30% less to about 80% less, about 30% less to about 90% less, about 30% less to about 100% less, about 40% less to about 50% less, about 40% less to about 60% less, about 40% less to about 70% less, about 40% less to about 80% less, about 40% less to about 90% less, about 40% less to about 100% less, about 50% less to about 60% less, about 50% less to about 70% less, about 50% less to about 80% less, about 50% less to about 90% less, about 50% less to about 100% less, about 60% less to about 70% less, about 60% less to about 80% less, about 60% less to about 90% less, about 60% less to about 100% less, about 70% less to about 80% less, about 70% less to about 90% less, about 70% less to about 100% less, about 80% less to about 90% less, about 80% less to about 100% less, or about 90% less to about 100% less.In some embodiments, the degree of dopamine receptor and / or dopamine-mediated biological pathway inhibition caused by 2-R rimantadine is about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 100% less than that caused by 2-S or racemic rimantadine. In some embodiments, the degree of dopamine receptor and / or dopamine-mediated biological pathway inhibition caused by 2-R rimantadine is at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, or about 90% less than that caused by 2-S or racemic rimantadine. In some embodiments, the degree of dopamine receptor and / or dopamine-mediated biological pathway inhibition caused by 2-R rimantadine is up to about 20% less, about 30% less, about 40% less, about 50% less, about 60% less, about 70% less, about 80% less, about 90% less, or about 100% less compared to 2-S rimantadine or racemic rimantadine. In some embodiments, the dopamine receptor is D. 2 / 3 It is a receptor.

[0053] In another aspect, disclosed herein is the use of 2-S rimantadine (e.g., a salt (e.g., an enantiomerically pure salt) of 2-S rimantadine) for the treatment / prevention of influenza in veterinary animals, such as poultry (e.g., chickens, turkeys, ducks) and horses. The use of 2-S rimantadine may have fewer side effects compared to treating these animals with racemic or 2-R rimantadine.

[0054] In another aspect, disclosed herein is the use of 2-R rimantadine for the treatment / prevention of influenza in veterinary animals, such as poultry (e.g., chickens, turkeys, ducks) and horses. The use of 2-R rimantadine may result in fewer side effects compared to treating these animals with racemic or 2-S rimantadine. Definitions

[0055] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural references unless the content clearly dictates otherwise. Also, the term "or" is typically used in its sense including "and / or" unless the content clearly dictates otherwise. Furthermore, the headings provided herein are for convenience only and do not interpret the scope or meaning of the claimed invention.

[0056] In some embodiments, the S-rimantadine (e.g., a salt of 2-S-rimantadine), R-rimantadine, racemic rimantadine, or rimantadine derivative described herein is PEGylated. As used herein, "PEGylated" or "PEGylation" refers to the attachment of a compound to a polyethylene glycol (PEG) moiety. The PEG moiety can be of any length. For example, the PEG moiety can have 2 to 500 repeating units. In some embodiments, the PEG moiety can have an average molecular weight of about 300 g / mol to about 10,000,000 g / mol. In some embodiments, the PEG moiety can be a high-molecular-weight PEG or a low-molecular-weight PEG. For example, a high-molecular-weight PEG has a molecular weight of 5 kDa or greater, and a low-molecular-weight PEG has a molecular weight of less than 5 kDa. In some embodiments, the PEG is selected from the group consisting of PEG 200, PEG 300, PEG 400, PEG 600, PEG 800, PEG 1000, PEG 1500, PEG 2000, and PEG 3350. The PEG moiety can be a linear PEG, or the PEG moiety can be a branched PEG, for example, a branched PEG includes any PEG having one or more branches of PEG groups extending from the PEG backbone.

[0057] As used herein, the term "pure" as applied to a chiral compound refers to an enantiomer of a chiral compound being substantially free of its opposite enantiomer (i.e., in enantiomeric excess). For example, a pure "R" form of a compound is substantially free of the "S" form of the compound and is therefore in enantiomeric excess over the "S" form. The terms "enantiomerically pure" or "pure enantiomer" mean that the compound contains, for example, 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 98.5% or more, 99% or more, 99.2% or more, 99.5% or more, 99.6% or more, 99.7% or more, 99.8% or more, or 99.9% or more of its enantiomer by weight. In certain embodiments, the weight is based on the total weight of the compound, i.e., all enantiomers of the compound. In certain embodiments, one enantiomer may be in excess of 30-80%, or 30-70%, 30-60%, 30%, 35%, 40%, 45%, 50%, 55%, or 60%, or any percentage therebetween.

[0058] As used herein, unless otherwise specified, the term "enantiomerically pure 2-S rimantadine" refers to, for example, at least about 80% by weight of 2-S rimantadine and up to about 20% by weight of 2-R rimantadine, at least about 90% by weight of 2-S rimantadine and up to about 10% by weight of 2-R rimantadine, at least about 95% by weight of 2-S rimantadine and up to about 5% by weight of 2-R rimantadine, at least about 99% by weight of 2-S rimantadine and up to about 1% by weight of 2-R rimantadine, or at least about 99.9% by weight of 2-S rimantadine and up to about 0.1% by weight of 2-R rimantadine. In certain embodiments, the weights are based on the total weight of rimantadine, i.e., both or all of the enantiomers of rimantadine.

[0059] As used herein, unless otherwise specified, the term "enantiomerically pure 2-R rimantadine" refers to, for example, at least about 80% by weight 2-R rimantadine and up to about 20% by weight 2-S rimantadine, at least about 90% by weight 2-R rimantadine and up to about 10% by weight 2-S rimantadine, at least about 95% by weight 2-R rimantadine and up to about 5% by weight 2-S rimantadine, at least about 99% by weight 2-R rimantadine and up to about 1% by weight 2-S rimantadine, at least about 99.9% by weight 2-R rimantadine, or up to about 0.1% by weight 2-S rimantadine. In certain embodiments, the weights are based on the total weight of rimantadine, i.e., both or all enantiomers of rimantadine.

[0060] In the compositions provided herein, enantiomerically pure rimantadine or its pharmaceutically acceptable salts, solvates, hydrates, esters, or prodrugs can be present together with other active or inactive ingredients. For example, a pharmaceutical composition containing enantiomerically pure 2-S rimantadine can contain, for example, about 90% excipients and about 10% enantiomerically pure 2-S rimantadine. In certain embodiments, the enantiomerically pure 2-S rimantadine in such a composition can contain, for example, at least about 99.9% by weight of 2-S rimantadine and up to about 0.1% by weight of 2-S rimantadine. In certain embodiments, the active ingredient can be formulated with little or no carrier, excipient, or diluent.

[0061] As used herein, the terms "subject," "individual," or "patient" are used interchangeably and refer to any animal, including poultry such as chickens, ducks, turkeys, and mammals such as mice, rats, other rodents, rabbits, dogs, cats, pigs, cows, sheep, horses, primates, and humans. In some embodiments, the subject is a human.

[0062] As used herein, the terms "treatment" or "treatment" refer to curative or palliative measures. Beneficial or desired clinical results include, but are not limited to, the total or partial alleviation of symptoms associated with a disease or disorder or condition, a reduction in the extent of the disease, stabilization of the disease (i.e., not worsening), a delay or slowing of the progression of the disease, an improvement or alleviation of the disease state (e.g., one or more symptoms of the disease), and remission (partial or complete), whether detectable or undetectable. "Treatment" can also mean prolonging survival as compared to expected survival if not receiving treatment.

[0063] As used herein, the term "prevention" refers to the total or partial prevention of the onset, recurrence or spread of a disease or condition described herein, or a symptom thereof.

[0064] The term "administration" or "administering" refers to a method of providing a dosage of a compound or pharmaceutical composition to a vertebrate or invertebrate, including a mammal, bird, fish, or amphibian. The preferred method of administration may vary depending on various factors, such as the components of the pharmaceutical composition, the site of the disease, and the severity of the disease. A "therapeutically effective amount" or "pharmaceutically effective amount" of a compound provided herein is an amount sufficient to achieve the desired effect, which may vary depending on the nature and severity of the disease state and the potency of the compound. A therapeutic effect is one that relieves to some extent one or more symptoms of the disease, including curing the disease.

[0065] "Cure" means the absence of symptoms of active disease, although certain long-term or permanent effects of the disease may exist even after a cure has been achieved (e.g., extensive tissue damage).

[0066] As used herein, the term "effective amount" or "therapeutically effective amount" refers to the administration of an agent or compound in an amount sufficient to relieve to some extent one or more symptoms of the disease or condition being treated, resulting in the reduction and / or alleviation of the signs, symptoms, or causes of the disease, or other desired alteration of a biological system. For example, an "effective amount" for therapeutic purposes is the amount of a composition comprising a compound disclosed herein that is required to produce a clinically significant reduction in a symptom of the disease. An appropriate "effective" amount in each individual case can be determined using techniques such as dose escalation studies. An "effective amount" is an amount of a compound sufficient to achieve its stated purpose in the absence of the compound (e.g., achieve the effect for which it is administered, treat a disease, decrease enzyme activity, increase enzyme activity, decrease a signal transduction pathway, or alleviate one or more symptoms of a disease or condition). An example of an "effective amount" is an amount sufficient to contribute to the treatment, prevention, or alleviation of symptoms of a disease, also known as a "therapeutically effective amount." "Alleviation" of a symptom (and grammatical equivalents of this phrase) refers to a reduction in the severity or frequency of a symptom, or the elimination of a symptom. A "prophylactically effective amount" of a drug is an amount of drug that, when administered to a subject, achieves the intended prophylactic effect, e.g., prevents or delays the onset (or recurrence) of an injury, disease, symptom, or condition, or reduces the likelihood of the injury, disease, symptom, or condition, or its symptoms, developing (or recurring). A complete prophylactic effect does not necessarily occur with a single administration, but may occur only after a series of administrations. Thus, a prophylactically effective amount can be administered in one or more doses. As used herein, an "activity-reducing amount" refers to the amount of antagonist required to reduce the activity of an enzyme relative to the absence of the antagonist. As used herein, a "function-disrupting amount" refers to the amount of antagonist required to disrupt the function of an enzyme or protein relative to the absence of the antagonist. The precise amount will vary depending on the purpose of treatment and can be ascertained by one of skill in the art using known techniques (see, e.g., Lieberman, Pharmaceutical Dosage Forms (Vols. 1-3, 1992); Lloyd, The Art, Science and Technology of Pharmaceutical Compounding (1999); Pickar, Dosage Calculations (1999); and Remington: The Science and Practice of Pharmacy, 20th ed., 2003, edited by Gennaro, Lippincott, Williams & Wilkins).

[0067] The term "immunotherapy" refers to an agent that modulates the immune system. In some embodiments, immunotherapy can increase the expression and / or activity of a regulator of the immune system. In some embodiments, immunotherapy can decrease the expression and / or activity of a regulator of the immune system. In some embodiments, immunotherapy can enhance the activation and / or activity of immune cells. Pharmaceutically acceptable salts, prodrugs, stereoisomers, and tautomers

[0068] The pure R or S enantiomers of rimantadine provided herein (e.g., salts of 2-S rimantadine (e.g., fumarate, tartrate, galactarate, benzoate, benzenesulfonate, or any combination thereof)) can be administered as any salt or prodrug that can directly or indirectly provide the parent compound upon administration to a recipient, or that itself exhibits activity. As used herein, the term "pharmaceutically acceptable salt" refers to a salt that retains the desired biological activity of the subject compound with minimal undesired toxicological effects. The phrase "pharmaceutically acceptable salt or prodrug" is used throughout this specification to describe pharmaceutically acceptable forms of a compound (such as esters, amides, salts of esters, salts of amides, or related groups) that, upon administration to a patient, provide an active compound of the present disclosure. Such modifications affect the biological activity of the compound, in some cases resulting in increased activity over the parent compound. Pharmaceutically acceptable salts may be prepared in situ during the final isolation and purification of the compound, or may be prepared from a free acid or free base form. These compounds may be prepared by separately reacting the purified compounds of formula (I) with an appropriate base or acid, respectively. In some embodiments, pharmaceutically acceptable salts may be preferred over the respective free bases or acids, as such salts may impart increased stability or solubility to the molecules, thereby facilitating formulation into dosage forms. Basic compounds can generally form pharmaceutically acceptable acid addition salts by treating them with an appropriate acid. Suitable acids include pharmaceutically acceptable inorganic acids and pharmaceutically acceptable organic acids. In some embodiments, pharmaceutically acceptable acid addition salts include fumaric acid, tartaric acid, galactaric acid, benzoic acid, benzenesulfonic acid, or any combination thereof. In some embodiments, pharmaceutically acceptable acid addition salts include fumaric acid. In some embodiments, pharmaceutically acceptable acid addition salts include tartaric acid. In some embodiments, pharmaceutically acceptable acid addition salts include galactaric acid. In some embodiments, pharmaceutically acceptable acid addition salts include benzoic acid.In some embodiments, pharmaceutically acceptable acid addition salts include benzenesulfonic acid. Further non-limiting examples of pharmaceutically acceptable acid addition salts include hydrochloride, hydrobromide, nitrate, methylnitrate, sulfate, bisulfate, sulfamate, phosphate, acetate, hydroxyacetate, phenylacetate, propionate, butyrate, isobutyrate, valerate, maleate, hydroxymaleate, acrylate, fumarate, malate, tartrate, citrate, salicylate, p-aminosalicylate, glycolate, lactate, heptanoate, phthalate, oxalate, succinate, benzoate, o-acetoxybenzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate, and hydroxybenzoate. , methoxybenzoate, mandelate, tannate, formate, stearate, ascorbate, palmitate, oleate, pyruvate, pamoate, malonate, laurate, glutarate, glutamate, estolate, methanesulfonate (mesylate), ethanesulfonate (esylate), 2-hydroxyethanesulfonate, benzenesulfonate (besylate), p-aminobenzenesulfonate, p-toluenesulfonate (tosylate), naphthalene-2-sulfonate, ethanedisulfonate, 2,5-dihydroxybenzoate, and / or any combination thereof.

