2-S and 2-R Rimantadine for Treating Cancer and Precancerous Papillomavirus Lesions

Abstract

The present invention relates to the use of purified 2-S rimantadine or purified 2-R rimantadine or racemic rimantadine or a pharma- ceutically acceptable salt thereof to treat cancer and precancerous lesions, including cancer and precancerous lesions associated with human papillomavirus (HPV), in a subject in need of such treatment.The present invention also relates to compositions comprising purified 2-S rimantadine or purified 2-R rimantadine or racemic rimantadine or a pharma-ceutically acceptable salt thereof, in combination with one or more immune checkpoint inhibitors.

Description

[Technical field]

[0001] cross reference This application claims the benefit of U.S. Provisional Patent Application No. 63 / 150,027, filed February 16, 2021, the entirety of which is incorporated herein by reference.

[0002] Incorporation by Reference 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 document, the term in the present specification shall control.

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

[0004] Genital human papillomavirus (HPV) is the most common sexually transmitted disease in the United States. The Centers for Disease Control and Prevention (CDC) states that 90% of HPV infections will not cause symptoms and will resolve spontaneously within two years. In some cases, however, HPV infections persist and result in either warts or precancerous lesions. These lesions increase the risk of cancer of the cervix, vulva, vagina, penis, anus, rectum, and oropharynx, depending on the affected area. HPV types associated with cervical tumorigenicity are classified into 15 "high-risk types" (HPV16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68, 73, and 82) and three "potentially high-risk types" (HPV26, 53, and 66). Researchers have also shown an association of HPV16 and 18 with breast cancer. HPV types (HPV6, 11, 40, 42, 43, 44, 54, 61, 70, 72, and 81) are classified as "low-risk types" and are known to cause benign, mild cervical changes, genital warts, and recurrent respiratory papillomatosis. Cutaneous types HPV5, 8, and 92 are associated with skin cancer.

[0005] Rimantadine hydrochloride (α-methyl-1-adamantane-methanamine hydrochloride) is an oral drug sold under the trade name Flumadine® used to treat influenza A. Rimantadine inhibits influenza activity by binding to amino acids in the viral M2 transmembrane channel and blocking proton transport through the M2 channel. Flumadine® contains a racemic mixture of rimantadine. One study found evidence that the R-enantiomer binds to the M2 channel pore with higher affinity than the S-enantiomer. However, that finding contradicts the results of some earlier studies that found no difference between the enantiomers for M2. The lack of distinction between the enantiomers for M2 was confirmed in later studies. It has been suggested that rimantadine also has some antiparkinsonian activity. However, the use of rimantadine for this indication has not been developed or approved.

[0006] Flumadine® has gastrointestinal and central nervous system adverse effects, including nausea, upset stomach, vomiting, loss of appetite, xerostomia, abdominal pain, asthenia, irritability, fatigue, lightheadedness, dizziness, headache, sleep disturbances, difficulty concentrating, confusion, and anxiety. Anxiety and insomnia are the toxicities most commonly cited as reasons for discontinuing treatment. Summary of the Invention

[0007] Formula I below shows the chemical structures of the 2-R enantiomer of rimantadine and the 2-S enantiomer of rimantadine.

[0008] [ka]

[0009] One aspect of the disclosure includes a method of treating cancer in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of either enantiomerically pure 2-S rimantadine or a pharma- ceutically acceptable salt thereof. In some embodiments, side effects associated with administration of 2-S rimantadine are reduced compared to side effects associated with racemic rimantadine. In some embodiments, the subject is administered a pharma- ceutically acceptable salt of 2-S rimantadine. In some embodiments, the pharma- ceutically acceptable salt is a hydrochloride salt. In some embodiments, the cancer is selected from one or more of melanoma, head and neck cancer, lung cancer, colon cancer, breast cancer, esophageal cancer, pancreatic cancer, prostate cancer, cervical cancer, and gastric cancer. In some embodiments, the cancer is a sarcoma, carcinoma, lymphoma, or leukemia. In some embodiments, the carcinoma is 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 selected from the group consisting of head and neck cancer, breast cancer, and melanoma. In some embodiments, the cancer is an HPV-associated cancer. In some embodiments, the HPV-associated cancer is associated with the alpha genus of HPV. In some embodiments, one or more cancer cells from the subject express a human papillomavirus (HPV) protein. In some embodiments, the HPV protein is an HPV E5 protein. In some embodiments, the HPV E5 protein is from one or more HPV subtypes selected from the group consisting of HPV6, HPV11, HPV16, HPV18, HPV31, HPV33, HPV35, HPV39, HPV45, HPV51, HPV52, HPV56, HPV58, HPV66, and HPV69. In some embodiments, the HPV protein is E5 of HPV16. In some embodiments, the HPV protein is HPV18 E5.

[0010] Another aspect of the present disclosure includes a method of treating cancer in a subject, the method comprising: (a) detecting cancer cells expressing a human papillomavirus (HPV) protein in a sample from the subject; and (b) administering to the subject a therapeutically effective amount of 2-S rimantadine or a pharma- ceutically acceptable salt thereof. In some embodiments, the cancer is associated with the alpha genus of HPV. In some embodiments, the HPV protein is one or more of HPV E5, E6, E7 proteins. In some embodiments, the HPV E5, E6, or E7 protein is from one or more HPV subtypes selected from the group consisting of HPV6, HPV11, HPV16, HPV18, HPV31, HPV33, HPV35, HPV39, HPV45, HPV51, HPV52, HPV56, HPV58, HPV66, and HPV69. In some embodiments, the cancer is selected from the group consisting of head and neck cancer, mucosal squamous cell carcinoma, cutaneous squamous cell carcinoma, cervical cancer, vaginal cancer, vulvar cancer, penile cancer, and anal cancer. In some embodiments, the method further comprises administering an additional anti-cancer agent. In some embodiments, the additional anti-cancer 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 anti-cancer agent is an immunotherapy. In some embodiments, the additional anti-cancer agent is an immune checkpoint inhibitor. In some embodiments, the immune checkpoint inhibitor targets one or more of CTLA-4, PD-1, PD-L1, BTLA, LAG-3, A2AR, TIM-3, B7-H3, VISTA, and IDO. In some embodiments, the immune checkpoint inhibitor is selected from the group consisting of ipilimumab, nivolumab, pembrolizumab, atezolizumab, avelumab, durvalumab, tremelimumab, cemiplimab, and combinations thereof. In some embodiments, the method further comprises subjecting the subject to radiation therapy, surgery, or a combination thereof. In some embodiments, the subject is a human.

[0011] Another aspect of the present disclosure includes a method of treating a precancerous HPV lesion in a subject in need of treatment, comprising administering a therapeutically effective amount of rimantadine. In some embodiments, the HPV lesion is associated with the alpha genus of HPV. In some embodiments, the rimantadine is a racemic mixture. In some embodiments, the rimantadine is purified 2-S rimantadine. In some embodiments, the rimantadine is purified 2-R rimantadine. In some embodiments, the precancerous HPV lesion is a lesion of the cervix, skin, urethra, nasal cavity, paranasal sinuses, larynx, tracheobronchial mucosa, or oral cavity. In some embodiments, the precancerous HPV lesion expresses one or more HPV proteins selected from one or more of HPV E5, E6, or E7 proteins. In some embodiments, the HPV E5, E6, or E7 protein is from one or more HPV subtypes selected from the group consisting of one or more of HPV6, HPV11, HPV16, HPV18, HPV31, HPV33, HPV35, HPV39, HPV45, HPV51, HPV52, HPV56, HPV58, HPV66, and HPV69. In some embodiments, the rimantadine is administered topically, orally, subcutaneously, or parenterally.

[0012] Another aspect of the present disclosure includes a method for treating or preventing avian influenza in poultry, comprising administering a therapeutically effective amount of pure 2-S rimantadine or pure 2-S rimantadine, or a pharmaceutically acceptable salt thereof. In some embodiments, the avian influenza is H5N1. In some embodiments, side effects associated with administration of 2-S rimantadine are reduced compared to side effects associated with racemic rimantadine or enantiomerically pure 2-R rimantadine.

[0013] Another aspect of the disclosure includes a method of treating cancer in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of enantiomerically pure 2-R rimantadine or a pharma- ceutically acceptable salt thereof. In some embodiments, side effects associated with administration of pure 2-R rimantadine are reduced compared to side effects associated with racemic rimantadine or 2-S rimantadine. In some embodiments, the subject is administered a pharma- ceutically acceptable salt of pure 2-R rimantadine. In some embodiments, the pharma- ceutically acceptable salt is a hydrochloride salt. In some embodiments, the cancer is selected from one or more of melanoma, head and neck cancer, lung cancer, colon cancer, breast cancer, esophageal cancer, pancreatic cancer, prostate cancer, cervical cancer, and gastric cancer. In some embodiments, the cancer is a sarcoma, carcinoma, lymphoma, or leukemia. In some embodiments, the carcinoma is a squamous cell carcinoma. In some embodiments, the squamous cell carcinoma is head and neck squamous cell carcinoma. In some embodiments, the cancer is selected from the group consisting of head and neck cancer, breast cancer, and melanoma. In some embodiments, the cancer is an HPV-associated cancer. In some embodiments, one or more cancer cells from the subject express a human papillomavirus (HPV) protein. In some embodiments, the HPV-associated cancer is associated with the alpha genus of HPV. In some embodiments, the HPV protein is an HPV E5 protein. In some embodiments, the HPV E5 protein is from one or more HPV subtypes selected from the group consisting of HPV6, HPV11, HPV16, HPV18, HPV31, HPV33, HPV35, HPV39, HPV45, HPV51, HPV52, HPV56, HPV58, HPV66, and HPV69. In some embodiments, the HPV protein is E5 of HPV16. In some embodiments, the HPV protein is E5 of HPV18.

[0014] Another aspect of the present disclosure includes 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 pure 2-R rimantadine or a pharma- ceutically acceptable salt thereof. In some embodiments, the HPV protein is associated with the alpha genus of HPV. In some embodiments, the HPV protein is one or more of HPV E5, E6, E7 proteins. In some embodiments, the HPV E5, E6, or E7 protein is from one or more HPV subtypes selected from the group consisting of HPV6, HPV11, HPV16, HPV18, HPV31, HPV33, HPV35, HPV39, HPV45, HPV51, HPV52, HPV56, HPV58, HPV66, and HPV69. In some embodiments, the cancer cells are from a cancer selected from the group consisting of head and neck cancer, mucosal squamous cell carcinoma, cutaneous squamous cell carcinoma, cervical cancer, vaginal cancer, vulvar cancer, penile cancer, and anal cancer. In some embodiments, the method further comprises administering an additional anti-cancer agent. In some embodiments, the additional anti-cancer 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 anti-cancer agent is an immunotherapeutic agent. In some embodiments, the additional anti-cancer agent is an immune checkpoint inhibitor. In some embodiments, the immune checkpoint inhibitor targets one or more of CTLA-4, PD-1, PD-L1, BTLA, LAG-3, A2AR, TIM-3, B7-H3, VISTA, and IDO. In some embodiments, the immune checkpoint inhibitor is selected from the group consisting of ipilimumab, nivolumab, pembrolizumab, atezolizumab, avelumab, durvalumab, tremelimumab, cemiplimab, and combinations thereof. In some embodiments, the method further comprises subjecting the subject to radiation therapy, surgery, or a combination thereof. In some embodiments, the subject is a human.In some embodiments, side effects associated with administration of pure 2-R rimantadine are reduced as compared to side effects associated with racemic rimantadine or pure 2-S rimantadine.

[0015] Another aspect of the present disclosure includes a composition comprising pure 2-R rimantadine or a pharma- ceutically acceptable salt thereof, pure 2-S rimantadine or a pharma- ceutically acceptable salt thereof, and one or more immune checkpoint inhibitors. In some embodiments, the one or more immune checkpoint inhibitors include CTLA-4, PD-1, PD-L1, BTLA, LAG-3, A2AR, TIM-3, B7-H3, VISTA, IDO, or any combination thereof. In some embodiments, the pure 2-R rimantadine or a pharma- ceutically acceptable salt thereof, the pure 2-S rimantadine or a pharma- ceutically acceptable salt thereof, or the racemic rimantadine or a pharma- ceutically acceptable salt thereof is formulated for injection. [Brief description of the drawings]

[0016] [Figure 1] Figure 1A-B show the peak current amplitude and steady-state current measurements of 2-S rimantadine (TGN-S15) and 2-R rimantadine (TGN-S16) for NR2A. Figure 1C-D show the peak current amplitude and steady-state current measurements of 2-S rimantadine (TGN-S15) and 2-R rimantadine (TGN-S16) for NR2B. [Diagram 2] Figure 1 shows proliferation of CAL-27 cells with varying concentrations of RS-rimantadine (TGN-S11), S-rimantadine (TGN-S15), R-rimantadine (TGN-S16), and memantine (TGN-S13). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0017] One aspect of the present disclosure is the use of enantiomerically pure 2-S rimantadine or enantiomerically pure 2-R rimantadine to treat cancer.In some embodiments, the use of 2-S rimantadine (also referred to as "S-rimantadine") or enantiomerically pure 2-R rimantadine to treat cancer associated with papillomavirus, such as human papillomavirus (HPV), is disclosed herein.In some embodiments, HPV is alpha genus HPV.

[0018] Another aspect of the present disclosure is the use of 2-S rimantadine or enantiomerically pure 2-R rimantadine to treat precancerous lesions associated with papilloma viruses, such as human papilloma virus (HPV).

[0019] Racemic rimantadine has side effects at currently prescribed doses, including central nervous system (CNS) side effects, sleep side effects, gastrointestinal side effects, and atropine side effects, including, but not limited to, lightheadedness, dizziness, depression, confusion, difficulty concentrating, anxiety (e.g., irritability), irritability, hallucinations, headache, insomnia, excessive fatigue, loss of appetite, nausea, vomiting, constipation, xerostomia, blurred vision, difficulty urinating, and difficulty swallowing. Anxiety and insomnia are the most commonly cited toxicity of racemic rimantadine as reasons for discontinuing treatment.

[0020] As disclosed herein, 2-S rimantadine inhibits the NR2B subunit of the N-methyl-D-aspartate (NMDA) subtype glutamate receptor subunit to a lesser extent than 2-R rimantadine and racemic rimantadine (see Table 2 in Example 2 below).

[0021] In some embodiments, the use of 2-S rimantadine to treat cancer, HPV-associated cancer, HPV-associated precancerous lesions, and / or influenza A is disclosed herein. Due to its lower ability to inhibit NR2B compared to racemic rimantadine, 2-S rimantadine may have fewer side effects compared to treating these diseases with racemic rimantadine or 2-R rimantadine. Due to its ability to inhibit NR2B to a greater extent than 2-S rimantadine, 2-R rimantadine may have fewer side effects compared to treating these diseases with racemic rimantadine or 2-S rimantadine.

[0022] In some embodiments, disclosed herein is the use of 2-R rimantadine to treat cancer, HPV-associated cancer, HPV-associated precancerous lesions, and / or influenza A. Due to its increased ability to inhibit NR2B compared to racemic rimantadine, 2-R rimantadine may have fewer side effects compared to treatment of these diseases with racemic or 2-S rimantadine.

