CROSS-REFERENCE TO RELATED APPLICATION

[0001] This application claims priority to U.S. Provisional Patent Application No. 62/621 ,079, filed on January 24, 2018, the disclosure of which is incorporated herein by reference in its entirety.

FIELD OF THE DISCLOSURE

[0002] The present disclosure relates to methods for preserving neurogenesis in a subject treated with chemotherapy by administering to the subject a thiosemicarbazone compound, or an analogue thereof. The disclosure also relates to methods for increasing the number of cells expressing doublecortin protein in a subject treated with chemotherapy by administering to a subject a thiosemicarbazone compound, or an analogue thereof. The chemotherapy may be an anti-cancer therapeutic. Furthermore, the disclosure relates to methods for correcting cancer therapy-impaired cognitive function by administering to a subject a thiosemicarbazone compound, or an analogue thereof.

BACKGROUND OF THE DISCLOSURE

[0003] Chemotherapies, such as anti-cancer drugs, are usually associated with severe side effects that can affect quality of life and pose difficulties for the continuation of therapy. One of the major adverse effects is the suppression of neurogenesis, which may lead to cognitive impairment in subjects treated with chemotherapy. There is a need for methods for preserving neurogenesis or preventing death of neural stem cells in chemotherapy-treated subjects.

SUMMARY OF THE DISCLOSURE

[0004] The disclosure provides a method for reducing chemotherapy-induced inhibition of neurogenesis in a subject, the method comprising administering to the subject a thiosemicarbazone compound, or an analogue thereof.

[0005] The disclosure further provides a method for preserving neurogenesis in a subject treated with chemotherapy, the method comprising administering to the subject a thiosemicarbazone compound, or an analogue thereof.

[0006] The disclosure further provides a method for increasing the number of cells expressing doublecortin (DCX) protein in a subject treated with chemotherapy, the method comprising administering to the subject a thiosemicarbazone compound, or an analogue thereof.

[0007] The disclosure further provides a method for improving memory or learning in a subject treated with chemotherapy, the method comprising administering to the subject a thiosemicarbazone compound, or an analogue thereof. [0008] In some embodiments, the thiosemicarbazone compound is 3-aminopyridine-2-carboxaldehyde thiosemicarbazone (PAN-811).

[0009] In some embodiments, the thiosemicarbazone compound is a compound of Formula (I):

or an analogue thereof,

wherein R, R ₁ , R ₂ , R ₃ , and R ₄ are independently selected from the group consisting of hydrogen, C1 - Salkyl, C2-8alkenyl, C2-8alkynyl, C3-8cycloalkyl, C1 -8haloalkyl, C6-10aryl, amino-C1 -8alkyl, hydroxy-C1 - 8alkyl, C1 -8alkoxye-C1-8alkyl, and C1-8alkanoyl, or NR1R2 taken in combination form a 3 to 7 member ring which may comprise 0, 1 , or 2 additional ring heteroatoms selected from N, O, and S;

R ₆ is hydrogen, hydroxy, amino, or C1-8alkyl; and

R ₅ and R ₇ are independently selected from the group consisting of hydrogen, halide, hydroxy, thiol, amino, hydroxyamino, mono-C1 -8alkylamino, di(C1-8alkyl)amino, C1 -8alkoxy, C1-8alkyl, C1-8alkenyl, and C2-8alkynyl.

[0010] In some embodiments, the thiosemicarbazone compound is

or an analogue thereof.

[0011] In some embodiments, the administration of the thiosemicarbazone compound, or an analogue thereof, is intravenous, intraperitoneal, subcutaneous, intramuscular, topical, transdermal or oral.

[0012] In some embodiments, the subject is treated with the chemotherapy before or after the administration of the thiosemicarbazone compound, or an analogue thereof. In some embodiments, the subject is treated with the chemotherapy simultaneously with the administration of the thiosemicarbazone compound, or an analogue thereof.

[0013] In some embodiments, the chemotherapy is an anti-cancer agent. In some embodiments, the anticancer agent is an immunotherapeutic agent (e.g., an immune checkpoint inhibitor). In some embodiments, the anti-cancer agent is a small molecule drug, a therapeutic antibody, an antibody-drug conjugate or a chimeric antigen receptor T-cell therapy. In some embodiments, the chemotherapy is a combination of two, three, four, or more than four anti-cancer agents. In some embodiments, the anti-cancer agent is an S- phase antimetabolite, G1/S phase inhibitor, topoisomerase II inhibitor, M phase inhibitors, G2/M phase inhibitor, alkylating agent, anthracycline, or topoisomerase I inhibitor. In some embodiments, the anticancer agent is 5-fluorouracil, methotrexate, leucovorin, cytarabine, fludarabine, gemcitabine, capecitabine, clofarabine, azacytidine, nelarabine, decitabine, pralatrexate, pemetrexed, etoposide, docetaxel, paclitaxel, ixabepilone, cabazitaxel, eribulin mesylate, vincristine, vinblastine, bleomycin, chlorambucil, procarbazine, dacarbazine, ifosfamide, temozolomide, oxaliplatin, bendamustine, daunorubicin, epirubicin, doxorubicin, irinotecan, topotecan, platinum analogue, cisplatin, pegaspargase, arsenic trioxide, lenalidomide or plerixafor. In some embodiments, the chemotherapy is a combination of 5-fluorouracil and methotrexate.