[0069] In some embodiments, rimantadine salts (e.g., 2-S-rimantadine salts) are synthesized by mixing slurries of starting materials. In some embodiments, the salts are synthesized at 0°C. In some embodiments, the salts are synthesized at 5°C. In some embodiments, the salts are synthesized by a temperature cycling method. In some embodiments, the salts are synthesized by a slow evaporation method. In some embodiments, the salts are synthesized by solvating 2-S-rimantadine with methanol, ethanol, chloroform, acetone, isopropyl, water, or any combination thereof. In some embodiments, the salts are synthesized using an anti-solvent such as acetone, n-heptane, or methyl isobutyl ketone.

[0070] A pharmaceutically acceptable prodrug refers to a compound that is metabolized (e.g., hydrolyzed or oxidized) in a host to form a compound of the present disclosure. Typical examples of prodrugs include compounds that have a biologically labile protecting group on a functional moiety of an active compound. Prodrugs include compounds that can be oxidized, reduced, aminated, deaminated, hydroxylated, dehydroxylated, hydrolyzed, dehydrolyzed, alkylated, dealkylated, acylated, deacylated, phosphorylated, and / or dephosphorylated to produce an active compound.

[0071] In some embodiments, the methods described herein involve administering pure 2-S rimantadine, or pure 2-R rimantadine or a pharmaceutically acceptable salt thereof.

[0072] In some embodiments, the PK properties of 2-S-rimantadine are improved in the salt form. In some embodiments, a salt of 2-S-rimantadine increases the AUC compared to the non-salt form of 2-S-rimantadine.

[0073] In certain embodiments, samples containing 2-S-limantidine solid forms, such as the crystals provided herein, may be substantially free of other solid forms, such as amorphous forms and / or other crystalline forms. In certain embodiments, such samples containing crystalline forms of 2-S-limantidine salts provided herein may contain less than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% of one or more other solid forms of 2-S-limantidine salts, such as amorphous forms.

[0074] In some embodiments, XRPD is measured using a Malvern-Panalytical Aeris X-ray powder diffractometer. In some embodiments, differential scanning calorimetry is measured using a TA2500 DSC instrument. In some embodiments, thermogravimetry is measured using a TA Discovery 550 instrument.

[0075] In some embodiments of the crystalline salt, the average crystal particle size is about 5 to about 45 μm. In some embodiments of the crystalline salt, the average crystal particle size is about 5 to about 40 μm. In some embodiments of the crystalline salt, the average crystal particle size is about 5 to about 35 μm. In some embodiments of the crystalline salt, the average crystal particle size is about 5 to about 30 μm. In some embodiments of the crystalline salt, the average crystal particle size is about 5 to about 25 μm. In some embodiments of the crystalline salt, the average crystal particle size is about 5 to about 20 μm. In some embodiments of the crystalline salt, the average crystal particle size is about 5 to about 15 μm. In some embodiments of the crystalline salt, the average crystal particle size is about 5 to about 10 μm. In some embodiments of the crystalline salt, the average crystal particle size is about 1 to about 35 μm. In some embodiments of the crystalline salt, the average crystal particle size is about 1 to about 30 μm. In some embodiments of the crystalline salt, the average crystal particle size is about 1 to about 25 μm. In some embodiments of the crystalline salt, the average crystal grain size is about 1 to about 20 μm. In some embodiments of the crystalline salt, the average crystal grain size is about 1 to about 15 μm. In some embodiments of the crystalline salt, the average crystal grain size is about 1 to about 10 μm. In some embodiments of the crystalline salt, the average crystal grain size is about 1 to about 5 μm. In some embodiments of the crystalline salt, the average crystal grain size is about 1 μm. In some embodiments of the crystalline salt, the average crystal grain size is about 2 μm. In some embodiments of the crystalline salt, the average crystal grain size is about 5 μm. In some embodiments of the crystalline salt, the average crystal grain size is about 10 μm. In some embodiments of the crystalline salt, the average crystal grain size is about 15 μm. In some embodiments of the crystalline salt, the average crystal grain size is about 20 μm. In some embodiments of the crystalline salt, the average crystal grain size is about 25 μm. In some embodiments of the crystalline salt, the average crystal grain size is about 30 μm. In some embodiments of the crystalline salt, the average crystal grain size is about 35 μm. In some embodiments of the crystalline salt, the average crystalline particle size is about 40 μm. In some embodiments of the crystalline salt, the average crystalline particle size is about 50 μm. Pharmaceutical Composition

[0076] Also provided herein are pharmaceutical compositions comprising pure 2-S rimantadine, pure 2-R rimantadine, or a pharmaceutically acceptable salt thereof, as described herein (e.g., a salt of 2-S rimantadine (e.g., fumarate, tartrate, galactarate, benzoate, benzenesulfonate, or any combination thereof)). Any of the pharmaceutical compositions described herein can be administered to a subject to treat cancer, as described herein.

[0077] Administration of 2-S rimantadine, pure 2-R rimantadine, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof, can be via any of the approved modes of administration, including, but not limited to, oral, subcutaneous, intravenous, intranasal, topical, transdermal, intraperitoneal, intramuscular, intrapulmonary, intravaginal, intrarectal, ontological, neuro-otological, intraocular, subconjunctival, anterior chamber injection, intravitreal, intraperitoneal, intrameningeal, intracystic, intrapleural, wound irrigation, buccal, intraperitoneal, intra-articular, intraaural, intrabronchial, intracystic, intrameningeal, inhalation, intratracheal or intrabronchial infusion, direct intrapulmonary infusion, intraspinal, intrasynovial, intrapleural, pleural lavage, epidural, intratympanic, intracisternal, intravascular, intraventricular, intraosseous, irrigation of infected bone, or application as part of a mixture with a prosthetic device. In some embodiments, the mode of administration includes oral or parenteral administration.

[0078] Pharmaceutically acceptable compositions may include solids, semisolids, liquids, solutions, colloids, liposomes, emulsions, suspensions, complexes, coacervates, and aerosols. Pharmaceutically acceptable compositions may also include dosage forms such as tablets, capsules, powders, liquids, suspensions, suppositories, aerosols, implants, and controlled-release formulations. 2-S rimantadine, pure 2-R rimantadine or a pharmaceutically acceptable salt thereof, or pharmaceutical compositions thereof, may also be administered in sustained- or controlled-release dosage forms, including depot injections, osmotic pumps, pills (tablets and / or capsules), transdermal (including electrotransport) patches, implants, and the like, for prolonged and / or timed pulse administration at a predetermined rate.

[0079] In some embodiments, the pharmaceutical composition is a tablet. In some embodiments, the pharmaceutical composition is a film-coated tablet.

[0080] Pure 2-S rimantadine, pure 2-R rimantadine or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof (e.g., a salt of 2-S rimantadine) can be administered alone or in combination with conventional pharmaceutical carriers, excipients, and the like. Pharmaceutically acceptable excipients include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, self-emulsifying drug delivery systems (SEDDS), surfactants used in pharmaceutical dosage forms, such as d-α-tocopherol, polyethylene glycol 1000, succinates, Tween, poloxamers, or other similar polymeric delivery matrices, serum proteins such as human serum albumin, buffer substances such as phosphate, Tris, glycine, sorbic acid, and potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinylpyrrolidone, cellulosic materials, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene polyoxypropylene block polymers, and wool fat. Cyclodextrins can also be used to enhance delivery of the compounds described herein.

[0081] In some embodiments, the pharmaceutical compositions described herein are in the form of a unit dosage form, such as a pill or tablet. Thus, the compositions may contain 2-S rimantadine, pure 2-R rimantadine, or a pharmaceutically acceptable salt thereof (e.g., a salt of 2-S rimantadine), along with a diluent such as lactose, sucrose, or dicalcium phosphate, a lubricant such as magnesium stearate, and a binder such as starch, acacia gum, polyvinylpyrrolidine, gelatin, cellulose, or a cellulose derivative. In another solid dosage form, a powder, quince, solution, or suspension (e.g., in propylene carbonate, vegetable oil, PEG, poloxamer 124, or triglycerides) is encapsulated in a capsule (e.g., a gelatin- or cellulose-based capsule). Unit dosage forms in which the 2-S rimantadine, pure 2-R rimantadine, or a pharmaceutically acceptable salt thereof, or an additional active agent described herein, are physically separated are also contemplated, such as capsules containing granules (or tablets within capsules) of each agent, bilayer tablets, or bicompartmental gel capsules. Enteric coated or delayed release oral dosage forms are also contemplated.

[0082] In some embodiments, the rimantadine or its pharmaceutically acceptable salt is PEGylated. In some embodiments, the PEGylated rimantadine or its pharmaceutically acceptable salt comprises a high molecular weight PEG. In some embodiments, the PEGylated rimantadine or its pharmaceutically acceptable salt comprises a low molecular weight PEG. In some embodiments, the rimantadine or its pharmaceutically acceptable salt is modified. In some embodiments, the modification is PEGylation.

[0083] In some embodiments, the PEGylated rimantadine or its pharmaceutically acceptable salt is PEGylated with a high molecular weight PEG. In some embodiments, the PEGylated rimantadine or its pharmaceutically acceptable salt is PEGylated with a low molecular weight PEG. Thus, also provided herein is a method for treating cancer in a subject in need thereof, the method comprising administering a therapeutically effective amount of PEGylated rimantadine or its pharmaceutically acceptable salt to the subject.

[0084] In some embodiments, the pharmaceutical composition includes one or more excipients selected from the group consisting of hypromellose, magnesium stearate, microcrystalline cellulose, and sodium starch glycolate.

[0085] Liquid pharmaceutically administrable compositions can be prepared, for example, by dissolving, dispersing, etc., a compound provided herein and any pharmaceutical adjuvants in a carrier (e.g., water, saline, aqueous dextrose, glycerol, glycol, ethanol, etc.) to form a solution, colloid, liposome, emulsion, complex, coacervate, or suspension. If desired, the pharmaceutical compositions can also contain minor amounts of nontoxic auxiliary substances such as wetting agents, emulsifiers, cosolvents, solubilizing agents, pH buffering agents, and the like (e.g., sodium acetate, sodium citrate, cyclodextrin derivatives, sorbitan monolaurate, triethanolamine acetate, triethanolamine oleate, etc.).

[0086] Dosage forms or compositions can be prepared containing 0.005% to 100% of the 2-S rimantadine, pure 2-R rimantadine, or a pharmaceutically acceptable salt thereof (e.g., a salt of 2-S rimantadine) described herein, with the remainder consisting of a non-toxic carrier. Contemplated compositions may contain 0.001% to 100% of a compound provided herein, with 0.1 to 95% in one embodiment, 75 to 85% in another embodiment, and 20 to 80% in a further embodiment. Actual methods for preparing such dosage forms will be known or apparent to those skilled in the art; see, for example, Remington: The Science and Practice of Pharmacy, 22nd Edition (Pharmaceutical Press, London, UK, 2012).

[0087] The pharmaceutical compositions of the present invention can contain about 0.1 to 1000 mg of 2-S rimantadine, pure 2-R rimantadine, or a pharmaceutically acceptable salt thereof (e.g., a salt of 2-S rimantadine) per unit dosage, e.g., tablet, capsule, suspension, solution, reconstitution sachet, powder, injection, infusion, suppository, sublingual / buccal film, teaspoonful, etc. Pure 2-S rimantadine, pure 2-R rimantadine, or a pharmaceutically acceptable salt thereof can be administered at a dosage of about 0.01 to 300 mg / kg / day, or any range therein, preferably about 0.5 to 50 mg / kg / day, or any range therein. In some embodiments, the pharmaceutical compositions provided herein contain about 25 mg to about 500 mg of a compound provided herein per unit dosage (e.g., about 25 mg to about 400 mg, about 25 mg to about 300 mg, about 25 mg to about 250 mg, about 25 mg to about 200 mg, about 25 mg to about 150 mg, about 25 mg to about 100 mg, about 25 mg to about 75 mg, about 50 mg to about 500 mg, about 100 mg to about 500 mg, about 150 mg to about 500 mg, about 200 mg to about 500 mg, about 250 mg to about 500 mg, about 300 mg to about 500 mg, about 400 mg to about 500 mg, about 50 mg to about 200 mg, about 100 mg to about 250 mg, or about 50 mg to about 150 mg). In some embodiments, the pharmaceutical compositions provided herein contain about 25 mg, about 50 mg, about 100 mg, about 150 mg, about 200 mg, about 250 mg, about 300 mg, about 400 mg, or about 500 mg of a compound provided herein per unit dose. However, dosages may vary depending on the patient's requirements, the severity of the condition being treated, and the compound being used. In some embodiments, dosages are administered once daily (QD) or twice daily (BID).

[0088] In some embodiments, the present disclosure includes compositions comprising pure 2-R rimantadine or a pharmaceutically acceptable salt thereof, pure 2-S rimantadine or a pharmaceutically acceptable salt thereof.

[0089] Also provided herein are methods for treating cancer in a subject. In some embodiments, the methods comprise administering to the subject a therapeutically effective amount of one or more of the pharmaceutical compositions described herein. In some embodiments, the pharmaceutical composition comprises enantiomerically pure 2-S rimantadine or a pharmaceutically acceptable salt thereof (e.g., a salt of 2-S rimantadine). In some embodiments, the pharmaceutical composition comprises enantiomerically pure 2-R rimantadine or a pharmaceutically acceptable salt thereof. In some embodiments, the pharmaceutically acceptable acid addition salt comprises fumaric acid, tartaric acid, galactaric acid, benzoic acid, benzenesulfonic acid, or any combination thereof. In some embodiments, the pharmaceutically acceptable acid addition salt comprises fumaric acid. In some embodiments, the pharmaceutically acceptable acid addition salt comprises tartaric acid. In some embodiments, the pharmaceutically acceptable acid addition salt comprises galactaric acid. In some embodiments, the pharmaceutically acceptable acid addition salt comprises benzoic acid. In some embodiments, the pharmaceutically acceptable acid addition salt comprises benzenesulfonic acid. In some embodiments, the pharmaceutically acceptable salt is a hydrochloride salt.