[0023] In some embodiments, the 2-S rimantadine disclosed herein may have fewer side effects compared to treatment of these diseases with racemic or 2-R rimantadine because 2-S rimantadine has a reduced ability 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 less than about 10% to less than about 100% relative to 2-R or racemic rimantadine. In some embodiments, the degree of NMDA receptor inhibition caused by 2-S rimantadine relative to 2-R rimantadine or racemic rimantadine is less than about 10% to less than about 20%, less than about 10% to less than about 30%, less than about 10% to less than about 40%, less than about 10% to less than about 50%, less than about 10% to less than about 60%, less than about 10% to less than about 70%, less than about 10% to less than about 80%, less than about 10% to less than about Less than 90%, less than about 10% to less than about 100%, less than about 20% to less than about 30%, less than about 20% to less than about 40%, less than about 20% to less than about 50%, less than about 20% to less than about 60%, less than about 20% to less than about 70%, less than about 20% to less than about 80%, less than about 20% to less than about 90%, less than about 20% to less than about 100%, less than about 30% to less than about 40%, less than about 30% to less than about 50%, less than about 30% to less than about 60%, less than about 30 Less than 30% to less than about 70%, Less than about 30% to less than about 80%, Less than about 30% to less than about 90%, Less than about 30% to less than about 100%, Less than about 40% to less than about 50%, Less than about 40% to less than about 60%, Less than about 40% to less than about 70%, Less than about 40% to less than about 80%, Less than about 40% to less than about 90%, Less than about 40% to less than about 100%, Less than about 50% to less than about 60%, Less than about 50% to less than about 70%, Less than about 50% to less than about 80% less than about 50% to less than about 90%, less than about 50% to less than about 100%, less than about 60% to less than about 70%, less than about 60% to less than about 80%, less than about 60% to less than about 90%, less than about 60% to less than about 100%, less than about 70% to less than about 80%, less than about 70% to less than about 90%, less than about 70% to less than about 100%, less than about 80% to less than about 90%, less than about 80% to less than about 100%, or about 90% to less than about 100%.In some embodiments, the degree of NMDA receptor inhibition caused by 2-S rimantadine relative to 2-R rimantadine or racemic rimantadine is less than about 10%, less than about 20%, less than about 30%, less than about 40%, less than about 50%, less than about 60%, less than about 70%, less than about 80%, less than about 90%, or less than about 100%. In some embodiments, the degree of NMDA receptor inhibition caused by 2-S rimantadine relative to 2-R rimantadine or racemic rimantadine is at least less than about 10%, less than about 20%, less than about 30%, less than about 40%, less than about 50%, less than about 60%, less than about 70%, less than about 80%, or less than about 90%. In some embodiments, the degree of NMDA receptor inhibition caused by 2-S rimantadine is less than about 20%, less than about 30%, less than about 40%, less than about 50%, less than about 60%, less than about 70%, less than about 80%, less than about 90%, or less than about 100% compared to 2-R rimantadine or racemic rimantadine.In some embodiments, the NMDA receptor is NR2A.In some embodiments, the NMDA receptor is NR2B.

[0024] In some embodiments, the 2-R rimantadine disclosed herein may have fewer side effects compared to treatment of these diseases with racemic or 2-S rimantadine, since 2-R rimantadine has a reduced ability 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 less than about 10% to less than about 100% relative to 2-S or racemic rimantadine. In some embodiments, the degree of NMDA receptor inhibition caused by 2-R rimantadine relative to 2-S rimantadine or racemic rimantadine is less than about 10% to less than about 20%, less than about 10% to less than about 30%, less than about 10% to less than about 40%, less than about 10% to less than about 50%, less than about 10% to less than about 60%, less than about 10% to less than about 70%, less than about 10% to less than about 80%, less than about 10% to less than about Less than 90%, less than about 10% to less than about 100%, less than about 20% to less than about 30%, less than about 20% to less than about 40%, less than about 20% to less than about 50%, less than about 20% to less than about 60%, less than about 20% to less than about 70%, less than about 20% to less than about 80%, less than about 20% to less than about 90%, less than about 20% to less than about 100%, less than about 30% to less than about 40%, less than about 30% to less than about 50%, less than about 30% to less than about 60%, less than about 30 Less than 30% to less than about 70%, Less than about 30% to less than about 80%, Less than about 30% to less than about 90%, Less than about 30% to less than about 100%, Less than about 40% to less than about 50%, Less than about 40% to less than about 60%, Less than about 40% to less than about 70%, Less than about 40% to less than about 80%, Less than about 40% to less than about 90%, Less than about 40% to less than about 100%, Less than about 50% to less than about 60%, Less than about 50% to less than about 70%, Less than about 50% to less than about 80% less than about 50% to less than about 90%, less than about 50% to less than about 100%, less than about 60% to less than about 70%, less than about 60% to less than about 80%, less than about 60% to less than about 90%, less than about 60% to less than about 100%, less than about 70% to less than about 80%, less than about 70% to less than about 90%, less than about 70% to less than about 100%, less than about 80% to less than about 90%, less than about 80% to less than about 100%, or about 90% to less than about 100%.In some embodiments, the degree of NMDA receptor inhibition caused by 2-R rimantadine relative to 2-S rimantadine or racemic rimantadine is less than about 10%, less than about 20%, less than about 30%, less than about 40%, less than about 50%, less than about 60%, less than about 70%, less than about 80%, less than about 90%, or less than about 100%. In some embodiments, the degree of NMDA receptor inhibition caused by 2-R rimantadine relative to 2-S rimantadine or racemic rimantadine is at least less than about 10%, less than about 20%, less than about 30%, less than about 40%, less than about 50%, less than about 60%, less than about 70%, less than about 80%, or less than about 90%. In some embodiments, the degree of NMDA receptor inhibition caused by 2-R rimantadine is less than about 20%, less than about 30%, less than about 40%, less than about 50%, less than about 60%, less than about 70%, less than about 80%, less than about 90%, or less than about 100% compared to 2-S rimantadine or racemic rimantadine.In some embodiments, the NMDA receptor is NR2A.In some embodiments, the NMDA receptor is NR2B.

[0025] In some embodiments, the 2-S rimantadine disclosed herein may have fewer side effects compared to treatment of these diseases with racemic or 2-R rimantadine, since 2-S rimantadine has a reduced ability to antagonize GABA receptors and / or inhibit GABA-mediated biological pathways. In some embodiments, the degree of inhibition of GABA receptors and / or GABA-mediated biological pathways caused by 2-S rimantadine relative to 2-R or racemic rimantadine is less than about 10% to less than about 100%. In some embodiments, the degree of inhibition of GABA receptors and / or GABA-mediated biological pathways caused by 2-S rimantadine relative to 2-R rimantadine or racemic rimantadine is less than about 10% to less than about 20%, less than about 10% to less than about 30%, less than about 10% to less than about 40%, less than about 10% to less than about 50%, less than about 10% to less than about 60%, less than about 10% to less than about 70%, less than about 10% ~Less than about 80%, Less than about 10% to less than about 90%, Less than about 10% to less than about 100%, Less than about 20% to less than about 30%, Less than about 20% to less than about 40%, Less than about 20% to less than about 50%, Less than about 20% to less than about 60%, Less than about 20% to less than about 70%, Less than about 20% to less than about 80%, Less than about 20% to less than about 90%, Less than about 20% to less than about 100%, Less than about 30% to less than about 40%, Less than about 30% to less than about 50%, Less than about 30% ~ Less than about 60%, Less than about 30% to less than about 70%, Less than about 30% to less than about 80%, Less than about 30% to less than about 90%, Less than about 30% to less than about 100%, Less than about 40% to less than about 50%, Less than about 40% to less than about 60%, Less than about 40% to less than about 70%, Less than about 40% to less than about 80%, Less than about 40% to less than about 90%, Less than about 40% to less than about 100%, Less than about 50% to less than about 60%, Less than about 50% to less than about 70%, Less than about 50% to less than about less than 80%, less than about 50% to less than about 90%, less than about 50% to less than about 100%, less than about 60% to less than about 70%, less than about 60% to less than about 80%, less than about 60% to less than about 90%, less than about 60% to less than about 100%, less than about 70% to less than about 80%, less than about 70% to less than about 90%, less than about 70% to less than about 100%, less than about 80% to less than about 90%, less than about 80% to less than about 100%, or less than about 90% to less than about 100%.In some embodiments, the degree of inhibition of GABA receptors and / or GABA-mediated biological pathways caused by 2-S rimantadine relative to 2-R rimantadine or racemic rimantadine is less than about 10%, less than about 20%, less than about 30%, less than about 40%, less than about 50%, less than about 60%, less than about 70%, less than about 80%, less than about 90%, or less than about 100%. In some embodiments, the degree of inhibition of GABA receptors and / or GABA-mediated biological pathways caused by 2-S rimantadine relative to 2-R rimantadine or racemic rimantadine is at least less than about 10%, less than about 20%, less than about 30%, less than about 40%, less than about 50%, less than about 60%, less than about 70%, less than about 80%, or less than about 90%. In some embodiments, the degree of inhibition of GABA receptors and / or GABA-mediated biological pathways caused by 2-S rimantadine relative to 2-R rimantadine or racemic rimantadine is up to less than about 20%, less than about 30%, less than about 40%, less than about 50%, less than about 60%, less than about 70%, less than about 80%, less than about 90%, or less than about 100%.

[0026] In some embodiments, the 2-R rimantadine disclosed herein may have fewer side effects compared to treatment of these diseases with racemic rimantadine or 2-S rimantadine, since 2-R rimantadine has a reduced ability to antagonize GABA receptors and / or inhibit GABA-mediated biological pathways. In some embodiments, the degree of inhibition of GABA receptors and / or GABA-mediated biological pathways caused by 2-R rimantadine is less than about 10% to less than about 100% 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 relative to 2-S rimantadine or racemic rimantadine is less than about 10% to less than about 20%, less than about 10% to less than about 30%, less than about 10% to less than about 40%, less than about 10% to less than about 50%, less than about 10% to less than about 60%, less than about 10% to less than about 70%, less than about 10% ~Less than about 80%, Less than about 10% to less than about 90%, Less than about 10% to less than about 100%, Less than about 20% to less than about 30%, Less than about 20% to less than about 40%, Less than about 20% to less than about 50%, Less than about 20% to less than about 60%, Less than about 20% to less than about 70%, Less than about 20% to less than about 80%, Less than about 20% to less than about 90%, Less than about 20% to less than about 100%, Less than about 30% to less than about 40%, Less than about 30% to less than about 50%, Less than about 30% ~ Less than about 60%, Less than about 30% to less than about 70%, Less than about 30% to less than about 80%, Less than about 30% to less than about 90%, Less than about 30% to less than about 100%, Less than about 40% to less than about 50%, Less than about 40% to less than about 60%, Less than about 40% to less than about 70%, Less than about 40% to less than about 80%, Less than about 40% to less than about 90%, Less than about 40% to less than about 100%, Less than about 50% to less than about 60%, Less than about 50% to less than about 70%, Less than about 50% to less than about less than 80%, less than about 50% to less than about 90%, less than about 50% to less than about 100%, less than about 60% to less than about 70%, less than about 60% to less than about 80%, less than about 60% to less than about 90%, less than about 60% to less than about 100%, less than about 70% to less than about 80%, less than about 70% to less than about 90%, less than about 70% to less than about 100%, less than about 80% to less than about 90%, less than about 80% to less than about 100%, or less than about 90% to less than about 100%.In some embodiments, the degree of inhibition of GABA receptors and / or GABA-mediated biological pathways caused by 2-R rimantadine relative to 2-S or racemic rimantadine is less than about 10%, less than about 20%, less than about 30%, less than about 40%, less than about 50%, less than about 60%, less than about 70%, less than about 80%, less than about 90%, or less than about 100%. In some embodiments, the degree of inhibition of GABA receptors and / or GABA-mediated biological pathways caused by 2-R rimantadine relative to 2-S or racemic rimantadine is at least less than about 10%, less than about 20%, less than about 30%, less than about 40%, less than about 50%, less than about 60%, less than about 70%, less than about 80%, or less than about 90%. In some embodiments, the degree of inhibition of GABA receptors and / or GABA-mediated biological pathways caused by 2-R rimantadine relative to 2-S rimantadine or racemic rimantadine is up to less than about 20%, less than about 30%, less than about 40%, less than about 50%, less than about 60%, less than about 70%, less than about 80%, less than about 90%, or less than about 100%.

[0027] In some embodiments, the 2-S rimantadine disclosed herein may have fewer side effects compared to treatment of these diseases with racemic or 2-R rimantadine, since 2-S rimantadine has a reduced ability to antagonize dopamine receptors and / or inhibit dopamine-mediated biological pathways. In some embodiments, the degree of inhibition of dopamine receptors and / or dopamine-mediated biological pathways caused by 2-S rimantadine relative to 2-R or racemic rimantadine is from about 10% to about 100% less.In some embodiments, the degree of inhibition of dopamine receptors and / or dopamine-mediated biological pathways caused by 2-S rimantadine relative to 2-R rimantadine or racemic rimantadine is less than about 10% to less than about 20%, less than about 10% to less than about 30%, less than about 10% to less than about 40%, less than about 10% to less than about 50%, less than about 10% to less than about 60%, less than about 10% to less than about 70%, less than about 10% Less than about 80%, Less than about 10% to less than about 90%, Less than about 10% to less than about 100%, Less than about 20% to less than about 30%, Less than about 20% to less than about 40%, Less than about 20% to less than about 50%, Less than about 20% to less than about 60%, Less than about 20% to less than about 70%, Less than about 20% to less than about 80%, Less than about 20% to less than about 90%, Less than about 20% to less than about 100%, Less than about 30% to less than about 40%, Less than about 30% to less than about 50%, Less than about 30% ~ Less than about 60%, Less than about 30% to less than about 70%, Less than about 30% to less than about 80%, Less than about 30% to less than about 90%, Less than about 30% to less than about 100%, Less than about 40% to less than about 50%, Less than about 40% to less than about 60%, Less than about 40% to less than about 70%, Less than about 40% to less than about 80%, Less than about 40% to less than about 90%, Less than about 40% to less than about 100%, Less than about 50% to less than about 60%, Less than about 50% to less than about 70%, Less than about 50% ~ less than about 80%, less than about 50% to less than about 90%, less than about 50% to less than about 100%, less than about 60% to less than about 70%, less than about 60% to less than about 80%, less than about 60% to less than about 90%, less than about 60% to less than about 100%, less than about 70% to less than about 80%, less than about 70% to less than about 90%, less than about 70% to less than about 100%, less than about 80% to less than about 90%, less than about 80% to less than about 100%, or less than about 90% to less than about 100%. In some embodiments, the degree of inhibition of dopamine receptors and / or dopamine-mediated biological pathways caused by 2-S rimantadine relative to 2-R rimantadine or racemic rimantadine is less than about 10%, less than about 20%, less than about 30%, less than about 40%, less than about 50%, less than about 60%, less than about 70%, less than about 80%, less than about 90%, or less than about 100%.In some embodiments, the degree of inhibition of dopamine receptors and / or dopamine-mediated biological pathways caused by 2-S rimantadine, relative to 2-R rimantadine or racemic rimantadine, is at least about 10% or less, about 20% or less, about 30% or less, about 40% or less, about 50% or less, about 60% or less, about 70% or less, about 80% or less, or about 90% or less. In some embodiments, the degree of inhibition of dopamine receptors and / or dopamine-mediated biological pathways caused by 2-S rimantadine, relative to 2-R rimantadine or racemic rimantadine, is at most about 20% or less, about 30% or less, about 40% or less, about 50% or less, about 60% or less, about 70% or less, about 80% or less, about 90% or less, or about 100% or less. In some embodiments, the dopamine receptor is D. 2 / 3 It is a receptor.

