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

Abstract

Disclosed herein is the use of purified 2-S rimantadine or purified 2-R rimantadine, or a pharmaceutically acceptable salt thereof, for the treatment of cancer and precancerous lesions in a subject in need thereof, including cancer and precancerous lesions associated with papilloma virus.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of U.S. Provisional Application No. 63 / 398,826, filed on August 17, 2022, the entire contents of which are incorporated herein by reference.

[0003] Incorporated by Reference

[0004] 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 in an incorporated reference, the terminology herein controls. Technical Field

[0005] The present disclosure relates to methods of treating or preventing cancer, including cancer caused by papillomavirus, comprising administering an enantiomerically pure salt of the 2-S enantiomer of rimantadine. Background Art

[0006] Genital human papillomavirus (HPV) is the most common sexually transmitted infection in the United States. The Centers for Disease Control and Prevention (CDC) states that 90% of HPV infections do not cause symptoms and resolve spontaneously within two years. However, in some cases, HPV infection persists and leads to warts or precancerous lesions. Depending on the site affected, these lesions increase the risk of cancer of the cervix, vulva, vagina, penis, anus, rectum, and oropharynx. The HPV types associated with cervical carcinogenicity are divided into 15 "high-risk types" (HPV 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68, 73, and 82) and three "possibly high-risk types" (HPV26, 53, and 66). Researchers have also shown an association between HPV 16 and 18 and breast cancer. HPV types (HPV 6, 11, 40, 42, 43, 44, 54, 61, 70, 72, and 81) are classified as "low risk" and are known to show benign low-grade cervical changes, genital warts, and recurrent respiratory papillomatosis. Cutaneous HPV types 5, 8, and 92 are associated with skin cancer.

[0007] Rimantadine hydrochloride (α-methyl-1-adamantane-hydroxypropylamine hydrochloride) is a An oral medication marketed for the treatment of influenza A. Rimantadine inhibits influenza activity by binding to amino acids in the viral M2 transmembrane channel and blocking proton transport across the M2 channel. A racemic mixture containing rimantadine. One study found evidence that the R-enantiomer binds to the M2 channel pore with greater affinity than the S-enantiomer. However, this finding conflicted with several earlier findings that there was no difference between the enantiomers against M2. In later studies, this finding was confirmed. Rimantadine is also thought to have some anti-Parkinson's disease activity. However, its use for this indication has not been developed or approved.

[0008] There are gastrointestinal and central nervous system side effects, including nausea, stomach upset, vomiting, anorexia, dry mouth, abdominal pain, weakness, nervousness, tiredness, lightheadedness, dizziness, headache, difficulty sleeping, difficulty concentrating, confusion, and anxiety. Anxiety and insomnia were the most commonly cited toxicities for discontinuation of treatment. Summary of the invention

[0009] In some aspects, the present disclosure relates to various salts and solid forms of 2-S-rimantadine. In some embodiments, the composition comprises a salt containing 2-S-rimantadine and fumaric acid. In some embodiments, the salt is an anhydrous salt. In some embodiments, the salt is crystalline. In some embodiments, the salt has an X-ray powder diffraction containing a peak at about 6.092θ. In some embodiments, the X-ray powder diffraction of the salt further comprises a peak at about 14.362θ, about 17.562θ, about 18.452θ, about 18.812θ and / or about 27.172θ. In some embodiments, the composition comprises a salt containing 2-S-rimantadine 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 containing a peak at about 7.592θ. In some embodiments, the X-ray powder diffraction pattern of the salt further comprises peaks at about 17.642θ, about 18.682θ, about 15.432θ, about 19.372θ and / or about 22.482θ. In some embodiments, the composition comprises a salt containing 2-S-rimantadine 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 comprising a peak at about 5.712θ. In some embodiments, the X-ray powder diffraction pattern of the salt further comprises peaks at about 15.852θ, about 16.962θ, about 19.762θ and / or about 19.432θ. In some embodiments, the salt has an X-ray powder diffraction pattern comprising a peak at 5.832θ. In some embodiments, the X-ray powder diffraction pattern of the salt further comprises peaks at about 14.892θ, about 16.872θ, about 17.622θ, about 39.872θ, and / or about 19.722θ. In some embodiments, the composition comprises a salt comprising 2-S-rimantadine 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 7.852θ. In some embodiments, the X-ray powder diffraction pattern of the salt further comprises peaks at about 9.722θ, about 11.4929, about 12.292θ, about 15.662θ, about 19.962θ, about 2θ.382θ, and / or about 2θ.912θ. In some embodiments, the composition comprises a salt comprising 2-S-rimantadine 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 comprising a peak at 6.212θ.In some embodiments, the salt has an X-ray diffraction powder pattern further comprising peaks at about 8.922θ, about 8.442θ, about 14.112θ, about 15.392θ, about 16.912θ, and / or about 19.982θ. In some embodiments, the salt has an X-ray diffraction powder pattern comprising a peak at 8.9429. In some embodiments, the salt has an X-ray diffraction powder pattern further comprising a peak at about 8.452θ, about 14.132θ, about 15.372θ, about 16.862θ, and / or about 19.192θ. In some embodiments, the composition comprises a crystalline form of 2-S-rimantadine, wherein the XRPD peaks are about 14.362θ, about 17.562θ, about 18.452θ, about 18.812θ, and / or about 27.1729. In some embodiments, the composition includes a composition containing a salt of 2-S rimantadine. In some embodiments, the composition includes fumarate, tartrate, galactarate salt, benzoate, benzenesulfonate or a combination thereof. In some embodiments, the composition includes fumarate. In some embodiments, fumarate is fumarate type A. In some embodiments, the composition is characterized by one or more of the following: a peak at about 6.99°2θ in an X-ray powder diffraction pattern ("XRPD") diffraction pattern; a peak at about 14.36°2θ in an XRPD diffraction pattern; a peak at about 17.56°2θ in an XRPD diffraction pattern; a peak at about 18.45°2θ in an XRPD diffraction pattern; a peak at about 18.81°2θ in an XRPD diffraction pattern; or a peak at about 27.17°2θ in an XRPD diffraction pattern. In some embodiments, the salt is tartrate. In some embodiments, tartrate is tartrate type A. In some embodiments, the composition is characterized by one or more of the following: a peak at about 6.99°2θ in an X-ray powder diffraction pattern ("XRPD") diffraction pattern; a peak at about 7.59°2θ in an XRPD diffraction pattern; a peak at about 17.64°2θ in an XRPD diffraction pattern; a peak at about 18.68°2θ in an XRPD diffraction pattern; a peak at about 15.43°2θ in an XRPD diffraction pattern; a peak at about 19.37°2θ in an XRPD diffraction pattern; or a peak at about 22.48°2θ in an XRPD diffraction pattern. In some embodiments, the salt is a galactaric acid salt. In some embodiments, the galactaric acid salt is a Type A galactaric acid salt. In some embodiments, the salt is characterized by one or more of: a peak in the XRPD diffraction pattern at about 5.71° 2θ; a peak in the XRPD diffraction pattern at about 15.85° 2θ; a peak in the XRPD diffraction pattern at about 16.96° 2θ; a peak in the XRPD diffraction pattern at about 19.76° 2θ; or a peak in the XRPD diffraction pattern at about 19.43° 2θ.In some embodiments, the galactaric acid salt is a B-type galactaric acid salt. In some embodiments, the composition is characterized by one or more of the following: a peak at about 5.83°2θ in an XRPD diffractogram; a peak at about 14.89°2θ in an XRPD diffractogram; a peak at about 16.87°2θ in an XRPD diffractogram; a peak at about 17.62°2θ in an XRPD diffractogram; a peak at about 30.87°2θ in an XRPD diffractogram; or a peak at about 19.72°2θ in an XRPD diffractogram. In some embodiments, the salt is a benzoate. In some embodiments, the benzoate is a Type A benzoate. In some embodiments, the composition is characterized by one or more of the following: a peak at about 7.85°2θ in an XRPD diffractogram; a peak at about 9.72°2θ in an XRPD diffractogram; a peak at about 11.49°2θ in an XRPD diffractogram; a peak at about 12.29°2θ in an XRPD diffractogram; a peak at about 15.66°2θ in an XRPD diffractogram; a peak at about 19.96°2θ in an XRPD diffractogram; a peak at about 2θ.38°2θ in an XRPD diffractogram; or a peak at about 2θ.91°2θ in an XRPD diffractogram. In some embodiments, the salt is a benzenesulfonate. In some embodiments, the benzenesulfonate is a benzenesulfonate type A. In some embodiments, the salt is characterized by one or more of the following: a peak at about 6.21°2θ in the XRPD diffractogram; a peak at about 8.92°2θ in the XRPD diffractogram; a peak at about 8.44°2θ in the XRPD diffractogram; a peak at about 14.11°2θ in the XRPD diffractogram; a peak at about 15.39°2θ in the XRPD diffractogram; a peak at about 16.91°2θ in the XRPD diffractogram; or a peak at about 19.98°2θ in the XRPD diffractogram. In some embodiments, the benzenesulfonate salt is Form B benzenesulfonate salt. In some embodiments, the salt is characterized by one or more of: an X-ray powder diffraction pattern ("XRPD") diffraction pattern at about 20°C; a peak in the XRPD diffraction pattern at about 8.94°2θ; a peak in the XRPD diffraction pattern at about 8.45°2θ; a peak in the XRPD diffraction pattern at about 14.13°2θ; a peak in the XRPD diffraction pattern at about 15.37°2θ; a peak in the XRPD diffraction pattern at about 16.86°2θ; or a peak in the XRPD diffraction pattern at about 19.19°2θ. In some embodiments, the composition is crystalline. In some embodiments, differential scanning calorimetry is substantially as described. Fig.11 In some embodiments, differential scanning calorimetry is performed substantially as follows Fig.12 In some embodiments, differential scanning calorimetry is performed substantially as follows Fig.13 In some embodiments, differential scanning calorimetry is performed substantially as follows Fig.14 In some embodiments, differential scanning calorimetry is performed substantially as follows Fig.15 In some embodiments, differential scanning calorimetry is performed substantially as follows Fig.16 In some embodiments, differential scanning calorimetry is performed substantially as follows Fig.17 In some embodiments, differential scanning calorimetry is performed substantially as follows Fig.18 In some embodiments, differential scanning calorimetry is performed substantially as follows Fig.19 In some embodiments, differential scanning calorimetry is performed substantially as follows Fig. 20 In some embodiments, wherein the differential scanning calorimetry is substantially as follows Fig.21As shown. In some embodiments, the composition comprises a salt of 2-S-rimantadine, characterized in that the sample weight loss in thermogravimetric analysis is about 9.91w / w% loss at a temperature of 0°C to 150°C. In some embodiments, the salt is a tartrate. In some embodiments, the composition comprises a salt of 2-S-rimantadine, characterized in that the sample weight loss between the temperature of 0°C to 150°C in thermogravimetric analysis is about 6.72w / w% loss. In some embodiments, the salt is a maleate. In some embodiments, the composition comprises a salt of 2-S-rimantadine, characterized in that the sample weight loss between the temperature of 0°C to 85°C in thermogravimetric analysis is about 10.30w / w% loss. In some embodiments, the salt is a hippurate. In some embodiments, the composition comprises a salt of 2-S-rimantadine, characterized in that the sample weight loss in thermogravimetric analysis at a temperature of 0°C to 150°C is about 0.15w / w%. In some embodiments, the salt is a galactarate. In some embodiments, the composition comprises a salt of 2-S-rimantadine, characterized in that the sample weight loss in thermogravimetric analysis between temperatures of 0°C and 150°C is about 0.00w / w%. In some embodiments, the salt is a galactarate. In some embodiments, the composition comprises a salt of 2-S-rimantadine, characterized in that the sample weight loss in thermogravimetric analysis between temperatures of 0°C and 100°C is about 0.26w / w%. In some embodiments, the salt is a benzoate. In some embodiments, the composition comprises a salt of 2-S-rimantadine, characterized in that the sample weight loss in thermogravimetric analysis between temperatures of 0°C and 110°C is about 14.77w / w%. In some embodiments, the salt is a benzoate. In some embodiments, the composition comprises a salt of 2-S-rimantadine, characterized in that the sample weight loss in thermogravimetric analysis at temperatures of 0°C to 150°C is about 1.68w / w% loss. In some embodiments, the salt is a gentisate. In some embodiments, the composition comprises a salt of 2-S-rimantadine, characterized in that at a temperature of 0°C to 200°C, the sample weight loss in thermogravimetric analysis is about 3.55w / w% loss. In some embodiments, the salt is a toluenesulfonate. In some embodiments, the composition comprises a salt of 2-S-rimantadine, characterized in that at a temperature of 0°C to 200°C, the sample weight loss in thermogravimetric analysis is about 0.02w / w% loss. In some embodiments, the salt is a benzenesulfonate. In some embodiments, the composition comprises a salt of 2-S-rimantadine, characterized in that at a temperature of 0°C to 200°C, the sample weight loss in thermogravimetric analysis is about 0.09w / w% loss. In some embodiments, the salt is a benzenesulfonate.In some embodiments, the composition comprises a salt of 2-S-rimantadine, wherein the salt exhibits a differential scanning calorimetry analysis 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-rimantadine, wherein the salt exhibits a differential scanning calorimetry analysis 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-rimantadine, wherein the salt exhibits a differential scanning calorimetry analysis comprising an endothermic peak at about 62°C. In some embodiments, the salt is a hippurate salt. In some embodiments, the composition comprises a salt of 2-S-rimantadine, wherein the salt exhibits a differential scanning calorimetry analysis comprising an endothermic peak at about 193°C. In some embodiments, the salt is a galactaric acid salt. In some embodiments, the composition comprises a salt of 2-S-rimantadine, wherein the salt exhibits a differential scanning calorimetry analysis comprising an endothermic peak at about 195°C. In some embodiments, the salt is a galactarate. In some embodiments, the composition comprises a salt of 2-S-rimantadine, wherein the salt exhibits a differential scanning calorimetry analysis comprising an endothermic peak at about 199°C. In some embodiments, the salt is a benzoate. In some embodiments, the composition comprises a salt of 2-S-rimantadine, wherein the salt exhibits a differential scanning calorimetry analysis comprising an endothermic peak at about 198°C. In some embodiments, the salt is a benzoate. In some embodiments, the composition comprises a salt of 2-S-rimantadine, wherein the salt exhibits a differential scanning calorimetry analysis comprising an endothermic peak at about 206°C. In some embodiments, the salt is a gentisate. In some embodiments, the composition comprises a salt of 2-S-rimantadine, wherein the salt exhibits a differential scanning calorimetry analysis comprising an endothermic peak at about 214°C. In some embodiments, a salt as described in the preceding claim, wherein the salt is a tosylate. In some embodiments, the composition comprises a salt of 2-S-rimantadine, wherein the salt exhibits a differential scanning calorimetry analysis comprising 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-rimantadine, wherein the salt exhibits a differential scanning calorimetry analysis comprising 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-rimantadine, characterized in that the crystal particle size is 1-50 μm. In some embodiments, the composition comprises a salt of 2-S-rimantadine, characterized in that the XRPD diffraction pattern is substantially as follows. Figure 4 In some embodiments, the composition comprises a salt of 2-S-rimantadine characterized by an XRPD diffraction pattern substantially as shown Figure 5 In some embodiments, the composition comprises a salt of 2-S-rimantadine characterized by an XRPD diffraction pattern substantially as shown Figure 6 In some embodiments, the composition comprises a salt of 2-S-rimantadine characterized by an XRPD diffraction pattern substantially as shown Figure 7 In some embodiments, the composition comprises a salt of 2-S-rimantadine characterized by an XRPD diffraction pattern substantially as shown Figure 8 In some embodiments, the composition comprises a salt of 2-S-rimantadine characterized by an XRPD diffraction pattern substantially as shown Fig. 9 In some embodiments, the composition comprises a salt of 2-S-rimantadine characterized by an XRPD diffraction pattern substantially as shown Fig.10As shown. In some embodiments, the salt of 2-S-rimantadine is the tartrate of 2-S-rimantadine. In some embodiments, the salt of 2-S-rimantadine is the fumarate of 2-S-rimantadine. In some embodiments, the salt of 2-S-rimantadine is the galactocarbamide of 2-S-rimantadine. In some embodiments, the salt of 2-S-rimantadine is the benzoate of 2-S-rimantadine. In some embodiments, the salt of 2-S-rimantadine is the benzenesulfonate of 2-S-rimantadine. In some embodiments, the method of the present disclosure includes a method comprising administering to a subject an effective therapeutic 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 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 selected from head and neck cancer, breast cancer and melanoma. In some embodiments, the cancer is an HPV-related cancer. In some embodiments, HPV-related cancers are associated with the alpha genus of HPV. In some embodiments, one or more cancer cells from a subject express 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, HPV58, HPV 66 and HPV 69. In some embodiments, the HPV protein is E5 from HPV 16, and in some embodiments, the HPV protein is E5 from HPV 18. In some embodiments, the methods described include a method of treating cancer in a subject, the method comprising: detecting cancer cells expressing human papillomavirus (HPV) proteins in a sample from the subject; and administering to the subject an effective therapeutic 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 the E5, E6, or E7 HPV proteins.In some embodiments, the HPV E5, E6 or E7 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 cancer is selected from the group consisting of head and neck cancer, mucosal squamous cell carcinoma, skin 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 the following: 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, cimiprilimumab and combinations thereof. In some embodiments, the method further comprises subjecting the subject to radiotherapy, surgery or a combination thereof. In some embodiments, the subject is a human. In some embodiments, the method described herein includes a method for treating a precancerous HPV lesion in a subject in need of treatment, the method comprising administering a salt or composition of an effective therapeutic amount of 2-S-rimantadine. In some embodiments, the HPV lesions are associated with the alpha genus of HPV. In some embodiments, the HPV precancer is a lesion of the cervix, skin, urethra, nasal cavity, paranasal sinus, larynx, tracheobronchial mucosa or oral cavity. In some embodiments, the HPV precancer expresses one or more HPV proteins selected from one or more of E5, E6 or E7 HPV proteins. In some embodiments, HPV E5, E6 or E7 proteins are from one or more HPV subtypes selected from the following: 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 a method for treating or preventing avian influenza in poultry, the method comprising administering an effective therapeutic amount of a salt or composition according to any one of the preceding claims.In some embodiments, the avian influenza is H5N1. In some embodiments, the side effects associated with the administration of a salt or composition of 2-S rimantadine are reduced compared to the side effects associated with racemic rimantadine or enantiopure 2-R rimantadine. In some embodiments, the composition includes a composition comprising: a salt or composition of 2-S-rimantadine, and one or more immune checkpoint inhibitors. In some embodiments, 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 includes enantiopure 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 methods disclosed herein include a method of 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 methanol, ethanol, chloroform and water. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Figure 1 AB show the peak current amplitude measurements and steady-state current measurements for 2-S rimantadine (TGN-S15) and 2-R rimantadine (TGN-S16) of NR2A. Figure 1 CD show the peak current amplitude measurements and steady-state current measurements for 2-S rimantadine (TGN-S15) and 2-R rimantadine (TGN-S16) of NR2B.