[0090] In some embodiments, the cancer is a sarcoma, carcinoma, melanoma, lymphoma, or leukemia. Non-limiting examples of sarcomas include osteosarcomas (e.g., angiosarcoma, fibrosarcoma, liposarcoma, chondrosarcoma, chordoma, Ewing's sarcoma, giant cell tumor, osteosarcoma, rhabdomyosarcoma, synovial sarcoma) and soft tissue sarcomas (e.g., fibrosarcoma, gastrointestinal stromal tumor (GIST), Kaposi's sarcoma, leiomyosarcoma, liposarcoma, rhabdomyosarcoma, and soft tissue Ewing's sarcoma). Non-limiting examples of carcinomas include basal cell carcinoma, squamous cell carcinoma, renal cell carcinoma, invasive ductal carcinoma, hepatocellular carcinoma, and adenocarcinoma. Non-limiting examples of lymphomas include non-Hodgkin's lymphomas (e.g., B-cell lymphoma, T-cell lymphoma, Burkitt's lymphoma, follicular lymphoma, mantle cell lymphoma, primary mediastinal B-cell lymphoma, small lymphocytic lymphoma, Waldenstrom's macroglobulinemia) and Hodgkin's lymphomas (e.g., lymphopenic Hodgkin's disease, lymphocytic Hodgkin's disease, mixed cellularity Hodgkin's lymphoma, nodular lymphocyte-predominant Hodgkin's disease, nodular sclerosing Hodgkin's lymphoma). Non-limiting examples of leukemias include acute hairy cell leukemia, acute lymphocytic leukemia, acute myeloid leukemia, acute promyelocytic leukemia, chronic lymphocytic leukemia, chronic myelogenous leukemia, myeloproliferative neoplasms, and systemic mastocytosis.

[0091] In some embodiments, the cancer is selected from the group consisting of melanoma, head and neck cancer, lung cancer, colon cancer, anal cancer, breast cancer, esophageal cancer, pancreatic cancer, prostate cancer, cervical cancer, liver cancer, and gastric cancer.

[0092] In some embodiments, the cancer is a carcinoma. In some embodiments, the carcinoma is selected from the group consisting of adenocarcinoma, squamous cell carcinoma, transitional cell carcinoma, hepatocellular carcinoma, and clear cell carcinoma. In some embodiments, the cancer is squamous cell carcinoma. In some embodiments, the squamous cell carcinoma is head and neck squamous cell carcinoma. In some embodiments, the cancer is hepatocellular carcinoma.

[0093] In some embodiments, the cancer is selected from the group consisting of head and neck cancer, breast cancer, and melanoma.

[0094] In some embodiments, pure 2-S rimantadine or pure 2-R rimantadine, or a pharmaceutically acceptable salt thereof (e.g., a salt of 2-S rimantadine) described herein can be used to treat hepatitis B virus (HBV)-associated cancer in a subject. As used herein, an "HBV-associated cancer" is a cancer in which one or more cancer cells express at least one HBV protein (see, e.g., Liu et al., Hepatitis B Virus X Protein Induces RHAMM-Dependent Motility in Hepatocellular Carcinoma Cells via PI3K-Akt-Oct-1 Signaling. Mol Cancer Res. 2020 Afar;18(3):375-389. doi:10.1158 / 1541-7786. MCR-19-0463. Epub 2019 Dec 2. PMID: 31792079.). For example, one or more cancer cells can express an HBV oncoprotein. In some embodiments, the HBV-associated cancer is liver cancer (e.g., hepatocellular carcinoma). In some embodiments, the HBV-associated cancer is cervical cancer.

[0095] In some embodiments, the pure 2-S rimantadine or pure 2-R rimantadine described herein, or a pharmaceutically acceptable salt thereof (e.g., a salt of 2-S rimantadine), can be used to treat a human papillomavirus (HPV)-associated cancer in a subject. As used herein, "HPV-associated cancer" refers to a cancer in which one or more cancer cells express at least one HPV protein. For example, one or more cancer cells may express an HPV oncoprotein. Human papillomavirus (HPV) can target, for example, important tumor suppressors p53 and Rb to cause malignant transformation (see, e.g., Conway and Meyers. J Dent Res. 2009 Apr;88(4):307-17, and Hoppe-Seyler. Trends Microbiol. 2018 Feb;26(2):158-168). HPV genes can also help HPV-infected cells evade immune responses (see, e.g., Senba.Oncol Rev.5 2012 Oct 5,6(2): e17). For example, HPV genes and proteins can target antigen processing and antigen presentation, which are necessary for an effective adaptive immune response (see, e.g., Senba.Oncol Rev.2012 Oct 5;6(2): e17; and O'Brien and Saveria Campo. Virus Res. 2002 Sep;88(l-2):103-17). HPV has many HPV oncoproteins, including (but not limited to) HPV16 E5, E6, E7, etc. For example, HPV E5 is a protein that has been reported to have multiple functions, including regulation of tumor cell differentiation and apoptosis, regulation of the H+ ATPase responsible for acidification of late endosomes, and immune regulation, including direct binding and downregulation of major histocompatibility complex (MHC) class I and MHC class II (see, e.g., Venuti. Mol Cancer. 2011, 10:140), which may affect antigen processing and presentation.

[0096] In some embodiments, one or more cancer cells from a subject express an HPV protein. In some embodiments, the HPV protein is one or more of HPV E5, E6, or E7 proteins. In some embodiments, the HPV E5, E6, or E7 proteins are from one or more HPV subtypes selected from the group consisting of HPV 6, HPV 11, HPV 16, HPV 18, HPV 3, HPV 33, HPV 35, HPV 39, HPV 45, HPV 51, HPV 52, HPV 56, HPV 58, HPV 66, and HPV 69. In some embodiments, the HPV protein is HPV16 E5. In some embodiments, the subject is afflicted with a cancer selected from the group consisting of head and neck cancer, mucosal squamous cell carcinoma, cutaneous squamous cell carcinoma, cervical cancer, vaginal cancer, vulvar cancer, penile cancer, and anal cancer.

[0097] In some embodiments, the cancer is an HPV-associated cancer. In some embodiments, the HPV-associated cancer is HPV-associated head and neck squamous cell carcinoma (HNSCC).

[0098] In some embodiments, pure 2-S rimantadine or pure 2-R rimantadine or a pharmaceutically acceptable salt thereof (e.g., a salt of 2-S rimantadine) described herein can be used to treat human papillomavirus precancerous lesions such as those associated with proliferative verrucous leukoplakia (PV1), oral leukoplakia, reverse smoker's nicotine uvula, oral erythroplakia, laryngeal keratosis, actinic cheilitis, smooth thick leukoplakia, smooth red Plummer-Vinson tongue, smokeless tobacco keratosis, oral submucous fibrosis syndrome, erythroleukoplakia, granular leukoplakia, oral lichen planus (erosive form), smooth thin leukoplakia, nicotinic stomatitis, tobacco pouch keratosis, cervix (cervical dysplasia), and penile intraepithelial neoplasia (PelN lesion). In the oral cavity, 24 HPV types (1, 2, 3, 4, 6, 7, 10, 11, 13, 16, 18, 30, 31, 32, 33, 35, 45, 52, 55, 57, 59, 69, 72, and 73) have been associated with benign lesions, and 12 types (2, 3, 6, 11, 13, 16, 18, 31, 33, 35, 52, and 57) have been associated with malignant lesions. Approximately 40% of invasive penile cancers are attributable to HPV 16, 18, and 6 / 11. HPV types associated with cervical carcinogenesis include 15 high-risk types (HPV 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68, 73, and 82) and three "probable high-risk" types (HPV 26, 53, and 66).

[0099] Tests for HPV are known in the art. See, for example, Coultlee, F., et. al., 2005, Can J Infect Dis Med Microbiol 16(2):83-91; careHPV Test Kit (QIAGEN, Redwood City, CA); Tang, KD, 2019, Unlocking the Potential of Saliva-Based Test to Detect HPV-16-Driven Oropharyngeal Cancer, Cancers (Basel), 11(4):473; HPV probes (BIOCARE MEDICAL, Pacheo, CA).

[0100] In some embodiments of the methods described herein, pure 2-S rimantadine, or pure 2-R rimantadine or a pharmaceutically acceptable salt thereof (e.g., a salt of 2-S rimantadine), is administered in combination with a therapeutically effective amount of at least one additional therapeutic agent selected from one or more additional anti-cancer therapies or therapeutic agents (e.g., chemotherapeutic agents). The use of different forms of treatment in combination to treat cancer patients is common practice in medical oncology. In addition to the 2-S rimantadine or a pharmaceutically acceptable salt thereof described herein, these other forms of co-treatment or therapy may include, for example, surgery, radiation therapy, and additional anti-cancer agents such as kinase inhibitors, signal transduction inhibitors, platinum-based chemotherapy, and / or monoclonal antibodies. In some embodiments, the method further comprises administering an additional anti-cancer agent.

[0101] Non-limiting examples of additional anti-cancer agents include carboplatin, cisplatin, gemcitabine, methotrexate, paclitaxel, pemetrexed, lomustine, temozolomide, and dacarbazine.

[0102] In some embodiments, the additional anticancer agent is an immunotherapy. Many types of immunotherapy can be used in combination with the pure 2-S rimantadine or pure 2-R rimantadine described herein, or its pharmaceutically acceptable salt. Non-limiting examples of immunotherapy include immune checkpoint inhibitors, antibody therapy, cellular immunotherapy, antibody-drug conjugates, cytokine therapy, mRNA-based immunotherapy, and cancer vaccines.

[0103] In some embodiments, the immunotherapy is one or more immune checkpoint inhibitors. In some embodiments, the immune checkpoint inhibitor targets one or more of CTLA-4, PD-1, PD-L1, BTLA, LAG-3, A2AR, TIM-3, B7-H3, VISTA, and IDO. In some embodiments, the checkpoint inhibitor is selected from the group consisting of ipilimumab, nivolumab, pembrolizumab, atezolizumab, avelumab, durvalumab, cemiplimab, tremelimumab, and combinations thereof.

[0104] In some embodiments, the immune checkpoint inhibitor is a CTLA-4 inhibitor, a PD-1 inhibitor, or a PD-L1 inhibitor. In some embodiments, the CTLA-4 inhibitor is ipilimumab (YERVOY®) or tremelimumab (CP-675,206). In some embodiments, the PD-1 inhibitor is pembrolizumab (KEYTRUDA®), cemiplimab (LIBTAYO®), or nivolumab (OPDIVO®). In some embodiments, the PD-L1 inhibitor is atezolizumab (TECENTRIQ®), avelumab (BAVENCIO®), or durvalumab (IMFINZI).

[0105] In some embodiments, the antibody therapy is bevacizumab (MVASTI™, AVASTIN®), trastuzumab (HERCEPTIN®), avelumab (BAVENCIO®), rituximab (MABTHERA™, RITUXAN®), edrecolomab (Panorex), daratumab (DARZALEX®), or olaratumab (LARTRUVO™). ofatumumab (ARZERRA®), alemtuzumab (CAMPATH®), cetuximab (ERBITUX®), oregovomab, pembrolizumab (KEYTRUDA®), dinuticimab (UNITUXIN®), obinutuzumab (GAZYVA®), tremelimumab (CP-675,206), ramucirumab (CYRAMZA®), ublituximab (TG-1101), panitumumab (VECTIBIX®), elotuzumab (E MPLICITI), avelumab (BAVENCIO®), necitumumab (PORTRAZZA), silimtuzumab (UC-961), ibritumomab (ZEVALIN®), isatuximab (SAR650984), nimotuzumab, fresolimumab (GC1008), lirilumab (INN), 5-mogamulizumab (POTELIGEO®), ficlatuzumab (AV-299), denosumab (XGEVA®), ganitumab, urelumab, pidilizumab, or amatuximab.

[0106] In some embodiments, the immunotherapy is a cellular immunotherapy (eg, adoptive T cell therapy, dendritic cell therapy, natural killer cell therapy).

[0107] In some embodiments, the immunotherapy is an antibody-drug conjugate. In some embodiments, the antibody-drug conjugate is gemtuzumab ozogamicin (MYLOTARG), inotuzumab ozogamicin (BESPONSA®), brentuximab vedotin (ADCETRIS®), adotrastuzumab emtansine (TDM-1, KADCYLA®), moxetumomab pasudotox (LUMOXITI®), polatuzumab vedotin piik (POLIVY®), mirvetuximab soravitansine (IMGN853), or anetumab ravitasine.

[0108] In some embodiments, the immunotherapy is cytokine therapy. In some embodiments, the cytokine therapy is interleukin-2 (IL-2) therapy, interleukin (IL-15) therapy, interleukin-7 (IL-7) therapy, interferon alpha (IFNα) therapy, agranulocyte colony-stimulating factor (G-CSF) therapy, interleukin-12 (IL-12) therapy, or erythropoietin alpha (EPO) therapy. In some embodiments, the IL-2 therapy is aldesleukin (Proleukin®). In some embodiments, the IFNα therapy is interferon alpha-2b (e.g., IntronA®) or interferon alpha-2a (e.g., Roferon-A®). In some embodiments, the G-CSF therapy is filgrastim (Neupogen®).

[0109] In some embodiments, the immunotherapy is an mRNA-based immunotherapy. In some embodiments, the mRNA-based immunotherapy is CV9104 (see, for example, Rausch et al. (2014) Human Vaccin Immunother 10(11):3146-52; Kubler et al. (2015) J. Immunother Cancer 3:26). Also see Pardi et al. Nat Rev Drug Discov. 2018 Apr, 17(4):261-279, which are incorporated herein by reference in their entirety.

[0110] In some embodiments, the method includes administering radiation therapy, surgery, or a combination thereof to the subject. For example, the surgery can be open surgery or minimally invasive surgery.

[0111] In some embodiments, the subject does not respond to standard therapy (e.g., standard of care). In some embodiments, the subject has no standard of care options. In some embodiments, the subject has recurred or progressed after standard of care. In some embodiments, the methods provided herein are useful for treating locally advanced or metastatic solid tumors that do not respond to standard of care. For example, HPV-associated cancers may not respond to immune checkpoint inhibitors such as those described herein.

[0112] In some embodiments, the subject has a cancer that is refractory or intolerant to standard therapy (e.g., administration of a chemotherapeutic agent, immunotherapy, or radiation). In some embodiments, the subject has a cancer (e.g., a locally advanced or metastatic tumor) that is refractory or intolerant to a previous therapy (e.g., administration of a chemotherapeutic agent, immunotherapy (e.g., an immune checkpoint inhibitor), or radiation). In some embodiments, the cancer that is refractory or intolerant to standard therapy is an HPV-associated cancer. In some embodiments, the subject has a cancer (e.g., a locally advanced or metastatic tumor) for which there is no standard therapy.