[0028] In some embodiments, the 2-R rimantadine disclosed herein may have fewer side effects compared to treatment of these diseases with racemic rimantadine or 2-S rimantadine, since 2-R rimantadine has a reduced ability to antagonize dopamine receptors and / or inhibit dopamine-mediated biological pathways. In some embodiments, the degree of inhibition of dopamine receptors and / or dopamine-mediated biological pathways caused by 2-R rimantadine is about 10% to about 100% less than that caused by 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 relative to 2-S rimantadine or racemic rimantadine is less than about 10% to less than about 20%, less than about 10% to less than about 30%, less than about 10% to less than about 40%, less than about 10% to less than about 50%, less than about 10% to less than about 60%, less than about 10% to less than about 70%, less than about 10% Less than about 80%, Less than about 10% to less than about 90%, Less than about 10% to less than about 100%, Less than about 20% to less than about 30%, Less than about 20% to less than about 40%, Less than about 20% to less than about 50%, Less than about 20% to less than about 60%, Less than about 20% to less than about 70%, Less than about 20% to less than about 80%, Less than about 20% to less than about 90%, Less than about 20% to less than about 100%, Less than about 30% to less than about 40%, Less than about 30% to less than about 50%, Less than about 30% ~ Less than about 60%, Less than about 30% to less than about 70%, Less than about 30% to less than about 80%, Less than about 30% to less than about 90%, Less than about 30% to less than about 100%, Less than about 40% to less than about 50%, Less than about 40% to less than about 60%, Less than about 40% to less than about 70%, Less than about 40% to less than about 80%, Less than about 40% to less than about 90%, Less than about 40% to less than about 100%, Less than about 50% to less than about 60%, Less than about 50% to less than about 70%, Less than about 50% ~ less than about 80%, less than about 50% to less than about 90%, less than about 50% to less than about 100%, less than about 60% to less than about 70%, less than about 60% to less than about 80%, less than about 60% to less than about 90%, less than about 60% to less than about 100%, less than about 70% to less than about 80%, less than about 70% to less than about 90%, less than about 70% to less than about 100%, less than about 80% to less than about 90%, less than about 80% to less than about 100%, or less than about 90% to less than about 100%. In some embodiments, the degree of inhibition of dopamine receptors and / or dopamine-mediated biological pathways caused by 2-R rimantadine relative to 2-S rimantadine or racemic rimantadine is less than about 10%, less than about 20%, less than about 30%, less than about 40%, less than about 50%, less than about 60%, less than about 70%, less than about 80%, less than about 90%, or less than about 100%.In some embodiments, the degree of inhibition of dopamine receptors and / or dopamine-mediated biological pathways caused by 2-R rimantadine, relative to 2-S rimantadine or racemic rimantadine, is at least about 10% or less, about 20% or less, about 30% or less, about 40% or less, about 50% or less, about 60% or less, about 70% or less, about 80% or less, or about 90% or less. In some embodiments, the degree of inhibition of dopamine receptors and / or dopamine-mediated biological pathways caused by 2-R rimantadine, relative to 2-S rimantadine or racemic rimantadine, is at most about 20% or less, about 30% or less, about 40% or less, about 50% or less, about 60% or less, about 70% or less, about 80% or less, about 90% or less, or about 100% or less. In some embodiments, the dopamine receptor is D. 2 / 3 It is a receptor.

[0029] In another aspect, disclosed herein is the use of 2-S rimantadine for the treatment / prevention of influenza in veterinary animals, such as poultry (e.g., chickens, turkeys, and ducks). The use of 2-S rimantadine may have fewer side effects compared to the treatment of these animals with racemic rimantadine or 2-R rimantadine.

[0030] In another aspect, disclosed herein is the use of 2-R rimantadine for the treatment / prevention of influenza in veterinary animals, such as poultry (e.g., chickens, turkeys, and ducks). The use of 2-R rimantadine may have fewer side effects compared to the treatment of these animals with racemic rimantadine or 2-S rimantadine.

[0031] definition As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless expressly stated 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. Additionally, the headings provided herein are for convenience only and do not interpret the scope or meaning of the subject invention.

[0032] In some embodiments, the S-rimantadine, R-rimantadine, racemic rimantadine, or rimantadine derivatives as described herein are PEGylated. As used herein, "PEGylated" or "PEGylated" describes a conjugate of a compound having a polyethylene glycol (PEG) moiety. The PEG moiety can be of any length. For example, the PEG moiety can have 2 to 500 repeating units. In some embodiments, the PEG moiety can have an average molecular weight of about 300 g / mol to about 10,000,000 g / mol. In some embodiments, the PEG moiety can be a high molecular weight PEG or a low molecular weight PEG. For example, a high molecular weight PEG has a molecular weight of 5 kDa or more, and a low molecular weight PEG has a molecular weight of less than 5 kDa. In some embodiments, the PEG is selected from the group consisting of PEG200, PEG300, PEG400, PEG600, PEG800, PEG1000, PEG1500, PEG2000, and PEG3350. The PEG moiety can be a linear PEG, or the PEG moiety can be a branched PEG. For example, a branched PEG includes any PEG having one or more branches of PEG groups extending from the PEG backbone.

[0033] As used herein, the term "pure," when applied to a chiral compound, refers to an enantiomer of a chiral compound that is substantially free of its opposite enantiomer (i.e., in enantiomeric excess). For example, a pure "R" form of a compound is substantially free of the "S" form of the compound and is thus in enantiomeric excess of the "S" form. The term "enantiomerically pure" or "pure enantiomer" means that the compound contains an excess of the enantiomer, e.g., greater than 75%, greater than 80%, greater than 85%, greater than 90%, greater than 91%, greater than 92%, greater than 93%, greater than 94%, greater than 95%, greater than 96%, greater than 97%, greater than 98%, greater than 98.5%, greater than 99%, greater than 99.2%, greater than 99.5%, greater than 99.6%, greater than 99.7%, greater than 99.8%, 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 may be in excess of 30-80%, or 30-70%, 30-60%, 30%, 35%, 40%, 45%, 50%, 55%, or 60%, or any percentage therebetween.

[0034] As used herein, and unless otherwise specified, the term "enantiomerically pure 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.

[0035] As used herein, and unless otherwise specified, the term "enantiomerically pure 2-R rimantadine" refers, for example, to at least about 80% by weight of 2-R rimantadine and up to about 20% by weight of 2-S rimantadine, at least about 90% by weight of 2-R rimantadine and up to about 10% by weight of 2-S rimantadine, at least about 95% by weight of 2-R rimantadine and up to about 5% by weight of 2-S rimantadine, at least about 99% by weight of 2-R rimantadine and up to about 1% by weight of 2-S rimantadine, or at least about 99.9% by weight of 2-R rimantadine and up to about 0.1% by weight of 2-S rimantadine. In certain embodiments, the weights are based on the total weight of rimantadine, i.e., both or all enantiomers of rimantadine.

[0036] In the compositions provided herein, enantiomerically pure rimantadine or its pharma- ceutically acceptable salt, solvate, hydrate, ester, or prodrug can be present with other active or inactive ingredients.For example, a pharmaceutical composition comprising enantiomerically pure 2-S rimantadine can comprise, for example, about 90% excipient and about 10% enantiomerically pure 2-S rimantadine.In certain embodiments, the enantiomerically pure 2-S rimantadine in such compositions can comprise, for example, at least about 99.9% by weight of 2-S rimantadine and up to about 0.1% by weight of 2-S rimantadine.In certain embodiments, the active ingredient can be formulated with little or no carrier, excipient, or diluent.

[0037] As used herein, the terms "subject," "individual," or "patient" are used interchangeably and refer to any animal, including poultry such as chickens, ducks, turkeys, and mammals such as mice, rats, other rodents, rabbits, dogs, cats, pigs, cows, sheep, horses, primates, and humans. In some embodiments, the subject is a human.

[0038] As used herein, the term "treat" or "treatment" refers to curative or palliative measures. Beneficial or desired clinical results include, but are not limited to, the total or partial alleviation of symptoms associated with a disease or disorder or condition, whether detectable or undetectable, the reduction in the extent of the disease, a stable (i.e., not worsening) state of the disease, the delay or slowing of the progression of the disease, the improvement or alleviation of the disease state (e.g., one or more symptoms of the disease), and remission (whether complete or partial). "Treatment" can also mean extending survival time compared to the expected survival time in the absence of treatment.

[0039] As used herein, the term "prevent" means preventing, in whole or in part, the onset, recurrence, or spread of a disease or disorder as described herein or a symptom thereof.

[0040] The term "administration" or "administering" refers to a method of giving a dosage of a compound or pharmaceutical composition to a vertebrate or invertebrate, including a mammal, a bird, a fish, or an amphibian. The preferred method of administration may vary depending on various factors, such as the components of the pharmaceutical composition, the site of the disease, and the severity of the disease. A "therapeutically effective amount" or a "pharmaceutical effective amount" of a compound as provided herein is an amount sufficient to achieve a desired effect, which may vary depending on the nature and severity of the disease state and the potency of the compound. A therapeutic effect is some relief of one or more symptoms of the disease, which may include curing the disease.

[0041] "Cure" means that the symptoms of active disease are eliminated, however, certain long-term or permanent effects of the disease may exist even after a cure has been achieved (e.g., extensive tissue damage, etc.).

[0042] The term "effective amount" or "therapeutically effective amount" as used herein refers to a sufficient amount of an agent or compound being administered that alleviates to some extent one or more of the symptoms of the disease or condition being treated. The result may be a reduction and / or alleviation of the signs, symptoms, or causes of the disease, or any other desired change in a biological system. For example, an "effective amount" for therapeutic use is the amount of a composition containing a compound as disclosed herein that is required to clinically significantly reduce a disease symptom. An appropriate "effective" amount in any individual may be determined using techniques such as dose escalation studies. An "effective amount" is an amount sufficient for the compound to achieve a stated purpose (e.g., achieve the effect for which the compound is administered, treat a disease, reduce enzyme activity, increase enzyme activity, reduce signal transduction pathways, reduce one or more symptoms of a disease or condition) compared to the absence of the compound. An example of an "effective amount" is an amount sufficient to cause treatment, prevention, or reduction of a symptom of a disease, which may also be referred to as a "therapeutically effective amount". A "reduction" of a symptom (and grammatical equivalents of this phrase) refers to a decrease in the severity or frequency of a symptom, or the elimination of a symptom. A "prophylactically effective amount" of a drug is an amount of a drug that, when administered to a subject, has an intended prophylactic effect, such as preventing or delaying the onset (or recurrence) of an injury, disease, condition, or illness, or reducing the likelihood of the onset (or recurrence) of an injury, disease, condition, or illness, or symptoms thereof. A complete prophylactic effect does not necessarily occur by administration of a single dose, but may occur only after administration of a series of doses. Thus, a prophylactically effective amount may be administered in one or more administrations. An "activity-reducing amount" as used herein refers to the amount of antagonist required to reduce the activity of an enzyme compared to the absence of the antagonist. A "function-interfering amount" as used herein refers to the amount of antagonist required to interfere with the function of an enzyme or protein compared to the absence of the antagonist.The exact amount will depend on the purpose of the treatment and will be ascertainable by one of ordinary skill in the art using known techniques (see, e.g., Lieberman, Pharmaceutical Dosage Forms (vols. 1-3, 1992); Lloyd, The Art, Science and Technology of Pharmaceutical Compounding (1999); Pickar, Dosage Calculations (1999); and Remington: The Science and Practice of Pharmacy, 20th Edition, 2003, Gennaro, Ed., Lippincott, Williams & Wilkins).

[0043] The term "immunotherapeutic agent" refers to an agent that modulates the immune system. In some embodiments, an immunotherapeutic agent can increase the expression and / or activity of a regulator of the immune system. In some embodiments, an immunotherapeutic agent can decrease the expression and / or activity of a regulator of the immune system. In some embodiments, an immunotherapeutic agent can replenish and / or enhance the activity of immune cells.

[0044] Pharmaceutically acceptable salts, prodrugs, stereoisomers, and tautomers The pure R or S enantiomers of rimantadine provided herein can be administered as any salt or prodrug that can directly or indirectly provide the parent compound upon administration to a recipient, or that exhibits activity itself. As used herein, the term "pharmaceutical acceptable salt" refers to a salt that retains the desired biological activity of the subject compound and exhibits minimal undesirable toxicological effects. The phrase "pharmaceutical acceptable salt or prodrug" is used throughout the present specification to describe any pharmaceutical acceptable form of a compound (e.g., ester, amide, salt of ester, salt of amide, or related group) that provides an active compound of the present disclosure upon administration to a patient. Modifications such as these can affect the biological activity of the compound, in some cases increasing its activity over the parent compound. Pharmaceutically acceptable salts can be prepared in situ during the final isolation and purification of the compound, or by separately reacting the purified compound in its free acid or free base form with a suitable base or acid, respectively. In some embodiments, pharma- ceutically acceptable salts may be preferred over the respective free bases or free acids, since such salts provide greater stability or solubility to the molecules, thereby facilitating formulation into dosage forms.Basic compounds can generally form pharma-ceutically acceptable acid addition salts by treatment with a suitable acid.Suitable acids include pharma-ceutically acceptable inorganic acids and pharma-ceutically acceptable organic acids.Non-limiting examples of pharma- ceutically acceptable acid addition salts include hydrochloride, hydrobromide, nitrate, methylnitrate, sulfate, hydrogensulfate, sulfamate, phosphate, acetate, hydroxyacetate, phenylacetate, propionate, butyrate, isobutyrate, valerate, maleate, hydroxymaleate, acrylate, fumarate, malate, tartrate, citrate, salicylate, p-aminosalicylate, glycolate, lactate, heptanoate, phthalate, oxalate, succinate, benzoate, o-acetoxybenzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate, hydrochloride, benzoic acid ... Examples of suitable salts include hydroxybenzoate, methoxybenzoate, mandelate, tannate, formate, stearate, ascorbate, palmitate, oleate, pyruvate, pamoate, malonate, laurate, glutarate, glutamate, estolate, methanesulfonate (mesylate), ethanesulfonate (esylate), 2-hydroxyethanesulfonate, benzenesulfonate (besylate), p-aminobenzenesulfonate, p-toluenesulfonate (tosylate), naphthalene-2-sulfonate, ethanedisulfonate, and 2,5-dihydroxybenzoate.

[0045] Pharmaceutically acceptable prodrug refers to a compound that is metabolized (e.g., hydrolyzed or oxidized) in the host to form the compound of the present disclosure. Typical examples of prodrugs include compounds that have a biologically labile protecting group on the functional moiety of the active compound. Prodrugs include compounds that can be oxidized, reduced, aminated, deaminated, hydroxylated, dehydroxylated, hydrolyzed, dehydrolyzed, alkylated, dealkylated, acylated, deacylated, phosphorylated, and / or dephosphorylated to generate the active compound.

[0046] In some embodiments, the methods as described herein comprise administering pure 2-S rimantadine or pure 2-R rimantadine, or a pharma- ceutically acceptable salt thereof.

[0047] Pharmaceutical Compositions Also provided herein is a pharmaceutical composition comprising pure 2-S rimantadine, pure 2-R rimantadine, or a pharma- ceutically acceptable salt thereof, as described herein.Any of the pharmaceutical compositions described herein can be administered to a subject to treat cancer as described herein.