[0011] Figure 2 Proliferation of CAL-27 cells with different concentrations of LPS are shown: RS-rimantadine (TGN-S11), S-rimantadine (TGN-S15), R-rimantadine (TGN-S16), and memantine (TGN-S13).

[0012] Figure 3 The interaction diagram for 2-S-rimantadine is shown.

[0013] Figure 4 The X-ray powder diffraction ("XRPD") diffraction pattern of 2-S-rimantadine fumarate Form A is shown.

[0014] Figure 5 The XRPD diffraction pattern of the tartrate salt Form A of 2-S-rimantadine is shown.

[0015] Figure 6The XRPD diffraction pattern of 2-S-rimantadine galactarate Form A is shown.

[0016] Figure 7 The XRPD diffraction pattern of 2-S-rimantadine galactarate Form B is shown.

[0017] Figure 8 Shown is the XRPD diffraction pattern of the benzoate salt Form A of 2-S-rimantadine.

[0018] Fig. 9 The XRPD diffraction pattern of 2-S-rimantadine benzenesulfonate Form A is shown.

[0019] Fig.10 The XRPD diffraction pattern of the benzenesulfonate salt Form B of 2-S-rimantadine is shown.

[0020] Fig.11 Shown are thermogravimetric and differential scanning calorimetric analyses of the tartrate salt Form A of 2-S-rimantadine.

[0021] Fig.12 Shown are thermogravimetric and differential scanning calorimetric analyses of the maleate salt Form A of 2-S-rimantadine.

[0022] Fig.13 Shown are thermogravimetric and differential scanning calorimetric analyses of the hippurate salt Form A of 2-S-rimantadine.

[0023] Fig.14 Shown are thermogravimetric analysis and differential scanning calorimetry of 2-S-rimantadine galactarate Form A.

[0024] Fig.15 Shown are thermogravimetric analysis and differential scanning calorimetry of 2-S-rimantadine galactarate Form B.

[0025] Fig.16 Shown are thermogravimetric and differential scanning calorimetric analyses of the benzoate salt Form A of 2-S-rimantadine.

[0026] Fig.17 Shown are thermogravimetric analysis and differential scanning calorimetry of benzoate salt Form B of 2-S-rimantadine.

[0027] Fig.18 Thermogravimetric analysis and differential scanning calorimetry of 2-S-rimantadine gentisate Form A are shown.

[0028] Fig.19 Shown are thermogravimetric and differential scanning calorimetric analyses of the tosylate salt Form A of 2-S-rimantadine.

[0029] Fig. 20Shown are thermogravimetric analysis and differential scanning calorimetry of the benzenesulfonate salt Form A of 2-S-rimantadine.

[0030] Fig.21 Shown are thermogravimetric analysis and differential scanning calorimetry of the benzenesulfonate salt Form B of 2-S-rimantadine.

[0031] Fig. 22 Polarized light microscopy analysis of the fumarate salt Form A of 2-S-rimantadine is shown.

[0032] Fig.23 Polarized light microscopy analysis of the tartrate salt Form A of 2-S-rimantadine is shown.

[0033] Fig.24 Polarized light microscopy analysis of the maleate salt Form A of 2-S-rimantadine is shown.

[0034] Fig.25 Polarized light microscopy analysis of the hippurate salt form A of 2-S-rimantadine is shown.

[0035] Fig.26 Polarized light microscopy analysis of the galactarate salt Form A of 2-S-rimantadine is shown.

[0036] Fig. 27 Polarized light microscopy analysis of 2-S-rimantadine galactarate Form B is shown. Fig.28 Polarized light microscopy analysis of the benzoate salt Form A of 2-S-rimantadine is shown.

[0037] Fig.29 Polarized light microscopy analysis of the benzoate salt Form B of 2-S-rimantadine is shown.

[0038] Fig.30 Polarized light microscopy analysis of 2-S-rimantadine gentisate Form A is shown.

[0039] Fig.31 Polarized light microscopy analysis of the tosylate salt Form A of 2-S-rimantadine is shown.

[0040] Fig.32 Polarized light microscopy analysis of the benzenesulfonate salt Form A of 2-S-rimantadine is shown.

[0041] Fig.33 Polarized light microscopy analysis of the benzenesulfonate salt Form B of 2-S-rimantadine is shown. DETAILED DESCRIPTION

[0042] One aspect of the present disclosure is the use of enantiomerically pure 2-S rimantadine (e.g., a salt of enantiomerically pure 2-S rimantadine (e.g., a fumarate, tartrate, galactarate, benzoate, besylate, or any combination thereof) or enantiomerically pure 2-R rimantadine for treating cancer. In some embodiments, disclosed herein is 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 for treating cancer associated with a papillomavirus, such as a human papillomavirus (HPV). In some embodiments, the HPV is an HPV from the genus alpha.

[0043] Another aspect of the present disclosure is the use of enantiomerically pure 2-S rimantadine (eg, a salt of 2-S rimantadine) or enantiomerically pure 2-R rimantadine for treating precancerous lesions associated with papillomavirus (eg, human papillomavirus).

[0044] Racemic rimantadine has side effects at currently prescribed doses. Side effects include central nervous system (CNS) side effects, sleep side effects, gastrointestinal side effects, and atropine-like side effects, such as, but not limited to, mild headache, dizziness, depression, confusion, difficulty concentrating, anxiety (e.g., nervousness), irritability, hallucinations, and headaches, 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 toxicities of racemic rimantadine at the time of discontinuation of treatment.

[0045] As disclosed herein, 2-S rimantadine inhibits the N-methyl-D-aspartate subtype glutamate receptor (NMDA) subunit NR2B subunit to a lesser extent than 2-R rimantadine and racemic rimantadine (see Table 2 in Example 2 below).

[0046] In some embodiments, disclosed herein is the use of 2-S rimantadine (e.g., a salt of 2-S rimantadine (e.g., an enantiopure salt)) for treating cancer, HPV-associated cancer, HPV-associated precancerous lesions, and / or influenza A. Due to the lower ability of 2-S rimantadine to inhibit NR2B compared to racemic rimantadine, 2-S rimantadine has fewer side effects compared to treating these conditions with racemic rimantadine or 2-R rimantadine. Due to the greater ability of 2-R rimantadine to inhibit NR2B than 2-S rimantadine, 2-R rimantadine may have fewer side effects compared to treating these conditions with racemic rimantadine or 2-S rimantadine.

[0047] In some embodiments, disclosed herein is the use of 2-R rimantadine for treating cancer, HPV-associated cancer, HPV-associated precancerous lesions, and / or influenza A. Because 2-R rimantadine has a greater ability to inhibit NR2B than racemic rimantadine, 2-R rimantadine may have fewer side effects than treating these conditions with racemic rimantadine or 2-S rimantadine.

[0048] In some embodiments, 2-S rimantadine (e.g., a salt (e.g., an enantiopure salt) of 2-S rimantadine) disclosed herein can have fewer side effects compared to treatment of these conditions with racemic rimantadine or 2-R rimantadine due to the lower 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 relative to 2-R rimantadine or racemic rimantadine. In some embodiments, the degree of NMDA receptor inhibition caused by 2-S rimantadine is about 10% to about 20% less, about 10% to about 30% less, about 10% to about 40% less, about 10% to about 50% less, about 10% to about 60% less, about 10% to about 70% less, about 10% to about 80% less, about 10% to about 90% less, about 10% to about 1 ... % to about 100% lower, about 20% to about 30% lower, about 20% to about 40% lower, about 20% to about 50% lower, about 20% to about 60% lower, about 20% to about 70% lower, about 20% to about 80% lower, about 20% to about 90% lower, about 20% to about 100% lower, about 30% to about 40% lower, about 30% to about 50% lower, about 30% to about 60% lower, about 30% to about 70%, about 30% to about 80% lower, about 30% to about 90% lower, about 30% to about 100% lower, about 40% to about 50% lower, about 40% to about 60% lower, about 40% to about 70% lower, about 40% to about 80% lower, about 40% to about 90% lower, about 40% to about 100% lower, about 50% to about 60% lower, about 50% to about 70% lower, about 50% to about 80% lower, 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, about 100% less, about 90% less, about 100% less, about 100% 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. In some embodiments, the degree of NMDA receptor inhibition caused by 2-S rimantadine is at least about 10% less, about 20% less, about 30% less, about 40% less, about 50% less, about 60% less, about 70% less, about 80% less, or about 90% less relative to 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% lower, about 30% lower, about 40% lower, about 50% lower, about 60% lower, about 70% lower, about 80% lower, about 90% lower, or about 100% lower relative to 2-R rimantadine or racemic rimantadine. In some embodiments, the NMDA receptor is NR2A. In some embodiments, the NMDA receptor is NR2B.

[0049] In some embodiments, the 2-R rimantadine disclosed herein may have fewer side effects than treatment of these conditions with racemic rimantadine or 2-S rimantadine due to the lower 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 relative to 2-S rimantadine or racemic rimantadine. In some embodiments, the degree of NMDA receptor inhibition caused by 2-R rimantadine is about 10% to about 20% less, about 10% to about 30% less, about 10% to about 40% less, about 10% to about 50% less, about 10% to about 60% less, about 10% to about 70% less, about 10% to about 80% less, about 10% to about 90% less, about 10% to about 1 ... % to about 100% lower, about 20% to about 30% lower, about 20% to about 40% lower, about 20% to about 50% lower, about 20% to about 60% lower, about 20% to about 70% lower, about 20% to about 80% lower, about 20% to about 90% lower, about 20% to about 100% lower, about 30% to about 40% lower, about 30% to about 50% lower, about 30% to about 60% lower, about 30% to about 70%, about 30% to about 80% lower, about 30% to about 90% lower, about 30% to about 100% lower, about 40% to about 50% lower, about 40% to about 60% lower, about 40% to about 70% lower, about 40% to about 80% lower, about 40% to about 90% lower, about 40% to about 100% lower, about 50% to about 60% lower, about 50% to about 70% lower, about 50% to about 80% lower, In some embodiments, the degree of NMDA receptor inhibition caused by 2-R rimantadine is about 10% lower, about 20% lower, about 30% lower, about 40% lower, about 50% lower, about 60% lower, about 70% lower, about 80% lower, about 90% lower, about 100% lower, about 90% lower, about 100% lower, about 80% lower, about 90% lower, about 100% lower, about 100% lower, about 100% lower, about 20% lower, about 30% lower, about 40% lower, about 50% lower, about 60% lower, about 70% lower, about 80% lower, about 90% lower, or about 100% lower. In some embodiments, 2-R rimantadine causes at least about 10% less, about 20% less, about 30% less, about 40% less, about 50% less, about 60% less, about 70% less, about 80% less, or about 90% less inhibition of NMDA receptors relative to 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% lower, about 30% lower, about 40% lower, about 50% lower, about 60% lower, about 70% lower, about 80% lower, about 90% lower, or about 100% lower relative to 2-S rimantadine or racemic rimantadine. In some embodiments, the NMDA receptor is NR2A. In some embodiments, the NMDA receptor is NR2B.

[0050] In some embodiments, 2-S rimantadine (e.g., a salt (e.g., an enantiopure salt) of 2-S rimantadine) can have fewer side effects than treatment of these conditions with racemic rimantadine or 2-R rimantadine due to the lower ability of 2-S rimantadine to antagonize GABA receptors and / or inhibit GABA-mediated biological pathways. In some embodiments, 2-S rimantadine causes about 10% less inhibition of GABA receptors and / or GABA-mediated biological pathways to about 100% less than 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 (e.g., a salt of 2-S rimantadine) is about 10% to about 20% less, about 10% to about 30% less, about 10% to about 40% less, about 10% to about 50% less, about 10% to about 60% less, about 10% to about 70% less, about 10% to about 80% less, about 10% to about 90% less, about 10% to about 10 ... about 80%, about 10% to about 90% below, about 10% to about 100% below, about 20% to about 30% below, about 20% to about 40% below, about 20% to about 50% below, about 20% to about 60% below, about 20% to about 70% below, about 20% to about 80% below, about 20% to about 90% below, about 20% to about 100% below, about 30% to about 40% below, about 30% to about 50% below, about 30% to about 60% below, about 20% to about 70% below, about 20% to about 80% below, about 20% to about 90% below, about 20% to about 100% below, about 30% to about 40% below, about 30% to about 50% below, about 30% to about About 60% lower, about 30% lower to about 70% lower, about 30% lower to about 80% lower, about 30% lower to about 90% lower, about 30% lower to about 100% lower, about 40% lower to about 50% lower, about 40% lower to about 60% lower, about 40% lower to about 70% lower, about 40% lower to about 80% lower, about 40% lower to about 90% lower, about 40% lower to about 100% lower, about 50% lower to about 60% lower, about 50% lower to about 70% lower, about 50% lower to about 80% below, about 50% below to about 90% below, about 50% below to about 100% below, about 60% below to about 70% below, about 60% below to about 80% below, about 60% below to about 90% below, about 60% below to about 100% below, about 70% below to about 80% below, about 70% below to about 90% below, about 70% below to about 100% below, about 80% below to about 90% below, about 80% below to about 100% below, or about 90% below to about 100% below. In some embodiments, the degree of inhibition of GABA receptors and / or GABA-mediated biological pathways 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 relative to 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% less, about 20% less, about 30% less, about 40% less, about 50% less, about 60% less, about 70% less, about 80% less, or about 90% less relative to 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 most 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 relative to 2-R rimantadine or racemic rimantadine.

[0051] In some embodiments, 2-R rimantadine disclosed herein may have fewer side effects compared to treatment of these conditions with racemic rimantadine or 2-S rimantadine due to the lower ability of 2-R rimantadine to antagonize GABA receptors and / or inhibit GABA-mediated biological pathways. In some embodiments, 2-R rimantadine causes about 10% less inhibition of GABA receptors and / or GABA-mediated biological pathways to about 100% less than 2-S rimantadine or racemic rimantadine. In some embodiments, the degree of inhibition of GABA receptors and / or GABA-mediated biological pathways caused by 2-R rimantadine is about 10% to about 20% less, about 10% to about 30% less, about 10% to about 40% less, about 10% to about 50% less, about 10% to about 60% less, about 10% to about 70% less, about 10% to about 80% less, about 10% to about 90% less, about 10% to about 100% less, about 10% to about 110% less, about 10% to about 120% less, about 10% to about 130% less, about 10% to about 140% less, about 10% to about 150% less, about 10% to about 160% less, about 10% to about 170% less, about 10% to about 180% less, about 10% to about 190% less, about 10% to about 200% less, about 10% to about 210% less, about 10% to about 220% less, about 10% to about 230% less, about 10% to about 240% less, about 10% to about 250% less, about 10% to about 260% less, about 10% to about 270% less, about 10% to about 280% less, about 10% to about 290% less, about 10% to about 300% less, about 10% to about 310% less, about 10% to about 310% less About 90% lower, about 10% lower to about 100% lower, about 20% lower to about 30% lower, about 20% lower to about 40% lower, about 20% lower to about 50% lower, about 20% lower to about 60% lower, about 20% lower to about 70% lower, about 20% lower to about 80% lower, about 20% lower to about 90% lower, about 20% lower to about 100% lower, about 30% lower to about 40% lower, about 30% lower to about 50% lower, about 30% lower to about 60% lower, about 30% to about 70% lower, about 30% to about 80% lower, about 30% to about 90% lower, about 30% to about 100% lower, about 40% to about 50% lower, about 40% to about 60% lower, about 40% to about 70% lower, about 40% to about 80% lower, about 40% to about 90% lower, about 40% to about 100% lower, about 50% to about 60% lower, about 50% to about 70% lower, about 50% to about 90% lower, about 40% to about 100% lower, about 50% to about 60% lower, about 50% to about 70% lower, about 50% to about From about 50% to about 90% below, from about 50% to about 100% below, from about 60% to about 70% below, from about 60% to about 80% below, from about 60% to about 90% below, from about 60% to about 100% below, from about 70% to about 80% below, from about 70% to about 90% below, from about 70% to about 100% below, from about 80% to about 90% below, from about 80% to about 100% below, or from about 90% to about 100% below. 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, 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 relative to 2-S rimantadine or racemic rimantadine.In some embodiments, the degree of inhibition of GABA receptors and / or GABA-mediated biological pathways caused by 2-R rimantadine is at least about 10% less, about 20% less, about 30% less, about 40% less, about 50% less, about 60% less, about 70% less, about 80% less, or about 90% less relative to 2-S rimantadine or racemic rimantadine. In some embodiments, the degree of inhibition of GABA receptors and / or GABA-mediated biological pathways caused by 2-R rimantadine is at most 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 relative to 2-S rimantadine or racemic rimantadine.

[0052] In some embodiments, 2-S rimantadine (e.g., a salt (e.g., an enantiopure salt) of 2-S-rimantadine) disclosed herein can have fewer side effects compared to treatment of these conditions with racemic rimantadine or 2-R rimantadine due to the lower ability of 2-S rimantadine to antagonize dopamine receptors and / or inhibit dopamine-mediated biological pathways. In some embodiments, 2-S rimantadine causes about 10% less inhibition of dopamine receptors and / or dopamine-mediated biological pathways to about 100% less than 2-R rimantadine or racemic rimantadine. In some embodiments, the degree of inhibition of dopamine receptors and / or dopamine-mediated biological pathways caused by 2-S rimantadine is about 10% to about 20% less, about 10% to about 30% less, about 10% to about 40% less, about 10% to about 50% less, about 10% to about 60% less, about 10% to about 70% less, about 10% to about 80% less, about 10% to about 90% less, about 10% to about 100% less, about 10% to about 110% less, about 10% to about 120% less, about 10% to about 130% less, about 10% to about 140% less, about 10% to about 150% less, about 10% to about 160% less, about 10% to about 170% less, about 10% to about 180% less, about 10% to about 190% less, about 10% to about 200% less, about 10% to about 210% less, about 10% to about 220% less, about 10% to about 230% less, about 10% to about 240% less, about 10% to about 250% less, about 10% to about 260% less, about 10% to about 270% less, about 10% to about 280% less, about 10% to about 290% less, about 10% to about 300% less, about 10% to about 310% less, about 10% to about 310% less about 90%, about 10% to about 100% lower, about 20% to about 30% lower, about 20% to about 40% lower, about 20% to about 50% lower, about 20% to about 60% lower, about 20% to about 70% lower, about 20% to about 80% lower, about 20% to about 90% lower, about 20% to about 100% lower, about 30% to about 40% lower, about 30% to about 50% lower, about 30% to about 60% lower, about 30% to about 70% lower, about 30% to about 80% lower, about 30% to about 90% lower, about 30% to about 100% lower, about 40% to about 50% lower, about 40% to about 60% lower, about 40% to about 70% lower, about 40% to about 80% lower, about 40% to about 90% lower, about 40% to about 100% lower, about 50% to about 60% lower, about 50% to about 70% lower, about 50% to about 80% lower, about 40% to about 90% lower, about 40% to about 100% lower, about 50% to about 60% lower, about 50% to about 70% lower, about 50% to about 80% lower 0%, about 50% to about 90% below, about 50% to about 100% below, about 60% to about 70% below, about 60% to about 80% below, about 60% to about 90% below, about 60% to about 100% below, about 70% to about 80% below, about 70% to about 90% below, about 70% to about 100% below, about 80% to about 90% below, about 80% to about 100% below, or about 90% to about 100% below. In some embodiments, 2-S rimantadine causes 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 inhibition of dopamine receptors and / or dopamine-mediated biological pathways relative to 2-R rimantadine or racemic rimantadine.In some embodiments, 2-S rimantadine causes at least about 10% less inhibition of dopamine receptors and / or dopamine-mediated biological pathways, relative to 2-R rimantadine or racemic rimantadine, about 20% less, about 30% less, about 40% less, about 50% less, about 60% less, about 70% less, about 80% less, or about 90% less inhibition. In some embodiments, 2-S rimantadine causes at most about 20% less inhibition of dopamine receptors and / or dopamine-mediated biological pathways, relative to 2-R rimantadine or racemic rimantadine, 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 inhibition. In some embodiments, the dopamine receptor is a D2 / 3 receptor.