[0113] In some embodiments, the subject has previously been treated with platinum-based chemotherapy, immune checkpoint inhibitors (e.g., PD-1 / PDL1 immunotherapy), radiation therapy, or a combination thereof, prior to treatment with 2-S rimantadine or a pharmaceutically acceptable salt thereof.

[0114] The optimal dosage of pure 2-S rimantadine or pure 2-R rimantadine or a pharmaceutically acceptable salt thereof (e.g., a salt of 2-S rimantadine) administered to a subject can be determined by one of skill in the art and will vary depending on the method of administration, the strength of the formulation, the mode of administration, and the progression of the disease state. In some embodiments, a subject is administered a dosage of about 0.01 to 10,000 mg of 2-S rimantadine or a pharmaceutically acceptable salt thereof per adult per day. For example, a pharmaceutical composition comprising pure 2-S rimantadine, or pure 2-R rimantadine, or racemic rimantadine, or a pharmaceutically acceptable salt thereof (e.g., a salt of 2-S rimantadine) can be formulated to provide a dosage of about 0.01, about 0.05, about 0.1, about 0.5, about 1, about 2.5, about 5, about 10, about 15, about 25, about 50, about 100, about 150, about 200, about 250, or about 500 mg of rimantadine or a pharmaceutically acceptable salt thereof. In some embodiments, an effective amount of pure 2-S rimantadine, pure 2-R rimantadine, or a pharmaceutically acceptable salt thereof can be provided at a dosage level of about 0.1 mg to about 1000 mg per kg of body weight per day, or any range therein. For example, about 0.5 to about 500 mg / kg body weight per day, about 1.0 to about 250 mg / kg body weight per day, about 0.1 to about 100 mg / kg body weight per day, 0.1 to about 50.0 mg / kg body weight per day, 15.0 mg / kg body weight per day, or about 0.5 to about 7.5 mg / kg body weight per day. Pure 2-S rimantadine, or pure 2-R rimantadine, or a pharmaceutically acceptable salt thereof, can be administered to a subject on a regimen of 1 to 5 times per day, or in a single daily dose.

[0115] In one aspect, the compounds disclosed herein are used in the manufacture of a medicament for the treatment of a disease or condition described herein. Additionally, a method of treating any of the diseases or conditions described herein in a subject in need of such treatment comprises administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising at least one compound disclosed herein or a pharmaceutically acceptable salt, active metabolite, prodrug, or solvate thereof.

[0116] In certain embodiments, compositions containing the compounds disclosed herein are administered for prophylactic and / or therapeutic treatment. In certain therapeutic applications, the compositions are administered to patients already suffering from a disease or condition in an amount sufficient to cure or at least partially suppress at least one symptom of the disease or condition. The amount effective for this application depends on the severity and course of the disease or condition, previous treatments, the patient's health status, weight, and response to the drug, and the judgment of the treating physician. Therapeutically effective amounts are optionally determined by methods including, but not limited to, dose escalation clinical trials.

[0117] In prophylactic applications, compositions containing the compounds disclosed herein are administered to a patient susceptible to or otherwise at risk of a particular disease, disorder, or condition.

[0118] In certain embodiments, the dose of the administered drug is temporarily reduced or temporarily discontinued for a period of time (i.e., a "drug holiday").

[0119] Another aspect of the present disclosure includes a method of treating cancer in a subject, comprising detecting cancer cells expressing an HPV protein in a sample from the subject, and then administering to the subject a therapeutically effective amount of any one of the pharmaceutical compositions described herein (e.g., a pharmaceutical composition comprising a salt of 2-S rimantadine). The detection methods described herein are based on determining the presence or absence of an HPV protein or a functionally equivalent variant thereof. In some embodiments, the presence of an HPV protein or a functionally equivalent variant thereof is detected in a sample from the subject, and the expression level of the HPV protein is determined. In some embodiments, the HPV protein is HPV16 E5. In some embodiments, the pharmaceutical composition comprises at least one additional therapeutic agent selected from one or more additional anti-cancer therapies or therapeutic agents (e.g., a chemotherapeutic agent).

[0120] Therefore, in another aspect, the present disclosure relates to an in vitro method for diagnosing a disease associated with the presence of an HPV protein in a subject, or for determining a subject's predisposition to suffer from said disease associated with the presence of an HPV protein, or for determining the stage or severity of said disease associated with the presence of an HPV protein in a subject, or for monitoring the effect of a treatment administered to a subject suffering from said disease associated with the presence of an HPV protein. The method comprises quantifying the expression level of an HPV protein or its functionally equivalent variant in a biological sample from the subject, wherein an increase in the expression of a gene encoding an HPV protein or its functionally equivalent variant compared to the expression level of a gene encoding an HPV protein or its functionally equivalent variant in a control sample indicates a disease associated with the presence of an HPV protein, or a greater predisposition of the subject to suffer from a disease associated with the presence of an HPV protein, or non-responsiveness to a treatment administered to the subject. In some embodiments, the HPV protein is HPV16 E5. In some embodiments, the pharmaceutical composition comprises at least one additional therapeutic agent selected from one or more additional anti-cancer therapies or therapeutic agents (e.g., chemotherapeutic agents).

[0121] Therefore, the term "functionally equivalent variant" as used herein also includes functionally equivalent fragments of the marker protein. The term "fragment" relates to a peptide comprising a part of the marker protein. In this case, a functionally equivalent fragment is a peptide or protein comprising a part of the marker protein and having essentially the same function as the protein. "Marker protein" preferably refers to, but is not limited to, an HPV protein.

[0122] As those skilled in the art will understand, detection may not be usually correct for 100% of subjects, but it is desirable to do so. However, this term requires that a statistically significant portion of subjects having a sufficient amount of the protein of interest so that the subjects are suffering from or predisposed to a disease associated with the presence of the protein of interest can be identified. Those skilled in the art can simply use one or more well-known statistical evaluation tools, such as determining a confidence interval, determining a p-value, Student's t-test, or Mann-Whitney test, to determine whether the portion is statistically significant. Details are described in Dowdy and Wearden, "Statistics for Research," John Wiley & Sons, New York, 1983. Preferred confidence intervals are at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, and at least 95%. P-values ​​of 0.2, 0.1, and 0.05 are preferred.

[0123] As used herein, the term "predisposition" means that a subject has not yet developed a disease or any of the above-mentioned symptoms or other diagnostic criteria of the disease, but will develop the disease at a certain probability in the future.The probability is significantly different from the statistical probability of developing a disease associated with the presence of HPV proteins.Preferably, the probability of developing a disease associated with the presence of HPV proteins is diagnosed as at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or 100% of predisposition.The diagnosis of predisposition may also be referred to as prognosis or prediction of the probability that a subject will develop a disease.

[0124] In the context of the present disclosure, "control sample" is understood as a reference sample used to determine the variation in the expression levels of genes and proteins used in the present disclosure. In one embodiment, the reference value is obtained from the signal provided by using a tissue sample taken from a healthy individual. Preferably, the samples are taken from the same tissue of multiple healthy individuals and combined, and the amount of polypeptide in the sample reflects the average value of said molecule in the population.

[0125] Thus, in certain embodiments of the present disclosure, the expression level of an HPV protein can be quantified. In some embodiments, the HPV protein is HPV16 E5.

[0126] As will be appreciated by those skilled in the art, protein expression levels can be quantified by any conventional method. As a non-limiting example, protein levels can be quantified, for example, by using an antibody capable of binding to the protein (or a fragment thereof containing an antigenic determinant) and then quantifying the complex formed. The antibodies used in these assays can be labeled or unlabeled. Specific examples of markers that can be used include radioisotopes, enzymes, fluorophores, chemiluminescent reagents, enzyme substrates or cofactors, enzyme inhibitors, particles, dyes, and the like. A wide variety of known assays using unlabeled antibodies (primary antibodies) and labeled antibodies (secondary antibodies) can be used in the present disclosure. These techniques include Western blots, enzyme-linked immunosorbent assays (ELISAs), radioimmunoassays (RIAs), competitive enzyme-linked immunosorbent assays (EIAs), double-antibody sandwich ELISAs (DAS-ELISAs), immunocytochemistry and immunohistochemistry techniques, techniques based on the use of biochips or microarrays of proteins containing specific antibodies, or colloidal precipitation-based assays in formats such as dipsticks. In another specific embodiment, quantification of protein levels is performed by immunoanalytical methods such as Western blot, immunohistochemistry, or ELISA. In some embodiments, the immunoanalytical method comprises an antibody specific for HPV16 E5.

[0127] Similarly, the detection method of the present disclosure can be applied to any disease associated with the presence of the above-defined HPV protein.In a preferred embodiment, the disease associated with the presence of the HPV protein is cancer, preferably cancer with high levels of HPV protein.In some embodiments, the HPV protein is HPV16 E5.

[0128] The method of the present disclosure includes obtaining a biological sample from a subject to be studied. Illustrative, non-limiting examples of the sample include various types of biological fluids, such as blood, serum, plasma, cerebrospinal fluid, ascites, feces, urine, and saliva, as well as tissue samples. Biological fluid samples, like tissue samples, can be obtained by any conventional method. For example, the tissue sample may be a biopsy sample obtained by surgical resection.

[0129] In another aspect, the present disclosure relates to a kit comprising reagents for quantifying the expression level of an HPV protein or its functionally equivalent variant, diagnosing cancer in a subject, determining a subject's predisposition to the cancer, determining the stage or severity of the cancer in a subject, or monitoring the effectiveness of a treatment administered to a subject with the cancer, wherein if the reagents detect an increase in expression of the gene or protein or its functionally equivalent variant compared to a control sample, the subject may be suffering from a disease associated with the presence of the HPV protein, or may be more predisposed to the disease associated with the presence of the HPV protein, or the disease may be more severe, or the administered treatment may be ineffective. In some embodiments, the HPV protein is HPV16 E5. In some embodiments, the pharmaceutical composition comprises at least one additional therapeutic agent selected from one or more additional anti-cancer therapies or therapeutic agents (e.g., chemotherapeutic agents).

[0130] The present disclosure also relates to the use of said kit.

[0131] All terms and expressions used in defining the use of the kit are described above and explained for other inventive aspects and specific embodiments of this disclosure and are also applicable to the use of the kit described herein. Methods for designing customized treatment regimens and selecting patients who may benefit from administration of 2-S rimantadine (e.g., salts of 2-S rimantadine) or 2-S rimantadine

[0132] In another aspect, the present disclosure relates to an in vitro method for designing a customized therapy for a patient suffering from a disease associated with the presence of an HPV protein, comprising the steps of: (a) quantifying the expression level of HPV proteins in said patient; (b) comparing the expression level to a control level; If the patient's HPV protein expression level is greater than the control value, the patient is administered a therapeutically effective amount of 2-S rimantadine or a pharmaceutically acceptable salt thereof, or 2-R rimantadine or a pharmaceutically acceptable salt thereof (e.g., a salt of 2-S rimantadine).

[0133] In some embodiments, the HPV protein is HPV16 E5. In some embodiments, at least one additional therapeutic agent selected from one or more additional anti-cancer therapies or therapeutic agents (e.g., chemotherapeutic agents) is administered to the patient.

[0134] In another aspect, the present disclosure relates to an in vitro method for selecting a patient suffering from a disease associated with the presence of an HPV protein to be treated with a therapeutically effective amount of 2-S rimantadine or a pharmaceutically acceptable salt thereof, or 2-R rimantadine or a pharmaceutically acceptable salt thereof (e.g., a salt of 2-S rimantadine), comprising: a) quantifying the expression level of the HPV protein in the patient; and b) comparing the expression level with a control level, wherein if the patient's expression level of the HPV protein is higher than the control value, the patient is selected to receive a therapeutically effective amount of 2-S rimantadine or a pharmaceutically acceptable salt thereof (e.g., a salt of 2-S rimantadine), or 2-R rimantadine or a pharmaceutically acceptable salt thereof.

[0135] In some embodiments, the HPV protein is HPV16 E5. In some embodiments, the patient is administered at least one additional therapeutic agent selected from one or more additional anti-cancer therapies or therapeutic agents (e.g., chemotherapeutic agents).

[0136] For further description of the components and methods disclosed herein, see, for example, U.S. Provisional Application No. 63 / 150,027, which is incorporated herein by reference in its entirety. [Example]

[0137] The present disclosure is further illustrated by the following examples, which should not be construed as limiting in any way. The experimental procedures for generating the data presented are described in more detail below. It is to be understood that the present disclosure has been described in an illustrative manner and that the terminology used is intended to be descriptive in nature rather than limiting. General experimental, equipment, and methodological details X-ray powder diffraction (XRPD)

[0138] A Malvem-Panalytical Aeris X-ray powder diffractometer was used for XRPD analysis. The XRPD parameters used were as follows: all samples were placed directly on the sample holder without any external force. The detector used was a PIXcel detector. The scan mode used was continuous scan. The radiation source was Cu(kα). No monochromator was used. The X-ray generator power was 45 kV, 15 mA, and the goniometer diameter was 290 mm. The step size was 0.0109, and the time per step was 0.0795 seconds. The scan range was 3–40. The slit was a fixed 1 / 8° divergence slit. Samples were measured in a zero-background holder and calibrated using a Panalytical Si reference standard disk. Differential scanning calorimetry (DSC) and thermogravimetric analysis (TGA)

[0139] TGA data were collected on a TA Discovery 550 using nickel calibration standards and platinum sample pans. After mass determination, the sample was placed in the measurement chamber and equilibrated with a stream of nitrogen gas.

[0140] DSC data were collected using a TA 2500 DSC instrument and calibrated using an indium standard. Samples were weighed, purged with nitrogen, and heated during analysis of mass change. Example 1. ScreenPatch® Assay for NR1 / NR2A and NR1 / NR2B

[0141] ScreenPatch ·NR1 / NR2A ionotropic receptors encoded by the human GRIN1 / GRIN2A genes and expressed in HEK293 cells. NR1 / NR2B ionotropic receptors encoded by the human GRIN1 / GRIN2B genes and expressed in HEK293 cells formulation

[0142] 2-S rimantadine, 2-R rimantadine, racemic rimantadine, and amantadine solutions were prepared daily by diluting stock solutions into the appropriate HEPES-buffered saline (HB-PS) solution. All test and control solutions contained 0.6% DMSO, as this does not affect channel currents. Test substance formulations were sonicated (Model 2510 / 5510, Branson Ultrasonics, Danbury, CT) for at least 20 minutes at room temperature to promote dissolution.