[0048] Administration of 2-S rimantadine, pure 2-R rimantadine, or a pharma- ceutically acceptable salt thereof, or a pharmaceutical composition thereof, may be oral, subcutaneous, intravenous, intranasal, topical, transdermal, intraperitoneal, intramuscular, intrapulmonary, intravaginal, intrarectal, ontological, neurological, intraocular, subconjunctival, via anterior chamber injection, intrathecal, intracystic, intrapleural, intraperitoneal, intrathecal, via wound irrigation, intrabuccal, intraperitoneal, intraarticular, intraaural, intrabronchial, intravesical, intravenous, intramuscular, intravenous ... It may be via any of the accepted modes of administration, including, but not limited to, intrathecal, via inhalation, via intratracheal or intrabronchial instillation, via direct instillation into the lung cavity, intraspinal, intrasynovial, intrathoracic, via thoracotomy lavage, epidural, intratympanic, intracisternal, intravascular, intraventricular, intraosseous, via lavage of infected bone, or via application as part of any mixture with a prosthetic device. In some embodiments, the method of administration comprises oral or parenteral administration.

[0049] 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 pharma- ceutically acceptable salt thereof, or a pharmaceutical composition thereof, may be administered in retained or controlled release dosage forms including depot injections, osmotic pumps, pills (tablets and / or capsules), transdermal (including electrotransport) patches, implants, and the like, for prolonged and / or periodic pulse administration at a pre-determined rate.

[0050] In some embodiments, the pharmaceutical composition is a tablet. In some embodiments, the pharmaceutical composition is a film-coated tablet.

[0051] Pure 2-S rimantadine, pure 2-R rimantadine, or a pharma- ceutically acceptable salt thereof, or a pharmaceutical composition thereof, can be administered alone or in combination with conventional pharmaceutical carriers, excipients, and the like. Pharmaceutically acceptable excipients include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, self-emulsifying drug delivery systems (SEDDS), such as d-α-tocopherol polyethylene glycol 1000 succinate, surfactants used in pharmaceutical dosage forms, such as Tween, poloxamer, or other similar polymeric delivery matrices, serum proteins such as human serum albumin, buffer substances, such as phosphates, 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 silica, magnesium trisilicate, polyvinylpyrrolidone, cellulose-based substances, polyethylene glycol, sodium sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, and wool fat.Cyclodextrins can also be used to enhance the delivery of the compounds described herein.

[0052] In some embodiments, the pharmaceutical compositions described herein are in the form of a unit dosage form, such as a pill or tablet, and thus the composition may contain, in addition to 2-S rimantadine, pure 2-R rimantadine, or a pharma- ceutically acceptable salt thereof, diluents such as lactose, sucrose, dicalcium phosphate, lubricants such as magnesium stearate, and binders such as starch, gum arabic, polyvinylpyrrolidine, gelatin, cellulose, cellulose derivatives, etc. In another solid dosage form, a powder, marume, solution, or suspension (e.g., in propylene carbonate, vegetable oil, PEG, poloxamer 124, or triglycerides) is encapsulated in a capsule (gelatin or cellulose-based capsule). Further contemplated are unit dosage forms in which the 2-S rimantadine, pure 2-R rimantadine, or a pharma- ceutically acceptable salt thereof provided herein, or additional active agents are physically separated, e.g., enteric coated or delayed release oral dosage forms such as capsules (or tablets within capsules) having granules of each drug, bilayer tablets, two-compartment gelatin capsules, and the like.

[0053] In some embodiments, the rimantadine or a pharma- ceutically acceptable salt thereof is PEGylated. In some embodiments, the PEGylated rimantadine or a pharma- ceutically acceptable salt thereof comprises a high molecular weight PEG. In some embodiments, the PEGylated rimantadine or a pharma- ceutically acceptable salt thereof comprises a low molecular weight PEG. In some embodiments, the rimantadine or a pharma- ceutically acceptable salt thereof is modified. In some embodiments, the modification is PEGylation.

[0054] In some embodiments, the PEGylated rimantadine or its pharmaceutically acceptable salt is PEGylated with high molecular weight PEG.In some embodiments, the PEGylated rimantadine or its pharmaceutically acceptable salt is PEGylated with low molecular weight PEG.Therefore, also provided herein is a method for treating cancer in a subject in need of cancer treatment, comprising administering a therapeutically effective amount of PEGylated rimantadine or its pharmaceutically acceptable salt to the subject.

[0055] In some embodiments, the pharmaceutical composition comprises an excipient selected from the group consisting of hypromellose, magnesium stearate, microcrystalline 5 cellulose, and sodium starch glycolate.

[0056] Pharmaceutically administrable liquid compositions can be prepared, for example, by dissolving and dispersing the compounds described herein and any pharmaceutical adjuvants in carriers (e.g., water, saline, aqueous dextrose, glycerin, glycols, ethanol, etc.) to form solutions, colloids, liposomes, emulsions, complexes, coacervates, or suspensions. If necessary, the pharmaceutical compositions can also contain small amounts of nontoxic auxiliary substances such as wetting agents, emulsifiers, cosolvents, solubilizing agents, pH buffers, and the like (e.g., sodium acetate, sodium citrate, cyclodextrin derivatives, sorbitan monolaurate, triethanolamine acetate, triethanolamine oleate, and the like).

[0057] Dosage forms or compositions may be prepared that contain 2-S rimantadine, pure 2-R rimantadine, or a pharma- ceutically acceptable salt thereof as described herein in the range of 0.005%-100%, with the remainder consisting of non-toxic carriers. Contemplated compositions may contain 0.001%-100%, in one embodiment 0.1-95%, in another embodiment 75-85%, and in a further embodiment 20-80% of the compounds provided herein. Actual methods of preparing such dosage forms are known or will be apparent to those skilled in the art. See, for example, Remington: The Science and Practice of Pharmacy, 22nd Edition (Pharmaceutical Press, London, UK. 2012).

[0058] The pharmaceutical compositions herein may contain about 0.1-1000 mg of 2-S rimantadine, pure 2-R, rimantadine, or a pharma- ceutically acceptable salt thereof per unit dose, e.g., tablet, capsule, suspension, solution, sachet for reconstitution, powder, injection, IV, suppository, sublingual / buccal film, teaspoon, etc. Pure 2-S rimantadine, pure 2-R rimantadine, or a pharma- ceutically acceptable salt thereof may be given 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 about 25 mg to about 500 mg of a compound provided herein per unit dose (e.g., about 25 mg to about 400 mg, about 25 mg to about 300 mg, about 25 mg to about 250 mg, about 25 mg to about 200 mg, about 25 mg to about 150 mg, about 25 mg to about 100 mg, about 25 mg to about 75 mg, about 50 mg to about 500 mg, about 100 mg to about 500 mg, about 150 mg to about 500 mg, about 200 mg to about 500 mg, about 250 mg to about 500 mg, about 300 mg to about 500 mg, about 400 mg to about 500 mg, about 50 to about 200 mg, about 100 to about 250 mg, about 50 to about 150 mg). In some embodiments, the pharmaceutical compositions provided herein contain about 25 mg, about 50 mg, about 100 mg, about 150 mg, about 200 mg, about 250 mg, about 300 mg, about 400 mg, or about 500 mg of a compound provided herein per unit dose. However, the dosage may vary depending on the patient's requirements, the severity of the disease being treated, and the compound being used. In some embodiments, the dosage is administered once a day (QD) or twice a day (BID).

[0059] In some embodiments, the disclosure includes compositions comprising pure 2-R rimantadine or a pharma- ceutically acceptable salt thereof, pure 2-S rimantadine or a pharma- ceutically acceptable salt thereof.

[0060] Treatment Method Also provided herein is a method for treating cancer in a subject. In some embodiments, the method comprises administering to a subject a therapeutically effective amount of one or more of the pharmaceutical compositions described herein. In some embodiments, the pharmaceutical composition comprises enantiomerically pure 2-S rimantadine or any of its pharmaceutically acceptable salts. In some embodiments, the pharmaceutical composition comprises enantiomerically pure 2-R rimantadine or any of its pharmaceutically acceptable salts. In some embodiments, the pharmaceutically acceptable salt is hydrochloride.

[0061] In some embodiments, the cancer is sarcoma, carcinoma, melanoma, lymphoma, or leukemia.Non-limiting examples of sarcoma include bone sarcoma (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, 5 gastrointestinal stromal tumor (GIST), Kaposi's sarcoma, leiomyosarcoma, liposarcoma, rhabdomyosarcoma, and Ewing's sarcoma of soft tissue).Non-limiting examples of carcinoma include basal cell carcinoma, squamous cell carcinoma, renal cell carcinoma, invasive ductal carcinoma, hepatocellular carcinoma, and adenocarcinoma. Non-limiting examples of lymphomas include non-Hodgkin's lymphomas (e.g., B-cell lymphoma, T-cell lymphoma, Burkitt's lymphoma, follicular lymphoma, mantle cell lymphoma, primary mediastinal B-cell lymphoma, small lymphocytic lymphoma, Waldenstrom's macroglobulinemia), and Hodgkin's lymphomas (e.g., lymphocyte-depleted Hodgkin's disease, lymphocyte-rich Hodgkin's disease, mixed cellularity Hodgkin's lymphoma, nodular lymphocyte-predominant Hodgkin's disease, and nodular sclerosing Hodgkin's lymphoma). Non-limiting examples of leukemias include acute hairy cell leukemia, acute lymphocytic leukemia, acute myeloid leukemia, acute promyelocytic leukemia, chronic lymphocytic leukemia, chronic myeloid leukemia, myeloproliferative neoplasms, and systemic mastocytosis.

[0062] 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.

[0063] In some embodiments, the cancer is a carcinoma. In some embodiments, the carcinoma is selected from the group consisting of adenocarcinoma, squamous cell carcinoma, transitional cell carcinoma, hepatocellular carcinoma, and clear cell carcinoma. In some embodiments, the cancer is squamous cell carcinoma. In some embodiments, the squamous cell carcinoma is head and neck squamous cell carcinoma. In some embodiments, the cancer is hepatocellular carcinoma.

[0064] In some embodiments, the cancer is selected from the group consisting of head and neck cancer, breast cancer, and melanoma.

[0065] In some embodiments, pure 2-S rimantadine or pure 2-R rimantadine, or a pharma- ceutically acceptable salt thereof, can be used to treat a subject's Hepatitis B Virus (HBV)-associated cancer, as described herein. As used herein, an "HBV-associated cancer" is a cancer in which one or more cancer cells express at least one HBV protein (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 Mar;18(3):375-389. doi:10.1158 / 1541-7786. MCR-19-0463. Epub 2019 Dec 2. PMID:31792079.). For example, one or more cancer cells can express an HBV oncoprotein. In some embodiments, the HBV-associated cancer is liver cancer (e.g., hepatocellular carcinoma).In some embodiments, the HBV-associated cancer is cervical cancer.

[0066] In some embodiments, pure 2-S rimantadine or pure 2-R rimantadine, or a pharma- ceutically acceptable salt thereof, can be used to treat a human papillomavirus (HPV)-associated cancer in a subject, as described herein. As used herein, an "HPV-associated cancer" is a cancer in which one or more cancer cells express at least one HPV protein. For example, one or more of the cancer cells can express HPV oncoproteins. Human papillomavirus (HPV) can cause malignant transformation, for example, by targeting p53 and Rb, which are important tumor suppressors (see, for example, 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 to evade immune responses (see, e.g., Senba.Oncol Rev.5 2012 Oct 5;6(2):e17). For example, HPV genes and HPV proteins can target antigen processing and antigen presentation, which are necessary for effective adaptive immune responses (see, e.g., Senba.Oncol Rev.2012 Oct 5;6(2):e17;and O'Brien and Saveria Campo.Virus Res.2002 Sep;88(1-2):103-17). There are many HPV oncoproteins, including but not limited to HPV16 E5, E6, and E7. For example, HPV E5 is a protein that has been reported to have multiple functions, including regulation of tumor cell differentiation and apoptosis, regulation of H+ ATPase, which is involved in 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).

[0067] In some embodiments, one or more cancer cells from the 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 one or more HPV subtypes selected from the group consisting of HPV6, HPV11, HPV16, HPV18, HPV31, HPV33, HPV35, HPV39, HPV45, HPV51, HPV52, HPV56, HPV58, HPV66, and HPV69. In some embodiments, the HPV protein is HPV16 E5. In some embodiments, the subject has a cancer selected from the group consisting of head and neck cancer, mucosal squamous cell carcinoma, cutaneous squamous cell carcinoma, cervical cancer, vaginal cancer, vulvar cancer, penile cancer, and anal cancer.

[0068] In some embodiments, the cancer is an HPV-associated cancer. In some embodiments, the HPV-associated cancer is HPV-associated head and neck squamous cell carcinoma (HNSCC).

[0069] In some embodiments, pure 2-S rimantadine or pure 2-R rimantadine, or a pharma- ceutically acceptable salt thereof, as described herein, may be used to treat a variety of conditions, including, but not limited to, vegetative verrucous leukoplakia (PVl), oral leukoplakia, nicotinic palatitis in back smokers, oral erythroplakia, laryngeal keratosis, actinic cheilitis, smooth thick leukoplakia, smooth red tongue due to Plummer-Vinson syndrome, smokeless tobacco keratosis, oral submucous fibrosis syndrome, erythroleukoplakia, granular leukoplakia, oral lichen planus (erosive type), smooth thin leukoplakia, nicotinic stomatitis, and tobacco pouch keratosis. It can be used to treat human papillomavirus precancerous lesions associated with the oral cavity, such as the cervix (cervical keratosis), cervix (cervical dysplasia), and penile intraepithelial neoplasia (PeIN lesions). In the oral cavity, 24 types of HPV (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 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 are caused by HPV16, 18, and 6 / 11. HPV types associated with cervical oncogenicity include 15 high-risk types (HPV16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68, 73, and 82) and three "potential high-risk types" (HPV26, 53, and 66).

[0070] Tests for HPV known in the art include, e.g., Coultlee, F., et. al., 2005, Can J Infect Dis Med Microbiol 16(2):83-91; careHPV Test Kit (QIAGEN, Redwood City, CA); Tang, KD, 2019, Unlocking the Potential of Saliva-Based Test to Detect HPV-16-Driven Oropharyngeal Cancer, Cancers (Basel), 11(4):473; HPV probes (BIOCARE MEDICAL, Pacheo, CA).

[0071] In some embodiments of the methods described herein, pure 2-S rimantadine or pure 2-R rimantadine, or a pharma- ceutically acceptable salt thereof, is administered in combination with a therapeutically effective amount of at least one additional therapeutic agent selected from one or more additional anti-cancer or therapeutic agents (e.g., chemotherapeutic agents). It is a common practice in oncology to use a combination of various forms of treatment to treat subjects with cancer. In addition to the 2-S rimantadine described herein, or a pharma-ceutically acceptable salt thereof, these other forms of combination treatment or therapy may include, for example, surgery, radiation therapy, and additional anti-cancer agents, such as kinase inhibitors, signal transduction inhibitors, platinum-based chemotherapy, and / or monoclonal antibodies. In some embodiments, the methods further include administering an additional anti-cancer agent.

[0072] Non-limiting examples of additional anti-cancer agents include carboplatin, cisplatin, gemcitabine, methotrexate, paclitaxel, pemetrexed, lomustine, temozolomide, and dacarbazine.