[0053] In some embodiments, 2-R rimantadine may have fewer side effects than treatment of these conditions with racemic rimantadine or 2-S rimantadine due to the lower ability of 2-R rimantadine to antagonize dopamine receptors and / or inhibit dopamine-mediated biological pathways. In some embodiments, 2-R rimantadine causes about 10% less inhibition of dopamine receptors and / or dopamine-mediated biological pathways to about 100% less than 2-S rimantadine or racemic rimantadine. In some embodiments, the degree of inhibition of dopamine receptors and / or dopamine-mediated biological pathways caused by 2-R rimantadine is about 10% to about 20% less, about 10% to about 30% less, about 10% to about 40% less, about 10% to about 50% less, about 10% to about 60% less, about 10% to about 70% less, about 10% to about 80% less, about 10% to about 90% less, about 10% to about 100% less, about 10% to about 110% less, about 10% to about 120% less, about 10% to about 130% less, about 10% to about 140% less, about 10% to about 150% less, about 10% to about 160% less, about 10% to about 170% less, about 10% to about 180% less, about 10% to about 190% less, about 10% to about 200% less, about 10% to about 210% less, about 10% to about 220% less, about 10% to about 230% less, about 10% to about 240% less, about 10% to about 250% less, about 10% to about 260% less, about 10% to about 270% less, about 10% to about 280% less, about 10% to about 290% less, about 10% to about 300% less, about 10% to about 310% less, about 10% to about 320% less, about 10% to about 330% less, about 90%, about 10% to about 100% lower, about 20% to about 30% lower, about 20% to about 40% lower, about 20% to about 50% lower, about 20% to about 60% lower, about 20% to about 70% lower, about 20% to about 80% lower, about 20% to about 90% lower, about 20% to about 100% lower, about 30% to about 40% lower, about 30% to about 50% lower, about 30% to about 60% lower, about 30% to about 70% lower, about 30% to about 80% lower, about 30% to about 90% lower, about 30% to about 100% lower, about 40% to about 50% lower, about 40% to about 60% lower, about 40% to about 70% lower, about 40% to about 80% lower, about 40% to about 90% lower, about 40% to about 100% lower, about 50% to about 60% lower, about 50% to about 70% lower, about 50% to about 80% lower, about 40% to about 90% lower, about 40% to about 100% lower, about 50% to about 60% lower, about 50% to about 70% lower, about 50% to about 80% lower 0%, about 50% to about 90% below, about 50% to about 100% below, about 60% to about 70% below, about 60% to about 80% below, about 60% to about 90% below, about 60% to about 100% below, about 70% to about 80% below, about 70% to about 90% below, about 70% to about 100% below, about 80% to about 90% below, about 80% to about 100% below, or about 90% to about 100% below. 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, 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 relative to 2-S rimantadine or racemic rimantadine.In some embodiments, 2-R rimantadine causes at least about 10% less inhibition of dopamine receptors and / or dopamine-mediated biological pathways, relative to 2-S rimantadine or racemic rimantadine, about 20% less, about 30% less, about 40% less, about 50% less, about 60% less, about 70% less, about 80% less, or about 90% less inhibition. In some embodiments, 2-R rimantadine causes at most about 20% less inhibition of dopamine receptors and / or dopamine-mediated biological pathways, relative to 2-S rimantadine or racemic rimantadine, 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 inhibition. In some embodiments, the dopamine receptor is a D2 / 3 receptor.

[0054] In another aspect, disclosed herein is the use of 2-S rimantadine (e.g., a salt (e.g., an enantiopure salt) of 2-S rimantadine) for treating / preventing influenza in veterinary animals such as poultry (e.g., chickens, turkeys, and ducks) and horses. Use of 2-S rimantadine can have fewer side effects than treatment of these animals with racemic rimantadine or 2-R rimantadine.

[0055] In another aspect, disclosed herein is the use of 2-R rimantadine for treating / preventing influenza in veterinary animals such as poultry (e.g., chickens, turkeys, and ducks) and horses. Use of 2-R rimantadine may have fewer side effects than treatment of these animals with racemic rimantadine or 2-S rimantadine.

[0056] definition

[0057] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the content clearly dictates otherwise. It should also be noted that the term "or" is generally 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.

[0058] In some embodiments, S-rimantadine (e.g., a salt of 2-S rimantadine), R-rimantadine, racemic rimantadine, or a rimantadine derivative as described herein is pegylated. As used herein, "pegylated" or "pegylation" describes the conjugation of a compound to a polyethylene glycol (PEG) moiety. The PEG moiety can be 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 greater than or equal to 5 kDa, and a low molecular weight PEG has a molecular weight less than 5 kDa. In some embodiments, PEG is selected from the following: 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 a PEG group extending from the PEG backbone.

[0059] As used herein, when applied to chiral compounds, the term "pure" refers to an enantiomer of a chiral compound that is substantially free of its opposite enantiomer (i.e., enantiomeric excess). For example, a pure "R" form of a compound is substantially free of the "S" form of the compound, so the "S" form is in enantiomeric excess. The term "enantiomerically pure" or "pure enantiomer" means that the compound contains an excess of enantiomer, such as greater than 75% by weight, greater than 80% by weight, greater than 85% by weight, greater than 90% by weight, greater than 91% by weight, greater than 92% by weight, greater than 93% by weight, greater than 94% by weight, greater than 95% by weight, greater than 96% by weight, greater than 97% by weight, greater than 98% by weight, greater than 98.5% by weight, greater than 99% by weight, greater than 99.2% by weight, greater than 99.5% by weight, greater than 99.6% by weight, greater than 99.7% by weight, greater than 99.8% by weight, or greater than 99.9% by weight of the enantiomer. 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 can be in excess of 30-80%, or 30-70%, 30-60%, 30%, 35%, 40%, 45%, 50%, 55% or 60%, or any percentage therebetween.

[0060] As used herein, unless otherwise indicated, the term "enantiopure 2-S rimantadine" refers, for example, to 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 enantiomers of rimantadine.

[0061] As used herein, unless otherwise indicated, the term "enantiopure" 2-A 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 weight is based on the total weight of rimantadine, i.e., both or all enantiomers of rimantadine.

[0062] In the compositions provided herein, enantiomerically pure rimantadine or a pharmaceutically acceptable salt, solvate, hydrate, ester or prodrug thereof can be present with other active or inactive ingredients. For example, a pharmaceutical composition comprising enantiomerically pure 2-S rimantadine can contain, for example, about 90% of an excipient and about 10% of enantiomerically pure 2-S rimantadine. In certain embodiments, enantiomerically pure 2-S rimantadine in such a composition can, for example, contain at least about 99.9% by weight of 2-S rimantadine and at most 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.

[0063] As used herein, the terms "subject," "individual," or "patient," which are used interchangeably, 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.

[0064] As used herein, the term "treat" or "treating" refers to therapeutic or palliative measures. Beneficial or desired clinical results include, but are not limited to, all or partial relief of symptoms associated with a disease or disorder or condition, reduction in disease extent, stabilization (i.e., non-exacerbation) of the disease state, delay or slowing of disease progression, improvement or alleviation of the disease state (e.g., one or more symptoms of the disease), and relief (whether partial or total), whether detectable or undetectable. "Treatment" may also mean prolonging survival compared to expected survival if not receiving treatment.

[0065] As used herein, the term "preventing" or "preventing" means completely or partially preventing the onset, recurrence, or spread of a disease or disorder as described herein, or symptoms thereof.

[0066] The term "administration" or "administering" refers to a method of giving a dose of a compound or pharmaceutical composition to a vertebrate or invertebrate, including mammals, birds, fish, or amphibians. 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. An "effective therapeutic amount" or "pharmaceutically effective amount" of a compound provided herein is an amount sufficient to achieve the desired effect, and may vary depending on the nature and severity of the disease condition and the potency of the compound. The therapeutic effect is to alleviate one or more symptoms of the disease to some extent, and may include curing the disease.

[0067] "Cure" refers to the elimination of symptoms of active disease. However, even after a cure is achieved, there may be certain long-term or permanent effects of the disease (such as, for example, extensive tissue damage).

[0068] As used herein, the term "effective amount" or "effective therapeutic amount" refers to a sufficient amount of the administered agent or compound, which will alleviate one or more symptoms of the disease or condition being treated to a certain extent. The result can be a reduction and / or alleviation of the signs, symptoms or causes of the disease, or any other desired change in the biological system. For example, an "effective amount" for therapeutic use is the amount of a composition comprising a compound as disclosed herein required to provide a clinically significant reduction in disease symptoms. Techniques such as dose escalation studies can be used to determine the appropriate "effective" amount in any individual case. An "effective amount" is an amount sufficient to achieve the purpose of the compound relative to the absence of the compound (e.g., to achieve the effect of its administration, to treat the disease, to reduce enzyme activity, to increase enzyme activity, to reduce signal transduction pathways, or to reduce one or more symptoms of the disease or condition). An example of an "effective amount" is an amount sufficient to contribute to the treatment, prevention or reduction of one or more symptoms of the disease, which may also be referred to as an "effective therapeutic amount". "Reduction" of one or more symptoms (and grammatical equivalents of the phrase) means a reduction in the severity or frequency of the symptoms, or the elimination of the symptoms. The "effective preventive amount" of a drug is the amount of the drug that will have the expected preventive effect when applied to a subject, such as preventing or delaying the onset (or recurrence) of an injury, disease, pathology or condition, or reducing the likelihood of the onset (or recurrence) of an injury, disease, pathology or condition or its symptoms. A complete preventive effect does not necessarily occur by administering one dose, and may occur only after a series of doses are administered. Therefore, an effective preventive amount may be administered in one or more administrations. As used herein, an "activity-reducing amount" refers to the amount of an antagonist required to reduce enzyme activity relative to the absence of an antagonist. As used herein, a "functionally disruptive amount" refers to the amount of an antagonist required to destroy the function of an enzyme or protein relative to the absence of an antagonist. The exact amount will depend on the purpose of the treatment, and will be determined by one skilled 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 Edition, 2003, Gennaro, Ed., Lippincott, Williams & Wilkins).

[0069] The term "immunotherapy" refers to an agent that modulates the immune system. In some embodiments, immunotherapy can increase the expression and / or activity of immune system modulators. In some embodiments, immunotherapy can reduce the expression and / or activity of immune system modulators. In some embodiments, immunotherapy can recruit and / or enhance the activity of immune cells.

[0070] Pharmaceutically acceptable salts, prodrugs, stereoisomers and tautomers

[0071] A pure R or S enantiomer of rimantadine (i.e., for example, a salt of 2-S rimantadine (e.g., fumarate, tartrate, galactarate, benzoate, besylate, or any combination thereof) provided herein can be administered as any salt or prodrug that is capable of providing the parent compound directly or indirectly upon administration to a recipient, or exhibits activity itself. As used herein, the term "pharmaceutically acceptable salt" refers to a salt that retains the desired biological activity of the subject compound and exhibits minimal undesirable toxicological effects. The phrase "pharmaceutically acceptable salt or prodrug" is used throughout the specification to describe any pharmaceutically acceptable form of a compound (e.g., ester, amide, salt of an ester, salt of an amide, or related groups) that provides an active compound of the disclosure upon administration to a patient. Modifications like these can affect the biological activity of the compound, in some cases increasing activity relative to the parent compound. Pharmaceutically acceptable salts can be prepared in situ during the final isolation and purification of the compound, or by separately subjecting the purified compound to a purification step. The compound is prepared by reacting its free acid or free base form with a suitable base or acid, respectively. In some embodiments, a pharmaceutically acceptable salt may be superior to the corresponding free base or free acid because such a salt imparts greater stability or solubility to the molecule, thereby facilitating formulation into a dosage form. Basic compounds are generally capable of forming pharmaceutically acceptable acid addition salts by treatment with a suitable 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.Other non-limiting examples of pharmaceutically acceptable acid addition salts include hydrochloride, hydrobromide, nitrate, methylnitrate, sulfate, bisulfate, sulfamate, phosphate, acetate, glycolate, phenylacetate, propionate, butyrate, isobutyrate, valerate, maleate, hydroxymaleate, acrylate, fumarate, malate, tartrate, citrate, salicylate, p-aminosalicylate, glycolate, lactate, heptanoate, phthalate, oxalate, succinate, benzoate, o-acetoxybenzoate, chlorobenzene, Formate, methylbenzoate, dinitrobenzoate, hydroxybenzoate, methoxybenzoate, mandelate, tannate, formate, stearate, ascorbate, palmitate, oleate, pyruvate, pamoate, malonate, laurate, glutarate, glutamate, estolate, methanesulfonate, ethanesulfonate, 2-hydroxyethanesulfonate, benzenesulfonate, p-aminobenzenesulfonate, p-toluenesulfonate, naphthalene-2-sulfonate, edisylate, 2,5-dihydroxybenzoate and / or any combination thereof.

[0072] In some embodiments, the rimantadine salt (e.g., 2-S-rimantadine salt) is synthesized by mixing a slurry of the starting materials. In some embodiments, the salt is synthesized at 0°C. In some embodiments, the salt is synthesized at 5°C. In some embodiments, the salt is synthesized by a temperature cycling method. In some embodiments, the salt is synthesized by a slow evaporation method. In some embodiments, the salt is synthesized by solvation of 2-S-rimantadine with methanol, ethanol, chloroform, acetone, isopropyl or water or any combination thereof. In some embodiments, the salt is synthesized using an antisolvent such as acetone, n-heptane or methyl isobutyl ketone.

[0073] Pharmaceutically acceptable prodrugs refer to compounds that are metabolized (i.e., for example, hydrolyzed or oxidized) in a host to form compounds of the present disclosure. Typical examples of prodrugs include compounds having biologically labile protecting groups on the functional moieties of the active compound. Prodrugs include compounds that can be oxidized, reduced, aminized, deaminated, hydroxylated, dehydroxylated, hydrolyzed, dehydrated, alkylated, dealkylated, acylated, deacylated, phosphorylated, and / or dephosphorylated to produce active compounds.

[0074] In some embodiments, the methods described herein comprise administering pure 2-S rimantadine or pure 2-S-rimantadine or a pharmaceutically acceptable salt thereof.

[0075] In some embodiments, the PK properties of 2-S-rimantadine in salt form are improved. In some embodiments, the AUC of the salt of 2-S-rimantadine is increased compared to the non-salt form of 2-S-rimantadine.

[0076] In certain embodiments, a sample containing a solid form (e.g., crystal) of 2-S-rimantadine provided herein may be substantially free of other solid forms, such as an amorphous form and / or other crystalline forms. In certain embodiments, such a sample containing a crystalline form of a 2-S-rimantadine salt 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, such as an amorphous form, of a 2-S-rimantadine salt.

[0077] In some embodiments, XRPD is measured by a Malvern-Panalytical Aeris X-ray powder diffractometer. In some embodiments, differential scanning calorimetry is measured by a TA2500DSC instrument. In some embodiments, thermogravimetric analysis is measured by a TA Discovery 550 instrument.

[0078] In some embodiments of crystalline salts, the average crystal size is between about 5 μm and about 45 μm. In some embodiments of crystalline salts, the average crystal size is between about 5 and about 40 μm. In some embodiments of crystalline salts, the average crystal size is between about 5 and about 35 μm. In some embodiments of crystalline salts, the average crystal size is between about 5 and about 30 μm. In some embodiments of crystalline salts, the average crystal size is between about 5 and about 25 μm. In some embodiments of crystalline salts, the average crystal size is between about 5 and about 20 μm. In some embodiments of crystalline salts, the average crystal size is between about 5 and about 15 μm. In some embodiments of crystalline salts, the average crystal size is between about 5 and about 10 μm. In some embodiments of crystalline salts, the average crystal size is between about 1 and about 35 μm. In some embodiments of crystalline salts, the average crystal size is between about 1 and about 30 μm. In some embodiments of crystalline salts, the average crystal size is between about 1 and about 25 μm. In some embodiments of crystalline salts, the average crystal size is between about 1 and about 20 μm. In some embodiments of crystalline salts, the average crystal size is between about 1 and about 15 μm. In some embodiments of crystalline salts, the average crystal size is between about 1 and about 10 μm. In some embodiments of crystalline salts, the average crystal size is between about 1 and about 5 μm. In some embodiments of crystalline salts, the average crystal size is about 1 μm. In some embodiments of crystalline salts, the average crystal size is about 2 μm. In some embodiments of crystalline salts, the average crystal size is about 5 μm. In some embodiments of crystalline salts, the average crystal size is about 10 μm. In some embodiments of crystalline salts, the average crystal size is about 15 μm. In some embodiments of crystalline salts, the average crystal size is about 20 μm. In some embodiments of crystalline salts, the average crystal size is about 25 μm. In some embodiments of crystalline salts, the average crystal size is about 30 μm. In some embodiments of crystalline salts, the average crystal size is about 35 μm. In some embodiments of the crystalline salt, the average crystal size is about 40 μm. In some embodiments of the crystalline salt, the average crystal size is about 50 μm.

[0079] Pharmaceutical composition

[0080] Also provided herein are pharmaceutical compositions comprising pure 2-S rimantadine, pure 2-R rimantadine, or a pharmaceutically acceptable salt thereof (e.g., a salt of 2-S rimantadine (e.g., a fumarate, tartrate, galactarate, benzoate, besylate, or any combination thereof), as described herein. Any pharmaceutical composition described herein can be administered to a subject to treat a cancer as described herein.