[0143] The effects of test substances were evaluated in an eight-point concentration-response format (four replicate wells per concentration). All test and control solutions contained 0.6% DMSO. Test substance formulations were loaded into a 384-well compound plate using an automated liquid handling system (Assist Plus, INTEGRA). Positive control treatment group

[0144] Stock solutions of positive control substances were prepared in batches, aliquoted for individual use, stored frozen, and used within 6 months. Positive control test solutions were prepared fresh daily. The final DMSO concentration was 0.6% (v / v). The NMDA receptor agonists L-glutamate and glycine were used as reference agonists in this study. The NMDA receptor antagonist amantadine was used as the reference antagonist in this study. Tests and concentrations:

[0145] Test substances were evaluated for their functional effects on ion channels. The test concentrations are shown in Table 1 below. Table 1. Test substance concentrations JPEG2025528834000002.jpg68140 Clone Testing System

[0146] Cells were maintained in a tissue culture incubator. Stocks were maintained in cryopreservation. Cells used for electrophysiology were seeded onto plastic culture dishes. HEK293 culture procedure

[0147] HEK293 cells were transfected with the appropriate ion channel or receptor cDNA encoding NR1 and NR2A-B. Stable transfectants were selected using G418 and Zeocin® resistance genes incorporated into the expression plasmids. Selection pressure was maintained with O418 and Zeocin® in the culture medium. Cells were cultured in Dulbecco's modified Eagle's medium / nutrient mixture F-12 (D-MEM / F-12) supplemented with 10% fetal bovine serum, 100 U / mL penicillin O sodium, 100 μg / mL streptomycin sulfate, 100 μg / mL Zeocin®, 5 μg / mL blasticidin, and 500 μg / mL O418. ScreenPatch test method:

[0148] All experiments were performed at room temperature. Target-specific testing procedures are described below. The following procedures were applied to all ScreenPatch assays.

[0149] Prior to testing, cells in the culture dish were washed twice with HBSS solution. Immediately before use in the lonWorks Barracuda, cells were washed with HB-PS containing 6 mM CaCh to enhance sealing.

[0150] Test articles were evaluated in an 8-point concentration-response format (four replicate wells / concentration, see Table 1). Previous results have shown that 0.6% DMSO does not affect channel currents. Therefore, all test and control solutions contained 0.6% DMSO unless otherwise specified. Test article formulations were loaded into a 384-well compound plate and placed into the wells of a lonWorks Barracuda plate.

[0151] Positive control substances were prepared in batches, aliquoted for individual use, stored frozen, and used within 6 months. Positive control test solutions were prepared fresh daily. The final DMSO concentration was 0.6%.

[0152] A 2x concentration of the test substance (specified in Table 2) was pre-applied 2 min before application of L-glutamate (Sigma-Aldrich) / glycine (Sigma-Aldrich (5 μM L-glutamate and 50 μM glycine)) mixed with a 1 / 10 concentration of the test substance.

[0153] To monitor the sensitivity of the assay, an antagonist positive control, amantadine hydrochloride (Sigma-Aldrich), was applied at eight half-log concentrations (range 0.3–1000 μM; n = 4, where n is the number of replicates per concentration). An agonist positive control (L-glutamate), along with 50 μM glycine, was applied at eight concentrations (0.03–100 μM; n = 4, where n is the number of replicates).

[0154] The "blocking" effect of a compound on the channel was calculated as follows:

number

[0155] Nonlinear least-squares fits were solved using the XLfit add-in for Excel 2016 (Boston, MA).

[0156] The effects of the compounds were assessed by two measures: 1. Peak current amplitude (PCA) measurement at the maximum current value. 2. Steady-state current amplitude (SSC) measurement as the average of 4 to 5 seconds after stimulation of the receptor with an agonist. procedure

[0157] Electrophysiological procedures: a) Intracellular solution (mM): 50 mM CsCl, 90 mM CsF, 2 mM MgCl, 5 mM EGTA, 10 mM HEPES. pH adjusted to 7.2 with CsOH. This solution was prepared in batches and stored refrigerated. In preparation for the recording session, the intracellular solution was loaded into the intracellular compartment of a PPC planar electrode. b) Extracellular solution, HB-PS (composition in mM): NaCl, 137; KCl, 1.0; CaCl2, 5; HEPES, 10; glucose, 10; adjusted to pH 7.4 with NaOH (refrigerated until use). c) Holding potential: -70 mV, potential during test substance application: -70 mV.

[0158] Recording Procedure: a) Extracellular buffer was loaded into the wells of a PPC plate (11 μL per well). The cell suspension was pipetted into the wells of a PPC planar electrode (9 μL per well). b) Whole-cell recording configuration was established by patch perforation, and membrane currents were recorded with an on-board patch-clamp amplifier. c) Two recordings (scans) were performed: the first during the addition of 2x concentrated test substance to detect potential agonist effects and during pre-incubation of the cells with the test substance (2 min), and the second during agonist stimulation of the receptor (5 μM L-glutamate and 50 μM glycine) simultaneously with 1x concentrated test substance to detect antagonist effects of the test substance.

[0159] Administration of test substance: Applications consisted of adding 20 pL of 1x concentrated test substance solution and agonist at 10 μL / s (total application time 2 seconds).

[0160] Positive control agonists: 0.03-100 μM L-glutamate (8 concentration dose response, semi-logarithmic scale) and 50 μM glycine

[0161] Positive control antagonist: 0.3-1000 μM amantadine (8 concentration-response, semi-log scale) co-applied with 5 μM glutamate and 50 μM glycine.

[0162] The efficacy assessment was based on peak current measurements. result

[0163] The agonist and antagonist properties of the four compounds were examined using the Ion Work Barracuda (IWB), an HTS electrophysiology-based approach. A two-application protocol was employed.

[0164] Agonist format The potential agonist effects of the test substances and the positive control antagonist, amantadine, were examined at the first application. Neither the test substances nor amantadine produced significant activation of NMDA receptors (data not shown).

[0165] Antagonist formatThe antagonist activity of the test substances was examined during the second application of the compounds after receptor stimulation with 5 μM L-glutamate and 50 μM glycine. All four test substances were found to cause significant, concentration-dependent inhibition of NMDA receptor function. To assess open channel block-type inhibition, peak current amplitude and steady-state current amplitude (between 4 and 5 seconds after agonist application) were measured (PCA and SSC, respectively). Table 2 shows the mean IC50 values ​​of compounds at NR1 / NR2A and NR1 / NR2B receptors from these two types of measurements.

[0166] Amantadine inhibited NR1 / NR2A receptors with IC50 PCA of -97.8 μM and IC50 ssc = 48.9 μM for peak and steady-state current amplitudes, respectively. NR1 / NR2B receptors were inhibited by amantadine with IC50 PCA of 22.0 μM and IC50 ssc = 17.9 μM for peak and steady-state current amplitudes, respectively. The leftward shift in amantadine potency for steady-state current measurements suggests, at least in part, an open-channel block mechanism of inhibition of NR1 / NR2A NMDA receptors.

[0167] Table 2. Summary of the inhibitory IC50s produced by the test substances and the reference antagonist amantadine. JPEG2025528834000004.jpg66145

[0168] Amantadine inhibited NR1 / NR2A receptors with IC50 PCA = 97.8 μM and IC50 ssc = 48.9 μM for peak and steady-state current amplitudes, respectively. NR1 / NR2B receptors were inhibited with IC 50 _ PCA = 22.0 μM and IC50_ssc = 17.9 μM. The leftward shift in amantadine potency relative to steady-state current measurements suggests, at least in part, an open-channel block mechanism of inhibition of NR1 / NR2A NMDA receptors.

[0169] The results of these assays are further illustrated in Figures 1A-D. Example 2. Effects of pure 2-S rimantadine or pure 2-R rimantadine in mouse cancer models. method

[0170] cell line

[0171] AT-84-E7 and B16-OVA are cultured in RPMI 1640 containing 10% FBS, 1% L-glutamine, 1% penicillin / streptomycin, 1% sodium pyruvate, and 200 μg / ml G418. DC2.4, RAW264.7, B3Z, 4T1, B16, and MC38 are cultured in RPMI 1640 containing 10% FBS, 1% L-glutamine, 1% penicillin / streptomycin, and 1% sodium pyruvate. HEK293T are cultured in DMEM containing 10% FBS, 1% L-glutamine, and 1% penicillin / streptomycin. 4MOSC1 cells are cultured on collagen-coated dishes in KSFM medium (Invitrogen, Carlsbad, CA) supplemented with 1% penicillin / streptomycin, 5 ng / ml EGF (Invitrogen), and 2 x 10-11 M cholera toxin (Sigma, St. Louis, MO) (27). CAL-27, CAL-33, and SCC-47 cells are cultured in DME containing 10% FBS, 1% L-glutamine, and 1% penicillin / streptomycin. Periodic monitoring for mycoplasma contamination is performed using the MycoAlert PLUS detection kit (Lonza, Basel, Switzerland). All cell lines are used within 10 passages after thawing.

[0172] Mouse studies

[0173] The right flank of the mouse was inoculated with 1.0–5.0 x 10 5 AT-84-E7, 1.5x10 5 B16-OVA, or 5.0x10 54T1 cells resuspended in 100 µL of PBS are injected subcutaneously. For the orthotopic model, 1.0x10 cells are injected subcutaneously. 5 AT-84-E7 or 1.0x10 6 4MOSC1 dissolved in 30 μL of PBS was injected into the tongue. The diameter of the tumor was measured every 2–3 days with an electronic caliper, and the tumor volume (mm) was calculated. 3 )=(length x width 2 Report tumor volume using the formula: ) / 2. Once tumors are palpable, mice receive 200 μg of anti-PDL1 antibody (BioXcell, West Lebanon, NH) by IP injection every 3 days for a total of 3 or 4 doses per mouse, or 5 mice receive 10–20 mg of pure 2-S rimantadine or pure 2-R rimantadine by IP injection daily for 7 days. For adoptive transfer experiments, single-cell suspensions of OT-1 mouse spleens are first cultured in medium containing 10 ng / ml OVA-SIINFEKL peptide (InvivoGen, San Diego, CA) and 2 ng / ml recombinant IL-2 (PeproTech, Rocky Hill, NJ) for several days, then diluted to 4.0 x 10 6 Inject the cells intravenously into B16-OVA-bearing mice.

[0174] Flow cytometry

[0175] Single-cell suspensions were prepared from lungs, livers, tumor-draining lymph nodes, and tumors by mechanical dissociation and filtered using a 70 μM filter. AT-84-E7 and M0C2 tumors were incubated with collagenase D (Roche, Basel, Switzerland) for 1 hour at 37°C before mechanical dissociation. Single-cell suspensions from tumors were subjected to density gradient centrifugation using a 40% / 80% Percoll (GE Healthcare, Chicago, IL) gradient. After obtaining single-cell suspensions, each sample was incubated with Fc blocking reagent (anti-CD16 / 32 antibody, BioLegend, San Diego, CA). After Fc blocking, cells were stained with fluorescently labeled antibodies [BioLegend, BD Bioscience (San Jose, CA), or eBiosciences (Thermo Fisher Scientific, Waltham, MA)]. The LIVE / DEAD Fixable Cell Staining Kit (Invitrogen) was used for viability staining. For intracellular staining, cells were treated with Foxp3 / transcription factor fixation / permeabilization concentrate and diluent (Invitrogen). Cells were analyzed using a BD FACS Aria II or LSR II flow cytometer (BD). Data were analyzed using FlowJo (FlowJo, LLC, Ashland, OR).The following clones were used for each antibody: CD45.2 (104), CD3e (145-2C11), CD4 (RM4-5), CD8a (5H10), CD25 (3C7, PC61), CD44 (IM7), CD62L (MEL-14), IFN-γ (XMG1.2), Foxp3 (MF23), H-2Kb (AF6-88.5), H-2Kk (36-7-5), H-2Kd (SF1-1.1), H-2Kb / SIINFEKL (eBio25-D1.16), IA / IE (2G9), CD49b (DX5), CD1 lb (MI / 70), FLAG (L5), CD31 (MEC13.3), NK-T / NK cell antigen (U5A2-13), and CD102. (3C4 (MIC2 / 4)), CD62P (RMP-1), CD105 (MJ7 / 18), CD106 (429 (MVCAM.A)), and CD162 (2PH1). H-2Kb / SIINFEKL tetramer was purchased from MBL International (Woburn, MA).

[0176] Cell cycle and proliferation assays

[0177] Cell cycle progression was analyzed based on BrdU incorporation after staining 1 cell with BrdU-APC and 7-AAD using the BD Pharmingen BrdU Flow Kit (BD, Franklin Lakes, NJ). Cells were analyzed using flow cytometry according to the manufacturer's protocol. Cell proliferation was assessed using MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide]. First, cells were seeded into 96-well plates and cultured for 2–3 days. Next, the culture medium was replaced with fresh medium containing 0.5 mg / ml MTT (Sigma), and the plates were incubated at 37°C for 4 hours. Next, the purple formazan crystals are dissolved in dissolution buffer (4 mM HCl and 0.1% NP-40 in isopropanol) and the absorbance is recorded on a TECAN infinite M200 microplate reader (Tecan, Mannedorf, Switzerland) at a wavelength of 570 nm with the absorbance at 650 nm as a reference.

[0178] B3Z activation assay

[0179] B16-OVA cells were seeded in 96-well plates and treated with 100 μM pure 2-R rimantadine, pure 2-R rimantadine, or racemic rimantadine for 24 hours before adding B3Z cells. After 24 hours of co-culture, the medium was removed and 100 μL of lysis buffer (0.155 mM chlorophenol red PD-galactopyranoside (CPRG) (Roche), 0.125% Nonidet P-40 substitute (EMDCalbiochem), and 9 mM MgCl2 (Sigma) dissolved in PBS) was added. After 4 hours of incubation at 37°C, the absorbance at 570 nm was measured using a TECAN infinite M200 microplate reader.

[0180] Reverse transcription and quantitative PCR

[0181] Total RNA was extracted using TRIzol Reagent (Invitrogen) and reverse transcribed using a qScript cDNA Synthesis Kit (Quanta BioSciences, Beverly, MA) according to the manufacturer's instructions. Quantitative PCR analysis was performed using KAPA SYBR 1 FAST (KAPA Biosystems, Wilmington, MA) on a 7900HT Fast Real-Time PCR System (Applied Biosystems, Foster City, CA).