[0073] In some embodiments, the additional anticancer agent is an immunotherapy agent.Many types of immunotherapy agents can be used in combination with the pure 2-S rimantadine or pure 2-R rimantadine or its pharma- ceutically acceptable salts described herein.Non-limiting examples of immunotherapy agents include immune checkpoint inhibitors, antibody therapy, cellular immunotherapy, antibody-drug conjugates, cytokine therapy, mRNA-based immunotherapy, and cancer vaccines.

[0074] In some embodiments, the immunotherapeutic agent is one or more immune checkpoint inhibitors. In some embodiments, the immune checkpoint inhibitor targets one or more of CTLA-4, PD-1, PD-L1, BTLA, LAG-3, A2AR, TIM-3, B7-H3, VISTA, and IDO. In some embodiments, the checkpoint inhibitor is selected from the group consisting of ipilimumab, nivolumab, pembrolizumab, atezolizumab, avelumab, durvalumab, cemiplimab, tremelimumab, and combinations thereof.

[0075] In some embodiments, the immune checkpoint inhibitor is a CTLA-4 inhibitor, a PD-1 inhibitor, or a PD-L1 inhibitor. In some embodiments, the CTLA-4 inhibitor is ipilimumab (YERVOY®) or tremelimumab (CP-675,206). In some embodiments, the PD-1 inhibitor is pembrolizumab (KEYTRUDA®), cemiplimab (LIBTAYO®), or nivolumab (OPDIVO®). In some embodiments, the PD-L1 inhibitor is atezolizumab (TECENTRIQ®), avelumab (BAVENCIO®), or durvalumab (IMFINZI™).

[0076] In some embodiments, the antibody therapy is bevacizumab (MVASTI™, AVASTIN®), trastuzumab (HERCEPTIN®), avelumab (BAVENCIO®), rituximab (MABTHERA™, RITUXAN®), edrecolomab (Panorex), daratumumab (DARZALEX®), olaratumab (LARTRUVO™), ofatumumab (ARZERRA®), alemtuzumab (CAMPATH®), cetuximab (ERBITUX®), oregovomab, pembrolizumab (KEYTRUDA®), dinutuximab (UNITUXIN®), obinutuzumab (GAZYVA®), orthopedic amiodarone (THX ... )), tremelimumab (CP-675,206), ramucirumab (CYRAMZA®), ublituximab (TG-1101), panitumumab (VECTIBIX®), elotuzumab (EMPLICITI™), avelumab (BAVENCIO®), necitumumab (PORTRAZZA™), cirumutuzumab (UC-961), ipilimumab (Juventus®), rituximab ... Britumomab (ZEVALIN®), isatuximab (SAR650984), nimotuzumab, fresolimumab (GC1008), lirilumab (INN), 5-mogamulizumab (POTELIGEO®), ficlatuzumab (AV-299), denosumab (XGEVA®), ganitumab, urelumab, pidilizumab, or amatuximab.

[0077] In some embodiments, the immunotherapeutic agent is a cellular immunotherapeutic agent (eg, adoptive T cell therapy, dendritic cell therapy, natural killer cell therapy).

[0078] In some embodiments, the immunotherapeutic agent is an antibody-drug conjugate. In some embodiments, the antibody-drug conjugate is gemtuzumab ozogamicin (MYLOTARG™), inotuzumab ozogamicin (BESPONSA®), brentuximab vedotin (ADCETRIS®), adotrastuzumab emtansine (TDM-1; KADCYLA®), moxetumomab pasudotox (LUMOXITI®), polatuzumab vedotin-piiq (POLIVY®), mirvetuximab soravtansine (IMGN853), or anetumab ravtansine.

[0079] In some embodiments, the immunotherapy is a cytokine therapy. In some embodiments, the cytokine therapy is interleukin 2 (IL-2) therapy, interleukin (IL-15) therapy, interleukin 7 (IL-7) therapy, interferon alpha (IFNα) therapy, agranulocyte colony stimulating factor (G-CSF) therapy, interleukin 12 (IL-12) therapy, or erythropoietin-alpha (EPO) therapy. In some embodiments, the IL-2 therapy is aldesleukin (Proleukin®). In some embodiments, the IFNα therapeutic agent is interferon α-2b (e.g., IntronA®) or interferon α-2a (e.g., Roferon-A®). In some embodiments, the G-CSF therapeutic agent is filgrastim (Neupogen®).

[0080] In some embodiments, the immunotherapeutic agent is an mRNA-based immunotherapeutic agent. In some embodiments, the mRNA-based immunotherapeutic agent is CV9104 (see, for example, Rausch et al. (2014) Human Vaccin Immunother 10(11):3146-52; and Kubler et al. (2015) J. Immunother Cancer 3:26). Also see Pardi et al. Nat Rev Drug Discov. 2018 Apr;17(4):261-279, which is incorporated herein by reference in its entirety.

[0081] In some embodiments, the method includes subjecting the subject to radiation therapy, surgery, or a combination thereof. For example, the surgery can be open or minimally invasive surgery.

[0082] In some embodiments, the subject is refractory to standard of care (e.g., standard of care). In some embodiments, the subject has no standard of care options. In some embodiments, the subject has relapsed or progressed after standard of care. In some embodiments, the methods provided herein are useful for treating locally advanced or metastatic solid tumors that are refractory to standard of care. For example, HPV-associated cancers can be refractory to immune checkpoint inhibitors, such as those described herein.

[0083] In some embodiments, the subject has a cancer that is refractory or intolerant to a standard of care treatment (e.g., administration of a chemotherapeutic agent, an immunotherapeutic agent, or radiation). In some embodiments, the subject has a cancer (e.g., a locally advanced or metastatic tumor) that is refractory or intolerant to a prior therapy (e.g., administration of a chemotherapeutic agent, an immunotherapeutic agent (e.g., an immune checkpoint inhibitor), or radiation). In some embodiments, the cancer that is refractory or intolerant to a standard of care treatment is an HPV-associated cancer. In some embodiments, the subject has a cancer (e.g., a locally advanced or metastatic tumor) for which there is no standard of care treatment.

[0084] In some embodiments, the subject has undergone a prior therapy, hi some embodiments, the subject has undergone treatment with a platinum-based chemotherapeutic agent, an immune checkpoint inhibitor (e.g., a PD-1 / PDL1 immunotherapeutic agent), radiation therapy, or a combination thereof, prior to treatment with 2-S rimantadine or a pharma- ceutical acceptable salt thereof.

[0085] The optimal amount of pure 2-S rimantadine or pure 2-R rimantadine, or a pharma- ceutically acceptable salt thereof, administered to a subject can be determined by one of skill in the art and will vary with the mode of administration, the strength of the preparation, the mode of administration, and the progression of the disease state. In some embodiments, a subject may be administered a dosage of about 0.01 to 10,000 mg of 2-S rimantadine or a pharma- ceutically acceptable salt thereof per day per 25-year-old adult. For example, a pharmaceutical composition comprising pure 2-S rimantadine, or pure 2-R rimantadine, or racemic rimantadine, or a pharma- ceutically acceptable salt thereof, may be formulated to provide a dosage of about 0.01, about 0.05, about 0.1, about 0.5, about 1, about 2.5, about 5, about 10, about 15, about 25, about 50, about 100, about 150, about 200, about 250, or about 500 milligrams of rimantadine or a pharma- ceutically acceptable salt thereof. In some embodiments, an effective amount of pure 2-S rimantadine, pure 2-R rimantadine, or a pharma- ceutically acceptable salt thereof may be provided at a dosage level of about 0.1 mg / kg to about 1000 mg / kg of body weight per day, or any range therein. For example, about 0.5 to about 500 mg / kg body weight per day, about 1.0 to about 250 mg / kg body weight per day, about 0.1 to about 100 mg / kg body weight per day, 0.1 to about 50.0 mg / kg body weight per day, 15.0 mg / kg body weight per day, or about 0.5 to about 7.5 mg / kg body weight per day. Pure 2-S rimantadine, or pure 2-R rimantadine, or a pharma- ceutically acceptable salt thereof, may be administered to a subject on a regimen of 1 to 5 times per day, or in a single daily dose.

[0086] In one embodiment, the compounds described herein are used to prepare a medicament for the treatment of the diseases or conditions described herein.In addition, the method for treating any of the diseases or conditions described herein in a subject in need of such treatment comprises administering to the subject a pharmaceutical composition comprising at least one compound described herein, or its pharmaceutically acceptable salt, active metabolite, prodrug, or solvate, in a therapeutically effective amount.

[0087] In certain embodiments, compositions containing the compounds described herein are administered for prophylactic and / or therapeutic treatment. In certain therapeutic applications, the compositions are administered to a patient already suffering 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. Amounts effective for this use will depend on the severity and course of the disease or condition, previous treatments, the patient's health status, weight, and response to drugs, and the judgment of the treating physician. Therapeutically effective amounts are optionally determined by methods including, but not limited to, dose escalation clinical trials.

[0088] In prophylactic applications, compositions containing the compounds described herein are administered to a patient susceptible to or at risk of a particular disease, disorder, or condition.

[0089] In certain embodiments, the dose of a drug being administered may be temporarily reduced or temporarily discontinued for a particular period of time (ie, a "drug holiday").

[0090] How HPV is detected Another aspect of the present disclosure includes a method of treating cancer in a subject, comprising detecting cancer cells expressing an HPV protein in a sample from the subject, and then administering a therapeutically effective amount of any one of the pharmaceutical compositions described herein to the subject. The detection method described herein is based on determining the presence or absence of an HPV protein or a functionally equivalent variant thereof. In some embodiments in which the presence of an HPV protein or a functionally equivalent variant thereof is detected in a sample from the subject, the expression level of the HPV protein is determined. In some embodiments, the HPV protein is HPV16 E5. In some embodiments, the pharmaceutical composition comprises at least one additional therapeutic agent selected from one or more additional anti-cancer therapeutic agents or therapeutic agents (e.g., chemotherapeutic agents).

[0091] Thus, 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 the predisposition of a subject to suffer from a disease associated with the presence of the above-mentioned HPV protein in a subject, or for determining the stage or severity of a disease associated with the presence of the above-mentioned HPV protein in a subject, or for monitoring the effect of a treatment administered to a subject having a disease associated with the presence of the above-mentioned HPV protein, said method comprising a step of quantifying the expression level of the HPV protein or a functionally equivalent variant thereof in a biological sample from the subject, wherein an increase in the expression of a gene encoding the HPV protein or a functionally equivalent variant thereof, relative to the expression level of a gene encoding the HPV protein or a functionally equivalent variant thereof in a control sample, indicates a greater predisposition of the subject to suffer from a disease associated with the presence of the HPV protein in a subject, or a disease associated with the presence of the above-mentioned HPV protein in a subject, or a non-response to a treatment administered to the subject. In some embodiments, the HPV protein is HPV16 E5. In some embodiments, the pharmaceutical composition comprises at least one additional therapeutic agent selected from one or more additional anti-cancer therapeutic agents or therapeutic agents (e.g., chemotherapeutic agents).

[0092] Therefore, when the term "functionally equivalent variant" is used herein, it also includes any functionally equivalent fragment of the above-mentioned marker protein. The term "fragment" refers to a peptide that contains a part of the above-mentioned marker protein. In this case, a functionally equivalent fragment is a peptide or protein that contains a part of the above-mentioned marker protein and has essentially the same function as the above-mentioned protein. "Marker protein" preferably refers to, but is not limited to, HPV protein.

[0093] As will be appreciated by those skilled in the art, the detection step is usually accurate but may not be accurate for 100% of subjects. However, the term requires that a statistically significant portion of subjects can be identified as having a sufficient amount of the protein of interest such that the subjects suffer from a disease associated with the presence of the protein of interest or have a predisposition to the same. By simply using one or more well-known statistical evaluation tools, such as determining a confidence interval, determining a p-value, Student's t-test, Mann-Whitney test, etc., the skilled artisan can determine whether a portion is statistically significant. Details are described in Dowdy and Wearden, Statistics for Research, John Wiley & Sons, New York 1983. Preferred confidence intervals are at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%. The p-value is preferably 0.2, 0.1, 0.05.

[0094] As used herein, the term "predisposition" means that a subject has not yet developed a disease or any of the symptoms of the disease described above, or other diagnostic criteria, but will develop the disease in the future with a certain probability. The above-mentioned probability is significantly different from the statistical probability of developing a disease associated with the presence of HPV proteins. Preferably, the probability of developing a disease associated with the presence of HPV proteins is diagnosed as at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or 100% of predisposition. Diagnosis of predisposition may be referred to as prognosis or prediction of the probability of a subject developing the disease.

[0095] In the context of this disclosure, "control sample" is understood as a reference sample used to determine the variation in the expression level of genes and proteins used in this disclosure.In one embodiment, the reference value is obtained from the signal provided using a tissue sample obtained from a healthy individual.Preferably, the samples are taken from the same tissue of multiple healthy individuals and are combined so that the amount of polypeptide in the sample reflects the average value of the above-mentioned molecules in the population.

[0096] Thus, in certain embodiments of the present disclosure, the amount of expression level of an HPV protein can be quantified. In some embodiments, the HPV protein is HPV16 E5.

[0097] As will be understood by those skilled in the art, the expression level of a protein can be quantified by any conventional method. As a non-limiting example, the level of a protein can be quantified, for example, by using an antibody capable of binding to the above-mentioned protein (or its fragment containing an antigenic determinant) and then quantifying the complex formed. The antibodies used in these assays can be labeled or unlabeled. 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 wide variety of known assays that can be used in the present disclosure using unlabeled antibodies (primary antibodies) and labeled antibodies (secondary antibodies), including Western blots, ELISA (enzyme-linked immunosorbent assays), RIA (radioimmunoassays), competitive EIA (competitive enzyme-linked immunosorbent assays), DAS-ELISA (double antibody sandwich ELISA), immunocytochemical and immunohistochemical techniques, techniques based on the use of biochips or microarrays of proteins containing specific antibodies, or colloidal precipitation-based assays in formats such as dipsticks. In another specific embodiment, quantification of protein levels is performed by an immunoanalytical method, such as Western blot, immunohistochemistry, or ELISA. In some embodiments, the immunoanalytical method comprises an antibody specific for HPV16 E5.

[0098] Similarly, the detection method of the present disclosure is applicable to any disease associated with the presence of HPV protein defined above.In a preferred embodiment, the disease associated with the presence of HPV protein is cancer, preferably cancer with high level of HPV protein.In some embodiments, the HPV protein is HPV16 E5.

[0099] The implementation of the method of the present disclosure includes obtaining a biological sample from the subject to be tested.The illustrative and non-limiting examples of the aforementioned samples include different types of body fluids, such as blood, serum, plasma, cerebrospinal fluid, ascites, feces, urine, and saliva, as well as tissue samples.The body fluid samples can be obtained by any conventional method, as with the tissue samples, and as an example, the aforementioned tissue samples can be biopsy samples obtained by surgical resection.

[0100] 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 a cancer in a subject, or for determining a predisposition of a subject to suffer from the above-mentioned cancer, or for determining the stage or severity of the above-mentioned cancer in a subject, or for monitoring the effect of a treatment administered to a subject having the above-mentioned cancer, and if the reagent detects an increase in the expression of the above-mentioned gene or protein or a functionally equivalent variant thereof with respect to a control sample, the above-mentioned subject may subsequently suffer from a disease associated with the presence of the HPV protein, may be more predisposed to suffer from a disease associated with the presence of the above-mentioned HPV protein, may be more severely affected by the above-mentioned disease, or the administered treatment 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 therapeutic agents or therapeutic agents (e.g., chemotherapeutic agents).