[0081] Administration of 2-S rimantadine, pure 2-R rimantadine or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof can be carried out by any acceptable mode of administration, including but not limited to oral, subcutaneous, intravenous, intranasal, topical, transdermal, intraperitoneal, intramuscular, intrapulmonary, vaginal, rectal, otologic, neurootologic, intraocular, subconjunctival, anterior chamber, intravitreal, intraperitoneal, intrathecal, intravesical, intrapleural, wound wash, intraoral, intraperitoneal, intraarticular, intraauricular, intrabronchial, intracapsular, intrameningeal, inhalation, intratracheal or intrabronchial instillation, direct instillation into the lung cavity, intraspinal, intrasynovial, intrathoracic, thoracic wash, epidural, intratympanic, intracisternal, intravascular, intraventricular, intraosseous, infected bone wash, or as part of a mixture with a prosthetic device. In some embodiments, the method of administration comprises oral or parenteral administration.

[0082] 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, controlled release, and the like. 2-S rimantadine, pure 2-R rimantadine or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof may also be administered in a sustained-release or controlled-release dosage form, including long-acting injections, osmotic pumps, pills (tablets and / or capsules), transdermal (including electrotransport) patches, implants, and the like, for extended and / or timed, pulsed administration at a predetermined rate.

[0083] In some embodiments, the pharmaceutical composition is a tablet. In some embodiments, the pharmaceutical composition is a film-coated tablet.

[0084] 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, etc. Pharmaceutically acceptable excipients include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, self-emulsifying drug delivery systems (SEDDS) such as d-α-tocopherol, polyethylene glycol 1000, succinates, surfactants for pharmaceutical dosage forms such as Tween, poloxamer or other similar polymer delivery matrices, serum proteins such as human serum albumin, buffer substances such as phosphate, tris, glycine, sorbic acid, 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 silicon dioxide, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethyl cellulose, polyacrylates, waxes, polyethylene polyoxypropylene block polymers, and lanolin. Cyclodextrins may also be used to enhance the delivery of the compounds described herein.

[0085] In some embodiments, the pharmaceutical compositions described herein will take the form of a unit dosage form, such as a pill or tablet, so the composition may contain 2-S rimantadine, pure 2-R rimantadine or a pharmaceutically acceptable salt thereof (e.g., a salt of 2-S rimantadine), a diluent, such as lactose, sucrose, dicalcium phosphate, etc.; a lubricant, such as magnesium stearate, etc.; and a binder, such as starch, gum arabic, polyvinyl pyrrolidone, gelatin, cellulose, cellulose derivatives, etc. In another solid dosage form, the powder, pill, solution or suspension (e.g., in propylene carbonate, vegetable oil, PEG, poloxamer 124 or triglyceride) is encapsulated in a capsule (gelatin or cellulose-based capsule). Unit dosage forms are also contemplated, in which 2-S rimantadine, pure 2-R rimantadine or a pharmaceutically acceptable salt thereof, or another active agent provided herein is physically separated; for example, a capsule with particles (or tablets in a capsule) of each drug; a double-layer tablet; enteric coating or delayed release oral dosage forms are also contemplated.

[0086] In some embodiments, rimantadine or a pharmaceutically acceptable salt thereof is pegylated. In some embodiments, the pegylated rimantadine or a pharmaceutically acceptable salt thereof comprises a high molecular weight PEG. In some embodiments, the pegylated rimantadine or a pharmaceutically acceptable salt thereof comprises a low molecular weight PEG. In some embodiments, rimantadine or a pharmaceutically acceptable salt thereof is modified. In some embodiments, the modification is pegylation.

[0087] In some embodiments, the PEGylated rimantadine or a pharmaceutically acceptable salt thereof is PEGylated with a high molecular weight PEG. In some embodiments, the PEGylated rimantadine or a pharmaceutically acceptable salt thereof is PEGylated with a low molecular weight PEG. Thus, also provided herein is a method of treating cancer in a subject in need thereof, the method comprising administering to the subject an effective therapeutic amount of PEGylated rimantadine or a pharmaceutically acceptable salt thereof.

[0088] In some embodiments, the pharmaceutical composition comprises one or more excipients selected from the group consisting of hypromellose, magnesium stearate, microcrystalline cellulose, and sodium starch glycolate.

[0089] Liquid pharmaceutically administrable compositions can be prepared, for example, by dissolving, dispersing, etc., the compounds provided herein and optional pharmaceutical adjuvants in a carrier (e.g., water, saline, aqueous dextrose solution, glycerol, glycol, ethanol, etc.) to form a solution, colloid, liposome, emulsion, complex, coacervate or suspension. If necessary, the pharmaceutical composition may also contain a small amount of non-toxic auxiliary substances, such as wetting agents, emulsifiers, cosolvents, solubilizers, pH buffers, etc. (e.g., sodium acetate, sodium citrate, cyclodextrin derivatives, sorbitan monolaurate, triethanolamine acetate, triethanolamine oleate, etc.).

[0090] Dosage forms or compositions containing 2-S rimantadine, pure 2-R rimantadine, or a pharmaceutically acceptable salt thereof (e.g., a salt of 2-S rimantadine) as described herein can be prepared in the range of 0.005% to 100%, with the balance being made up of non-toxic carriers. The contemplated composition may contain 0.001%-100% of a compound provided herein, in one embodiment 0.1-95%, in another embodiment 75-85%, and in a further embodiment 20-80%. Actual methods of preparing such dosage forms are known to those skilled in the art, or will be apparent; for example, see Remington: The Science and Practice of Pharmacy, 22nd Edition (Pharmaceutical Press, London, UK. 2012).

[0091] The pharmaceutical composition herein may contain about 0.1-1000 mg of 2-S rimantadine, pure 2-R rimantadine or a pharmaceutically acceptable salt thereof (e.g., a salt of 2-S rimantadine) per dosage unit, such as tablets, capsules, suspensions, solutions, sachets for reconstitution, powders, injections, intravenous injections, suppositories, sublingual / buccal films, teaspoonfuls, etc. Pure 2-S rimantadine, pure 2-R rimantadine or a pharmaceutically acceptable salt thereof may be administered at a dose of about 0.01-300 mg / kg / day or any range therein, preferably about 0.5-50 mg / kg / day or any range therein. In some embodiments, the pharmaceutical compositions provided herein contain from about 25 mg to about 500 mg of a compound provided herein per dosage unit (e.g., from about 25 mg to about 400 mg, from about 25 mg to about 300 mg, from about 25 mg to about 250 mg, from about 25 mg to about 200 mg, from about 25 mg to about 150 mg, from about 25 mg to about 100 mg, from about 25 mg to about 75 mg, from about 50 mg to about 500 mg, from about 100 mg to about 500 mg, from about 150 mg to about 500 mg, from about 50 mg to about 500 mg, from about 100 mg to about 500 mg, from about 150 mg to about 500 mg, from about 200 mg to about 500 mg, from about 250 mg to about 500 mg, from about 300 mg to about 500 mg, from about 400 mg to about 500 mg, from about 50 mg to about 200 mg, from about 100 mg to about 250 mg, from 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 the compounds provided herein per unit dosage unit. However, the dosage can vary according to the needs of the patient, the severity of the disease being treated and the compound used. In some embodiments, the dosage is administered once a day (QD) or twice a day (BID).

[0092] 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

[0093] Treatment

[0094] Also provided herein is a method for treating cancer in a subject. In some embodiments, the method comprises administering to the subject an effective therapeutic amount of one or more pharmaceutical compositions described herein. In some embodiments, the pharmaceutical composition comprises enantiopure 2-S rimantadine or a pharmaceutically acceptable salt thereof (e.g., a salt of 2-S rimantadine). In some embodiments, the pharmaceutical composition comprises enantiopure 2-R rimantadine or a pharmaceutically acceptable salt thereof. 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. In some embodiments, pharmaceutically acceptable salts are hydrochlorides.

[0095] In some embodiments, the cancer is a sarcoma, carcinoma, melanoma, lymphoma or leukemia. Non-limiting examples of sarcomas include: osteosarcoma (e.g., angiosarcoma, fibrosarcoma, liposarcoma, chondrosarcoma, chordoma, Ewing's sarcoma, giant cell tumor, osteosarcoma, rhabdomyosarcoma and synovial sarcoma) and soft tissue sarcoma (e.g., fibrosarcoma, gastrointestinal stromal tumor (GIST), Kaposi's sarcoma, leiomyosarcoma, liposarcoma, rhabdomyosarcoma and soft tissue Ewing's sarcoma). Non-limiting examples of cancer 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 lymphomas (e.g., B-cell lymphomas, T-cell lymphomas, Burkitt's lymphomas, follicular lymphomas, mantle cell lymphomas, primary mediastinal B-cell lymphomas, small lymphocytic lymphomas, Waldenstrom's macroglobulinemia) and Hodgkin lymphomas (e.g., lymphocyte-depleted Hodgkin's disease, lymphocyte-rich Hodgkin's disease, mixed cell type Hodgkin's lymphoma, nodular lymphocyte-predominant Hodgkin's disease, and nodular sclerosis 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 myeloid leukemia, myeloproliferative neoplasms, and systemic mastocytosis.

[0096] 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.

[0097] 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 a squamous cell carcinoma. In some embodiments, the squamous cell carcinoma is a head and neck squamous cell carcinoma. In some embodiments, the cancer is a hepatocellular carcinoma.

[0098] In some embodiments, the cancer is selected from head and neck cancer, breast cancer, and melanoma.

[0099] 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) as described herein can be used to treat a subject's hepatitis B virus (HBV)-related cancer. As used herein, "HBV-related cancer" is a cancer in which one or more cancer cells express at least one HBV protein (e.g., see 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 may express HBV oncoproteins. In some embodiments, the HBV-related cancer is liver cancer (e.g., hepatocellular carcinoma). In some embodiments, the HBV-related cancer is cervical cancer.

[0100] 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) as described herein can be used to treat human papillomavirus (HPV)-related cancers in a subject. As used herein, "HPV-related cancers" are cancers in which one or more cancer cells express at least one HPV protein. For example, one or more cancer cells may express HPV oncoproteins. Human papillomavirus (HPV) can cause malignant transformation by, for example, targeting key tumor suppressor genes p53 and Rb (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 escape immune responses (see, e.g., Senba. Oncol Rev. 5 October 5, 2012, 6 (2): e17). For example, HPV genes and proteins can target antigen processing and antigen presentation required for effective adaptive immune responses (see, e.g., Senba. Oncol Rev. 2012 Oct 5; 6(2): e17; and O'Brien and Virus Res. 2002 Sep; 88(l-2): 103-17). There are many HPV oncoproteins, including but not limited to HPV16 E5, E6, and E7. For example, HPV E5 is a protein reported to have multiple functions, including regulation of tumor cell differentiation and apoptosis, regulation of 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 can affect antigen processing and presentation.

[0101] 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 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 HPV16 E5. In some embodiments, the subject suffers from a cancer selected from the group consisting of head and neck cancer, mucosal squamous cell carcinoma, skin squamous cell carcinoma, cervical cancer, vaginal cancer, vulvar cancer, penile cancer, and anal cancer.

[0102] In some embodiments, the cancer is an HPV-related cancer. In some embodiments, the HPV-related cancer is HPV-related head and neck squamous cell carcinoma (HNSCC).

[0103] 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) as described herein can be used to treat human papillomavirus precancerous lesions, such as lesions associated with: hyperplastic verrucous leukoplakia (PV1), oral leukoplakia, anti-smoker's nicotine palatitis, oral erythema, laryngeal keratosis, actinic leukoplakia, plummer-vinson smooth red tongue, smokeless tobacco keratosis, syndromic oral submucous fibrosis, erythroplakia, granular leukoplakia, oral lichen planus (erosive form), smooth thin leukoplakia, nicotine stomatitis and pouch keratosis, cervix (cervical dysplasia); and penile intraepithelial neoplasia (PelN lesions). 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) are associated with benign lesions and 12 HPV types (2, 3, 6, 11, 13, 16, 18, 31, 33, 35, 52, and 57) are associated with malignant lesions. Approximately 40% of invasive penile cancers can be attributed to HPV 16, 18, and 6 / 11. The HPV types associated with cervical carcinogenicity are 15 high-risk types (HPV 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68, 73, and 82) and 3 "possibly high-risk types" (HPV 26, 53, and 66).

[0104] Detection of HPV is known in the art, for example, see Coultlee, F., et al., 2005, Can J Infect Dis Med Microbiol 16(2):83-91; tzwc HPV 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 probe (BIOCARE MEDICAL, Pacheo, CA).

[0105] 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 an effective therapeutic amount of at least one additional therapeutic agent selected from one or more additional anti-cancer therapies or therapeutic agents (e.g., chemotherapeutic agents). It is common practice in medical oncology to treat subjects with cancer using a combination of different forms of therapy. In addition to 2-S rimantadine or a pharmaceutically acceptable salt thereof described herein, these other forms of combined therapy or treatment may include, for example, surgery, radiation therapy, and other 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.

[0106] Non-limiting examples of additional anticancer agents include: carboplatin, cisplatin, gemcitabine, methotrexate, paclitaxel, pemetrexed, lomustine, temozolomide, and dacarbazine.

[0107] In some embodiments, the additional anticancer agent is an immunotherapy. Many types of immunotherapy can be used in combination with pure 2-S rimantadine or pure 2-R rimantadine or a pharmaceutically acceptable salt thereof as described herein. Non-limiting examples of immunotherapy include: immune checkpoint inhibitors, antibody therapy, cellular immunotherapy, antibody-drug conjugates, cytokine therapy, mRNA-based immunotherapy, and cancer vaccines.

[0108] In some embodiments, the immunotherapy is one or more immune checkpoint inhibitors. In some embodiments, the immune checkpoint inhibitor targets one or more of the following: 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: ipilimumab, nivolumab, pembrolizumab, atezolizumab, avelumab, durvalumab, cemiplizumab, tremelimumab, and combinations thereof.

[0109] 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. or tremelimumab (CP-675,206). In some embodiments, the PD-1 inhibitor is pembrolizumab Cimiprilimab Nivolumab In some embodiments, the PD-L1 inhibitor is atezolizumab Avelumab or durvalumab (IMFINZI TM ).

[0110] In some embodiments, the antibody therapy comprises bevacizumab (MVASTI TM , ), trastuzumab Avelumab Rituximab (MABTHERA TM , ), edrecolomab (Panorex), daratumumab Olaratumab (LARTRUVO TM ), Ofatumumab Alemtuzumab Cetuximab Oregovomab, pembrolizumab Denuximab Obinutuzumab Tremelimumab (CP-675,206), Ramucirumab Ubliximab (TG-1101), panitumumab Elotuzumab (EMPLICITI TM ), Avelumab Nexituzumab (PORTRAZZA TM ), Cetuzumab (UC-961), Ibrutinib Ixatuximab (SAR650984), nimotuzumab, fresoluzumab (GC1008), lirelumab (INN), moglizumab Fituzumab (AV-299), denosumab ganitumab, urelumab, pidilizumab, or amatuximab.

[0111] In some embodiments, the immunotherapy is cellular immunotherapy (eg, adoptive T cell therapy, dendritic cell therapy, natural killer cell therapy).

[0112] In some embodiments, the immunotherapy is an antibody-drug conjugate. In some embodiments, the antibody-drug conjugate is gemtuzumab ozogamicin (MYLOTARG TM ), Inotuzumab Ozomibin Brentuximab vedotin Trastuzumab emtansine (T-DM1; ), Moxifloxacin, Pasutomucil Plotuzumab vedotin-PIIQ Mituximab soravtansine (IMGN853) or anetumab ravtansine.

[0113] 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, granulocyte colony stimulating factor (G-CSF) therapy, interleukin 12 (IL-12) therapy, or erythropoietin-α (EPO) therapy. In some embodiments, IL-2 therapy is aldesleukin In some embodiments, the IFNα therapy is interferon α-2b (e.g., ) or interferon alfa-2a (e.g., In some embodiments, the G-CSF therapy is filgrastim.

[0114] In some embodiments, the immunotherapy is an mRNA-based immunotherapy. In some embodiments, the mRNA-based immunotherapy is CV9104 (see, e.g., Rausch et al. (2014) Human Vaccin Immunother 10(11): 3146-52; and Kubler et al. (2015) J. Immunother Cancer 3: 26). See also Pardi et al. Nat Rev Drug Disc Ov. 2013; April 2018; 17(4): 261-279, which is incorporated herein by reference in its entirety.

[0115] In some embodiments, the method comprises subjecting the subject to radiation therapy, surgery, or a combination thereof. For example, the surgery may be open surgery or minimally invasive surgery.

[0116] In some embodiments, the subject is refractory to standard therapy (e.g., standard of care). In some embodiments, the subject does not have standard therapy options. In some embodiments, the subject relapses or progresses after standard therapy. In some embodiments, the methods provided herein can be used to treat locally advanced or metastatic solid tumors that are refractory to standard therapy. For example, HPV-related cancers can be difficult to treat with immune checkpoint inhibitors (such as those described herein).

[0117] In some embodiments, the subject has a cancer that is refractory or intolerant to standard therapy (e.g., administration of chemotherapeutic agents, immunotherapy, or radiation). In some embodiments, the subject has a cancer (e.g., locally advanced or metastatic tumor) that is refractory or intolerant to prior therapy (e.g., administration of chemotherapeutic agents, immunotherapy (e.g., immune checkpoint inhibitors), or radiation). In some embodiments, the cancer that is refractory or intolerant to standard therapy is an HPV-related cancer. In some embodiments, the subject has a cancer (e.g., locally advanced or metastatic tumor) for which there is no standard therapy.

[0118] In some embodiments, the subject has undergone prior therapy. In some embodiments, prior to treatment with 2-S rimantadine or a pharmaceutically acceptable salt thereof, the subject has been treated with platinum-based chemotherapy, immune checkpoint inhibitors (e.g., PD-1 / PDL1 immunotherapy), radiotherapy, or a combination thereof.

[0119] The optimal dose of pure 2-S rimantadine or pure 2-R rimantadine or a pharmaceutically acceptable salt thereof (e.g., a salt of 2-S rimantadine) to be administered to a subject can be determined by one skilled in the art and will vary with the mode of administration, the strength of the formulation, the mode of administration, and the progression of the disease condition. In some embodiments, a dose of about 0.01 mg to 10,000 mg of 2-S rimantadine or a pharmaceutically acceptable salt thereof per 25 adults per day can be administered to a subject. 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 dose 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 / kg to about 1000 mg / kg 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.

[0120] In one aspect, the compounds disclosed herein are used to prepare a medicament for treating a disease or condition described herein. In addition, a method for treating any disease or condition described herein in a subject in need of such treatment involves administering to the subject a pharmaceutical composition in an effective therapeutic amount, the pharmaceutical composition comprising at least one compound disclosed herein or a pharmaceutically acceptable salt, active metabolite, prodrug or solvate thereof.