[0182] result

[0183] Pure 2-S rimantadine and pure 2-R rimantadine alone have antitumor activity and significantly reduce tumor growth. 5 Mice were inoculated with AT84-E7 / E5 tumor cells and treated with 10 mg of pure 2-S rimantadine or pure 2-R rimantadine per kg of body weight intraperitoneally (IP) for a total of seven injections starting on day 8. Tumor volumes were measured over the course of the experiment. Mice receiving pure 2-S rimantadine or pure 2-R rimantadine showed statistically significant reductions in tumor size compared to the control group. Six mice were inoculated with 1.5x10 cells. 5 Mice were inoculated with 1000 B16-OVA tumor cells and injected IP with 10 mg of pure 2-S or pure 2-R rimantadine per kg of body weight, starting on day 10, for a total of seven injections. Tumor volume was measured over the course of the experiment. Mice receiving pure 2-S or pure 2-R rimantadine showed statistically significant reductions in tumor size compared to the control group. This experiment was repeated three times with similar results. Five mice were inoculated with 5x10 cells. 54T1 tumor cells were inoculated into the mice, and starting on day 6, 10 mg of pure 2-S or pure 2-R rimantadine per kg of body weight was injected IP once daily for a total of seven doses. Tumor volumes were measured throughout the experiment. Mice treated with 2-S or pure 2-R rimantadine showed statistically significant reductions in tumor size compared to the control group. The antitumor effects of pure 2-S or pure 2-R rimantadine were reduced in AT-84-E7 tumors, which do not express E5. Significant increases in MHC surface expression were observed in multiple cell lines. Cell surface expression of MHC1 on E5-positive AT-84-E7 tumors was restored by treatment with pure 2-S or pure 2-R rimantadine.

[0184] To test the ability of 2-S rimantadine to enhance functional antigen presentation on tumor cells, B16 cells expressing OVA were used as a model tumor antigen and cocultured with B3Z cells that react with the OVA SINNFKL peptide. Treatment of B16-OVA cells with pure 2-S rimantadine or pure 2-R rimantadine significantly increased the recognition of this model tumor antigen by B3Z cells by threefold. Combining anti-PDL1 immunotherapy with pure 2-S rimantadine or pure 2-R rimantadine significantly improved the survival of B16-OVA tumor-bearing mice.

[0185] Using the RAW264.7 cell line, we tested the ability of pure 2-S rimantadine or pure 2-R rimantadine to increase MHC expression on antigen-presenting cells and observed a significant increase in the surface expression of both MHC class I and MHC class II. These results demonstrate that pure 2-S rimantadine or pure 2-R rimantadine possess novel antitumor activity in multiple preclinical tumor models and may function to enhance antigen presentation by upregulating MHC.

[0186] To investigate the direct cytotoxic activity of pure 2-S or pure 2-R rimantadine, we performed an in vitro BrdU incorporation assay to quantify the effects of pure 2-S or pure 2-R rimantadine on the cell cycle of human HNSCC cell lines. Administration of pure 2-S or pure 2-R rimantadine alone significantly increased G0 / G1 cell cycle arrest and significantly reduced S phase in both the AT-84-E7 and B16-OVA models. Inhibition of cell proliferation was also observed. Analysis of the effects of pure 2-S or pure 2-R rimantadine on T cell proliferation was tested, but no significant effects were observed.

[0187] We used RTqPCR to screen for changes in gene expression of cell cycle proteins induced by pure 2-S rimantadine or pure 2-R rimantadine, and found significant decreases in microtubules and the cell cycle regulatory molecule stathmin. We also observed a decrease in the microtubule-associated molecule tau.

[0188] To confirm the activity of pure 2-S or pure 2-R rimantadine against human head and neck tumor lines, BrdU incorporation and proliferation assays were performed. Significant cell cycle arrest and reduced proliferation with rimantadine alone were observed in human CAL-27, CAL-33, and SCC-47 squamous cell carcinoma cell lines. Finally, pure 2-S or pure 2-R rimantadine induced cell cycle arrest in mouse and human cell lines engineered to express HPV16 E5. This demonstrates that pure 2-S or pure 2-R rimantadine can functionally reverse the effects of HPV E5. Example 3. HPV genotyping

[0189] HPV genotyping is known in the art, see, for example, Sichero et al., 2017, Cancer Epidemiol Biomarkers, 26(8):1312-1320. For example, DNA is extracted from exfoliated cervical cells by spin column chromatography. Mucosal alpha HPV is tested using PCR amplification with primers such as MY09 / 11 and PGMY09 / 11 (see Table 3), followed by genotyping by hybridization with HPV type-specific oligonucleotide probes and restriction fragment length polymorphism analysis. Negative and positive controls are used to confirm the quality of the template DNA. Table 3 - Primer sequences JPEG2025528834000005.jpg209135JPEG2025528834000006.jpg78134 The degenerate basic chords are: M=A or C, W=A or T, Y=C or T, R=A or G. Example 4. In vivo toxicity of 2-S and 2-R rimantadine

[0190] A series of in vivo experiments will be conducted to determine whether 2-S rimantadine or 2-R rimantadine has higher binding selectivity for glutamate, GABA, or dopamine receptors, or a combination thereof. Compared to racemic rimantadine, the improved selectivity of 2-S rimantadine or 2-R rimantadine for glutamate, GABA, or dopamine receptors, or a combination thereof, does not result in the central nervous system side effects commonly associated with racemic rimantadine, such as nausea, upset stomach, vomiting, loss of appetite, dry mouth, abdominal pain, asthenia, irritability, fatigue, lightheadedness, dizziness, headache, sleep disturbances, decreased concentration, confusion, and anxiety.

[0191] To test the above, mice were treated with pure 2-S rimantadine, pure 2-R rimantadine, racemic rimantadine (control), or amantadine (control) at 10–20 mg / kg body weight via IP injection daily for 7 days. A series of SPECT analyses were then performed as described by Schramm, N., et al. (2000). A miniature, high-resolution detector for small-animal SPECT was used to evaluate the binding selectivity of 2-S rimantadine and 2-R rimantadine for glutamate, GABA, and dopamine receptors, respectively.

[0192] SPECT analysis involves treating mice with radioligands specific for each receptor. 123 I]IBZM is D 2 / 3 Radioligands specific for glutamate receptors and GABA receptors are known to those skilled in the art. Appropriate amounts of radioligands for glutamate receptors, GABA receptors, and dopamine receptors are injected into the lateral tail vein of mice, and SPECT measurements are initiated 45 minutes after radioligand administration.

[0193] Surprisingly, 2-R rimantadine has significantly higher binding selectivity or agonistic activity for glutamate, GABA, and dopamine receptors or pathways, or any combination thereof, compared to 2-S rimantadine. Therefore, compared to 2-S rimantadine, 2-R rimantadine has a higher incidence of central nervous system side effects, such as nausea, upset stomach, vomiting, loss of appetite, dry mouth, abdominal pain, asthenia, irritability, fatigue, dizziness, headache, sleep disturbance, difficulty concentrating, confusion, and anxiety. Considered in conjunction with Example 2, 2-S rimantadine is significantly less toxic than 2-R rimantadine while still being effective as a cancer treatment. Example 5. In vivo toxicity of 2-S and 2-R rimantadine

[0194] A series of in vivo experiments were conducted to determine whether 2-S or 2-R rimantadine have higher binding selectivity for glutamate, GABA, or dopamine receptors, or a combination thereof. The increased selectivity of 2-S or 2-R rimantadine for glutamate, GABA, or dopamine receptors, compared with racemic rimantadine, eliminates the central nervous system side effects commonly associated with racemic rimantadine, such as nausea, upset stomach, vomiting, loss of appetite, dry mouth, abdominal pain, asthenia, irritability, fatigue, dizziness, headache, sleep disturbances, impaired concentration, confusion, and anxiety.

[0195] To test this, mice were administered pure 2-S rimantadine, pure 2-R rimantadine, racemic rimantadine (control), or amantadine (control) at 10–20 mg / kg body weight via IP injection daily for 7 days. A series of SPECT analyses were then performed at glutamate, GABA, and dopamine receptors to assess the binding selectivity of 2-S rimantadine versus 2-R rimantadine, as described by Schramm, N., et al. (2000).

[0196] SPECT analysis involves treating mice with radioligands specific for each receptor. 123 I]IBZM is D 2 / 3 Radioligands specific for glutamate receptors and GABA receptors are known to those skilled in the art. Appropriate amounts of radioligands for glutamate receptors, GABA receptors, and dopamine receptors are injected into the lateral tail vein of mice, and SPECT measurements are initiated 45 minutes after radioligand administration.

[0197] Surprisingly, 2-S rimantadine has significantly higher binding selectivity or agonism for glutamate, GABA, and dopamine receptors or pathways, or any combination thereof, compared to 2-R rimantadine. Therefore, compared to 2-R rimantadine, 2-S rimantadine has a higher incidence of central nervous system side effects, such as nausea, upset stomach, vomiting, loss of appetite, dry mouth, abdominal pain, asthenia, irritability, fatigue, dizziness, headache, sleep disturbance, impaired concentration, confusion, and anxiety. Considered together with Example 2, 2-S rimantadine is significantly less toxic than 2-R rimantadine while still remaining effective as a cancer treatment. Example 6. In vitro growth of 2-S and 2-R rimantadine

[0198] Experiments were conducted to determine the ability of 2-S rimantadine (also referred to herein as "S-rimantadine"), 2-R rimantadine (also referred to herein as "R-rimantadine"), racemic (RS) rimantadine, and memantine to affect proliferation in CAL-27 cells. S-rimantadine resulted in enhanced or equivalent cancer cell proliferation compared to R-rimantadine or racemic rimantadine.

[0199] On day 1, CAL-27 cells were seeded in 96-well plates (2-4x10 3Cells were plated overnight (100 μL of medium per well) to allow cells to adhere to the plate. On day 2, various concentrations of rimantadine (0 μM, 100 μM, 250 μM, or 500 μM) were added to the cells and incubated for 24 or 48 hours. On day 3 or 4, the culture medium was aspirated, and 100 μL of MTT solution (MTT concentration 0.5 mg / ml, prepared by diluting thiazolyl blue tetrazolium bromide solution (Sigma, catalog number M2128) with a stock solution (5 mg / ml in PBS at -20°C)) was added to the culture medium. The cells were incubated at 37°C in a CO2 incubator for 3 hours, after which the MTT solution was aspirated. Next, 100 μL of DMSO was added per well, and the cells were incubated for approximately 5 minutes. The OD570nm (Ref650nm) was then read. The results of the experiment are shown in Figure 2. Example 7. In vivo tumor model / antitumor activity method

[0200] The activity of 2-S rimantadine, 2-R rimantadine, and racemic rimantadine is tested against HPV-associated tumors using in vivo mouse syngeneic tumor models. S-rimantadine exhibits equal or greater antitumor activity compared to racemic rimantadine and / or R-rimantadine. Plasmid construction and HPV16 E5-expressing stable cell lines

[0201] Codon-optimized HPV16 E5 was amplified. C- or N-terminal FLAG-tagged full-length HPV16 E5 and deletion mutants were cloned into the MIP (MSCV-IRES-Puro) or pMSCV-Blasticidin vector. All constructs were verified by DNA sequencing. To establish HPV16 E5-expressing cell lines, HEK293T cells were co-transfected with MIP-HPV16 E5 and Ecopac (pIK6.1MCV.ecopac.UTd) using PEI reagent (Sigma-Aldrich). Retroviruses from the culture medium of these cells were then used to infect AT-84-E7, M0C2, and CAL-27 cells, and infected cells were selected with puromycin. pMSCV-Blasticidin-HPV16 E5 was used to transfect MEER cells. Mouse studies

[0202] For the experiments, 6-8 week old female mice were used. C3H / HeN, C57BL / 6, and BALB / c mice were used. The right flank of the mice was inoculated with 1.0-5.0x10 PBS-resuspended 100 mL of PBS. 5 AT-84-E7, 1.5xl0 5 B16-0VA, 5.0xl0 5 4T1, or 1.0xl0 5 Inject M0C2 cells subcutaneously. For the orthotopic model, inject 1.0x10 cells dissolved in 30 mL of PBS. 5 AT-84-E7 or 1.0x10 6 4MOSC1 is injected into the tongue. Once tumors become palpable, mice are given 200 mg of anti-PD-L1 antibody (Bio X Cell) intraperitoneally every 3 days for a total of 3 or 4 injections per mouse, or mice are given 10 mg of R-rimantadine, S-rimantadine, and / or racemic rimantadine per kg body weight intraperitoneally daily for 7 days. For adoptive transfer experiments, single-cell suspensions of spleens from OT-1 mice are cultured in medium containing 10 ng / mL OVA-SIINFEKL peptide (InvivoGen) and 2 ng / mL recombinant IL2 (PeproTech) for 5 days, followed by 4.0x0 6Cells were intravenously injected into B16-OVA-bearing mice. Tumor diameters were measured every 2-3 days with electronic calipers, and tumor volumes (mm) were calculated. 3 )=(length x width 2 Report as volume using the formula: ) / 2.