[0101] The present disclosure further relates to the use of the above-mentioned kit.

[0102] All terms and expressions used in the definition of the use of the kit are described above and explained for other inventive aspects and specific embodiments of the present disclosure, and are also applicable to the use of the kit described herein.

[0103] Methods for designing customized treatments and for selecting patients who may benefit from administration of 2-S or 2-R rimantadine In another aspect, the present disclosure provides a method for producing a method for manufacturing a pharmaceutical composition comprising: (a) quantifying the expression level of HPV proteins in said patient; (b) comparing said expression level with a control level; In accordance with the present invention, there is provided an in vitro method for designing a customized treatment for a patient suffering from a disease associated with the presence of an HPV protein, comprising: if the expression level of the HPV protein in said patient is greater than a control value, then a therapeutically effective amount of 2-S rimantadine or a pharma- ceutical acceptable salt thereof or 2-R rimantadine or a pharma-ceutical acceptable salt thereof is administered to said patient.

[0104] 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 therapeutic agents or agents (e.g., chemotherapeutic agents) is administered to the patient.

[0105] In another aspect, the present disclosure provides a method for producing a method for manufacturing a pharmaceutical composition comprising: a) quantifying the expression level of HPV proteins in said patient; b) comparing said expression levels with a control level; The present invention relates to an in vitro method for selecting a patient suffering from a disease associated with the presence of an HPV protein to be treated with a therapeutically effective amount of 2-S rimantadine or a pharma- ceutical acceptable salt thereof, or 2-R rimantadine or a pharma-ceutical acceptable salt thereof, comprising: if the expression level of the HPV protein in said patient is greater than a control value, said patient is selected to receive a therapeutically effective amount of 2-S rimantadine or a pharma-ceutical acceptable salt thereof, or 2-R rimantadine or a pharma-ceutical acceptable salt thereof.

[0106] 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 therapeutic agents or agents (e.g., chemotherapeutic agents) is administered to the patient. EXAMPLES

[0107] Example 1. ScreenPatch® Assay for NR1 / NR2A and NR1 / NR2B ScreenPatch ·NR1 / NR2A ionotropic receptors encoded by the human GRIN1 / GRIN2A genes expressed in HEK293 cells ·NR1 / NR2B ionotropic receptors encoded by the human GRIN1 / GRIN2B genes expressed in HEK293 cells

[0108] Formulation Solutions of 2-S rimantadine, 2-R rimantadine, racemic rimantadine, and amantadine were prepared daily by diluting stock solutions into the appropriate HEPES-buffered saline (HB-PS) solutions. All test and control solutions contained 0.6% DMSO, as this has no effect on channel currents. Test article formulations were sonicated (model 2510 / 5510, Branson Ultrasonics, Danbury, CT) for at least 20 minutes at room temperature to facilitate dissolution.

[0109] 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 formulations were loaded into 384-well compound plates using an automated liquid handling system (Assist Plus, INTEGRA).

[0110] Positive control treatment group Stock solutions of the positive control articles were prepared in batches, aliquoted for individual use, stored frozen, and used within 6 months. Positive control test solutions were prepared fresh daily. The final DMSO concentration was 0.6% (v / v). NMDA receptor agonists, L-glutamic acid, and glycine were used as reference agonists in this study. The NMDA receptor antagonist (amantadine) was used as the reference antagonist in this study.

[0111] Tests and Concentrations Test substances were evaluated for their functional effects on ion channels. The test concentrations are shown in Table 1 below.

[0112] [Table 1]

[0113] Cloning Test System Cells were maintained in a tissue culture incubator. Stocks were maintained in cryogenic storage. Cells used for electrophysiology were plated in plastic culture dishes.

[0114] HEK293 culture procedure HEK293 cells were transfected with the appropriate ion channel or receptor cDNAs encoding NRl 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's Medium / Nutrient Mixture F-12 (D-MEM / F-12) supplemented with 10% fetal bovine serum, 100 U / mL penicillin O sodium, 100 μg / mL streptomycin sulfate, 100 ug / mL Zeocin, 5 ug / mL blasticidin, and 500 μg / mL 0418.

[0115] ScreenPatch Test Method All experiments were performed at ambient temperature. Target-specific test procedures are described below. The following procedures apply to all ScreenPatch assays.

[0116] Prior to testing, the cells in the culture dish were washed twice with HBSS solution. Immediately prior to use in the IonWorks Barracuda™, the cells were washed in HB-PS containing 6 mM CaCh to improve sealing.

[0117] 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 specified. Test article formulations were loaded in a 384-well compound plate and placed into IonWorks Barracuda™ plate wells.

[0118] The positive control article was prepared in batches, aliquoted for individual use, stored frozen and then used within 6 months. The positive control test solution was prepared fresh each day. The final DMSO concentration was 0.6%.

[0119] A 2x concentration of the test article (listed in Table 2) was pre-applied 2 min prior to application of L-glutamic acid (Sigma-Aldrich) / glycine (Sigma-Aldrich) (5 μM L-glutamic acid and 50 μM glycine) mixed with a 1x concentration of the test article.

[0120] To monitor the sensitivity of the assay, an antagonist positive control, amantadine hydrochloride (Sigma-Aldrich), was applied in eight half-log concentrations (ranging from 0.3 to 1000 μM; n = 4, where n = number of replicates per concentration). An agonist positive control (L-glutamic acid) was applied in eight (8) concentrations (0.03 to 100 μM, n = 4, where n = number of replicates) along with 50 μM glycine.

[0121] The inhibitory effect of the compound on the channel was calculated as follows.

[0122]

number

[0123] Nonlinear least-squares fits were solved using XLfit (Boston, MA), an add-in for Excel 2016.

[0124] The effect of the compound, 1. Peak Current Amplitude (PCA) measurements at maximum current 2. Steady-state current amplitude (SSC) measurements as an average over 4-5 seconds following stimulation of the receptor with an agonist The evaluation was carried out using two types of measurements:

[0125] procedure Electrophysiological procedures: a) Intracellular solution (mM): 50 mM CsCl, 90 mM CsF, 2 mM MgCl2, 5 mM EGTA, 10 mM HEPES. pH was adjusted to 7.2 with CsOH. This solution was prepared in batches and stored refrigerated. In preparation for the recording session, the intracellular solution was loaded into the intracellular compartment of a PPC planar electrode. b) Extracellular solution, HB-PS (composition in mM): NaCl, 137; KCl, 1.0; CaCl2, 5; HEPES, 10; Glucose, 10; pH adjusted to 7.4 with NaOH (refrigerated until use). c) Holding potential: -70 mV, potential during application of test substance: -70 mV.

[0126] Recording Procedure: a) Extracellular buffer was loaded into the PPC plate wells (11 μL per well). The cell suspension was dispensed into the wells of the PPC planar electrodes (9 μL per well). b) A whole-cell recording configuration was established by patch perforation with membrane currents recorded by an integrated patch clamp amplifier. 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 for preincubation of the test substance with the cells (2 min); the second was during agonist stimulation of the receptor (5 μM L-glutamate and 50 μM glycine) co-applied with the test substance at 1x concentration to detect antagonist effects of the test substance.

[0127] Test article administration: Applications consisted of 20 μL of 1× concentrated test article solution and addition of agonist at 10 μL / s (total application time of 2 seconds).

[0128] Positive control agonists: 0.03 to 100 μM L-glutamate (8 concentration dose response, semi-log scale) and 50 μM glycine

[0129] Positive control antagonist: 0.3 to 1000 µM amantadine co-applied with 5 µM glutamate and 50 µM glycine (8 concentration responses, semi-log scale).

[0130] Efficacy assessment was based on peak current measurements.

[0131] result The agonist and antagonist properties of the four compounds were examined using the Ion Work Barracuda™ (IWB), an HTS electrophysiology-based approach. Two application protocols were used.

[0132] Agonist format: The potential agonist effects of the test articles and the positive control antagonist amantadine were examined during the first application. Neither the test articles nor amantadine caused significant activation of the NMDA receptor (data not shown).

[0133] Antagonist format: Antagonist activity of the test substances was examined during the second application of the compounds after stimulation of the receptors with 5 μM L-glutamate and 50 μM glycine. It was found that all four test substances produced significant concentration-dependent inhibition of NMDA receptor function. To access the open channel block type of inhibition, peak and steady state current amplitudes (between 4-5 s after agonist application) were measured (in PCA and SSC, respectively). Table 2 shows the IC of the compounds at NR1 / NR2A and NR1 / NR2B receptors according to these two types of measurements. 50 The average of is shown.

[0134] Amantadine increased the IC 50_PCA = 97.8 μM and IC 50_SSC = 48.9 μM produced inhibition of NR1 / NR2A receptors. NR1 / NR2B receptors produced inhibition of 5.1 μM for peak and steady-state current amplitudes, respectively, with IC 50_PCA = 22.0 μM and IC 50_SSC = 17.9 μM. The left shift in amantadine potency relative to steady-state current measurements suggests, at least in part, an open channel block mechanism of inhibition at the NR1 / NR2A NMDA receptors.

[0135] [Table 2]

[0136] Amantadine increased the IC 50_PCA = 97.8 μM and IC 50_SSC = 48.9 μM produced inhibition of NR1 / NR2A receptors. NR1 / NR2B receptors produced inhibition of 5.1 μM for peak and steady-state current amplitudes, respectively, with IC 50_PCA = 22.0 μM and IC 50_SSC= 17.9 μM. The left shift in amantadine potency relative to steady-state current measurements suggests, at least in part, an open channel block mechanism of inhibition at the NR1 / NR2A NMDA receptors.

[0137] The results of these assays are further illustrated in Figure 1, A-D.

[0138] Example 2. Effects of pure 2-S rimantadine or pure 2-R rimantadine on mouse cancer models method

[0139] cell line

[0140] AT-84-E7 and B16-OVA are grown in RPMI1640 containing 10% FBS, 1% L-flutamine, 1% penicillin / streptomycin, 1% sodium pyruvate, and 200 μg / ml G418. DC2.4, RAW264.7, B3Z, 4T1, B16, and MC38 are grown in RPMI1640 20 containing 10% FBS, 1% L-glutamine, 1% penicillin / streptomycin, and 1% sodium pyruvate. HEK293T are grown in DMEM containing 10% FBS, 1% L-glutamine, and 1% penicillin / streptomycin. 4MOSC1 are cultured in collagen-coated dishes in 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 are grown in DMEM containing 10% FBS, 22 1% L-glutamine, and 1% penicillin / streptomycin. Regular monitoring for mycoplasma contamination is performed using the MycoAlert PLUS detection kit (Lonza, Basel, Switzerland). All cell lines are used within 10 passages after thawing.

[0141] Mouse studies

[0142] 1.0-5.0 x 10 resuspended in 100 µl PBS 5 AT-84-E7, 1.5 × 10 5 B16-OVA, or 5.0 × 10 5 4T1 cells are injected subcutaneously into the right flank of the mice. For the orthotopic model, 1.0 x 10 cells in 30 μl PBS are injected subcutaneously into the right flank of the mice. 5 AT-84-E7 or 1.0×10 6 4MOSC1 was injected into the tongue. Tumor diameters were measured every 2-3 days with an electronic caliper, and tumor volumes (mm 3 )=(length x width 2 Tumors are reported as volume using the formula: ) / 2. Once tumors were palpable, mice were injected with 200 μg of anti-PDL1 antibody (BioXcell, West Lebanon, NH) per mouse every 3 days for a total of 3 or 4 doses, or 5 mice were treated with 10-20 mg of pure 2-S or pure 2-R rimantadine per kg body weight IP injections for 7 days. For adoptive transfer experiments, single cell suspensions of spleens from OT-1 mice were first cultured in medium containing 10 ng / ml OVA SIINFEKL peptide (InvivoGen, San Diego, CA) and 2 ng / ml recombinant IL-2 (PeproTech, Rocky Hill, NJ) for several days and then cultured with 4.0 × 10 6 The cells are injected intravenously into mice bearing B16-OVA.

[0143] Flow cytometry

[0144] Single cell suspensions are prepared from lung, liver, tumor-draining lymph nodes, and tumor by mechanical dissociation and filtered using a 70 μm filter. AT-84-E7 and MOC2 tumors are incubated in collagenase D (Roche, Basel, Switzerland) for 1 h at 37° C. prior to mechanical dissociation. Density gradient centrifugation on a 40% / 80% Percoll (GE Healthcare, Chicago, IL) gradient is performed on the single cell suspension of tumor. After obtaining a single cell suspension, each sample is incubated with Fc-blocking reagent (anti-CD16 / 32 antibody; BioLegend, San Diego, CA). After Fc-blocking, cells are stained with fluorescently labeled antibodies (BioLegend, BD Bioscience (San Jose, CA), or eBiosciences (Thermo Scientific Fisher, Waltham, MA)). The LIVE / DEAD Fixable Cell Staining Kit (Invitrogen) is 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 LSRII 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 (DX5), CD11b (M1 / 70), FLAG (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).

[0145] Cell cycle and cell proliferation assays

[0146] Cell cycle progression was analyzed based on BrdU incorporation after staining single cells with BrdU-APC and 7-AAD using the BD Pharmingen BrdU Flow Kit (BD, Franklin Lakes, NJ) according to the manufacture's protocol. 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. Then, the culture medium was replaced with fresh medium containing 0.5 mg / ml MTT (Sigma) and the plates were incubated at 37 °C for 4 h. The purple formazan crystals are then dissolved in lysis buffer (4 mM HCl and 0.1% NP-40 in isopropanol) and the absorbance is recorded on a TECAN infinite M200 microplate reader (Tecan, Männedorf, Switzerland) at a wavelength of 570 nm, with the absorbance at 650 nm as the reference.

[0147] B3Z activation assay

[0148] B16-OVA cells were seeded in 96-well plates and treated with 100 μM pure 2-R rimantadine, pure 2-R rimantadine, or racemic rimantadine for 24 h before adding B3Z cells. After 24 h of co-culture, the medium was removed and 100 μl of lysis buffer [0.155 mM chlorophenol red β-D-galactopyranoside (CPRG) (Roche), 0.125% Nonidet P-40 replacement (EMDCalbiochem), and 9 mM MgCl2 (Sigma) in PBS] was added. After 4 h of incubation at 37°C, the absorbance at 570 nm was determined on a TECAN infinite M200 microplate reader.

[0149] Reverse transcription and quantitative PCR

[0150] Total RNA is 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 is performed by using KAPA SYBR 1 FAST (KAPA Biosystems, Wilmington, MA) on a 7900HT Fast Real-Time PCR System (Applied Biosystems, Foster City, CA).