[0014] In some embodiments, the cells expressing DCX protein are located in the hippocampus. In some embodiments, the cells expressing DCX protein are in the dentate gyrus. In some embodiments, the cells expressing DCX protein are neural precursor cells, neural stem cells or immature neurons.

[0015] In some embodiments, the memory improved by the methods described herein is spatial memory, short term memory, long term memory, working memory, episodic memory, semantic memory, remote memory, verbal memory, visual memory, procedural memory, topographic memory, autobiographical memory, implicit memory, retrospective memory or prospective memory.

[0016] In some embodiments, the learning improved by the methods described herein is rule learning or discrimination learning.

[0017] In some embodiments, the subject treated by the methods described herein has cancer. In some embodiments, the cancer is a solid tumor or a hematologic malignancy. In some embodiments, the cancer is a metastatic cancer.

BRIEF DESCRIPTION OF THE DRAWINGS

[0018] FIG. 1 is an experimental timeline for the study described in Example 1 . A combination of 5- fluorouracil (5-FU) and methotrexate (MTX) or equal volume of physiological saline was administered to 3- month old rats, i.p. (intraperitoneal), 3 times at 10-day intervals, and PAN-81 1 or equal volume of saline was injected, i.p., 10 min following each anticancer drug administration. Chemo: MTX/5-FU; PAN: PAN- 81 1 ; Inject: injection; Orient.: Orientation; SM: spatial memory test; PT: probe test; NMTS: nonmatching-to- sample test; DL: discrimination learning test; Perfus.: perfusion; IHC: imrnunohistochemistry.

[0019] FIG. 2A - FIG. 2D are line graphs and bar graphs showing that PAN-81 1 reverses MTX/5-FU- induced cognitive deficits. Rats received 3 i.p. injections of TX/5-FU or equal volume of saline at 10-day intervals, followed with PAN-81 1 or saline i.p. delivery. (FIG. 2A) Effects of MTX/5-FU (Chemo) and PAN- 81 1 (Saline+PAN and Chemo+PAN) on spatial memory training. Data are presented as geometric means ± SEM. (FIG. 2B) Effects of MTX/5-FU and PAN-811 on performance in spatial memory probe test. Data are expressed as arithmetic mean ± SEM. (FIG. 2C) Effects of MTX/5-FU and PAN-81 1 on performance in nonmatching-to-sample test. Data are expressed as arithmetic mean ± SEM. (FIG. 2D) Effects of MTX/5- FU and PAN-811 on discrimination learning. Data from 4 groups per day were statistically analyzed with One-Way ANOVA followed by Tukey HSD test, and paired group also analyzed with T-Test. Figure symbols are #, p < 0.05, ##, p < 0.01 and ###, p < 0.005 compared with Saline control; * p < 0.05, ** p < 0.01 and *** p < 0.005 compared with Chemo group.

[0020] FIG. 3A - FIG. 3B are a set of fluorescent images and a bar graph showing that PAN-81 1 preserves neurogenesis against MTX/5-FU insult. DCX (doublecortin) staining of hippocampus on Day 97. (FIG. 3A) Representative fluorescent image of DCX, in which the abbreviations are: SGZ, subgranular zone; GCL, granule cell layer; and ML, molecular layer. Scale bars for photos in the top row, 100 μm; Scale bars for photos in the bottom row, 50 μm. (FIG. 3B) Quantification of DCX+ cells in SGZ. Chemo+PAN: Chemo+PAN-81 1. Data were analyzed with both One- Way ANOVA as well as T-Test. Figure symbols are ##, P < 0.01 compared with Saline control; ** , P < 0.01 compared with Chemo group.

DETAILED DESCRIPTION OF THE DISCLOSURE

[0021] The disclosure provides methods for preserving neurogenesis in subjects treated with chemotherapy by administering to the subjects a thiosemicarbazone compound, or an analogue thereof.

[0022] Provided herein is a method for reducing chemotherapy-induced inhibition of neurogenesis in a subject, the method comprising administering to the subject a thiosemicarbazone compound, or an analogue thereof.

[0023] Further provided herein is a method for preserving neurogenesis in a subject treated with chemotherapy, the method comprising administering to the subject a thiosemicarbazone compound, or an analogue thereof.

[0024] Still further provided herein is a method for increasing the number of cells expressing doublecortin (DCX) protein in a subject treated with chemotherapy, the method comprising administering to the subject a thiosemicarbazone compound, or an analogue thereof.

[0025] Yet further provided herein a method for improving memory or learning in a subject treated with chemotherapy, the method comprising administering to the subject a thiosemicarbazone compound, or an analogue thereof.

[0026] Each of the aforementioned methods is a "method of the disclosure".

[0027] In any of the methods of the disclosure, a subject may be a human, a non-human primate, a horse, a cow, a sheep, a goat, a pig, a dog, a cat, a rabbit, a hamster, a guinea pig, a rat or a mouse. In some embodiments, a subject is a human adult. In some embodiments, the subject is a human adolescent. In some embodiments, a subject is a human child.