[0203] The information and procedures used and disclosed in Miyauchi S., et al., Cancer Res. 2020 Feb 15;80(4):732-746 are incorporated herein by reference in their entirety. The disclosed information and procedures (e.g., protocols) are implemented in studies of S-rimantadine, R-rimantadine, and / or racemic rimantadine. Example 8. In vitro antiviral assay

[0204] The direct antiviral activity of rimantadine enantiomers (e.g., S-rimantadine) against HPV viral replication is tested using an in vitro HPV viral replication assay. S-rimantadine exhibits equivalent or greater direct HPV antiviral activity compared to racemic rimantadine or R-rimantadine. Plasmid

[0205] The Snls-Cre expression plasmid pCAGGS-nlsCre was used. The pNeo-loxP HPV-18 and pNeo-loxP HPV-18 E6*I plasmids were used. In both plasmids, 34-bp loxP sites flank linear HPV-18 sequences upstream of nucleotide 7474 and downstream of nucleotide 7473. The vectors carry a neomycin resistance marker gene selectable in bacterial and mammalian cells. In the HPV-18 E6*I mutant, the intronic coding sequence (nucleotides 234-415) of the major E6*I mRNA is deleted. For trans-complementation experiments, the empty vector-only retrovirus pLC and the pLJ HPV-18 URR-Ed or URR-E6 / E7 retroviruses were used. Each expresses a neomycin resistance gene (Cheng et al. 1995. Differentiation-dependent up-regulation of the human papillomavirus E7 gene reactivates cellular DNA replication in suprabasal differentiated keratinocytes. Genes & Dev. 9:2335-2349; Chien et al. 2002. Alternative fats of keratinocytes transduced by human papillomavirus type 18 E7 during squamous differentiation. J. Virol. 76: 2θ64-2972). All plasmids are purified by banding (e.g., CsCl-ethidium bromide equilibrium density gradient). Recovery and titration of HPV-18 viral particles

[0206] HPV-18 viral particles are harvested from the epithelium on day 14 or 16 as described (Favre, M. 1975. Structural polypeptides of rabbit, bovine, and human papillomaviruses. J. Virol. 15:1239-1247). To measure viral titer, a portion of the viral stock is digested with DNase I (Invitrogen) and inactivated by heating at 100°C for 5 minutes. Packaged viral DNA is purified by digestion with proteinase K and phenol / chloroform extraction. Serial dilutions of viral DNA are analyzed by real-time quantitative PCR, for example, using SYBR GreenER qPCR SuperMix (Invitrogen) and primers J and K listed in Supplementary Table 1 of Wang HK. et al., Genes Dev. 2009 Jan 15;23(2):181-194. As a standard, purified pNeo-LoxP HPV-18 plasmid DNA is added at approximately 40–4x10 per well. 8 Serially dilute the samples into copies. Perform 40-cycle PCR amplification reactions in triplicate (e.g., in a 384-well plate using an ABI 7900HT). Then, process the data (e.g., using SDS2.1 software (Applied Biosystems)). HPV-18 infectivity assay

[0207] Approximately 1x10 5Primary human keratinocytes (PHK) were inoculated with various amounts of virus stock corresponding to MOIs of 5200, 1040, 208, 42, 10, 2, 1, or 0 in 1 mL of K-SFM and incubated overnight. The medium was replaced, and the cells were cultured for an additional 4 days. Total RNA was then extracted (e.g., using Trizol (Invitrogen)). Reverse transcription was performed on 10 mg of RNA in a 50 mL reaction. Next, 1 mL of RT reaction was subjected to 30 cycles of PCR or nested PCR amplification (30 cycles each) in a 35 mL reaction mixture to generate cDNA fragments of spliced ​​HPV-18 E6-E7-E1AE4, RNA, or b-actin mRNA (Meyers et al., 2002. Infectious virions generated from human papillomavirus type 18 / 16 genomic DNA chimeras. J. Virol. 76:4723-4733). 15 mL of each reaction was electrophoresed on a 2% agarose gel and visualized by ethidium bromide staining. PHKs were also infected at various MOIs overnight in K-SFM, grown into raft cultures, and fixed on day 14 and processed as described.

[0208] PHKs administered various amounts of virus stock are exposed to various concentrations of R-rimantadine, S-rimantadine, and / or racemic rimantadine over a period of time (e.g., 1, 2, 3, 5, 7, and / or 10 days). The disclosed information and procedures (e.g., protocols) are implemented for studies of S-rimantadine, R-rimantadine, and / or racemic rimantadine.

[0209] The information and procedures used and disclosed in Wang HK. et al., Genes Dev. 2009 Jan 15;23(2):181-194 are incorporated herein by reference in their entirety. The disclosed information and procedures (e.g., protocols) are implemented in studies of S-rimantadine, R-rimantadine, and / or racemic rimantadine. Example 9. In Vivo Central Nervous System ("CNS") Assay

[0210] Studies are being conducted to examine the effects of R-rimantadine, S-rimantadine, and racemic rimantadine on the CNS of live animals (e.g., mice and rats). Various doses of R-rimantadine, S-rimantadine, and racemic rimantadine are being studied, with the following tests being conducted: Animals administered S-rimantadine show less CNS toxicity at similar doses of R-rimantadine and racemic rimantadine. Furthermore, animals administered S-rimantadine can receive higher doses of each drug before signs and symptoms of CNS toxicity appear compared to animals administered R-rimantadine or racemic rimantadine. Furthermore, mice administered S-rimantadine are more resistant to signs and symptoms of CNS toxicity compared to mice administered similar doses of R-rimantadine and racemic rimantadine. a) Rotarod

[0211] CNS toxicity associated with the use of R-rimantadine, S-rimantadine, and racemic rimantadine is studied using a rotarod system (e.g., Rotor Rod System, San Diego Instruments). The Rotor Rod system allows observation of motor coordination in animals (e.g., mice or rats), allowing for the study of CNS toxicity potentially caused by R-rimantadine, S-rimantadine, and racemic rimantadine.

[0212] Animals receive doses (e.g., varying doses) of R-rimantadine, S-rimantadine, or racemic rimantadine. After a period of time (e.g., 1 hour, 2 hours, 3 hours, 5 hours, 10 hours, 1 day, 2 days, 3 days, 5 days, 7 days, and / or 10 days) following administration, potential CNS effects are measured using a rotarod system. Animals administered S-rimantadine experience fewer adverse CNS effects and toxicity compared to R-rimantadine and racemic rimantadine. In particular, animals (e.g., mice or rats) administered S-rimantadine are less likely to exhibit abnormal motor coordination. The information and procedures used and disclosed in Rotor Rod, San Diego Instruments, https: / / sandiegoinstruments.com / product / rotor-rod / ; ROTOR-ROD System, Biomedical and Obesity Research Core, College of Education and Human Sciences, University of Nebraska-Lincoln, available at https: / / cehs.unl.edu / borc / rotor-rod%E2%84%A2-system / ; Castagne et al., CNS Safety Pharmacology, Reference Module in Biomedical Research, 2014; Dunham NW and Miya TS (1957) A note on a simple apparatus for detecting neurological deficit in rats and mice. Journal of the American Pharmaceutical Association, American Pharmaceutical Association (Baltimore) 46: 208‐209; Bohlen et al., Calibration of rotational acceleration for the rotarod test of rodent motor coordination, Journal of Neuroscience Methods (2009) 178: 10‐14; Shiotsuki et al., A rotarod test for evaluation of motor skill learning. J Neurosci Methods. 2010 Jun 15;189(2):180-5. doi: 10.1016 / j.jneumeth.2010.03.026. Epub 2010 Mar 30.PMID: 20359499; and Rustay NR, Wahlsten D, and Crabbe JC (2003) Influence of task parameters on rotarod performance and sensitivity to ethanol in mice. Behavioral Brain Research 141: 237-249, incorporated herein by reference in its entirety. The disclosed information and procedures (e.g., protocols) are used to study S-rimantadine, R-rimantadine, and / or racemic rimantadine. b) Photobeam activity system - home cage

[0213] CNS toxicity associated with the use of R-rimantadine, S-rimantadine, and racemic rimantadine is studied using a Photobeam Activity System-Home Cage (San Diego Instruments). The Photobeam Activity System-Home Cage allows for the study of animal locomotor activity. Animals administered R-rimantadine exhibit less CNS toxicity as evidenced by the Photobeam Activity System-Home Cage study.

[0214] Animals (e.g., mice and rats) are administered R-rimantadine, S-rimantadine, or racemic rimantadine (e.g., varying doses). After a set period of time (1 hour, 2 hours, 3 hours, 5 hours, 10 hours, 1 day, 2 days, 3 days, 5 days, 7 days, and / or 10 days) following administration, potential CNS effects are measured using a home cage photobeam activity system. Animals administered S-rimantadine exhibit less adverse CNS effects and toxicity compared to R-rimantadine or racemic rimantadine. Notably, animals (e.g., mice and rats) administered S-rimantadine exhibit less abnormal motor activity. The information and procedures used and disclosed in the Photobeam Activity System-Home Cage, San Diego Instruments, are available at https: / / sandiegoinstruments.com / product / pas-homecage / , and Tatem et al., Behavioral and locomotor measurements using an open field activity monitoring system for skeletal muscle diseases. J Vis Exp. 2014 Sep 29;(91):51785. doi: 10.3791 / 51785. PMID: 25286313; PMCID: PMC4672952, and are incorporated herein by reference in their entireties. The information and protocols described in these disclosures are used to study R-rimantadine, S-rimantadine, and racemic rimantadine. This study may be used, and possibly modified, to analyze aspects of animal physiological responses related to the central nervous system, such as circadian rhythms and anxiety. c) Irwin Test / Functional Observation Battery (FOB)

[0215] CNS toxicity associated with the use of R-rimantadine, S-rimantadine, and racemic rimantadine is investigated using the Irwin test and FOB. The Irwin test and FOB allow for qualitative investigation of the effects of R-rimantadine, S-rimantadine, and racemic rimantadine. Animals administered R-rimantadine exhibit less CNS toxicity as evidenced by the Irwin test / FOB test.

[0216] Animals (e.g., mice or rats) are administered various doses (e.g., four different doses) of R-rimantadine, S-rimantadine, or racemic rimantadine. After a set period of time (1 hour, 2 hours, 3 hours, 5 hours, 10 hours, 1 day, 2 days, 3 days, 5 days, 7 days, and / or 10 days) following administration, the behavior and physiology of the animals (e.g., mice or rats) are examined. Animals administered S-rimantadine exhibit fewer adverse effects and toxicity on the central nervous system compared to R-rimantadine or racemic rimantadine. Notably, animals administered S-rimantadine exhibit fewer abnormal behaviors and physiology at similar doses, while animals administered R-rimantadine tolerate higher doses before showing visible effects on behavior and physiology or before exhibiting obvious behavioral toxicity.Castagne et al., CNS Safety Pharmacology, Reference Module in Biomedical Research, 2014, Irwin S (1968), Comprehensive observational assessment: Ia. A systematic, quantitative procedure for assessing the behavioral and physiologic state of the mouse, Psychopharmacologia 13: 222‐257, Esteve J, Farre AJ, and Roser R (1988) Pharmacological profile of droxicam, General Pharmacology 19: 49‐54, Mattson et al., (1996) A performance standard for clinical and functional observational battery examination of rats. Journal of the American College of Toxicology, 15: 239‐250, and Roux et al., Primary observation (Irwin) test in rodents for assessing acute toxicity of a test agent and its effects on behavior and physiological function. Curr. Protoc. Pharmacol. 2005 Jan 1; Chapter 10:Unit 10.10. doi: 10.1002 / 0471141755.ph1010s27. PMID: 22294127. The information and procedures used and disclosed in these disclosures are incorporated herein by reference in their entirety. The information and protocols described in these disclosures are used to study R-rimantadine, S-rimantadine, and racemic rimantadine. d) Morris water maze test

[0217] CNS toxicity associated with the use of R-rimantadine, S-rimantadine, and racemic rimantadine is studied using the Morris water maze test. The Morris water maze test allows researchers to study potential CNS toxicity experienced by animals (such as mice and rats) by testing their spatial learning abilities. Animals administered R-rimantadine exhibit less CNS toxicity as evidenced by the Morris water maze test.

[0218] Animals (e.g., mice or rats) are administered various doses (e.g., four different doses) of R-rimantadine, S-rimantadine, or racemic rimantadine. After a set period of time (1 hour, 2 hours, 3 hours, 5 hours, 10 hours, 1 day, 2 days, 3 days, 5 days, 7 days, and / or 10 days) following administration, the animals are placed in a maze. Animals administered S-rimantadine exhibit less adverse effects and toxicity on the central nervous system compared to R-rimantadine or racemic rimantadine. In particular, animals administered S-rimantadine show less impairment of spatial learning ability. The information and procedures used and disclosed in Vorhees et al., Morris water maze: procedures for assessing spatial and related forms of learning and memory, Nat Protoc 1, 848-858 (2006). https: / / doi.org / 10.1038 / nprot.2006.116, and Castagne et al., CNS Safety Pharmacology, Reference Module in Biomedical Research, 2014, Morris RGM (1981) Spatial localization does not require the presence of local cues, Learning and Motivation 12: 239-260 are incorporated herein by reference in their entirety. The information and protocols described in these disclosures are used to study R-rimantadine, S-rimantadine, and racemic rimantadine. e) Electroencephalogram (EEG) scan

[0219] CNS toxicity associated with the use of R-rimantadine, S-rimantadine, and racemic rimantadine is studied using EEG scans. EEG scans allow the study of electrical activity in the brain of animals (such as mice and rats). Animals administered R-rimantadine show less CNS toxicity as evidenced by EEG testing.

[0220] Animals (e.g., mice or rats) are administered R-rimantadine, S-rimantadine, or racemic rimantadine at various doses (e.g., four different doses). After a set period of time (1 hour, 2 hours, 3 hours, 5 hours, 10 hours, 1 day, 2 days, 3 days, 5 days, 7 days, and / or 10 days) following administration, the animals' EEG signals are recorded. Animals administered S-rimantadine experience fewer adverse CNS effects and toxicity compared to animals administered R-rimantadine or racemic rimantadine. Notably, animals administered S-rimantadine exhibit fewer abnormal EEG signals compared to animals administered R-rimantadine or racemic rimantadine. Vogler et al., Low Cost Electrod Assembly for EEg Recordings in Mice, Front. Neurosci., 14 November 2017, https: / / doi.org / 10.3389 / fnins.2017.00629; Danhof M and Visser SA (2002) Pharmaco- electroencephalography and pharmacokinetic-pharmacodynamic modeling in drug development: focus on preclinical steps. Methods & Findings in Experimental & Clinical Pharmacology 24((Suppl D): 127‐128; Itil TM and Itil KZ (1995) Quantitative EEG Brain Mapping In Psychotropic Drug Development, Drug Treatment Selection, and Monitoring. American Journal of Therapy 2:359‐367; and Protocol for Rat Sleep EEG, NeuroDetective International, The information and procedures used and disclosed in https: / / www.ndineuroscience.com / userfiles / Rat_Sleep_EEG_Methods.pdf, are incorporated herein by reference in their entirety. The information and protocols described in these disclosures are used in studies of R-rimantadine, S-rimantadine, and racemic rimantadine. Example 10. In vitro central nervous system ("CNS") assay

[0221] Studies will be conducted to determine the effects of R-rimantadine, S-rimantadine, and racemic rimantadine on anatomical and / or physiologic changes associated with CNS toxicity. Tissues obtained from animals (e.g., surviving or dead) (e.g., mice and / or rats) administered S-rimantadine will demonstrate less alteration relative to baseline or normal (e.g., within acceptable limits) tissues when compared to tissues obtained from animals administered R-rimantadine or racemic rimantadine.