[0121] In certain embodiments, compositions containing compounds disclosed herein are used for preventive and / or therapeutic treatment. In certain therapeutic applications, the composition is applied to a patient who has already suffered from a disease or condition in an amount sufficient to cure or at least partially prevent at least one symptom of the disease or condition. The amount effective for this purpose depends on the severity and course of the disease or condition, previous treatment, the patient's health, body weight and response to the drug, and the judgment of the treating physician. The effective therapeutic amount is optionally determined by methods including but not limited to dose escalation clinical trials.

[0122] In prophylactic applications, compositions containing compounds disclosed herein are administered to a patient susceptible to or otherwise at risk of a particular disease, disorder or condition.

[0123] In certain embodiments, the dose of an administered drug may be temporarily reduced or temporarily suspended for a certain length of time (ie, a "drug holiday").

[0124] HPV testing methods

[0125] Another aspect of the present disclosure includes a method for treating cancer in a subject, the method comprising detecting cancer cells expressing HPV proteins in a sample from the subject, and then administering to the subject an effective therapeutic 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 HPV proteins or functionally equivalent variants thereof. In some embodiments, wherein the presence of HPV proteins or functionally equivalent variants thereof is detected in a sample from the subject, the expression level of the HPV proteins 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., chemotherapeutic agents).

[0126] 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 propensity to suffer from the disease associated with the presence of an HPV protein, or for determining the stage or severity of the disease associated with the presence of an HPV protein in a subject, or for monitoring the effect of a therapy administered to a subject suffering from the disease associated with the presence of an HPV protein, comprising quantifying the expression level of an HPV protein or its functionally equivalent variant in a biological sample from the subject, wherein an increased expression of a gene encoding an HPV protein or its functionally equivalent variant relative 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 an HPV protein. Or the subject is more susceptible to a disease associated with the presence of an HPV protein or is unresponsive to a therapy 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).

[0127] Therefore, as used herein, the term "functionally equivalent variant" also includes any functionally equivalent fragment of the marker protein. The term "fragment" refers to a peptide comprising a portion of the marker protein. In this case, a functionally equivalent fragment is a peptide or protein comprising a portion of the marker protein and having substantially the same function as the protein. "Marker protein" preferably refers to an HPV protein, but is not limited thereto.

[0128] As will be appreciated by those skilled in the art, detection may not be correct for 100% of the subjects, although it is preferred. However, the term requires the ability to identify a statistically significant portion of the subjects as having a sufficient amount of the target protein so that the subject suffers from a disease associated with the presence of the target protein or has a susceptibility to it. Those skilled in the art can determine whether a portion is statistically significant by simply using one or more well-known statistical evaluation tools (e.g., determination of confidence intervals, determination of p values, Student's t-test, Mann-Whitney test, etc.). For details, see 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%, at least 95%. The p value is preferably 0.2, 0.1, 0.05.

[0129] As used herein, the term "susceptibility" means that the subject has not yet developed any symptoms or other diagnostic criteria for the disease or the above-mentioned disease, but will develop the disease in the future with a certain probability. The probability will be significantly different from the statistical probability of the onset of the disease associated with the presence of HPV proteins. It is preferably diagnosed that the probability of developing a disease associated with the presence of HPV proteins is at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or 100% of the susceptibility. The diagnosis of susceptibility can sometimes be referred to as a prognosis or prediction of the probability of a subject developing a disease.

[0130] In the context of the present disclosure, a "control sample" is understood to be a reference sample that is used to determine changes in the expression levels of genes and proteins used in the present disclosure. In one embodiment, a reference value is obtained from a provided signal using a tissue sample obtained from a healthy individual. Preferably, the sample is taken from the same tissue of several healthy individuals and combined so that the amount of the polypeptide in the sample reflects the average value of the molecule in the population.

[0131] Therefore, in a specific embodiment of the present disclosure, the expression level of HPV protein can be quantified. In some embodiments, the HPV protein is HPV16 E5.

[0132] As will be appreciated by those skilled in the art, the expression level of a protein can be quantified by any conventional method. By way of non-limiting illustration, the level of a protein can be quantified, for example, by using an antibody having the ability to bind to the protein (or a fragment thereof containing an antigenic determinant) and then quantifying the complex formed. The antibodies used in these assays may be labeled or may not be labeled. Illustrative examples of markers that can be used include radioisotopes, enzymes, fluorophores, chemiluminescent reagents, enzyme substrates or cofactors, enzyme inhibitors, particles, dyes, and the like. There are a variety of known assays that can be used in the present disclosure, which use unlabeled antibodies (primary antibodies) and labeled antibodies (secondary antibodies); these techniques include Western blots, ELISA (enzyme-linked immunosorbent assay), RIA (radioimmunoassay), competitive EIA (competitive enzyme immunoassay), DAS-ELISA (double antibody sandwich ELISA), immunocytochemistry and immunohistochemistry techniques, techniques based on biochips or microarrays of proteins including specific antibodies, or assays based on colloidal precipitation forms such as test paper. In another specific embodiment, the quantification of protein levels is performed by immunoassay methods, such as Western blots, immunohistochemistry, or ELISA. In some embodiments, the immunoassay comprises an antibody specific for HPV16 E5.

[0133] Likewise, the detection methods disclosed herein can be applied to any disease associated with the presence of the HPV proteins defined above. In preferred embodiments, the disease associated with the presence of the HPV proteins is cancer, preferably a cancer with high levels of HPV proteins. In some embodiments, the HPV protein is HPV16 E5.

[0134] Putting the methods of the present disclosure into practice includes obtaining a biological sample from a subject to be studied. Illustrative, non-limiting examples of the sample include different types of biological fluids, such as blood, serum, plasma, cerebrospinal fluid, peritoneal fluid, feces, urine, and saliva, as well as tissue samples. The biological fluid sample can be obtained by any conventional method, such as a tissue sample; for example, the tissue sample can be a biopsy sample obtained by surgical resection.

[0135] In another aspect, the present disclosure relates to a kit comprising a reagent for quantifying the expression level of an HPV protein or a functionally equivalent variant thereof, for diagnosing cancer in a subject or for determining a subject's propensity to suffer from the cancer, or for determining the stage or severity of the cancer in a subject, or for monitoring the effect of a therapy administered to a subject suffering from the cancer, wherein if the reagent detects an increased expression of the gene or the protein or a functionally equivalent variant thereof relative to a control sample, the subject may suffer from a disease associated with the presence of the HPV protein, or exhibit a greater propensity to suffer from the disease associated with the presence of the HPV protein, or exhibit a greater severity of the disease, or the administered therapy is 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).

[0136] The present disclosure also relates to uses of the kit.

[0137] All terms and expressions used in the definition of the use of the kit have been described above and explained with respect to other inventive aspects and specific embodiments of the present disclosure, and also apply to the use of the kit described herein.

[0138] Methods for designing customized therapies and selecting patients who may benefit from administration of 2-S rimantadine (e.g., a salt of 2-S rimantadine) or 2-R rimantadine

[0139] 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 HPV proteins, comprising:

[0140] (a) quantifying the expression level of HPV protein in the patient, and

[0141] (b) comparing the expression level to a control level,

[0142] If the expression level of HPV protein in the patient is greater than the control value, an effective therapeutic 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) is administered to the patient.

[0143] 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 (eg, chemotherapeutic agents) is administered to the patient.

[0144] 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 HPV proteins 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), the method comprising:

[0145] a) quantifying the expression level of HPV protein in said patient, and

[0146] b) comparing the expression level to a control level,

[0147] If the expression level of HPV protein in the patient is greater than the control value, the patient is selected to receive an effective therapeutic 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.

[0148] 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 (eg, chemotherapeutic agents) is administered to the patient.

[0149] For further description of the compositions and methods disclosed herein, see, for example, US Provisional Patent Application No. 63 / 150,027, the entire contents of which are incorporated herein by reference.

[0150] Example

[0151] The present disclosure is further illustrated by the following examples, which should not be construed as limiting in any way. The experimental procedures that produce the data shown are discussed in more detail below. The present disclosure has been described in an illustrative manner, and it should be understood that the terms used are intended to have a descriptive nature rather than a limitation.

[0152] General experimental, instrumental, and methodological details

[0153] X-ray powder diffraction (XRPD)

[0154] For XRPD analysis, a Malverm-Panalytical Aeris X-ray powder diffractometer was used. The XRPD parameters used were as follows; All samples were placed directly on the sample holder without external forces. The detector used was a PIXcel detector. The scanning mode used was continuous scanning. 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 scanning range was 3-40. The slit was a fixed 1 / 8° divergence slit. The samples were measured on a zero background holder and calibrated using a Panalytical Si reference standard disk.

[0155] Differential Scanning Calorimetry (DSC) and Thermogravimetric Analysis (TGA)

[0156] TGA data were collected using a TADiscovery 550 using a nickel calibration standard and a platinum sample pan. For mass measurements, the sample was placed in the measurement chamber and equilibrated in a nitrogen flow.

[0157] DSC data were collected using a TA2500DSC instrument and calibrated using indium standards. The samples were weighed, purged with nitrogen, and heated while analyzing the mass change.

[0158] Example 1. NR1 / NR2A and NR1 / NR2B Determination

[0159] ScreenPatch

[0160] NR1 / NR2A are ionotropic receptors encoded by human GRIN1 / GRIN2A genes and expressed in HEK293 cells.

[0161] Expression of NR1 / NR2B ionotropic receptors encoded by human GRIN1 / GRIN2B genes in HEK293 cells

[0162] preparation

[0163] Solutions of 2-S rimantadine, 2-R rimantadine, racemic rimantadine, and rimantadine were prepared daily and prepared by diluting the stock solutions into the appropriate HEPES-buffered saline (HB-PS) solutions. All test and control solutions contained 0.6% DMSO because 0.6% DMSO did not affect channel currents. Test article preparations were sonicated (Model 2510 / 5510, Branson Ultrasonics, Danbury, CT) at room temperature for at least 20 minutes to facilitate dissolution.

[0164] The test article effects were evaluated in an 8-point concentration-response format (4 replicate wells / concentration). All test and control solutions contained 0.6% DMSO. Test article preparations were loaded into 384-well compound plates using an automated liquid handling system (Assist Plus, INTEGRA).

[0165] Positive control treatment group

[0166] The stock solution of the positive control preparation was prepared in batches, aliquoted for single use, stored frozen, and used within six months. The positive control test solution was 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 rimantadine was used as a reference antagonist in this study.

[0167] Tests and Concentrations:

[0168] The test substances were evaluated for their effects on ion channel function. The test concentrations are shown in Table 1 below.

[0169] Table 1. Test product concentrations

[0170]

[0171] Cloning test system

[0172] Maintain cells in a tissue culture incubator. Keep stock solutions in cryogenic storage. Cells for electrophysiology are plated in plastic culture dishes.

[0173] HEK293 culture procedures

[0174] HEK293 cells were transfected with 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 plasmid. Selection pressure was maintained with 0418 and Zeocin in the culture medium. Cells were cultured in Dulbecco's modified Eagle medium / nutrient mixture F-12 (D-MEM / F-12) supplemented with 10% fetal bovine serum, 100U / mL penicillin O sodium, 100μg / mL streptomycin sulfate, 100μg / mL bleomycin, 5μg / mL blasticidin and 500μg / mL O418.

[0175] ScreenPatch test method:

[0176] All experiments were performed at ambient temperature. The target specificity testing procedure is described below. The following procedure applies to all ScreenPatch assays.

[0177] Before testing, cells in the culture dish were washed twice with HBSS solution. TM Beforehand, cells were washed in HB-PS containing 6 mM CaCl2 to improve sealing.

[0178] Test articles were evaluated in an 8-point concentration-response format (4 replicate wells / concentration, see Table 1). Previous results showed that 0.6% DMSO did not affect channel currents; therefore, all test and control solutions contained 0.6% DMSO unless otherwise stated. Test article preparations were loaded into 384-well compound plates and placed on the IonWorks Barracuda TM Plate hole.

[0179] The positive control preparation was prepared in batches, aliquoted for individual use, stored frozen, and used within six months. The positive control test solution was prepared fresh daily. The final DMSO concentration was 0.6%.

[0180] Test articles at 2X concentration (as specified in Table 2) were pre-applied 2 minutes prior to application of L-glutamate (Sigma-Aldrich) / glycine (Sigma-Aldrich) mixed with 1X concentration of test article (5 μM L-glutamate and 50 μM glycine).

[0181] To monitor the sensitivity of the assay, the antagonist positive control, rimantadine hydrochloride (Sigma-Aldrich), was applied at 8 half-log concentrations (range 0.3-1000 μM); n=4, where n=number of replicates per concentration. The agonist positive control (L-glutamate) was applied at eight (8) concentrations (0.03-100 μM: n=4, where n=number of replicates) together with 50 μM glycine.

[0182] The inhibitory effect of the compound on the channel was calculated as follows:

[0183]

[0184] Where baseline is the response at low concentration of the test article. Max is the maximum response at high concentration, and xhalf is the EC50 or IC 50 , i.e., the concentration of the test substance that produces half-maximal activation or inhibition, and the rate is the Hill coefficient. Nonlinear least squares fits were performed assuming a simple binding model. Fits were weighted by standard deviations, if appropriate. No assumptions were made about the fit parameters; the fit parameters were determined by the algorithm.

[0185] Nonlinear least squares fitting was performed using the XLfit add-in for Excel 2016 (Boston, MA).

[0186] Two types of measurements are used to assess the effects of compounds:

[0187] 1. Peak current amplitude (PCA) measurement at the current maximum.

[0188] 2. Steady-state current amplitude (SSC) was measured as the average value between 4 and 5 seconds after receptor stimulation with agonist.

[0189] program

[0190] Electrophysiological procedures:

[0191] a) Intracellular solution (mM): 50 mM CsCl, 90 mM CsF, 2 mM MgCl2, 5 mM EGTA, 10 mM HEPES. Adjust to pH 7.2 with CsOH. Prepare this solution in batches and store refrigerated. In preparation for the recording session, load the intracellular solution into the intracellular compartment of the PPC planar electrode.

[0192] b) Extracellular solution, HB-PS (composition in mM): NaCl, 137; KCl, 1.0; CaCl2, 5; HEPES, 10; glucose, 10; pH adjusted to 7.4 with NaOH (refrigerated until use).

[0193] c) Holding potential: -70 mV, potential during application of the test substance: -70 mV.

[0194] Recording Procedure:

[0195] a) Load the extracellular buffer into the wells of the PPC plate (11 μL per well). Pipette the cell suspension into the wells of the PPC planar electrode (9 μL per well).

[0196] b) The whole-cell recording configuration was established by patch perforation, where membrane currents were recorded by an onboard patch clamp amplifier.

[0197] c) Two recordings (scans) were performed. The first scan was during the addition of the test substance at 2X concentration to detect potential agonist effects and pre-incubation of the test substance with the cells (2 minutes). Second, during agonist stimulation of the receptor (5 μM L-glutamate and 50 μM glycine) with co-application of the test substance at 1X concentration to detect antagonist effects of the test substance.

[0198] Test article administration: Administration consisted of adding 20 μL of IX concentrated test article solution and 10 μL / s of agonist (2 sec total application time).

[0199] Positive control agonists: 0.03-100 μM L-glutamate (8 concentration dose response, semi-log scale) and 50 μM glycine

[0200] Positive control antagonist: 0.3-1000 μM rimantadine co-applied with 5 μM glutamate and 50 μM glycine (8 concentration-response, semi-log scale).

[0201] The evaluation of the effect is based on peak current measurement.

[0202] result

[0203] Using HTS electrophysiology-based methods, IonWorkBarracuda TM (IWB) Four (4) compounds were examined for their agonist and antagonist properties. A two-dose regimen was employed.

[0204] Agonist form : Potential agonist effects of the test article and the positive control antagonist rimantadine were examined during the first administration. Neither the test article nor rimantadine produced significant activation of NMDA receptors (data not shown).

[0205] Antagonist form: After stimulating the receptors with 5 μM L-glutamate and 50 μM glycine, the antagonist activity of the test articles was examined during the second application of the compound. It was found that all four test articles produced significant concentration-dependent inhibition of NMDA receptor function. In order to obtain the inhibition of the open channel blocking type, the peak and steady-state current amplitudes (between the 4th and 5th seconds after the agonist application) were measured (PCA and SSC, respectively). Table 2 shows the IC values ​​of the compounds for NR1 / NR2A and NR1 / NR2B receptors using these two types of measurements. 50 The average value of .

[0206] Rimantadine inhibits NR1 / NR2A receptors, with peak current amplitude and steady-state current amplitude being IC 50_PCA =97.8μM and IC 50_SSC =48.9μM. NR1 / NR2B receptors were inhibited by rimantadine, with peak current amplitude and steady-state current amplitude being IC 50_PCA =22.0μM and IC 50_SSC = 17.9 μM. The leftward shift in rimantadine potency in steady-state current measurements suggests, at least in part, an open channel blockade inhibition mechanism at NR1 / NR2 AMNDA receptors.

[0207] Table 2. Inhibition IC produced by test substances and reference antagonist rimantadine 50 Summary.

[0208]

[0209] Rimantadine inhibits NR1 / NR2A receptors, with peak current amplitude and steady-state current amplitude being IC 50_PCA =97.8μM and IC 50_SSC =48.9μM. NR1 / NR2B receptors were inhibited by rimantadine, with peak current amplitude and steady-state current amplitude being IC 50_PCA =22.0μM and IC 50_SSC = 17.9 μM. The leftward shift in rimantadine potency in steady-state current measurements suggests, at least in part, an open channel blockade inhibition mechanism at NR1 / NR2 AMNDA receptors.

[0210] The results of these measurements are Figure 1 Further examples are given in AD.

[0211] Example 2. Effects of pure 2-S rimantadine or pure 2-R rimantadine in mouse cancer models.

[0212] method

[0213] Cell lines

[0214] AT-84-E7 and B16-OVA were grown in RPMI 1640 containing 10% FBS, 1% L-flutamide, 1% penicillin / streptomycin, 1% sodium pyruvate, and 200 μg / ml G418. DC2.4, RAW264.7, B3Z, 4T1, B16, and MC38 were grown in RPMI 164020 containing 10% FBS, 1% L-glutamine, 1% penicillin / streptomycin, and 1% sodium pyruvate. HEK293T were grown in DMEM containing 10% FBS, 1% L-glutamine, and 1% penicillin / streptomycin. 4MOSC1 was cultured in collagen-coated culture dishes with KSFM medium (Invitrogen, Carlsbad, CA) supplemented with 1% penicillin / streptomycin, 5 ng / ml EGF (Invitrogen), and 2×10-11 M cholera toxin (Sigma, St. Louis, MO) (27). CAL-27, CAL-33, and SCC-47 were grown in DMEM containing 10% FBS, 221% L-glutamine, and 1% penicillin / streptomycin. Routine monitoring for mycoplasma contamination was performed using the MycoAlert PLUS detection kit (Lonza, Basel, Switzerland). All cell lines were used within ten passages after thawing.