[0028] In any of the methods of the disclosure, the thiosemicarbazone compound may be 3-aminopyridine- 2-carboxaldehyde thiosemicarbazone (3-AP, also called Triapine), or a pharmaceutically acceptable salt thereof. This compound is referred to herein as PAN-81 1. PAN-81 1 is a ribonucleotide reductase inhibitor (see, e.g. , Finch et al., Biochem Pharmacol. 2000; 59: 983-991).

[0029] In any of the methods of the disclosure, the thiosemicarbazone compound may be a compound of Formula (I):

or an analogue thereof,

wherein R, R ₁ , R ₂ , R ₃ , and R ₄ are independently selected from the group consisting of hydrogen, C1 - 8alkyl, C2-8alkenyl, C2-8alkynyl, C3-8cycloalkyl, C1 -8haloalkyl, C6-10aryl, amino-C1 -8alkyl, hydroxy-C1 - 8alkyl, C1 -8alkoxye-C1-8alkyl, and C1-8alkanoyl, or NR1R2 taken in combination form a 3 to 7 member ring which may comprise 0, 1 , or 2 additional ring heteroatoms selected from N, O, and S;

R ₆ is hydrogen, hydroxy, amino, or C1-8alkyl; and

R ₅ and R7 are independently selected from the group consisting of hydrogen, halide, hydroxy, thiol, amino, hydroxyamino, mono-C1 -8alkylamino, di(C1-8alkyl)amino, C1 -8alkoxy, C1-8alkyl, C1-8alkenyl, and C2-8alkynyl.

[0030] In some embodiments, the thiosemicarbazone compound is

or an analogue thereof.

[0031] In some embodiments, the administration to the subject of the thiosemicarbazone compound (for example, PAN-81 1), or an analogue thereof, is intravenous, intraperitoneal, subcutaneous, intramuscular, topical, transdermal or oral. In some embodiments, a pharmaceutical composition comprising the thiosemicarbazone compound (for example, PAN-811), or an analogue thereof, and a pharmaceutically acceptable carrier, excipient or diluent is administered to the subject. In some embodiments, the pharmaceutical composition is formulated for intravenous, intraperitoneal, subcutaneous, intramuscular, topical, transdermal or oral delivery. As used herein "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. The type of carrier can be selected based upon the intended route of administration. In some embodiments, the carrier is suitable for intravenous, intraperitoneal, subcutaneous, intramuscular, topical, transdermal or oral administration. Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the pharmaceutical compositions of the invention is contemplated. Supplementary active compounds can also be incorporated into the compositions.

[0032] Therapeutic compositions typically must be sterile and stable under the conditions of manufacture and storage. The composition can be formulated as a solution, microemulsion, liposome, or other ordered structure suitable to high drug concentration. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, monostearate salts and gelatin. Moreover, the compounds can be administered in a time release formulation, for example in a composition which includes a slow release polymer. The active compounds can be prepared with carriers that will protect the compound against rapid release, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, polylactic acid and polylactic, polyglycolic copolymers (PLG). Many methods for the preparation of such formulations are generally known to those skilled in the art.

[0033] Sterile injectable solutions can be prepared by incorporating the thiosemicarbazone compound (for example, PAN-81 1), or an analogue thereof, in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying which yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

[0034] Depending on the route of administration, the thiosemicarbazone compound (for example, PAN- 81 1), or an analogue thereof, may be coated in a material to protect it from the action of enzymes, acids and other natural conditions which may inactivate the agent. For example, the compound can be administered to a subject in an appropriate carrier or diluent co-administered with enzyme inhibitors or in an appropriate carrier such as liposomes. Pharmaceutically acceptable diluents include saline and aqueous buffer solutions. Enzyme inhibitors include pancreatic trypsin inhibitor, diisopropylfluoro-phosphate (DEP) and trasylol. Liposomes include water-in-oil-in-water emulsions as well as conventional liposomes. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations may contain a preservative to prevent the growth of microorganisms.

[0035] In some embodiments, the thiosemicarbazone compound (for example, PAN-811), or an analogue thereof, is formulated in the composition in a therapeutically effective amount. A "therapeutically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic result to thereby influence the therapeutic course of a particular disease or disorder state. A therapeutically effective amount of the thiosemicarbazone compound (for example, PAN-81 1), or an analogue thereof, may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the agent to elicit a desired response in the individual. Dosage regimens may be adjusted to provide the optimum therapeutic response. A therapeutically effective amount is also one in which any toxic or detrimental effects of the agent are outweighed by the therapeutically beneficial effects. In another embodiment, the thiosemicarbazone compound (for example, PAN-811), or an analogue thereof, is formulated in the composition in a prophylactically effective amount. A "prophylactically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result. Typically, since a prophylactic dose is used in subjects prior to or at an earlier stage of disease, the prophylactically effective amount will be less than the therapeutically effective amount.