[0222] Animals administered S-rimantadine show fewer physiological and / or anatomical changes due to CNS toxicity compared to animals administered similar doses of R-rimantadine and racemic rimantadine. S-rimantadine-treated animals can be administered higher doses than R-rimantadine and racemic rimantadine-treated animals before exhibiting physiological and / or anatomical changes associated with CNS toxicity. Various doses of R-rimantadine, S-rimantadine, and racemic rimantadine have been studied, and at least the following tests have been used: The information and protocols described in these disclosures are used in studies of R-rimantadine, S-rimantadine, and racemic rimantadine. Brain slice / whole cell patch clamp

[0223] CNS toxicity associated with the use of R-rimantadine, S-rimantadine, and racemic rimantadine is studied using brain slice / whole-cell patch clamp studies. Brain slice / whole-cell patch clamp electrophysiology allows for analysis of biophysical mechanisms (e.g., ionic currents) of neuronal computation and pathology in neurons. Animals administered R-rimantadine experience less CNS toxicity (e.g., reduced anatomical and / or physiological changes) as evidenced by brain slice / whole-cell patch clamp testing.

[0224] Animals (e.g., mice or rats) are administered various doses (e.g., four different doses) of R-rimantadine, S-rimantadine, or racemic rimantadine. After a set period of time (1 hour, 2 hours, 3 hours, 5 hours, 10 hours, 1 day, 2 days, 3 days, 5 days, 7 days, and / or 10 days) following administration, the animals are euthanized, and brain sections are collected and analyzed. Alternatively, whole-cell patch analysis may be performed in vivo. In this case, after a set period of time following administration, the animals are analyzed without euthanasia. Animals administered S-rimantadine exhibit fewer adverse CNS effects and toxicity compared to animals administered R-rimantadine or racemic rimantadine. Notably, animals administered S-rimantadine exhibit fewer abnormal neuronal computations and biophysical mechanisms of pathology (e.g., ionic currents) than animals administered R-rimantadine or racemic rimantadine. The information and procedures used and disclosed in Kodandaramaiah et al., Automated whole-cell patch-clamp electrophysiology of neurons in vivo, Nat Methods. 2012 Jun;9(6):585-7. doi:10.1038 / nmeth.1993. Epub 2012 May 6. PMID: 22561988; PMCID: PMC3427788 are incorporated herein by reference in their entirety. The information and protocols described in these disclosures are used to study R-rimantadine, S-rimantadine, and racemic rimantadine. Example 11: X-ray powder diffraction of crystalline Form A fumarate salt of 2-S-rimantidine

[0225] Crystalline Form A fumarate of 2-S-rimantidine was synthesized using the methods described herein. The resulting crystalline sample was analyzed by X-ray powder diffraction. Table 4 shows the XRPD diffraction pattern observed when analyzing crystalline Form A fumarate. Additionally, Figure 4 shows the same data in diffractogram format. Table 4. JPEG2025528834000007.jpg138154 Example 12: X-ray powder diffraction of crystalline Form A tartrate salt of 2-S-rimantidine

[0226] Crystalline Form A tartrate salt of 2-S-rimantidine was synthesized by the method described herein. The resulting crystalline sample was subjected to X-ray powder diffraction for analysis. Table 5 shows the XRPD diffraction pattern observed when analyzing crystalline Form A tartrate salt. Additionally, Figure 5 shows the same data in diffractogram format. Table 5. JPEG2025528834000008.jpg149135 Example 13: X-ray powder diffraction of crystalline Form A galactarate salt of 2-S-rimantidine

[0227] Crystalline Form A galactarate of 2-S-rimantidine was synthesized by the method described herein. The resulting crystalline sample was subjected to X-ray powder diffraction for analysis. Table 6 shows the XRPD diffraction pattern observed when analyzing crystalline Form A galactarate. Additionally, Figure 6 shows the same data in diffractogram format. Table 6. JPEG2025528834000009.jpg138136 Example 14: X-ray powder diffraction of crystalline Form B galactarate salt of 2-S-rimantidine

[0228] Crystalline Form B galactarate of 2-S-rimantidine was synthesized using the methods described herein. The resulting crystalline sample was analyzed by X-ray powder diffraction. Table 7 shows the XRPD diffraction pattern observed when analyzing crystalline Form B galactarate. Additionally, Figure 7 shows the same data in diffractogram format. Table 7. JPEG2025528834000010.jpg142135Example 15: X-ray powder diffraction of crystalline Form A benzoate of 2-S-rimantidine Crystalline Form A of 2-S-rimantidine benzoate was synthesized by the method described herein. The resulting crystalline sample was subjected to X-ray powder diffraction for analysis. Table 8 shows the XRPD diffraction pattern observed when analyzing crystalline Form A of benzoate. Additionally, Figure 8 shows the same data in diffractogram format. Table 8. JPEG2025528834000011.jpg136133 Example 16: X-ray powder diffraction of crystalline Form A benzenesulfonate of 2-S-rimantidine

[0229] Crystalline benzenesulfonate salt of 2-S-rimantidine was synthesized by the method described herein. The resulting crystalline sample was subjected to X-ray powder diffraction for analysis. Table 9 shows the XRPD diffraction pattern observed when analyzing crystalline Form A of benzenesulfonate salt. Additionally, Figure 9 shows the same data in diffractogram format. Table 9. JPEG2025528834000012.jpg131138 Example 17: X-ray powder diffraction of crystalline Form B benzenesulfonate of 2-S-rimantidine

[0230] Crystalline Form B salt of 2-S-rimantidine was synthesized by the method described herein. The resulting crystalline sample was subjected to X-ray powder diffraction for analysis. Table 10 shows the XRPD diffraction pattern observed when analyzing the crystalline Form B benzenesulfonate salt. Additionally, Figure 10 shows the same data in diffractogram format. Table 10. JPEG2025528834000013.jpg161136Other embodiments

[0231] While the present invention has been described in conjunction with its detailed description, it is to be understood that the foregoing description is intended to illustrate, but not to limit, the scope of the invention, which is defined by the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

1. A salt comprising 2-S-rimantidine and a salt of fumaric acid, tartaric acid, galactaric acid, benzoic acid, or benzenesulfonic acid.

2. A salt according to claim 1, wherein the salt is anhydrous.

3. A salt according to claim 2, wherein the salt is crystalline.

4. The salt according to claim 3, (i) If the salt is a salt of 2-S-rimantidine and fumaric acid, the salt has an X-ray powder diffraction pattern with a peak at approximately 6.09 2θ, (ii) If the salt is a salt of 2-S-rimantidine and tartaric acid, the salt has an X-ray powder diffraction pattern that includes a peak at approximately 7.50 2θ. (iii) If the salt is a salt of 2-S-rimantidine and galactaric acid, the salt has an X-ray powder diffraction pattern with a peak at approximately 5.71 2θ, (iv) If the salt is a salt of 2-S-rimantidine and benzoic acid, the salt has an X-ray powder diffraction pattern with a peak at 7.85 2θ, and (v) If the salt is a salt of 2-S-rimantidine and benzenesulfonic acid, the salt has an X-ray diffraction powder pattern including a peak at 6.21 2θ.

5. The salt according to claim 4, wherein the salt is (i), the salt further includes peaks at approximately 14.36 2θ, approximately 17.56 2θ, approximately 18.45 2θ, approximately 18.81 2θ, and / or approximately 27.17 2θ. If the salt is (ii), the salt further includes peaks at approximately 17.64 2θ, approximately 18.68 2θ, approximately 15.43 2θ, approximately 19.37 2θ, and / or approximately 22.48 2θ. If the salt is (iii), the salt further includes peaks at approximately 15.85 2θ, approximately 16.96 2θ, approximately 19.76 2θ, and / or approximately 19.43 2θ. If the salt is (iv), the salt further includes peaks at approximately 9.72 2θ, approximately 11.49 2θ, approximately 12.29 2θ, approximately 15.66 2θ, approximately 19.06 2θ, approximately 20.38 2θ, and / or approximately 29.91 2θ, and If the salt is (v), the salt further includes peaks at approximately 8.02 2θ, approximately 8.44 2θ, approximately 14.11 2θ, approximately 15.39 2θ, approximately 16.01 2θ, and / or approximately 19.08 2θ.

6. Crystals of 2-S-rimantadine, wherein the XRPD peaks are approximately 14.36 2θ, 17.56 2θ, 18.45 2θ, 18.81 2θ, and / or 27.17 2θ.

7. A composition comprising a salt of 2-S rimantadine as described in claim 1.

8. The composition according to claim 7, wherein the salt is a fumarate, tartrate, galactarate, benzoate, benzenesulfonate, or a combination thereof.

9. The composition according to claim 8, wherein the fumarate is a type A fumarate.

10. A composition according to claim 9, characterized by one or more of the following: a. Powder X-ray diffraction pattern ("XRPD") shows a peak at approximately 6.09°2θ; b. In the XRPD diffraction pattern, there is a peak at approximately 14.36°2θ; c. XRPD diffraction pattern shows a peak at approximately 17.56°2θ; d. In the XRPD diffraction pattern, there is a peak at approximately 18.45°2θ; e. In the XRPD diffraction pattern, there is a peak at approximately 18.81°2θ; or In the f.XRPD diffraction pattern, there is a peak at approximately 27.17°2θ.

11. The composition according to claim 8, wherein the tartrate is of type A tartrate.

12. A composition according to claim 11, characterized by one or more of the following: a. Powder X-ray diffraction pattern ("XRPD") shows a peak at approximately 6.09°2θ; b. XRPD diffraction pattern shows a peak at approximately 7.50°2θ; c. XRPD diffraction pattern shows a peak at approximately 17.64°2θ; d. In the XRPD diffraction pattern, the curve is approximately 18.68°2θ; e. In the XRPD diffraction pattern, the curve is approximately 15.43°2θ; f. In the XRPD diffraction pattern, there is a peak at approximately 19.37°2θ; or In the g.XRPD diffraction pattern, there is a peak at approximately 22.48°2θ.

13. The composition according to claim 8, wherein the galactarate is galactarate type A.

14. A composition according to claim 13, characterized by one or more of the following: a. XRPD diffraction pattern shows a peak at approximately 5.71°2θ; b. XRPD diffraction pattern shows a peak at approximately 15.85°2θ; c. XRPD diffraction pattern shows a peak at approximately 16.96°2θ; d. In the XRPD diffraction pattern, there is a peak at approximately 19.76°2θ; or In the XRPD diffraction pattern, there is a peak at approximately 19.43°2θ.

15. The composition according to claim 8, wherein the galactarate is galactarate type B.

16. A composition according to claim 15, characterized by one or more of the following: a. XRPD diffraction pattern shows a peak at approximately 5.83°2θ; b. XRPD diffraction pattern shows a peak at approximately 14.89°2θ; c. XRPD diffraction pattern shows a peak at approximately 16.87°2θ; d. In the XRPD diffraction pattern, there is a peak at approximately 17.62°2θ; e. In the XRPD diffraction pattern, there is a peak at approximately 30.87°2θ; or In the f.XRPD diffraction pattern, there is a peak at approximately 19.72°2θ.

17. The composition according to claim 8, wherein the benzoate is a type A benzoate.

18. A composition according to claim 17, characterized by one or more of the following: a. XRPD diffraction pattern shows a peak at approximately 7.85°2θ; b. XRPD diffraction pattern shows a peak at approximately 9.72°2θ; c. XRPD diffraction pattern shows a peak at approximately 11.49°2θ; d. In the XRPD diffraction pattern, there is a peak at approximately 12.29°2θ; e. In the XRPD diffraction pattern, there is a peak at approximately 15.66°2θ; f. In the XRPD diffraction pattern, there is a peak at approximately 19.06°2θ; g. XRPD diffraction pattern shows a peak at approximately 20.38°2θ; or In the h.XRPD diffraction pattern, there is a peak at approximately 29.91°2θ.

19. The composition according to claim 8, wherein the benzenesulfonate is a benzenesulfonate type A.

20. A composition according to claim 19, characterized by one or more of the following: a. XRPD diffraction pattern shows a peak at approximately 6.21°2θ; b. XRPD diffraction pattern shows a peak at approximately 8.02°2θ; c. XRPD diffraction pattern shows a peak at approximately 8.44°2θ; d. In the XRPD diffraction pattern, there is a peak at approximately 14.11°2θ; e. In the XRPD diffraction pattern, there is a peak at approximately 15.39°2θ; f. In the XRPD diffraction pattern, there is a peak at approximately 16.01°2θ; or In the g.XRPD diffraction pattern, there is a peak at approximately 19.08°2θ.

21. The composition according to claim 8, wherein the benzenesulfonate is a benzenesulfonate type B.

22. The composition according to claim 21, characterized by one or more of the following: a. Powder X-ray diffraction pattern ("XRPD") shows a peak at approximately 8.04°2θ; b. XRPD diffraction pattern shows a peak at approximately 8.45°2θ; c. XRPD diffraction pattern shows a peak at approximately 14.13°2θ; d. In the XRPD diffraction pattern, there is a peak at approximately 15.37°2θ; e. In the XRPD diffraction pattern, there is a peak at approximately 16.86°2θ; or In the f.XRPD diffraction pattern, there is a peak at approximately 19.10°2θ.

23. A composition according to claim 7, wherein the composition is crystalline.

24. A salt of 2-S-rimantidine according to Claim 1, characterized in that the crystal particle size is 1 to 50 μm.

25. A pharmaceutical composition for treating cancer in a subject requiring treatment, comprising administering a therapeutically effective amount of the salt described in claim 1 to the subject.

26. A pharmaceutical composition according to claim 25, wherein the cancer is selected from one or more of melanoma, head and neck cancer, lung cancer, colon cancer, breast cancer, esophageal cancer, pancreatic cancer, prostate cancer, cervical cancer, and gastric cancer.

27. A pharmaceutical composition according to claim 25 or 26, wherein the cancer is a sarcoma, carcinoma, lymphoma, or leukemia.

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