[0215] Mouse studies

[0216] Mice were injected subcutaneously with 1.0 to 5.0 × 10 5 AT-84-E7, 1.5×10 5 B16-OVA, or 5.0×10 5 4T1 cells were resuspended in 100 μL PBS on the right flank. For the orthotopic model, 1.0×10 5 AT-84-E7 or 1.0×10 6 4MOSC1 were injected into the tongue. Tumor diameter was measured every 2 to 3 days with electronic calipers and reported as volume using the formula; tumor volume (mm 3) = (length x width2) / 2. Once tumors were palpable, mice were treated with 200 μg of anti-PDL1 antibody (BioXcell, West Lebanon, NH) by IP injection every 3 days for a total of three or four injections per mouse, or 5 mice were treated with 10-20 mg / kg body weight of pure 2-S rimantadine or pure 2-R rimantadine by IP injection every day for 7 days. For adoptive transfer experiments, single cell suspensions from spleens of OT-1 mice were first cultured in culture medium containing 10 ng / ml OVASIINFEKL peptide (InvivoGen, San Diego, CA) and 2 ng / ml recombinant IL-2 (PeproTech, Rocky Hill, NJ) for several days, and then 4.0×10 6 The cells were injected intravenously into B16-OVA-bearing mice.

[0217] Flow cytometry

[0218] Single cell suspensions were prepared from lungs, liver, tumor draining lymph nodes and tumors by mechanical dissociation and filtered using a 70 μm filter. Before mechanical dissociation, AT-84-E7 and MOC2 tumors were incubated at 37 ° C for 1 hour in collagenase D (Roche, Basel, Switzerland). Density gradient centrifugation was performed on a 40% / 80% Percoll (GE Healthcare, Chicago, IL) gradient from a single cell suspension of tumor. After obtaining a single cell suspension, 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)]. 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 on FlowJo (FlowJo, LLC, Ashland, OR). For each antibody, the following clones were used: 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 (L5), CD31 (MEC13.3), NK-T / NK cell antigen (U5A2-13), 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).

[0219] Cell cycle and proliferation assays

[0220] After staining 1 cell with BrdU-APC and 7-AAD using the BD Pharmingen BrdU Flow kit, the cell cycle progression was analyzed based on BrdU incorporation. (BD, Franklin Lakes, NJ). Cells were analyzed using flow cytometry. Cell proliferation was assessed by using MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide]. First, cells were seeded in 96-well plates and cultured for 2-3 days. Next, the culture medium was replaced with fresh culture medium containing 0.5 mg / ml MTT (Sigma), and the plates were incubated at 37°C for 4 hours. Then, the purple formazan crystals were dissolved in lysis buffer (4 mM HCl and 0.1% NP-40 in isopropanol) and the absorbance was recorded at a wavelength of 570 nm on a TECAN infiniteM200 microplate reader (Tecan, Mannedorf, Switzerland), with the absorbance at 650 nm as a reference.

[0221] B3Z activation assay

[0222] B16-OVA cells were seeded into 96-well plates and treated with 100 μM pure 2-R rimantadine, pure 2-R rimantadine or racemic rimantadine for 24 hours, and then B3Z cells were added. After 24 hours of co-culture, the medium was removed and 100 μL of lysis buffer [0.155 mM chlorophenol red PD-pyranogalactoside (CPRG) (Roche), 0.125% Nonidet P-40 substitute (EMD Calbiochem) and 9 mM MgCl2 (Sigma) in PBS solution] was added. After incubation at 37°C for 4 hours, the absorbance at 570 nm was measured on a TECANinfinite M200 microplate reader.

[0223] Reverse transcription and quantitative PCR

[0224] Total RNA was extracted using TRIzol reagent (Invitrogen) and reverse transcribed using the qScript cDNA synthesis kit (Quanta BioSciences, Beverly, MA) according to the manufacturer's instructions. Quantitative PCR analysis was performed using KAPA SYBR 1FAST (KAPA Biosystems, Wilmington, MA) on a 7900HT fast real-time PCR system (Applied Biosystems, Foster City, CA).

[0225] result

[0226] Pure 2-S rimantadine and pure 2-R rimantadine alone had antitumor activity and significantly inhibited tumor growth. Six mice were inoculated with 5×10 5 AT84-E7 / E5 tumor cells were injected intraperitoneally (IP) once a day with 10 mg / kg body weight of pure 2-S rimantadine or pure 2-R rimantadine for a total of 7 injections starting on day 8. Tumor volume was measured during the experiment. Mice receiving pure 2-S rimantadine or pure 2-R rimantadine showed a statistically significant decrease in tumor size compared to the control group. Six mice were inoculated with 1.5×10 5 B16-OVA tumor cells were treated with 10 mg / kg body weight of pure 2-S rimantadine or pure 2-R rimantadine IP injections once daily for a total of 7 injections starting on day 10. Tumor volume was measured during the experiment. Mice receiving pure 2-S rimantadine or pure 2-R rimantadine showed a statistically significant reduction in tumor size compared to the control group. The experiment was repeated three times with similar results. Five mice were inoculated with 5 × 10 5 4T1 tumor cells were treated with IP injections of 10 mg / kg body weight of pure 2-S rimantadine or pure 2-R rimantadine once daily for a total of 7 injections starting on day 6. Tumor volume was measured during the experiment. Mice receiving 2-S rimantadine or pure 2-R rimantadine showed a statistically significant reduction in tumor size compared to the control group. The antitumor effect of pure 2-S rimantadine or pure 2-R rimantadine was reduced in AT-84-E7 tumors that do not express E5. A significant increase in the surface expression of MHC was observed in multiple cell lines. Treatment with pure 2-S rimantadine or pure 2-R rimantadine restored cell surface expression of MHC I on E5-positive AT-84-E7.

[0227] 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 co-cultured with B3Z cells responding to the OVA SINNFKL peptide. Treatment of B16-OVA cells with pure 2-S rimantadine or pure 2-R rimantadine resulted in a significant 3-fold increase in B3Z cell recognition of this model tumor antigen. Pure 2-S rimantadine or pure 2-R rimantadine with anti-PDL1 immunotherapy resulted in a significant improvement in the survival of mice bearing B16-OVA tumors.

[0228] The ability of pure 2-S rimantadine or pure 2-R rimantadine to increase MHC expression on antigen-presenting cells was tested using the RAW 264.7 cell line, and significant increases in surface expression of both MHC class I and MHC class II were observed. These findings suggest that pure 2-S rimantadine or pure 2-R rimantadine has novel antitumor activity in multiple preclinical tumor models and acts to enhance antigen presentation by upregulating MHC.

[0229] To investigate the direct cytotoxic activity of pure 2-S rimantadine or pure 2-R rimantadine, an in vitro BrdU incorporation assay was performed to quantify the effect of pure 2-S rimantadine or pure 2-R rimantadine on the cell cycle in human HNSCC cell lines. Pure 2-S rimantadine or pure 2-R rimantadine alone resulted in a significant increase in G0 / G1 cell cycle arrest and a significant decrease in S phase in the AT-84-E7 and B16-OVA models. Inhibition of cell proliferation was also observed. An analysis of the effect of pure 2-S rimantadine or pure 2-R rimantadine on T cell proliferation was tested, but there was no significant effect.

[0230] RTqPCR was used to screen the changes in gene expression of cell cycle proteins caused by pure 2-S rimantadine or pure 2-R rimantadine, and a significant decrease in microtubule and cell cycle regulatory molecule Stathmin was observed. A decrease in the microtubule-associated molecule Tau was also observed.

[0231] To confirm that pure 2-S rimantadine or pure 2-R rimantadine has activity against human head and neck tumor lines, BrdU incorporation assays and proliferation assays were performed. Significant cell cycle arrest and decreased proliferation with rimantadine alone were observed in human CAL-27, CAL-33, and SCC-47 squamous cell carcinoma cell lines. Finally, pure 2-S rimantadine or pure 2-R rimantadine induced cell cycle arrest in murine and human cell lines engineered to express HPV16 E5, indicating that pure 2-S rimantadine or pure 2-R rimantadine can functionally reverse the effects of HPV E5.

[0232] Example 3. HPV genotyping

[0233] HPV genotyping is known in the art, for example, see 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 α-HPVs are PCR amplified with primers such as MY09 / 11 and PGMY09 / 11 (see Table 3) and then tested by genotyping with HPV type-specific oligonucleotide probe hybridization and restriction fragment length polymorphism analysis. Negative and positive controls are used to determine the quality of the template DNA.

[0234] Table 3 - Primer sequences

[0235]

[0236]

[0237] The degenerate base code is as follows: M=A or C, W=A or T, Y=C or T, and R=A or G.

[0238] Example 4. In vivo toxicology of 2-S rimantadine and 2-R rimantadine

[0239] A series of in vivo experiments were conducted to determine whether 2-S rimantadine or 2-R rimantadine has a higher binding selectivity for any one of the glutamate, GABA, dopamine receptors or any combination thereof. The enhanced selectivity of 2-S rimantadine or 2-R rimantadine for any one of the glutamate, GABA, dopamine receptors or any combination thereof compared to racemic rimantadine results in the absence of central nervous system side effects typically associated with racemic rimantadine, including nausea, stomach upset, vomiting, anorexia, dry mouth, abdominal pain, weakness, nervousness, tiredness, dizziness, headache, difficulty sleeping, difficulty concentrating, confusion and anxiety.

[0240] To test the above, mice were treated with 10-20 mg / kg body weight of pure 2-S rimantadine, pure 2-R rimantadine, racemic rimantadine (control), or rimantadine (control) daily by IP injection for 7 days. Next, a series of SPECT analyses were performed as described by Schramm, N. et al. (2000). Glutamate receptors, GABA receptors, and dopamine receptors were detected using a small high-resolution small animal SPECT detector to evaluate the selectivity of 2-S rimantadine and 2-R rimantadine.

[0241] SPECT analysis involves treating mice with radioligands specific for each receptor. 123 I] IBZM has been shown to have high affinity to D2 / 3 dopamine receptors. Specific radioligands to glutamate and GABA receptors are known to those skilled in the art. The respective radioligands of appropriate amounts of glutamate, GABA, and dopamine receptors are injected into the lateral tail vein of mice, and SPECT measurements are started 45 minutes after the radioligand is used.

[0242] Surprisingly, 2-R rimantadine has significantly higher binding selectivity or agonistic behavior for glutamate, GABA, dopamine receptors or pathways, or any combination thereof, than 2-S rimantadine. Thus, 2-R rimantadine causes a higher incidence of central nervous system side effects, including nausea, stomach upset, vomiting, anorexia, dry mouth, abdominal pain, weakness, nervousness, tiredness, lightheadedness, dizziness, headache, difficulty sleeping, difficulty concentrating, confusion, and anxiety, than 2-S rimantadine. Together with Example 2, 2-S rimantadine has significantly reduced toxicity as a cancer therapeutic agent, while still being effective, compared to 2-R rimantadine.

[0243] Example 5. In vivo toxicology of 2-S rimantadine and 2-R rimantadine

[0244] A series of in vivo experiments were conducted to determine whether 2-S rimantadine or 2-R rimantadine has a higher binding selectivity for any one of the glutamate, GABA, dopamine receptors or any combination thereof. The enhanced selectivity of 2-S rimantadine or 2-R rimantadine for any one of the glutamate, GABA, dopamine receptors or any combination thereof compared to racemic rimantadine results in the absence of central nervous system side effects typically associated with racemic rimantadine, including nausea, stomach upset, vomiting, anorexia, dry mouth, abdominal pain, weakness, nervousness, tiredness, dizziness, headache, difficulty sleeping, difficulty concentrating, confusion and anxiety.

[0245] To test the above, mice were treated with 10-20 mg / kg body weight of pure 2-S rimantadine, pure 2-R rimantadine, racemic rimantadine (control), or rimantadine (control) daily by IP injection for 7 days. Next, a series of SPECT analyses were performed as described by Schramm, N. et al. (2000). Here, a compact, high-resolution detector for small animal SPECT was described, and each of the glutamate receptors, GABA receptors, and dopamine receptors was examined to evaluate the binding selectivity of 2-S rimantadine and 2-R rimantadine.

[0246] SPECT analysis involves treating mice with radioligands specific for each receptor. 123 I] IBZM has been shown to have high affinity to D2 / 3 dopamine receptors. Specific radioligands to glutamate and GABA receptors are known to those skilled in the art. The respective radioligands of appropriate amounts of glutamate, GABA, and dopamine receptors are injected into the lateral tail vein of mice, and SPECT measurements are started 45 minutes after the radioligand is used.

[0247] Surprisingly, 2-S rimantadine has significantly higher binding selectivity or agonistic behavior for glutamate, GABA, dopamine receptors or pathways, or any combination thereof, than 2-R rimantadine. Thus, 2-S rimantadine causes a higher incidence of central nervous system side effects, including nausea, stomach upset, vomiting, anorexia, dry mouth, abdominal pain, weakness, nervousness, tiredness, lightheadedness, dizziness, headache, difficulty sleeping, difficulty concentrating, confusion, and anxiety, than 2-R rimantadine. Together with Example 2, 2-S rimantadine has significantly reduced toxicity as a cancer therapeutic agent, but is still effective, compared to 2-R rimantadine.

[0248] Example 6. In vitro proliferation of 2-S rimantadine and 2-R rimantadine

[0249] Experiments were performed to determine the ability of 2-S rimantadine (also referred to throughout the application as "S-rimantadine"), 2-R rimantadine (also referred to throughout the application as "R-rimantadine"), racemic (RS) rimantadine, and memantine to effect proliferation in CAL-27 cells. S-rimantadine resulted in enhanced or equivalent cancer cell proliferation compared to R-rimantadine or racemic rimantadine.

[0250] On day 1, CAL-27 cells were seeded in 96-well plates (2-4×10 3 cells / well, 100 μL / well of culture medium) and left overnight to allow the cells to attach to the plate. On day 2, different concentrations of rimantadine (0 μM, 100 μM, 250 μM or 500 μM) were added to the cells and incubated for 24 hours or 48 hours. On day 3 or 4, the culture medium was aspirated, and 100 μL / well of MTT solution (containing an MTT concentration of 0.5 mg / ml; a solution formed by diluting a thiazolyl blue tetrazolium bromide solution (SIGMA, catalog #M2128) with a stock solution (5 mg / ml, in PBS (-20°C)) was added to the culture medium. The cells were incubated in a CO2 incubator at 37°C for 3 hours, and the MTT solution was aspirated. 100 μL / well of DMSO was then added, and the cells were incubated for about 5 minutes. The OD570nm (reference 650nm) was then read. The experimental results are shown in Figure 2 shown.

[0251] Example 7. In vivo tumor model / anti-tumor activity method

[0252] The activity of 2-S rimantadine, 2-R rimantadine and racemic rimantadine against HPV-associated tumors was tested using an in vivo murine syngeneic tumor model. S-rimantadine will exhibit comparable or increased anti-tumor activity compared to racemic rimantadine and / or R-rimantadine.

[0253] Construction of stable expression plasmid of human papillomavirus type 16 E5 gene and establishment of cell line

[0254] The HPV16 E5 of codon optimization will be amplified. The full-length HPV16 E5 and deletion mutants of C-terminal or N-terminal FLAG tags will be cloned into MIP (MSCV-IRES-Puro) or pMSCV-blasticidin vector. All constructs will be confirmed by DNA sequencing. In order to establish the cell line expressing HPV16 E5, HEK293T cells were co-transfected with MIP-HPV16 E5 and Ecopac (pIK6.1MCV.ecopac.UTd) using PEI reagent (Sigma-Aldrich). Then the retrovirus from the culture medium of these cells is used to infect AT-84-E7, MOC2 and CAL-27 cells, and the cells infected are selected by puromycin. pMSCVblasticidin-HPV16 E5 will be used for MEER cells.

[0255] Mouse studies

[0256] Female 6 to 8 week old mice will be used for the experiments. C3H / HeN mice and C57BL / 6 and BALB / c mice will be used. Mice will be injected subcutaneously in the right flank with 1.0 to 5.0 × 10 resuspended in 100 ml PBS. 5 AT-84-E7, 1.5×10 5 B16-0VA, 5.0×10 5 4T1 or 1.0×10 5 For the orthotopic model, 1.0 × 10 M0C2 cells were cultured in 30 mL of PBS. 5 AT-84-E7 or 1.0×10 6 4MOSC1 were injected into the tongue. Once the tumors became palpable, the mice were treated with 200 mg of anti-PD-L1 antibody (Bio X Cell) by ip. Injections were given every 3 days for a total of three or four injections per mouse, or the mice were treated with 10 mg / kg body weight of R-rimantadine, S-rimantadine and / or racemic rimantadine by ip. Injections were given daily for 7 days. For adoptive transfer experiments, single cell suspensions from spleens of OT-1 mice were cultured in medium containing 10 ng / mL OVA SIINFEKL peptide (InvivoGen) and 2 ng / mL recombinant IL2 (PeproTech) for 5 days, and then 4.0×10 6 cells were injected intravenously into B16-OVA-bearing mice. Tumor diameter was measured every 2 to 3 days with an electronic caliper and reported as volume using the formula; tumor volume (mm 3 )(length and width 2) / 2.

[0257] Miyauchi S., et al., Cancer Res. 2020 Feb 15; 80(4): 732-746 is incorporated herein by reference in its entirety. The disclosed information and procedures (eg, protocols) will be used to study S-rimantadine, R-rimantadine and / or racemic rimantadine.

[0258] Example 8. In vitro antiviral method

[0259] The enantiomers of rimantadine (e.g., S-rimantadine) were tested for their direct antiviral activity against HPV viral replication using an in vitro HPV viral replication assay. S-rimantadine will exhibit comparable or increased direct HPV antiviral activity compared to racemic rimantadine or R-rimantadine.

[0260] Plasmids

[0261] The Snls-Cre expression plasmid pCAGGS-nlsCre will be used. The pNeo-loxP HPV-18 and pNeo-loxPHPV-18E6*I plasmids will be used. For both plasmids, 34-bp loxP sites will flank the linear HPV-18 sequence upstream of nucleotide 7474 and downstream of nucleotide 7473. The vector will carry a neomycin resistance marker gene selectable in bacteria and mammalian cells. In the HPV-18E6*I mutant, the intron coding sequence (nucleotides 234-415) in the major E6*I mRNA will be deleted. For trans-complementation experiments, empty vector-only retroviruses pLC and pLJ HPV-18URR-Ed or URR-E6 / E7 retroviruses will be used. Chien et al. 2002. Alternative fates of keratinocytes transduced by human papillomavirus type 18E7 during squamous differentiation. J. Virol. 76: 2064-2972.) All plasmids were purified by banding (eg, in a CsCl-ethidium bromide equilibrium density gradient).