[0036] The amount of the thiosemicarbazone compound (for example, PAN-81 1), or an analogue thereof, in the composition may vary according to factors such as the disease state, age, sex, and weight of the individual. Dosage regimens may be adjusted to provide the optimum therapeutic response. For example, a single bolus may be administered, several divided doses may be administered over time or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. It is especially advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the mammalian subjects to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the dosage unit forms used in the methods of the disclosure are dictated by and directly dependent on (a) the unique characteristics of the thiosemicarbazone compound (for example, PAN-81 1), or an analogue thereof, and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of compounding such an active compound for the treatment of sensitivity in individuals.

[0037] In some embodiments, the thiosemicarbazone compound (for example, PAN-811), or an analogue thereof, is administered to a subject (e.g,, a human subject) at a dose ranging from about 75 mg/rn ² /day to about 100 mg/m ² /day, from about 80 mg/m ² /day to about 100 mg/m ² /day or ranging from about 90 mg/m ² /day to about 100 mg/m ² /day. In some embodiments, the thiosemicarbazone compound (for example, PAN-811), or an analogue thereof, is administered to a subject (e.g, , a human subject) at a dose of about 96 mg/m ² /day.

[0038] In any of the methods of the disclosure, the chemotherapy is an anti-cancer agent (e.g., a cancer chemotherapeutic drug). In some embodiments, the anti-cancer agent is an immunotherapeutic agent (e.g., an immune checkpoint inhibitor). In some embodiments, an immunotherapeutic agent is a small molecule drug or a therapeutic antibody with immunomodulatory effects. Non-limiting examples of immune checkpoint inhibitors include inhibitors of PD-1 , PD-L1 , CTLA-4 and Lymphocyte Activation Gene 3 (LAG- 3). In some embodiments, the anti-cancer agent is a small molecule drug, a therapeutic antibody, an antibody-drug conjugate or a chimeric antigen receptor T-cell therapy. In some embodiments, the anticancer agent is, without limitation, an S-phase antimetabolite, G1/S phase inhibitor, topoisomerase II inhibitor, phase inhibitors, G2/ phase inhibitor, alkylating agent, anthracycline, or topoisomerase I inhibitor. In some embodiments, without limitation, the anti-cancer agent is 5-fluorouracil, methotrexate, leucovorin, cytarabine, fludarabine, gemcitabine, capecitabine, clofarabine, azacytidine, nelarabine, decitabine, pralatrexate, pemetrexed, etoposide, docetaxel, paclitaxel, ixabepilone, cabazitaxel, eribulin mesylate, vincristine, vinblastine, bleomycin, chlorambucil, procarbazine, dacarbazine, ifosfamide, temozolomide, oxaliplatin, bendamustine, daunorubicin, epirubicin, doxorubicin, irinotecan, topotecan, platinum analogue, cisplatin, pegaspargase, arsenic trioxide, lenalidomide or plerixafor.

[0039] In some embodiments of the methods of the disclosure, the chemotherapy is a combination of two or more, three or more, four or more, or five or more anti-cancer agents (i.e., different anti-cancer agents). In some embodiments, the chemotherapy is a combination of 5-fluorouracil and methotrexate. In some embodiments, the chemotherapy is a combination of 5-fluorouracil and cisplatin. In some embodiments, the chemotherapy is a combination of methotrexate and cisplatin.

[0040] In some embodiments of the methods of the disclosure, the subject is treated with the chemotherapy before or after the administration of the thiosemicarbazone compound (for example, PAN- 81 1), or an analogue thereof. In some embodiments, administration of the thiosemicarbazone compound and administration of the chemotherapy may be separated by about 0.5 hours, about 1 hour or about 2 hours.

[0041] In some embodiments, the subject is treated with the chemotherapy simultaneously with the administration of the thiosemicarbazone compound (for example, PAN-811), or an analogue thereof. In some embodiments of methods where the subject is treated with the chemotherapy simultaneously with the administration of the thiosemicarbazone compound, the chemotherapy and the thiosemicarbazone compound may be in the same composition. In some embodiments of methods where the subject is treated with the chemotherapy simultaneously with the administration of the thiosemicarbazone compound, the chemotherapy and the thiosemicarbazone compound may be in different compositions.

[0042] In some embodiments, the thiosemicarbazone compound (for example, PAN-811), or an analogue thereof, has no significant effect on the efficacy of the chemotherapy. For example, an anti-cancer agent may inhibit or kill cancer cells at a substantially similar level in the absence of and in the presence of the thiosemicarbazone compound (for example, PAN-81 1), or an analogue thereof.