[0151] result

[0152] Pure 2-S rimantadine and pure 2-R rimantadine only showed antitumor activity, significantly reducing tumor growth. 5Mice were inoculated with AT84-E7 / E5 tumor cells and treated with 10 mg / kg body weight pure 2-S or pure 2-R rimantadine intraperitoneally (IP) injections once a day, starting on day 8, for a total of seven injections. Tumor volumes were measured during the experiment. Mice receiving pure 2-S or pure 2-R rimantadine had a statistically significant reduction in tumor size compared to the control group. Six mice were inoculated with 1.5×10 5 Mice were inoculated with B16-OVA tumor cells and treated with 10 mg / kg body weight pure 2-S rimantadine or pure 2-R rimantadine IP injections once a day starting on day 10 for a total of seven injections. Tumor volumes were measured during the experiment. Mice receiving pure 2-S rimantadine or pure 2-R rimantadine had a statistically significant reduction in tumor size compared to the control group. This experiment was repeated three times with similar results. Five mice were inoculated with 1.5 × 10 5 4T1 tumor cells were inoculated and treated with 10 mg / kg body weight pure 2-S or pure 2-R rimantadine IP injections once a day starting on day 6 for a total of 7 injections. Tumor volumes are measured during the experiment. Mice receiving 2-S or pure 2-R rimantadine had a statistically significant reduction in tumor size compared to the control group. The antitumor effect of pure 2-S or pure 2-R rimantadine is reduced in AT-84-E7 tumors that do not express E5. A significant increase in surface expression of MHC is observed in multiple cell lines. Cell surface expression of MHC I on E5-positive AT-84-E7 is restored by treatment with pure 2-S or pure 2-R rimantadine.

[0153] To test the ability of 2-S rimantadine to enhance functional antigen presentation on tumor cells, OVA-expressing B16 cells are used as a model tumor antigen and co-cultured with B3Z cells that respond to the OVA SINNFKL peptide. Treatment of B16-OVA cells with pure 2-S rimantadine or pure 2-R rimantadine results in a significant three-fold increase in recognition of this model tumor antigen by B3Z cells. Combining pure 2-S rimantadine or pure 2-R rimantadine with anti-PDL1 immunotherapy results in a significant improvement in survival of mice bearing B16-OVA tumors.

[0154] When testing the ability of pure 2-S rimantadine or pure 2-R rimantadine to increase the expression of MHC on antigen-presenting cells using the RAW264.7 cell line, a significant increase in the surface expression of both MHC class I and MHC class II is observed. These findings demonstrate that pure 2-S rimantadine or pure 2-R rimantadine has novel antitumor activity against multiple preclinical tumor models and functions to enhance antigen presentation by upregulating MHC.

[0155] To test the direct cytotoxic activity of pure 2-S or pure 2-R rimantadine, an in vitro BrdU incorporation assay is performed to quantify the effect of pure 2-S or pure 2-R rimantadine on the cell cycle of human HNSCC cell lines. Pure 2-S or pure 2-R rimantadine alone significantly increases G0 / G1 cell cycle arrest and significantly reduces S-phase in both AT-84-E7 and B16-OVA models. Inhibition of cell proliferation is also observed. Analysis of the effect of pure 2-S or pure 2-R rimantadine on T cell proliferation was tested, but no significant effect is observed.

[0156] Screening of gene expression changes of cell cycle proteins induced by pure 2-S rimantadine or pure 2-R rimantadine using RTqPCR confirmed a significant decrease in the microtubule and cell cycle regulatory molecule stathmin. A decrease in the microtubule-associated molecule tau (Tau) was also observed.

[0157] To confirm that pure 2-S or pure 2-R rimantadine exhibits activity against human head and neck tumor lines, BrdU incorporation and proliferation assays are performed. Significant cell cycle arrest and reduced proliferation with rimantadine alone is observed in human CAL-27, CAL-33, and SCC-47 squamous cell carcinoma cell lines. Finally, pure 2-S or pure 2-R rimantadine induces cell cycle arrest in mouse and human cell lines engineered to express HPV16 E5, demonstrating that pure 2-S or pure 2-R rimantadine can functionally reverse the effects of HPV E5.

[0158] Example 3. HPV genotyping HPV genotyping is known in the art, see, for example, Sichero et al., 2017, Cancer Epidemiol Biomarkers, 26(8):1312-1320. For example, DNA is extracted from exfoliated cervical cells by spin column chromatography. Mucosal α-HPV is tested using PCR amplification with primers such as MY09 / 11 and PGMY09 / 11 (see Table 3), followed by genotyping by hybridization with HPV type-specific oligonucleotide probes and restriction fragment length polymorphism analysis. Negative and positive controls are used to confirm the quality of the template DNA.

[0159] [Table 3-1]

[0160] [Table 3-2]

[0161] Example 4. Toxicity of 2-S and 2-R Rimantadine in vivo A series of in vivo experiments are performed to determine whether 2-S or 2-R rimantadine has higher binding selectivity for any one of glutamate receptors, GABA receptors, dopamine receptors, or any combination thereof. The enhanced selectivity for any one of glutamate receptors, GABA receptors, dopamine receptors, or any combination thereof by 2-S or 2-R rimantadine compared to racemic rimantadine results in the absence of central nervous system adverse effects, including nausea, upset stomach, vomiting, loss of appetite, dry mouth, abdominal pain, asthenia, irritability, fatigue, lightheadedness, dizziness, headache, sleep disorders, difficulty concentrating, confusion, and anxiety, commonly associated with racemic rimantadine.

[0162] 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 amantadine (control) by daily IP injection for 7 days. A series of SPECT studies as described in Schramm, N., et al. (2000) were then performed. A compact high-resolution detector for small animal SPECT was performed for glutamate receptors, GABA receptors, and dopamine receptors, respectively, to evaluate the binding selectivity of 2-S rimantadine and 2-R rimantadine.

[0163] SPECT studies involve treating mice with radioligands specific for each receptor. 123 I]IBZM is D 2 / 3It has been documented to have high affinity for dopamine receptors. Radioligands specific for glutamate and GABA receptors are known to those skilled in the art. Appropriate amounts of each radioligand for glutamate, GABA, and dopamine receptors are injected into the lateral tail vein of mice, and SPECT measurements are started 45 minutes after administration of the radioligand.

[0164] Surprisingly, 2-R rimantadine has significantly higher binding selectivity or agonist behavior with respect to glutamate, GABA, dopamine receptors or pathways, or any combination thereof, compared with 2-S rimantadine.Therefore, 2-R rimantadine has a higher incidence of central nervous system adverse effects, including nausea, upset stomach, vomiting, anorexia, dry mouth, abdominal pain, asthenia, irritability, fatigue, lightheadedness, dizziness, headache, sleep disorder, difficulty concentrating, confusion, and anxiety, compared with 2-S rimantadine.In combination with Example 2, 2-S rimantadine has significantly lower toxicity compared with 2-R rimantadine, but is still effective as a cancer treatment.

[0165] Example 5. Toxicity of 2-S and 2-R Rimantadine in vivo A series of in vivo experiments are performed to determine whether 2-S or 2-R rimantadine has higher binding selectivity for any one of glutamate receptors, GABA receptors, dopamine receptors, or any combination thereof. The enhanced selectivity for any one of glutamate receptors, GABA receptors, dopamine receptors, or any combination thereof by 2-S or 2-R rimantadine compared to racemic rimantadine results in the absence of central nervous system adverse effects, including nausea, upset stomach, vomiting, loss of appetite, dry mouth, abdominal pain, asthenia, irritability, fatigue, lightheadedness, dizziness, headache, sleep disorders, difficulty concentrating, confusion, and anxiety, commonly associated with racemic rimantadine.

[0166] 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 amantadine (control) by daily IP injection for 7 days. A series of SPECT studies as described in Schramm, N., et al. (2000) were then performed. A compact high-resolution detector for small animal SPECT was performed for glutamate receptors, GABA receptors, and dopamine receptors, respectively, to evaluate the binding selectivity of 2-S rimantadine and 2-R rimantadine.

[0167] SPECT studies involve treating mice with radioligands specific for each receptor. 123 I]IBZM is D 2 / 3 It has been documented to have high affinity for dopamine receptors. Radioligands specific for glutamate and GABA receptors are known to those skilled in the art. Appropriate amounts of each radioligand for glutamate, GABA, and dopamine receptors are injected into the lateral tail vein of mice, and SPECT measurements are started 45 minutes after administration of the radioligand.

[0168] Surprisingly, 2-S rimantadine has significantly higher binding selectivity or agonist behavior with respect to glutamate, GABA, dopamine receptors or pathways, or any combination thereof, compared with 2-R rimantadine.Therefore, 2-S rimantadine has a higher incidence of central nervous system adverse effects, including nausea, upset stomach, vomiting, anorexia, dry mouth, abdominal pain, asthenia, irritability, fatigue, lightheadedness, dizziness, headache, sleep disorder, difficulty concentrating, confusion, and anxiety, compared with 2-R rimantadine.In combination with Example 2, 2-S rimantadine has significantly lower toxicity compared with 2-R rimantadine, but is still effective as a cancer treatment.

[0169] Example 6. Growth of 2-S and 2-R Rimantadine in vitro Experiments were performed to determine the ability of 2-S rimantadine (also referred to throughout this application as "S-rimantadine"), 2-R rimantadine (also referred to throughout this application as "R-rimantadine"), racemic (RS) rimantadine, and memantine to induce proliferation in CAL-27 cells. S-rimantadine results in enhanced or equivalent cancer cell proliferation compared to R-rimantadine or racemic rimantadine.

[0170] On day 1, CAL-27 cells were plated in 96-well plates (2–4 × 10 3 Cells / well, 100 μl / well of medium were seeded and left overnight to allow the cells to attach to the plate. On day 2, various concentrations of rimantadine (0 μM, 100 μM, 250 μM, or 500 μM) were added to the cells and incubated for 24 or 48 hours. On either day 3 or 4, the culture medium was aspirated and 100 μl / well of MTT solution (containing a MTT concentration of 0.5 mg / ml, made by dilution of thiazolyl blue tetrazolium bromide solution (SIGMA, catalog number 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. Then, 100 μl / well of DMSO was added and the cells were incubated for about 5 minutes. Then, the OD at 570 nm (reference 650 nm) was read. The experimental results are shown in Figure 2.

[0171] Example 7. In vivo tumor model / antitumor activity method Using an in vivo mouse syngeneic tumor model, the activity of 2-S rimantadine, 2-R rimantadine, and racemic rimantadine is tested against HPV-associated tumors. S-rimantadine demonstrates comparable or increased antitumor activity compared to racemic rimantadine and / or R-rimantadine.

[0172] Plasmid construction and stable cell lines expressing HPV16 E5 Codon-optimized HPV16 E5 is amplified. Either C-terminally or N-terminally FLAG-tagged full-length HPV16 E5 and deletion mutants are cloned into MIP (MSCV-IRES-Puro) or pMSCV-Blasticidin vectors. All constructs are verified by DNA sequencing. For the establishment of cell lines expressing HPV16 E5, HEK293T cells are co-transfected with MIP-HPV16 E5 and Ecopac (pIK6.1MCV.ecopac.UTd) using PEI reagent (Sigma-Aldrich). Retroviruses from the culture medium of these cells are then used to infect AT-84-E7, MOC2, and CAL-27 cells, and infected cells are selected by puromycin. pMSCV Blasticidin-HPV16 E5 is used for MEER cells.

[0173] Mouse studies Use 6-8 week old female mice for the experiment. Use C3H / HeN mice and C57BL / 6 and BALB / c mice. 1.0-5.0 × 10 resuspended in 100 mL of PBS 5 AT-84-E7, 1.5 × 10 5 B16-OVA, 5.0 × 10 5 4T1, or 1.0×10 5 MOC2 cells are injected subcutaneously into the right flank of the mice. For the orthotopic model, 1.0 x 10 MOC2 cells in 30 mL of PBS are injected subcutaneously into the right flank of the mice. 5 AT-84-E7 or 1.0×10 6 4MOSC1 are injected into the tongue. Once the tumors are palpable, the mice are treated with 200 mg of anti-PDL1 antibody (BioXcell) per mouse every 3 days for a total of 3 or 4 injections, or with 10 mg of pure R- or S-rimantadine and / or racemic rimantadine IP injections per kg body weight for 7 days. In adoptive transfer experiments, single-cell suspensions of spleens from OT-1 mice were cultured in medium containing 10 ng / ml OVA SIINFEKL peptide (InvivoGen) and 2 ng / ml recombinant IL-2 (PeproTech) for 5 days, after which 4.0 × 10 6 Inject cells intravenously into mice bearing B16-OVA. Measure tumor diameters with an electronic caliper every 2-3 days and estimate tumor volumes (mm 3 )(Length x Width 2 Report as volume using the formula: 1 / 2.

[0174] The information and procedures used and disclosed in Miyauchi S., et al., Cancer Res. 2020 Feb 15;80(4):732-746 are incorporated herein by reference in their entirety. The disclosed information and procedures (e.g., protocols) are carried out for testing on S-rimantadine, R-rimantadine, and / or racemic rimantadine.

[0175] Example 8: In vitro antiviral method Using an in vitro HPV viral replication assay, the direct antiviral activity of enantiomers of rimantadine (e.g., S-rimantadine) is tested against HPV viral replication. S-rimantadine demonstrates equivalent or increased direct HPV antiviral activity compared to racemic rimantadine or R-rimantadine.

[0176] Plasmids The Snls-Cre expression plasmid pCAGGS-nlsCre is used. pNeo-loxP HPV-18 and pNeo-loxP HPV-18 E6 * The HPV-18 E6 plasmid is used. For both plasmids, 34 bp loxP sites flank the linear HPV-18 sequence upstream of nucleotide 7474 and downstream of nucleotide 7473. The vector carries a neomycin resistance marker gene that is selectable in bacteria and mammalian cells. * In I mutants, the predominant E6 *Intron coding sequences (nucleotides 234-M15) in I mRNA are deleted. For transcomplementation experiments, use the empty vector-only retrovirus pLC and the pLJ HPV-18 URR-E6 or URR-E6 / E7 retroviruses, each of which expresses a neomycin resistance gene (Cheng et al. 1995. Differentiation-dependent up-regulation of the human papillomavirus E7 gene reactivates cellular DNA replication in suprabasal differentiated keratinocytes. Genes&Dev. 9:2335-2349; Chien et al. 2002. Alternative fates of keratinocytes transduced by human papillomavirus type 18 E7 during squamous differentiation. J. Virol. 76:2964-2972). All plasmids are purified by banding (e.g., on a CsCl-ethidium bromide equilibrium density gradient).

[0177] HPV-18 virion recovery and titer determination HPV-18 virions are harvested from epithelia on day 14 or day 16 as described (Favre, M. 1975. Structural polypeptides of rabbit, bovine, and human papillomaviruses. J. Virol. 15:1239-1247). To titer the virus, an aliquot of the virus stock is digested with DNase I (Invitrogen) and then inactivated by heating at 100°C for 5 minutes. Packaged viral DNA is then purified by digestion with proteinase K and phenol / chloroform extraction. Serial dilutions of viral DNA are analyzed by real-time quantitative PCR using, for example, SYBR GreenER qPCR SuperMix (Invitrogen) and primers J and K as disclosed in Supplementary Table 1 of Wang HK.etal., Genes Dev. 2009 Jan 15;23(2):181-194. As a standard, purified pNeo-LoxPHPV-18 plasmid DNA was added at approximately 40–4 × 10 per well. 8 Serial dilutions are performed to copies of each 40-cycle PCR amplification reaction in triplicate (e.g., in a 384-well plate using an ABI 7900HT), and data is then processed (e.g., using SDS2.1 software (Applied Biosystems)).