[0262] HPV-18 Virus Particle Recovery and Titer Determination

[0263] HPV-18 virions were recovered from day -14 or day -16 epithelium as described (Favre, M. 1975. Structural polypeptides of rabbit, bovine, and human papillomaviruses. J. Virol. 15: 1239-1247). To titrate the virus, an aliquot of the virus stock was digested with DNase I (Invitrogen) and then inactivated by heating at 100°C for 5 minutes. The packaged viral DNA was then purified by digestion with proteinase K and phenol / chloroform extraction. Serial dilutions of viral DNA were analyzed by real-time quantitative PCR using, for example, SYBR GreenER qPCR SuperMix (Invitrogen) and primers J and K disclosed in Supplementary Table 1 of Wang HK et al. (Genes Dev. 2013; 2009 Jan 15; 23(2): 181-194). As a standard, purified pNeo-LoxP HPV-18 plasmid DNA was serially diluted to ∼40 to 4 × 10 8 40 cycles of PCR amplification reactions will be performed in triplicate (eg, using ABI 7900HT in 384-well plates). The data will then be processed (eg, using SDS2.1 software (Applied Biosystems)).

[0264] HPV-18 infectivity assay

[0265] Various amounts of virus stock were used to inoculate approximately 1 × 10 5Primary human keratinocytes (PHKs) were added with the virus stock corresponding to an MOI of 5200, 1040, 208, 42, 10, 2, 1 or 0 in 1 ml K-SFM and incubated overnight. The medium was replaced and the cells were cultured for another 4 days. Total RNA was then extracted (e.g., using Trizol (Invitrogen)). Reverse transcription was performed on 10 mg RNA in a 50-mL reaction. One microliter of the RT reaction was then subjected to 30 cycles of PCR or nested PCR amplification (30 cycles each) in a 35-mL reaction mixture to produce spliced ​​cDNA fragments of HPV-18E6-E7-E1AE4, RNA or b-actin mRNA, as described (Meyers et al. 2002. Infectious virions produced from a human papillomavirus type 18 / 16 genomic DNA chimera. J. Virol. 76: 4723-4733). 15 microliters of each reaction will be resolved by electrophoresis in a 2% agarose gel and visualized by ethidium bromide staining. PHK will also be infected overnight at various MOIs in K-SFM and developed into raft cultures, fixed on day 14, and processed as described.

[0266] The PHKs receiving different amounts of virus stock are then exposed to different concentrations of R-rimantadine, S-rimantadine and / or racemic rimantadine for a period of time (e.g., 1 day, 2 days, 3 days, 5 days, 7 days and / or 10 days). The disclosed information and procedures (e.g., protocols) will be used to study S-rimantadine, R-rimantadine and / or racemic rimantadine.

[0267] The information and procedures used and disclosed by Wang HK et al. (Genes Dev. 2013; 2009 Jan 15; 23(2): 181-194) are incorporated herein by reference in their entirety. The disclosed information and procedures (e.g., protocols) will be used to study S-rimantadine, R-rimantadine and / or racemic rimantadine.

[0268] Example 9. In Vivo Central Nervous System ("CNS") Assays

[0269] Studies will be conducted to determine the effects of R-rimantadine, S-rimantadine, and racemic rimantadine on the CNS of living animals (e.g., mice and / or rats). Different doses of R-rimantadine, S-rimantadine, and racemic rimantadine will be studied and the following experiments will be performed. Animals receiving S-rimantadine exhibit lower CNS toxicity at similar doses of R-rimantadine and racemic rimantadine. In addition, animals receiving S-rimantadine will be able to receive higher doses of the corresponding agent before exhibiting signs and / or symptoms of CNS toxicity compared to animals receiving R-rimantadine or racemic rimantadine. In addition, mice receiving S-rimantadine will better tolerate signs and symptoms of CNS toxicity compared to mice receiving similar doses of R-rimantadine and racemic rimantadine.

[0270] a) Rotating rod

[0271] A rotating rod system (e.g., Rotor Rod System, San Diego Instruments) will be used to study CNS toxicity associated with the use of R-rimantadine, S-rimantadine, and racemic rimantadine. By allowing observation of motor coordination in animals (e.g., mice or rats), the use of a rotor rod system will allow the study of possible CNS toxicity caused by R-rimantadine, S-rimantadine, and racemic rimantadine.

[0272] Animals will receive doses (e.g., different doses) of R-rimantadine, S-rimantadine, or racemic rimantadine. Potential CNS effects will be measured using a rotating rod system 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) after receiving the dose. Animals receiving S-rimantadine will exhibit fewer adverse CNS effects and toxicity compared to R-rimantadine and racemic rimantadine. In particular, animals (e.g., mice or rats) receiving S-rimantadine will exhibit less abnormal motor coordination. Available at Rotor Rod, San Diego Instruments, https: / / sandiegoinstruments.com / product / rotor-rod / ; ROTOR-ROD TMSystem, Biomedical and Obesity Research Core, College of Education and Human Sciences, University of Nebraska-Lincoln, 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 asimple apparatus for detecting neurological disease 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. JNeurosci Methods. 2010Jun 15;189(2):180-5.doi:10.1016,j.jneumeth.2010.03.026.Epub 2010Mar 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. Behavioural Brain Research 141:237-249, the information and procedures used and disclosed in which are incorporated herein by reference in their entirety.The disclosed information and procedures (eg, protocols) will be used to study S-rimantadine, R-rimantadine and / or racemic rimantadine.

[0273] b) Photobeam Activity System - Home Cage

[0274] The CNS toxicity associated with the use of R-rimantadine, S-rimantadine, and racemic rimantadine will be studied using the Photoelectric Activity System-Home Cage (San Diego Instruments). The use of the Photoelectric Activity System-Home Cage will allow the study of the animals' locomotor activity. Animals receiving R-rimantadine will exhibit lower CNS toxicity, as demonstrated by the Light Beam Activity System-Home Cage test.

[0275] Animals (e.g., mice or rats) will receive a dose (e.g., different 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) after receiving the dose, potential CNS effects will be measured using the Light Beam Activity System-Home Cage. Animals receiving S-rimantadine will exhibit fewer adverse CNS effects and toxicity compared to R-rimantadine and racemic rimantadine. In particular, animals (e.g., mice or rats) receiving S-rimantadine will exhibit less abnormal motor activity. Photoelectric Sensor Activity System-Home Cage, San Diego Instruments, information and procedures for use and disclosure are available at https: / / sandiegoinstruments.com / product / pas-homecage / , and Tatem et al., Behavioral and locomotor measurements using an open field activity monitoring system forskeletal musclediseases. J Vis Exp. 2014 Sep 20;(91):51785. doi:10.3791 / 51785. PMID:2528613; PMCID:PMC4672952, which is incorporated herein by reference in its entirety. The information and protocols set forth in these publications will be used for studies of R-rimantadine, S-rimantadine, and racemic rimantadine. The studies will be used and potentially modified to analyze aspects of animal physiological responses related to the CNS, such as circadian rhythms and anxiety.

[0276] c) Irwin test / Functional Observation Battery (FOB)

[0277] The Irwin test and FOB were used to study the CNS toxicity associated with the use of R-rimantadine, S-rimantadine, and racemic rimantadine. The use of the Irwin test and FOB will allow the qualitative effects of R-rimantadine, S-rimantadine, and racemic rimantadine to be studied. Animals receiving R-rimantadine showed lower CNS toxicity as demonstrated by the Irwin test / FOB test.

[0278] Animals (e.g., mice or rats) will receive doses (e.g., different doses (e.g., four different 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) after receiving the dose, the behavior and physiological function of the animals (e.g., mice or rats) will be studied. Animals receiving S-rimantadine will exhibit fewer adverse CNS effects and toxicity compared to R-rimantadine and racemic rimantadine. In particular, animals receiving S-rimantadine will exhibit fewer abnormal behaviors and physiological functions with similar doses, and animals receiving R-rimantadine will tolerate higher doses before exhibiting observable effects on behavior and physiological function and / or tolerate higher doses before exhibiting overt behavioral toxicity.In Castagne et al., CNS Safety Pharmacology, Reference Module in Biomedical Research, 2014, Irwin S (1968), Comprehensive observational assessment: la. droxicam, General Pharmacology 19:49-54, Mattson et al., (1996) A performance standard for clinical and functional observationalbattery examination ofrats. 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 physiologicalfunction.Curr.Protoc.Pharmacol.2005Jan 1; Chapter 10: Unit 10.10.doi:10.1002 / 0471141755.phl010s27.PMID:22294127, the entire contents of which are incorporated herein by reference. The information and protocols set forth in these publications will be used in the study of R-rimantadine, S-rimantadine and racemic rimantadine.

[0279] d) Morris water maze test

[0280] The Morris water maze test will be used to study the CNS toxicity associated with the use of R-rimantadine, S-rimantadine, and racemic rimantadine. The Morris water maze test will allow the study of potential CNS toxicity experienced by animals (e.g., mice or rats) by testing the animals' spatial learning abilities. Animals receiving R-rimantadine will exhibit lower CNS toxicity, as demonstrated by the Morris water maze test.

[0281] Animals (e.g., mice or rats) will receive doses (e.g., different doses (e.g., four different 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) after administration, the animals are placed in a maze. Animals that received S-rimantadine showed fewer adverse CNS effects and toxicity compared to R-rimantadine and racemic rimantadine. In particular, animals that received S-rimantadine had less inhibition of their spatial learning ability. The information and steps used and disclosed in Vorhees et al., Morris watermaze: 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 set forth in these disclosures will be used for the study of R-rimantadine, S-rimantadine and racemic rimantadine.

[0282] e) Electroencephalogram (EEG) scan

[0283] The CNS toxicity associated with the use of R-rimantadine, S-rimantadine, and racemic rimantadine will be studied by using EEG scanning. EEG scanning will allow the study of electrical activity in the brain of animals (e.g., mice or rats). Animals receiving R-rimantadine will show lower CNS toxicity, as demonstrated by EEG testing.

[0284] Animals (e.g., mice or rats) will receive a dose (e.g., different doses (e.g., four different 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) after receiving the dose, EEG signals from the animals will be recorded. Animals receiving S-rimantadine exhibit fewer adverse CNS effects and toxicity compared to R-rimantadine and racemic rimantadine. In particular, animals receiving S-rimantadine exhibit fewer abnormal EEG signals compared to animals receiving R-rimantadine and racemic rimantadine. In Vogler et al., Low Cost Electrod Assembly for EEgRecordings 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 onpreclinical steps. Methods & Findings in Experimental & Clinical Pharmacology 24 ((Suppl D): 127-128; Itil TM and Itil KZ (1995) Quantitative EEG Brain Mapping InPsychotropic Drug Development, Drug Treatment Selection, and Monitoring. American Journal of Therapy 2: 359-367; and Protocol for Rat SleepEEG, NeuroDetective International, see https: / / www.ndineuroscience.com / userfiles / Rat_Sleep_EEG_Methods.pdf, the information and procedures used and disclosed are incorporated herein by reference in their entirety. The information and protocols set forth in these disclosures will be used for studies of R-rimantadine, S-rimantadine, and racemic rimantadine.

[0285] Example 10. In vitro central nervous system ("CNS") assay

[0286] Studies will be conducted to determine the effects of R-rimantadine, S-rimantadine, and racemic rimantadine on anatomical and / or physiological changes associated with CNS toxicity. Tissue obtained from animals (e.g., living or dead) (e.g., mice and / or rats) receiving S-rimantadine exhibits less changes compared to baseline or normal (e.g., within acceptable limits) tissue when compared to tissue obtained from animals receiving R-rimantadine or racemic rimantadine.

[0287] Animals receiving S-rimantadine will exhibit fewer physiological and / or anatomical changes due to CNS toxicity when compared to animals receiving similar doses of R-rimantadine and racemic rimantadine. Animals receiving S-rimantadine will be able to receive higher doses of S-rimantadine than animals receiving R-rimantadine and racemic rimantadine before exhibiting physiological and / or anatomical changes associated with CNS toxicity. Different doses of R-rimantadine, S-rimantadine, and racemic rimantadine will be studied, and at least the following tests will be used. The information and protocols set forth in these disclosures will be used for studies of R-rimantadine, S-rimantadine, and racemic rimantadine.

[0288] Brain slice / whole cell patch clamp

[0289] Brain slice / whole cell patch clamp studies will be used to study CNS toxicity associated with the use of R-rimantadine, S-rimantadine, and racemic rimantadine. Brain slice / whole cell patch clamp electrophysiology will allow analysis of biophysical mechanisms (e.g., ionic currents) of neural computation and pathology in neuronal cells. Animals receiving R-rimantadine will exhibit less CNS toxicity (e.g., less anatomical and / or physiological changes) as demonstrated by brain slice / whole cell patch clamp testing.

[0290] Animals (e.g., mice or rats) will receive doses (e.g., different doses (e.g., four different 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) after receiving the dose, the animals will be euthanized and brain slices will be obtained and analyzed. Alternatively, whole-cell patch clamp can be performed in vivo. In this case, the animals will be analyzed without euthanasia after a period of time after receiving the dose. Animals receiving S-rimantadine will exhibit fewer adverse CNS effects and toxicity than animals receiving R-rimantadine or racemic rimantadine. In particular, animals receiving S-rimantadine will exhibit fewer abnormal biophysical mechanisms of neural computation and pathology (e.g., ionic currents) than animals receiving 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. 2013; 2012 Jun; 9(6):585-7. doi: 10.1038 / nmeth.1993. Epub 2012 May 6. PMID: 22561988; PMCID: PMC3427788 are hereby incorporated by reference in their entirety. The information and protocols set forth in these publications will be used in the study of R-rimantadine, S-rimantadine and racemic rimantadine.

[0291] Example 11: X-ray powder diffraction of crystalline form A fumarate of 2-N-rimantadine

[0292] The crystals of the Form A fumarate of 2-S-rimantadine were synthesized by the method described herein. The resulting crystalline sample was subjected to X-ray powder diffraction for analysis. Table 4 shows the XRPD diffraction pattern observed when the crystals Form A fumarate were analyzed. In addition, Figure 4 The same data are presented in diffraction pattern format.

[0293] Table 4.

[0294]

[0295]

[0296] Example 12: X-ray powder diffraction of crystalline tartrate type A of 2-N-rimantadine

[0297] The crystalline Form A tartrate of 2-S-rimantadine 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 the crystalline Form A tartrate was analyzed. In addition, Figure 5 The same data is demonstrated in diffraction pattern format.

[0298] Table 5.

[0299]

[0300]

[0301] Example 13: X-ray powder diffraction of crystalline A-galactarate salt of 2-N-rimantadine Crystalline A-galactarate salt of 2-S-rimantadine 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 the crystalline A-galactarate salt. In addition, Figure 6 The same data are presented in diffraction pattern format.

[0302] Table 6.

[0303]

[0304] Example 14: X-ray powder diffraction of crystalline B-type galactarate salt of 2-N-rimantadine

[0305] The crystals of Form B galactarate of 2-S-rimantadine were synthesized by the method described herein. The resulting crystalline samples were subjected to X-ray powder diffraction for analysis. Table 7 shows the XRPD diffraction patterns observed when the crystals Form B galactarate were analyzed. In addition, Figure 7 The same data are presented in diffraction pattern format.

[0306] Table 7.

[0307]

[0308] Example 15: X-ray powder diffraction of crystalline A-type benzoate of 2-N-rimantadine

[0309] The crystalline A-type benzoate of 2-S-rimantadine was synthesized by the method described herein. The resulting crystalline sample was subjected to X-ray powder diffraction for analysis.

[0310] Table 8 shows the XRPD diffraction patterns observed when analyzing the crystalline Form A benzoate salt. Figure 8 The same data are presented in diffraction pattern format.

[0311] Table 8.

[0312]

[0313] Example 16: X-ray powder diffraction of crystalline benzenesulfonate type A of 2-N-rimantadine

[0314] The crystalline benzenesulfonate salt of 2-S-rimantadine was synthesized by the method described herein. The resulting crystalline sample was subjected to X-ray powder diffraction for analysis. Table X shows the XRPD diffraction pattern observed when the crystalline Form A benzenesulfonate salt was analyzed. In addition, Fig. 9 The same data are presented in diffraction pattern format.

[0315] Table 9.

[0316]

[0317]

[0318] Example 17: X-ray powder diffraction of crystalline 2-N-rimantadine type B benzenesulfonate

[0319] The Form B crystalline salt of 2-S-rimantadine 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 the crystalline Form B benzenesulfonate was analyzed. In addition, Fig.10 The same data are presented in diffraction pattern format.

[0320] Table 10.

[0321]

[0322]

[0323] Other Implementations

[0324] It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages and modifications are within the scope of the following claims.

Claims

1. A salt comprising 2-S-rimantadine and fumaric acid.

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

3. The salt according to claim 1 or 2, wherein the salt is crystalline.

4. The salt of claim 3 having an X-ray powder diffraction pattern comprising a peak at about 6.0920.

5. The salt of claim 3, 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.

6. A salt comprising 2-S-rimantadine and tartaric acid.

7. The salt according to claim 6, wherein the salt is anhydrous.

8. The salt according to claim 6 or 7, wherein the salt is crystalline.

9. The salt of claim 8 having an X-ray powder diffraction pattern comprising a peak at about 7.5020.

10. The salt of claim 9, 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.

11. A salt comprising 2-S-rimantadine and galactaric acid.

12. The salt of claim 11, wherein the salt is anhydrous.

13. The salt according to claim 11 or 12, wherein the salt is crystalline.

14. The salt of claim 13 having an x-ray powder diffraction pattern comprising a peak at about 5.712θ.

15. The salt of claim 14, further comprising peaks at about 15.85 2θ, about 16.96 2θ, about 19.76 2θ, and / or about 19.43 2θ.

16. The salt of claim 13, having an X-ray powder diffraction pattern comprising a peak at 5.83 2θ.

17. The salt of claim 16, further comprising peaks at about 14.89 2θ, about 16.87 2θ, about 17.62 2θ, about 39.87 2θ, and / or about 19.72 2θ.

18. A salt comprising 2-S-rimantadine and benzoic acid.

19. The salt of claim 18, wherein the salt is anhydrous.

20. The salt according to claim 18 or 19, wherein the salt is crystalline.

21. The salt of claim 29 having an X-ray powder diffraction pattern comprising a peak at 7.85 2θ.

22. The salt of claim 21, further comprising peaks at about 9.72 2θ, about 11.49 2θ, about 12.29 2θ, about 15.66 2θ, about 19.96 2θ, about 29.38 2θ, and / or about 29.91 2θ.

23. A salt comprising 2-S-rimantadine and benzenesulfonate.

24. The salt of claim 23, wherein the salt is anhydrous.

25. The salt according to claim 23 or 24, wherein the salt is crystalline.

26. The salt of claim 25 having an x-ray diffraction powder pattern comprising a peak at 6.21 2θ.

27. The salt of claim 26, further comprising peaks at about 8.02 2θ, about 8.44 2θ, about 14.11 2θ, about 15.39 2θ, about 16.91 2θ, and / or about 19.98 2θ.