[0043] In some embodiments of methods for increasing the number of cells expressing doublecortin (DCX) protein in a subject treated with chemotherapy by administration of the thiosemicabazone compound (for example, PAN-81 1), or an analogue thereof, the cells expressing DCX protein are located in the hippocampus. In some embodiments, the cells expressing DCX protein are in the dentate gyrus (for example, in the subgranular zone, the granule cell layer or the molecular layer of the dentate gyrus). In some embodiments, the DCX protein is human DCX protein. In some embodiments, the cells expressing DCX protein are neural precursor cells, neural stem cells or immature neurons. Immature neurons may be adult or embryonic immature neurons. In some embodiments, administration of the thiosemicarbazone compound (for example, PAN-811), or an analogue thereof to a subject treated with chemotherapy increases the number of cells expressing DCX by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, or at least about 70% compared to the number of cells expressing DCX in a subject treated with the same chemotherapy and not administered the thiosemicarbazone compound. [0044] In some embodiments, the memory improved by any of the methods of the disclosure is spatial memory, short term memory, long term memory, working memory, episodic memory, semantic memory, remote memory, verbal memory, visual memory, procedural memory, topographic memory, autobiographical memory, implicit memory, retrospective memory or prospective memory, or any combination of types of memory. In some embodiments, the memory improved by any of the methods of the disclosure may be evaluated in human subjects with clinically used tests (e.g., the Mini-Mental State Exam (MMSE) or the Mini-Cog test), or computerized tests using CANTAB Mobile, COGNIGRAM®, COGNIVUE®, COGNISION® and Automated Neuropsychological Assessment Metrics (ANAM) devices.

[0045] In some embodiments, the learning improved by the methods described herein is rule learning or discrimination learning, or a combination of these.

[0046] In some embodiments, the subject treated by any of the methods of the disclosure has cancer. In some embodiments, the cancer is a solid tumor. In some embodiments, without limitation, the cancer is skin, brain, head-and-neck, lung, stomach, colon, pancreatic, liver, kidney, bladder, breast, ovarian, uterine, cervical, prostate or testicular cancer. In some embodiments, the cancer is melanoma or basal cell carcinoma. In some embodiments, the cancer is a hematologic malignancy. In some embodiments, the cancer is leukemia, lymphoma or multiple myeloma. In some embodiments, the cancer is metastatic cancer.

[0047] In some embodiments, the subject has experienced cognitive impairment (for example, memory loss, attention deficit, learning impairment or confused thought processes, or any combination of these) before being treated by a method of the disclosure.

[0048] The disclosure also provides a use of a thiosemicarbazone compound (for example PAN-81 1), or an analogue thereof, for preserving neurogenesis in a subject treated with chemotherapy. The disclosure further provides a use of a thiosemicarbazone compound (for example PAN-81 1), or an analogue thereof, for reducing chemotherapy-induced inhibition of neurogenesis in a subject. The disclosure further provides a use of a thiosemicarbazone compound (for example PAN-81 1), or an analogue thereof, for increasing the number of cells expressing doublecortin (DCX) protein in a subject treated with chemotherapy. The disclosure yet further provides a use of a thiosemicarbazone compound (for example PAN-81 1), or an analogue thereof, for improving memory or learning in a subject treated with chemotherapy.

[0049] The term "about" when immediately preceding a numerical value means ± 0% to 10% of the numerical value, ± 0% to 10%, ± 0% to 9%, ± 0% to 8%, ± 0% to 7%, ± 0% to 6%, ± 0% to 5%, ± 0% to 4%, ± 0% to 3%, ± 0% to 2%, ± 0% to 1 %, ± 0% to less than 1 %, or any other value or range of values therein. For example, "about 40" means ± 0% to 10% of 40 (i.e., from 36 to 44).

[0050] The disclosure will be further clarified by the following example, which is intended to be purely exemplary of the disclosure and in no way limiting.

EXAMPLE [0051] EXAMPLE 1 : EFFECTS OF PAN-811 ON COGNITIVE TASKS AND NEUROGENESIS IN RATS TREATED WITH 5-FLUOROURACIL (5-FU) AND METHOTREXATE (MTX)

[0052] Materials and methods

[0053] Materials

[0054] PAN-811 Cl·H ₂ O was produced by Kimia Corp, Santa Clara for Panacea Pharmaceuticals Inc. MTX and 5-FU were purchased from Wyeth Canada, Thornhill, Ontario, and Mayne Pharma, Kirkland, Quebec, respectively.

[0055] Animal model and treatment

[0056] The study was conducted using healthy female adult Long Evan rats (Charles River Laboratories, St. Constant, Quebec), 3 months old at the beginning of the experiment. After one week in quarantine, they were transferred to standard laboratory cages with food and water always available. All rats were maintained on a 12-hour light-dark schedule, with lights on between 8:00PM and 8:00AM.

[0057] The experimental protocol and all handling procedures were approved by the Trent University and University of Toronto Animal Care Committees, and conformed to requirements of the Canadian Council on Animal Care. The rats were examined regularly by a veterinarian throughout the experiment.

[0058] Initially, 46 rats were assigned randomly to 4 groups - saline plus saline (Saline, N = 10), chemotherapy plus saline (Chemo, N = 13), saline plus PAN-811 (Saline+PAN, N = 10), and chemotherapy plus PAN-811 (Chemo+PAN, N = 13). Rats receiving chemotherapy in Chemo or Chemo+PAN groups were administered 37.5mg/kg MTX and 50mg/kg 5-FU dissolved in physiological (normal) saline, i.p., 3 times with 10-day intervals. Control rats in Saline and Saline+PAN groups were injected with equal volumes of saline at the same time points. For treatment, in Chemo+PAN and Saline+PAN groups, rats were administered 12mg/kg PAN-81 1 · Cl·H ₂ O (a salt format of PAN-81 1) dissolved in normal saline, i.p., also 3 times, but 10 min following each administration of the anticancer drugs or saline. Likewise, rats in Chemo and Saline groups were administered equal volumes of the normal saline, i.p., 3 times, 10 min following each administration of anticancer drugs or saline (Table 1). FIG. 1 provides the overall study timeline.