[0178] HPV-18 infectivity assay Approximately 1×10 5Primary human keratinocytes (PHK) are inoculated with various amounts of virus stocks corresponding to MOIs of 5200, 1040, 208, 42, 10, 2, 1, or 0 in 1 mL of K-SFM and incubated overnight. The medium is replaced and the cells are cultured for an additional 4 days. Total RNA is then extracted (e.g., using Trizol (Invitrogen)). Reverse transcription is performed in a 50 mL reaction for 10 mg of RNA. One microliter of the RT reaction is then subjected to 30 cycles of PCR or nested PCR amplification (30 cycles each) in a 35 mL reaction mixture to generate cDNA fragments of spliced ​​HPV-18 E6-E7-E1^E4, 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). Fifteen microliters of each reaction are separated by electrophoresis in a 2% agarose gel and visualized by ethidium bromide staining. PHKs are also infected overnight at various MOIs in K-SFM and developed into raft cultures, which are fixed on day 14 and processed as described.

[0179] The PHKs receiving various amounts of virus stock are then exposed to various 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). Disclosed information and procedures (e.g., protocols) are implemented for testing with S-rimantadine, R-rimantadine, and / or racemic rimantadine.

[0180] The information and procedures used and disclosed in Wang HK., et al., Genes Dev. 2009 Jan 15;23(2):181-194 are incorporated herein by reference in their entirety. The disclosed information and procedures (e.g., protocols) are implemented for testing on S-rimantadine, R-rimantadine, and / or racemic rimantadine.

[0181] Example 9. In vivo central nervous system ("CNS") assays Tests are conducted to determine the effect of R-rimantadine, S-rimantadine, and racemic rimantadine on the CNS of live animals (e.g., mice and / or rats). Various doses of R-rimantadine, S-rimantadine, and racemic rimantadine are tested to perform the following tests: Animals receiving S-rimantadine demonstrate less CNS toxicity than similar doses of R-rimantadine and racemic rimantadine. Furthermore, animals receiving S-rimantadine can receive higher doses of each drug before showing signs and / or symptoms of CNS toxicity compared to animals receiving R-rimantadine or racemic rimantadine. Furthermore, mice receiving S-rimantadine tolerate signs and symptoms of CNS toxicity better compared to mice receiving similar doses of R-rimantadine and racemic rimantadine.

[0182] a) Rotarod CNS toxicity associated with the use of R-rimantadine, S-rimantadine, and racemic rimantadine is tested using a rotarod system (e.g., Rotor Rod System, San Diego Instruments). The use of the Rotor Rod system allows for the observation of motor coordination in animals (e.g., mice or rats), thereby allowing for the testing of CNS toxicity potentially caused by R-rimantadine, S-rimantadine, and racemic rimantadine.

[0183] Animals receive doses (e.g., various 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 are measured using a rotarod system. Animals receiving S-rimantadine show less adverse CNS effects and toxicity compared to R-rimantadine and racemic rimantadine. In particular, animals receiving S-rimantadine (e.g., mice or rats) show less abnormal motor coordination. Rotor Rod, San Diego Instruments, available at https: / / sandiegoinstruments.com / product / rotor-rod / ; ROTOR-ROD™ System, available at https: / / cehs.unl.edu / borc / rotor-rod%E2%84%A2-system / , Biomedical and Obesity Research Core, College of Education and Human Sciences, University of Nebraska-Lincoln,; Castagne et al., CNS Safety Pharmacology,Reference Module in Biomedical Research,2014;Dunham NW and Miya TS(1957)A note on a simple apparatus for detecting neurological deficit in rats and mice.Journal of the American Pharmaceutical Association,American Pharmaceutical Association(Baltimore)46:208-209;Bohlen et al.,Calibration of rotational acceleration for the rotarod test of rodent motor coordination,Journal of Neuroscience Methods(2009)178:10-14;Shiotsuki et al.,A rotarod test for evaluation of motor skill learning.J Neurosci Methods.2010 Jun 15;189(2):180-5.doi:10.1016 / j.jneumeth.2010.03.026.Epub 2010 Mar 30.PMID:20359499;and Rustay NR,Wahlsten D,and Crabbe JC(2003)Influence of task parameters on rotarod performance and sensitivity to ethanol in mice.Behavioural Brain Research 141:237-249 are incorporated herein by reference in their entireties. The disclosed information and procedures (e.g., protocols) are implemented for testing of S-rimantadine, R-rimantadine, and / or racemic rimantadine.

[0184] b)Photobeam Activity System-Home Cage CNS toxicity associated with the use of R-rimantadine, S-rimantadine, and racemic rimantadine is tested using a Photobeam Activity System-Home Cage (San Diego Instruments). The use of the photobeam activity system-home cage allows for testing of locomotor activity of animals. Animals receiving R-rimantadine exhibit lower CNS toxicity as evidenced by the photobeam activity system-home cage test.

[0185] Animals (e.g., mice or rats) receive doses (e.g., various 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 are measured using a photobeam activity system-home cage. Animals receiving S-rimantadine exhibit less adverse CNS effects and toxicity compared to R-rimantadine and racemic rimantadine. In particular, animals (e.g., mice or rats) receiving S-rimantadine exhibit less locomotor activity. The information and procedures used and disclosed in Photobeam Activity System-Home Cage, San Diego Instruments, available at https: / / sandiegoinstruments.com / product / pas-homecage / and Tatem et al., Behavioral and locomotor measurements using an open field activity monitoring system for skeletal muscle diseases. J Vis Exp. 2014 Sep 29;(91):51785.doi:10.3791 / 51785.PMID:25286313;PMCID:PMC4672952 are incorporated herein by reference in their entirety. The information and protocols described in these disclosures are used for testing R-rimantadine, S-rimantadine, and racemic rimantadine. This testing is used to analyze and potentially modify aspects of the animal's physiological responses related to the CNS, such as circadian rhythms and anxiety.

[0186] c) Irwin test / Functional Observation Board (FOB) CNS toxicity associated with the use of R-rimantadine, S-rimantadine, and racemic rimantadine is tested using the Irwin test and FOB. The use of the Irwin test and FOB allows for the testing of the qualitative effects of R-rimantadine, S-rimantadine, and racemic rimantadine. Animals receiving R-rimantadine exhibit lower CNS toxicity as evidenced by the Irwin test / FOB test.

[0187] Animals (e.g., mice or rats) receive doses (e.g., various doses (e.g., 4 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) are tested. 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 behaviors and physiological function and similar doses, while animals receiving R-rimantadine tolerate higher doses before showing observable effects on either behavior and physiological function and / or tolerate higher doses before showing clear behavioral toxicity.Castagne et al., CNS Safety Pharmacology, Reference Module in Biomedical Research, 2014, Irwin S (1968), Comprehensive observational assessment: Ia. A systematic, quantitative procedure for assessing the behavioral and physiological state of the mouse, Psychopharmacologia 13:222-257, Esteve J, Farre AJ, and Roser R (1988) Pharmacological profile of droxicam, General Pharmacology 19:49-54, Mattson et al.,(1996)A performance standard for clinical and functional observational battery examination of rats.Journal of the American College of Toxicology,15:239-250, and Roux et al.,Primary observation(Irwin)test in rodents for assessing acute toxicity of a test agent and its effects on behavior and physiological function.Curr.Protoc.Pharmacol.2005 Jan The information and procedures used and disclosed in the above publications are incorporated herein by reference in their entirety. The information and protocols described in these disclosures are used for testing R-rimantadine, S-rimantadine, and racemic rimantadine.

[0188] d) Morris Water Maze Test The CNS toxicity associated with the use of R-rimantadine, S-rimantadine, and racemic rimantadine is tested using the Morris water maze test. The Morris water maze test allows for testing of potential CNS toxicity experienced by animals (e.g., mice or rats) by testing the animal's spatial learning ability. Animals receiving R-rimantadine show lower CNS toxicity as evidenced by the Morris water maze test.

[0189] Animals (e.g., mice or rats) receive doses (e.g., various doses (e.g., 4 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 receiving S-rimantadine show less adverse CNS effects and toxicity compared to R-rimantadine and racemic rimantadine. In particular, animals receiving S-rimantadine show less inhibition of their spatial learning ability. The information and procedures used and disclosed in Vorhees et al., Morris water maze: procedures for assessing spatial and related forms of learning and memory, Nat Protoc 1, 848-858 (2006). https: / / doi.org / 10.1038 / nprot.2006.116, and Castagne et al., CNS Safety Pharmacology, Reference Module in Biomedical Research, 2014, Morris RGM (1981) Spatial localization does not require the presence of local cues, Learning and Motivation 12: 239-260 are incorporated herein by reference in their entirety. The information and protocols described in these disclosures are used for testing R-rimantadine, S-rimantadine, and racemic rimantadine.

[0190] e) Electroencephalogram (EEG) Scan The CNS toxicity associated with the use of R-rimantadine, S-rimantadine, and racemic rimantadine is tested using EEG scanning. EEG scanning allows the examination of electrical activity in the brain of animals (e.g., mice or rats). Animals receiving R-rimantadine show lower CNS toxicity as evidenced by EEG testing.

[0191] Animals (e.g., mice or rats) receive doses (e.g., various 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 are 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.Vogler et al.,Low Cost Electrod Assembly for EEg Recordings in Mice,Front.Neurosci.,14 November 2017,https: / / doi.org / 10.3389 / fnins.2017.00629;Danhof M and Visser SA(2002)Pharmaco-electroencephalography and pharmacokinetic-pharmacodynamic modeling in drug development:focus on preclinical steps.Methods&Findings in Experimental&Clinical Pharmacology 24((Suppl D):127-128;Itil TM and Itil KZ(1995)Quantitative EEG Brain Mapping In Psychotropic Drug Development,Drug Treatment Selection,and Monitoring.American Journal of Therapy 2:359-367; and the information and procedures used and disclosed in Protocol for Rat Sleep EEG, NeuroDetective International, available at https: / / www.ndineuroscience.com / userfiles / Rat_Sleep_EEG_Methods.pdf, are incorporated herein by reference in their entireties. The information and protocols described in these disclosures are used for testing R-rimantadine, S-rimantadine, and racemic rimantadine.

[0192] Example 10. In Vitro Central Nervous System ("CNS") Assays Tests are performed to determine the effects of R-rimantadine, S-rimantadine, and racemic rimantadine on changes in anatomy and / or physiology associated with CNS toxicity. Tissues obtained from animals (e.g., living or dead) (e.g., mice and / or rats) receiving S-rimantadine show less change compared to baseline or normal (e.g., within tolerance) tissues when compared to tissues obtained from animals receiving R-rimantadine or racemic rimantadine.

[0193] Animals receiving S-rimantadine show less physiological and / or anatomical changes due to CNS toxicity compared to animals receiving similar doses of R-rimantadine and racemic rimantadine. Animals receiving S-rimantadine can receive higher doses of S-rimantadine than animals receiving R-rimantadine and racemic rimantadine before showing physiological and / or anatomical changes associated with CNS toxicity. Various doses of R-rimantadine, S-rimantadine, and racemic rimantadine are tested using at least the following tests. The information and protocols described in these disclosures are used for testing R-rimantadine, S-rimantadine, and racemic rimantadine.

[0194] Brain slice / whole cell patch clamp CNS toxicity associated with the use of R-rimantadine, S-rimantadine, and racemic rimantadine is examined using brain slice / whole cell patch clamp studies. Brain slice / whole cell patch clamp electrophysiology allows for the analysis of biophysical mechanisms (e.g., ionic currents) of neuronal computation and pathology in neurons. Animals receiving R-rimantadine show lower CNS toxicity (e.g., fewer anatomical and / or physiological changes) as evidenced by brain slice / whole cell patch clamp studies.

[0195] Animals (e.g., mice or rats) receive doses (e.g., various 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 are euthanized and brain slices are obtained and analyzed. Alternatively, whole-cell patch clamp may be performed in vivo. In such cases, after a period of time after receiving the dose, the animals are not euthanized and analyzed. Animals receiving S-rimantadine exhibit fewer adverse CNS effects and toxicity compared to animals receiving R-rimantadine or racemic rimantadine. In particular, animals receiving S-rimantadine exhibit fewer abnormal biophysical mechanisms of neural computation and pathology (e.g., ionic currents) than animals receiving either R-rimantadine or racemic rimantadine. The information and procedures used and disclosed in Kodandaramaiah et al., Automated whole-cell patch-clamp electrophysiology of neurons in vivo, Nat Methods. 2012 Jun;9(6):585-7. doi:10.1038 / nmeth.1993. Epub 2012 May 6. PMID:22561988; PMCID:PMC3427788 are incorporated herein by reference in their entirety. The information and protocols described in these disclosures are used for testing R-rimantadine, S-rimantadine, and racemic rimantadine.

[0196] Other embodiments Although the present invention has been described in conjunction with its detailed description, it should be understood that the foregoing description is intended to be illustrative, and not limiting, of the scope of the invention as defined by the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

1. Use of enantiomerically pure 2-S rimantadine, or either a pharma- ceutically acceptable salt thereof, in the manufacture of a medicament for use in treating cancer in a subject in need of such treatment, wherein said treating comprises administering to said subject a therapeutically effective amount of said enantiomerically pure 2-S rimantadine, or either a pharma- ceutically acceptable salt thereof.

2. 2. The use of claim 1, wherein the side effects associated with administration of 2-S rimantadine are reduced compared to the side effects associated with racemic rimantadine.

3. The use of claim 1, wherein the subject is administered a pharma- ceutically acceptable salt of 2-S rimantadine.

4. The use according to claim 1, wherein the pharma- ceutically acceptable salt is a hydrochloride salt.

5. 2. The use of claim 1, 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, mucosal squamous cell carcinoma, cutaneous squamous cell carcinoma, liver cancer, vaginal cancer, vulvar cancer, penile cancer, anal cancer, and gastric cancer.

6. The use of claim 1 , wherein the cancer is a sarcoma, carcinoma, lymphoma, or leukemia.

7. The use according to claim 6, wherein the carcinoma is squamous cell carcinoma.

8. The use according to claim 7, wherein the squamous cell carcinoma is head and neck squamous cell carcinoma.

9. 6. The use of claim 5, wherein the cancer is selected from the group consisting of head and neck cancer, cervical cancer, vaginal cancer, vulvar cancer, penile cancer, anal cancer, breast cancer, and melanoma.

10. The use according to claim 1 , wherein the cancer is an HPV-associated cancer.

11. The use according to claim 10, wherein the HPV-associated cancer is associated with the alpha genus of HPV.

12. The use described in claim 1, wherein the treating further comprises administering an additional anticancer agent.

13. The use of claim 12, wherein the additional anticancer agent is selected from the group consisting of carboplatin, cisplatin, gemcitabine, methotrexate, paclitaxel, pemetrexed, lomustine, temozolomide, dacarbazine, and combinations thereof.

14. The use described in claim 12, wherein the additional anticancer agent is an immunotherapeutic agent.

15. The use described in claim 14, wherein the additional anticancer agent is an immune checkpoint inhibitor.

16. The use of claim 15, 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.

17. The use of claim 16, wherein the immune checkpoint inhibitor is selected from the group consisting of ipilimumab, nivolumab, pembrolizumab, atezolizumab, avelumab, durvalumab, tremelimumab, cemiplimab, and combinations thereof.

18. The use described in claim 16, wherein the immune checkpoint inhibitor targets PD-1 or PD-L1.

19. The use described in claim 18, wherein the immune checkpoint inhibitor comprises pembrolizumab.

20. The use of claim 1, wherein one or more cancer cells from the subject express human papillomavirus (HPV).

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