28. The salt of claim 25 having an X-ray diffraction powder pattern comprising a peak at 8.94 2θ.

29. The salt of claim 26, further comprising peaks at about 8.45 2θ, about 14.13 2θ, about 15.37 2θ, about 16.86 2θ, and / or about 19.19 2θ.

30. A crystalline form of 2-S-rimantadine, wherein the XRPD peaks are about 14.36 2θ, about 17.56 2θ, about 18.45 2θ, about 18.81 2θ and / or about 27.17 2θ.

31. A composition comprising a salt of 2-S rimantadine.

32. The composition of claim 31, wherein the salt is a fumarate, tartrate, galactarate, benzoate, benzenesulfonate, or a combination thereof.

33. The composition of claim 32, wherein the salt is a fumarate salt.

34. The composition of claim 33, wherein the fumarate salt is fumarate type A.

35. A composition according to any one of claims 31-34, characterised in that One or more of the following: a X-ray powder diffraction pattern ("XRPD") having a peak at about 6.99° 2θ in the diffraction pattern; b. a peak at about 14.36° 2θ in the XRPD diffraction pattern; c. a peak at about 17.56° 2θ in the XRPD diffraction pattern; d. a peak at about 18.45° 2θ in the XRPD diffraction pattern; e. a peak at about 18.81° 2θ in the XRPD diffraction pattern; or f. Peak at about 27.17° 2θ in the XRPD diffraction pattern.

36. The composition of claim 32, wherein the salt is a tartrate salt.

37. The composition of claim 36, wherein the tartrate is tartrate Type A.

38. A composition according to any one of claims 31 or 36-37, characterized in that One or more of the following: a X-ray powder diffraction pattern ("XRPD") having a peak at about 6.99° 2θ in the diffraction pattern; b. A peak at about 7.59° 2θ in the XRPD diffraction pattern; c. a peak at about 17.64° 2θ in the XRPD diffraction pattern; d. a peak at about 18.68° 2θ in the XRPD diffraction pattern; e. a peak at about 15.43° 2θ in the XRPD diffraction pattern; f. a peak at about 19.37° 2θ in an XRPD diffraction pattern; or g. Peak at about 22.48° 2θ in the XRPD diffraction pattern.

39. The composition of claim 32, wherein the salt is a galactarate salt.

40. The composition of claim 39, wherein the galactarate is type A galactarate.

41. A composition according to any one of claims 31 or 39-49, characterized in that One or more of the following: a. A peak at about 5.71° 2θ in the XRPD diffraction pattern; b. a peak at about 15.85° 2θ in the XRPD diffraction pattern; c. a peak at about 16.96° 2θ in the XRPD diffraction pattern; d. a peak at about 19.76° 2θ in the XRPD diffraction pattern; or e. Peak at about 19.43° 2θ in the XRPD diffraction pattern.

42. The composition of claim 39, wherein the galactarate is Type B galactarate.

43. A composition according to any one of claims 31 or 39-49 or 42, characterized in that One or more of the following: a. A peak at about 5.83° 2θ in the XRPD diffraction pattern; b. A peak at about 14.89° 2θ in the XRPD diffraction pattern; c. a peak at about 16.87° 2θ in the XRPD diffraction pattern; d. a peak at about 17.62° 2θ in the XRPD diffraction pattern; e. a peak at about 39.87° 2θ in the XRPD diffraction pattern; or f. Peak at about 19.72° 2θ in the XRPD diffraction pattern.

44. The composition according to claim 32, wherein The salt is the benzoate salt.

45. The composition of claim 44, wherein the benzoate salt is Form A benzoate.

46. ​​A composition according to any one of claims 31 or 44-45, characterized in that One or more of the following: a. A peak at about 7.85° 2θ in the XRPD diffraction pattern; b. A peak at about 9.72° 2θ in the XRPD diffraction pattern; c. a peak at about 11.49° 2θ in the XRPD diffraction pattern; d. a peak at about 12.29° 2θ in the XRPD diffraction pattern; e. a peak at about 15.66° 2θ in the XRPD diffraction pattern; f. a peak at about 19.96° 2θ in the XRPD diffraction pattern; g. a peak at about 29.38° 2θ in the XRPD diffraction pattern; or h. Peak at about 29.91° 2θ in the XRPD diffraction pattern.

47. The composition of claim 32, wherein the salt is besylate.

48. The composition of claim 47, wherein the benzenesulfonate salt is Form A benzenesulfonate salt.

49. A composition according to any one of claims 31 or 44-45, characterized in that One or more of the following: a. A peak at about 6.21° 2θ in the XRPD diffraction pattern; b. A peak at about 8.92° 2θ in the XRPD diffraction pattern; c. a peak at about 8.44° 2θ in the XRPD diffraction pattern; d. a peak at about 14.11° 2θ in the XRPD diffraction pattern; e. a peak at about 15.39° 2θ in the XRPD diffraction pattern; f. a peak at about 16.91° 2θ in an XRPD diffraction pattern; or g. Peak at about 19.98° 2θ in the XRPD diffraction pattern.

50. The composition of claim 47, wherein the benzenesulfonate salt is Form B benzenesulfonate salt.

51. The composition according to claim 31 or 50, characterized in that One or more of the following: a X-ray powder diffraction pattern ("XRPD") having a peak at about ; b. A peak at about 8.04° 2θ in the XRPD diffraction pattern; c. a peak at about 8.45° 2θ in the XRPD diffraction pattern; d. a peak at about 14.13° 2θ in the XRPD diffraction pattern; e. a peak at about 15.37° 2θ in the XRPD diffraction pattern; f. a peak at about 16.86° 2θ in the XRPD diffraction pattern; or g. Peak at about 19.10° 2θ in the XRPD diffraction pattern.

52. The composition of any one of claims 31-51, wherein the composition is crystalline.

53. The salt of claim 1, wherein the differential scanning calorimetry analysis is substantially as shown in Figure 11.

54. The salt of claim 1, wherein the differential scanning calorimetry analysis is substantially as shown in Figure 12.

55. The salt of claim 1, wherein the differential scanning calorimetry analysis is substantially as shown in Figure 13.

56. The salt of claim 1, wherein the differential scanning calorimetry analysis is substantially as shown in Figure 14.

57. The salt of claim 1, wherein the differential scanning calorimetry analysis is substantially as shown in Figure 15.

58. The salt of claim 1, wherein the differential scanning calorimetry analysis is substantially as shown in Figure 16.

59. The salt of claim 1, wherein the differential scanning calorimetry analysis is substantially as shown in Figure 17.

60. The salt of claim 1, wherein the differential scanning calorimetry analysis is substantially as shown in Figure 18.

61. The salt of claim 1, wherein the differential scanning calorimetry analysis is substantially as shown in Figure 19.

62. The salt of claim 1, wherein the differential scanning calorimetry analysis is substantially as shown in Figure 20.

63. The salt of claim 1, wherein the differential scanning calorimetry analysis is substantially as shown in Figure 21.

64. A salt of 2-S-rimantamine, characterized in that In thermogravimetric analysis, the sample weight loss was about 0.01 w / w% at temperatures ranging from 0°C to 150°C.

65. The salt according to the preceding claim, wherein the salt is a tartrate salt.

66. A salt of 2-S-rimantamine, characterized in that The sample weight loss in thermogravimetric analysis was about 6.72 w / w % loss at temperatures ranging from 0°C to 150°C.

67. The salt according to the preceding claim, wherein the salt is a maleate salt.

68. A salt of 2-S-rimantamine, characterized in that In thermogravimetric analysis, the sample weight loss was about 10.30 w / w% at temperatures ranging from 0°C to 85°C.

69. The salt according to the preceding claim, wherein the salt is hippurate.

70. A salt of 2-S-rimantidine, characterized in that In thermogravimetric analysis, the sample weight loss was about 0.15 w / w% at temperatures ranging from 0°C to 150°C.

71. The salt according to the preceding claim, wherein the salt is a galactarate salt.

72. A salt of 2-S-rimantamine, characterized in that In thermogravimetric analysis, the sample weight loss was about 0.00 w / w% at temperatures ranging from 0°C to 150°C.

73. The salt according to the preceding claim, wherein the salt is a galactarate salt.

74. A salt of 2-S-rimantamine, characterized in that In thermogravimetric analysis, the sample weight loss was about 0.26 w / w % at a temperature of 0°C to 100°C.

75. The salt according to the preceding claim, wherein the salt is a benzoate salt.

76. A salt of 2-S-rimantamine, characterized in that In thermogravimetric analysis, the sample weight loss was about 14.77 w / w % at temperatures ranging from 0°C to 110°C.

77. The salt according to the preceding claim, wherein the salt is a benzoate salt.

78. A salt of 2-S-rimantamine, characterized in that In thermogravimetric analysis, the sample weight loss was about 1.68 w / w % loss at temperatures of 0°C and 150°C.

79. The salt according to the preceding claim, wherein the salt is gentisate.

80. A salt of 2-S-rimantadine, characterized in that In thermogravimetric analysis, the sample weight loss was about 3.55 w / w % loss at temperatures from 0°C to 200°C.

81. The salt according to the preceding claim, wherein the salt is a tosylate salt.

82. A salt of 2-S-rimantamine, characterized in that In thermogravimetric analysis, the sample weight loss was about 0.02 w / w % loss at temperatures from 0°C to 200°C.

83. The salt according to the preceding claim, wherein the salt is a besylate salt.

84. A salt of 2-S-rimantamine, characterized in that In thermogravimetric analysis, the sample weight loss was about 0.09 w / w% at temperatures ranging from 0°C to 200°C.

85. The salt according to the preceding claim, wherein the salt is a besylate salt.

86. A salt of 2-S-rimantadine, wherein the salt exhibits differential scanning calorimetry comprising an endothermic peak at about 221°C.

87. The salt according to the preceding claim, wherein the salt is a tartrate salt.

88. A salt of 2-S-rimantadine, wherein differential scanning calorimetry analysis of the salt comprises endothermic peaks at about 63°C, 74°C, and 121°C.

89. The salt according to the preceding claim, wherein the salt is a maleate salt.

90. A salt of 2-S-rimantadine, wherein differential scanning calorimetry analysis of the salt comprises an endothermic peak at about 62°C.

91. The salt according to the preceding claim, wherein the salt is hippurate.

92. A salt of 2-S-rimantadine, wherein differential scanning calorimetry analysis of the salt comprises an endothermic peak at about 193°C.

93. The salt according to the preceding claim, wherein the salt is a galactarate salt.

94. A salt of 2-S-rimantadine, wherein differential scanning calorimetry analysis of the salt comprises an endothermic peak at about 195°C.

95. The salt according to the preceding claim, wherein the salt is a galactarate salt.

96. A salt of 2-S-rimantadine, wherein differential scanning calorimetry analysis of the salt comprises an endothermic peak at about 199°C.

97. The salt according to the preceding claim, wherein the salt is a benzoate salt.

98. A salt of 2-S-rimantadine, wherein differential scanning calorimetry analysis of the salt comprises an endothermic peak at about 198°C.

99. The salt according to the preceding claim, wherein the salt is a benzoate salt.

100. A salt of 2-S-rimantadine, wherein differential scanning calorimetry analysis of the salt comprises an endothermic peak at about 206°C.

101. The salt according to the preceding claim, wherein the salt is gentisate.

102. A salt of 2-S-rimantadine, wherein differential scanning calorimetry analysis of the salt comprises an endothermic peak at about 214°C.

103. The salt according to the preceding claim, wherein the salt is a tosylate salt.

104. A salt of 2-S-rimantadine, wherein differential scanning calorimetry analysis of the salt comprises an endothermic peak at about 223°C.

105. The salt according to the preceding claim, wherein the salt is a besylate salt.

106. A salt of 2-S-rimantadine, wherein differential scanning calorimetry analysis of the salt comprises an endothermic peak at about 232°C.

107. The salt according to the preceding claim, wherein the salt is a besylate salt.

108. A salt of 2-S-rimantamine, characterized in that The crystal particle size is 1-50μm.

109. A salt of 2-S-rimantamine, characterized in that The XRPD diffraction pattern is essentially as shown in Figure 4.

110. A salt of 2-S-rimantamine, characterized in that The XRPD diffraction pattern is essentially as shown in Figure 5.

111. A salt of 2-S-rimantamine, characterized in that The XRPD diffraction pattern is substantially as shown in FIG6 .

112. A salt of 2-S-rimantamine, characterized in that The XRPD diffraction pattern is substantially as shown in FIG7 .

113. A salt of 2-S-rimantamine, characterized in that The XRPD diffraction pattern is substantially as shown in FIG8 .

114. A salt of 2-S-rimantamine, characterized in that The XRPD diffraction pattern is substantially as shown in FIG9 .

115. A salt of 2-S-rimantamine, characterized in that The XRPD diffraction pattern is substantially as shown in FIG10 .

116. The salt of 2-S-rimantadine according to claim 110, wherein the salt of 2-S-rimantadine is tartrate salt of 2-S-rimantadine.

117. The salt of 2-S-rimantadine according to claim 109, wherein the salt of 2-S-rimantadine is a fumarate salt of 2-S-rimantadine.

118. The salt of 2-S-rimantadine according to claim 111 or 112, wherein the salt of 2-S-rimantadine is galactaric acid salt of 2-S-rimantadine.

119. The salt of 2-S-rimantadine according to claim 113, wherein the salt of 2-S-rimantadine is benzoate of 2-S-rimantadine.

120. The 2-S-rimantadine salt according to any one of claims 114-115, wherein the 2-S-rimantadine salt is 2-S-rimantadine benzenesulfonate.

121. A method of treating cancer in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a salt or composition according to any one of the preceding claims.

122. The method of claim 121, 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.

123. The method of any one of claims 121-122, wherein the cancer is a sarcoma, carcinoma, lymphoma, or leukemia.

124. The method of claim 123, wherein the cancer is squamous cell carcinoma.

125. The method of claim 124, wherein the squamous cell carcinoma is head and neck squamous cell carcinoma.

126. The method of claim 122, wherein the cancer is selected from the group consisting of head and neck cancer, breast cancer, and melanoma.

127. The method of any one of claims 121-126, wherein the cancer is an HPV-related cancer.

128. The method of claim 127, wherein the HPV-related cancer is associated with the alpha genus of HPV.

129. The method of any one of claims 121-128, wherein one or more cancer cells from the subject express human papillomavirus (HPV) proteins.

130. The method of claim 129, wherein the HPV protein is HPV E5 protein.

131. The method of claim 130, wherein 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.

132. The method of claim 131, wherein the HPV protein is E5 from HPV 16 133. The method of claim 131, wherein the HPV protein is E5 from HPV 18.

134. A method of treating cancer in a subject, the method comprising: a. detecting cancer cells expressing human papillomavirus (HPV) proteins in a sample from the subject; and b. administering to the subject a therapeutically effective amount of the salt or composition of any one of the preceding claims.

135. The method of claim 134, wherein the cancer is associated with the alpha genus of HPV.

136. The method of claim 134 or 135, wherein the HPV protein is one or more of HPV E5, E6 or E7 proteins.

137. The method of claim 136, wherein the HPV E5, E6 or E7 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.

138. The method of any one of claims 134-137, wherein the cancer is selected from the group consisting of head and neck cancer, mucosal squamous cell carcinoma, skin squamous cell carcinoma, liver cancer, cervical cancer, vaginal cancer, vulvar cancer, penile cancer, and anal cancer.

139. The method of any one of claims 121-138, wherein the method further comprises administering an additional anti-cancer agent.

140. The method of claim 139, wherein the additional anticancer agent is selected from the group consisting of carboplatin, cisplatin, gemcitabine, methotrexate, paclitaxel, pemetrexed, lomustine, temozolomide, dacarbazine, and combinations thereof.

141. The method of any one of claims 139-140, wherein the additional anti-cancer agent is an immunotherapy.

142. The method of claim 141, wherein the additional anti-cancer agent is an immune checkpoint inhibitor.

143. The method of any one of claims 139-140, wherein 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.

144. The method of claim 142 or 143, wherein the immune checkpoint inhibitor is selected from the group consisting of: ipilimumab, nivolumab, pembrolizumab, atezolizumab, avelumab, durvalumab, tremelimumab, cemiplizumab, and combinations thereof.

145. The method of any one of claims 121-144, wherein the method further comprises subjecting the subject to radiation therapy, surgery, or a combination thereof.

146. The method of any one of claims 121-144, wherein the subject is a human.

147. A method of treating precancerous HPV lesions in a subject in need of treatment comprising administering a therapeutically effective amount of a salt or composition according to any one of the preceding claims.

148. The method of claim 147, wherein the HPV lesions are associated with the alpha genus of HPV.

149. The method of any one of claims 147-148, wherein the HPV precancerous lesion is a lesion of the cervix, skin, urethra, nasal cavity, paranasal sinuses, larynx, tracheobronchial mucosa, or oral cavity.

150. The method of any one of claims 147-149, wherein the HPV precancerous lesion expresses one or more HPV proteins selected from one or more of HPV E5, E6, or E7 proteins.

151. The method of claim 150, wherein the HPV E5, E6 or E7 protein is from one or more HPV subtypes selected from one or more of HPV 6, HPV 11, HPV 16, HPV 18, HPV 31, HPV 33, HPV 35, HPV 39, HPV45, HPV 51, HPV 52, HPV 56, HPV 58, HPV 66 and HPV 69.

152. The method of any one of claims 121-151, wherein rimantadine is administered topically, orally, subcutaneously, or parenterally.

153. A method of treating or preventing avian influenza in poultry, the method comprising administering a therapeutically effective amount of a salt or composition according to any one of the preceding claims.

154. The method of claim 153, wherein the avian influenza is H5N1.

155. The method of any one of claims 121-154, wherein 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.

156. A composition comprising: a salt or composition according to any one of the preceding claims, and one or more immune checkpoint inhibitors.

157. The composition of claim 156, wherein the one or more immune checkpoint inhibitors comprise CTLA-4, PD-1, PD-L1, BTLA, LAG-3, A2AR, TIM-3, B7-H3, VISTA, IDO, or any combination thereof.

158. The salt or composition of any preceding claim, wherein the salt or composition comprises enantiomerically pure 2-S-rimantadine.

159. The salt or composition of any preceding claim, wherein the salt or composition does not comprise R-rimantadine.

160. The composition of any of the preceding claims, wherein the salt or composition is formulated for injection.

161. A method for preparing a 2-S-rimantamine salt, comprising: i. dissolving 2-S-rimantamine in a first solvent to form a first solution; ii. adding a second solvent to the solution to form a second solution; iii. cooling the second solution; and iv. Evaporating the second solution.

162. The method of claim 161, wherein the first solvent is selected from methanol, ethanol, chloroform and water.

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