[0059] The doses for all drugs were selected on the basis of dose-response tests fortolerance and toxicity. The dosages were well tolerated and did not influence appetite or activity levels. The only noticeable effect was a small amount of hair loss in a few rats that had received MTX/5-FU.

Table 1. Regime at each treatment day (3 sets of injections at 10-day intervals).

[0060] Cognitive tests

[0061] The study utilized the following cognitive tests to investigate various aspects of learning and memory. [0062] Spatial learning and memory (SM). The SM test is a widely used, highly sensitive test of hippocampal dysfunction [Morris et al., Nature. 1982; 297: 681-683]. The test was conducted in a circular pool (130 cm diameter and 30 cm high), located in the center of a standard testing room. The pool was filled with opaque water and maintained at 21 °C. An inverted flowerpot (10cm diameter), situated a few cm below the surface, served as a platform on which the rats could climb to escape the water. Throughout testing, the water was cleaned after each trial and changed every 2-3 days. The pool was divided into 6 zones of approximately equal size. Swimming patterns were monitored by an overhead video camera connected to a recorder and data processing system. The system recorded swimming routes that were used to count errors. An error was recorded each time the rat entered a zone not containing the platform. On Day 31 , the rats received one day of orientation training (5 trials/day) in which they learned to swim to the platform that was visible and in a different location on each trial. SM testing began the following day (Day 32). The platform was now submerged and always located in the center of the northeast zone. At the start of each trial, the rat was placed in the water at the edge of the pool, facing the wall, at a different location, but never in the northeast zone. A trial continued until the rat mounted the platform with all four paws. If it failed to find the platform in 60 sec, it was guided to the platform, and assigned an error score of 15. After 20 sec. on the platform, the rat was placed under a heat lamp in a holding cage to await the next trial. Each rat received 5 such trials/day for 5 consecutive days (Days 32-36). One rat assigned to the Chemo+PAN group died following of MTX/FU injection, and one rat was removed from Saline group due to a handling error. Therefore, 44 rats completed SM test and probe test (PT. Chemo: n = 13; Chemo+PAN: n = 12; Saline+PAN: n = 10; Saline: n = 9).

[0063] PT or probe trial provided an additional test of memory for the location of the platform. On Day 37, trials 1 & 2 were conducted in the usual manner. On the third trial, the platform was removed and the rats were allowed to swim for 60 sec. Time spent in the target zone for each group was expressed as a percentage of the 60-sec period. The number of rats in each group was same as that in SM test. Data were expressed as arithmetic means ± SEM.

[0064] Nonmatching-to-sample learning (NMTS). The NMTS test consists of a series of paired sample (or study) - test trials in a water maze. The stimuli for the sample and test trials were black and white cylinders (30 cm long x 3 cm in diameter), suspended 5 centimeters above the surface of the water. In the sample trials, one of the stimuli signaled the platform's location. In the subsequent test trial, the sample stimulus was presented along with the other stimulus in new locations. In the test trial, the cylinder that was not present during the preceding sample trial now signaled the platform's location. NMTS and related rule- learning tasks incorporate conditional and working memory components and are known to be sensitive to frontal-lobe impairment [Winocur, Neuropsychologia. 1992; 30: 769-781].

[0065] NMTS testing began on Day 39. For the sample trials, the rat was placed in the southeast zone of the pool and allowed to swim to the submerged platform under a sample cylinder. The rat remained on the platform for 20 sec. and then placed under the heat lamp while the platform together with the cylinders were re-located to different zones. For the test trial, which began 10 sec. later, the rat was placed in the pool at a different location (with the exceptions of the zone containing either cylinder and the target zone in the preceding sample trial), and allowed to swim to the submerged platform. If the rat failed to find the platform within 60 sec, it was guided to the platform and given an error score of 15. After 20 sec. on the platform the rat was placed under the heat lamp, to await the next pair of trials. Ten sessions, each consisting of 5 pairs of sample and test trials, were administered each day over 10 days (Days 39-48). One rat assigned to the Chemo+PAN group died, 17 days following final drug injection and, as a result, did not participate in NMTS and DL testing. Therefore, 43 rats completed NMTS test (Chemo: n = 13; Chemo+PAN: n = 1 1 ; Saline+PAN: n = 10; Saline: n = 9). Data from test trials (not sample trials) were used for result judgement.

[0066] Discrimination learning (PL). The DL test requires the rat to discriminate between horizontal vs vertical, black and white striped cylinders (30 cm long x 3 cm in diameter) in order to find the submerged platform. The task measures non-conditional, stimulus-response learning and is sensitive to impairment in the striatal system [McDonald et al., Functional dissociation of bain regions in learning and memory: Evidence for multiple systems. In: Foster JK, Jelicic M, editors. Memory: Systems, Process or Function. New York: Oxford University Press; 1999. p. 66-103].

[0067] In this test, the pool was fitted with a gray, plastic cross-maze with walls that extended 10 cm above the surface of the water. Each arm of the maze was 55 cm long. Orientation training started on Day 50 (Day 30 following the final drug administration), and lasted for 2 days. For each orientation trial, the rat was placed in the pool at the end of one of the arms and allowed to swim to a submerged platform which was located at the end of each goal arm. Each orientation session consisted of 5 trials/day. For these trials, there was no discrete cue to direct the animal.

[0068] DL began the day following orientation (on Day 52 or Day 32 following the final drug administration), and consisted of 10 trials/day. On each trial, the rat was placed in the pool at the end of one of the arms and allowed to swim to the choice point, where it encountered the black and white cylinders. For half the rats, the cylinder with horizontal stripes was positive, and for the other half, the cylinder with vertical stripes was positive. The selection of the start arm for each trial and the positioning of the cylinders were determined by a random schedule. A submerged platform was located at the end of the correct arm. The rat made a correct response if, at the choice point, it turned in the direction of the correct stimulus and swam down that arm. An error was scored when a rat entered an incorrect arm with its whole body (less the tail) or left the correct arm. Between trials of orientation training and discrimination learning, animals were placed under the heat lamp to await the next trial.

[0069] Rats were tested on the DL task until they reached a criterion of 0.5 errors/trial/day averaged over two consecutive days. Testing was terminated if an animal failed to reach this criterion after 15 days (by the end of Day 66 orthe end of Day 46 following final drug administration), and a score of 150 was assigned. The number of rats in each group was same as that in NMTS test.

[0070] Immunohistochemistry

[0071] In order to examine and compare the level of adult neurogenesis in the 4 treatment groups, the brain sections were stained by an immunohistochemistry method using Doublecortin (DCX) antibody. Doublecortin is a reliable marker of immature neurons and has become a standard method for quantification of neurogenesis. Rats were sacrificed on Day 97 by an overdose i.p. injection of Euthansol (Day 77 following the final drug administration). Brain tissue was fixed by pre-intracardiac perfusion and post-fixed with 4% paraformaldehyde for 24 hrs. Brains were cut in half and the hippocampus was isolated from the right hemisphere of each rat. Isolated hippocampi were sectioned serially along the dorso-ventral axis using a vibratome (VT1000S, Leica Microsystems, Heidelberg, Germany) into sections 30 μηι thick. The sections were stored in phosphate-buffered saline (PBS) with 0.1% sodium azide for further processing. Twelve sections from each animal were sampled using a systematic random sampling procedure previously described [Morris et al., Nature. 1982; 297: 681-683]. DCX staining was performed on free-floating sections. Importantly, sections were rinsed extensively in PBS before processing and between each incubation. All primary and secondary antibody incubations were conducted in PBS containing 0.3% Triton X-100. The sections were incubated with a primary goat anti-DCX antibody (1 :200, sc-8066, Santa Cruz Biotechnology, 24 hours at 4°C), followed by secondary antibody donkey anti-goat IgG Alexa 488 (1 :200; A11055, Life Technologies; 2 hours at room temperature (RT) in the dark). Then sections were mounted onto glass slides using double-distilled water (ddH20), and coverslipped using PermaFluo (Thermo Scientific, Fremont, CA, USA).

[0072] Immunohistochemical controls included the omission of primary antibody, which resulted in lack of staining at the corresponding wavelength. Sections were examined and immunolabelled cells in the dentate gyrus (DG) of hippocampus were counted using a Leica TCS-SL confocal microscope (Leica Microsystems (Canada) Inc.; Richmond Hill, ON, Canada) with a 63x oil immersion objective lens. The experimenter was blinded with respect to the group and animal identification number to avoid bias. Immuno-positive cells (DCX+) were counted in the subgranular zone (SGZ) of DG. The SGZ was defined as a two-cell width wide (approximately 20 μπι) region just below the granule cell layer (GCL). All cells within each section were counted, but excluding top and bottom surfaces of the sections in order to avoid counting cells that were dissected or damaged. The average number of cells per section was multiplied by the total number of sections to obtain total cell numbers per DG [Morris et al., Nature. 1982; 297: 681-683; Wojtowicz et al., Nat Protoc. 2006; 1 : 1399-1405].

[0073] Data expression and statistical analysis

[0074] The data from rats in each group were expressed as geometric mean ± standard error (SEM) for the SM test, based on a log distribution profile of the data, and as arithmetic means ± SEM for all other tests. The data in all tests were verified normal distributions and results were statistically evaluated at a significance level of 1% with One-Way ANOVA followed by the Tukey HSD test for each day of a time- course test and also with T-Test by using software VASSARSTATS (vassarstats.net/index.html). Figure symbols are as follows: #, p < 0.05; ##, p < 0.01 ; ###, p < 0.005 for comparisons with the Saline group; * , p < 0.05; ** , p < 0.01 ; *** , p < 0.005 for comparisons with the Chemo group.

[0075] Results [0076] PAN-811 significantly reduces MTX/5-FU-induced impairment in the spatial memory (SM) test

[0077] Spatial learning and memory are closely related to hippocampus function. There were no differences between groups in terms of latency to reach and climb on the visible platform during orientation training (Table 2; F _{3, 41} = 0.47, p = 0.71). Similarly, on the first day following orientation, no statistically significant difference existed between the 4 groups (FIG. 2A. T21 = -0.63, p > 0.05 by T-Test; F _{3, 41} = 0.23, p > 0.05 by ANOVA). However, on Days 2-4 of the SM test, the Chemo group made more errors than the Saline or other groups in finding the platform on Day 2 (T21 = -1 .95, p < 0.05 by T-Test; F3, 41 = 3.52, p < 0.05 by ANOVA), Day 3 (T21 = -3.23, p < 0.005 by T-Test; ft, 41 = 3.27, p < 0.05 by ANOVA) and Day 4 (T21 = -5.46, p < 0.005 by T-Test; ft, 41 = 16.93, p < 0.005 by ANOVA). The Chemo group improved dramatically on Day 5, but still showed a statistically significant difference from the Saline or other groups (T21 = -2.9, p < 0.005 by T-Test; ft, 41 = 3.63, p < 0.05 by ANOVA). These data demonstrate that chemotherapy impaired performance during the spatial memory learning stage. By contrast, the Saline+PAN group did not differ from the Saline control group (no statistically significant difference between these two groups) on most test days, except that the error rate in the Saline +PAN group was slightly higher than that of Saline group on Day 3 (T18 = -1.89, p < 0.05 by T-Test, but not by Tukey HSD Test following One-Way ANOVA). Interestingly, in the Chemo+PAN group, the errors in finding the platform were significantly lower than those in the Chemo group on Day 2 (723 = 2.72, p < 0.01 by T-Test; ft, 41 = 3.52, p < 0.05 by ANOVA) and Day 4 (723 = 3.99, p < 0.005 by T-Test; ft, 41 = 16.93, p < 0.01 Tukey HSD Test following ANOVA). Although there was no statistically significant difference between these two groups on Day 3 and Day 5, the mean number of errors made by the Chemo+PAN group was much lower than that in the Chemo group and not significantly different from the Saline group. These indicated a beneficial effect of PAN-81 1 on chemotherapy-treated rats.

[0078] The PT, which was carried out on Day 6 of the SM test, measured time spent in the zone where the platform was previously present, provides an additional measure of hippocampus-sensitive spatial memory (FIG. 2B). As can be seen in FIG. 2B, the Chemo group spent significantly less time in the target zone than the Saline group (T21 = 2.5, p < 0.05 by T-Test). As in the SM test, PAN-81 1 treatment protected rats against the effects of chemotherapy on spatial memory. The % time spent by the Chemo+PAN group in the target zone was significantly higher than that in Chemo group (T22 = -2.86, p < 0.005 by T-Test), and did not differ from the Saline group (T19 = 0.7, p > 0.05 by T-Test). Thus, PAN-81 1 efficiently protected SM function in against MTX/5-FU insult.

Table 2. Mean latencies (sec.) for all groups to find visible platform over 5 trials of orientation training.

[0079] PAN-811 reduces MTX/5-FU-induced deficits in NMTS rule learning [0080] The NMTS test was used to measure conditional learning and working memory, which reflects frontal lobe function. From Days 3-10 of NMTS testing, the Chemo group demonstrated significantly higher errors than the Saline group (FIG. 2C). PAN-811 treatment did not affect performance in comparison with the Saline control (no statistically significant difference between the two groups) on test Days 1-9, except for a slight increase in errors on Day 10 (T ₁₈ = -2.65, p < 0.01 by T-Test but not by One-Way ANOVA). Coadministration of PAN-811 significantly reduced MTX/5-FU-induced errors to the levels of those in the Saline group on Days 2-6 and Day 8. The numbers of errors in the Chemo+PAN group were lower than those in the Chemo group on Days 7, 9 and 10, but were not statistically significant. Generally, then, PAN- 811 robustly suppressed MTX/5-FU- induced errors in the NMTS test.

[0081] PAN-811 reduces MTX/5-FU-induced deficit in discrimination learning

[0082] The DL test assessed discrimination capability that is sensitive to impairment of the striatal system. The measure of learning was the number of trials required to achieve a criterion of 0.5 errors/trial on two consecutive days of testing. The number of trials to criterion in the Chemo group was significantly greater than that in the Saline group (FIG. 2D; T ₂₀ = -2.733, p < 0.01 by T-Test; F _{3, 39} = 4.65, p < 0.05 by Tukey HSD Test following One-Way ANOVA and). PAN-811 by itself did not affect discrimination learning in comparison with Saline control (no statistically significant difference between the two groups). PAN-811 significantly reduced the number of the trials to criterion in the Chemo+PAN group (T ₂₂ = 2.49, p < 0.05 by T-Test; F _{3, 39} = 4.65, p < 0.05 by Tukey HSD Test following ANOVA, compared with Chemo group) down to the level in the Saline group (no statistically significant difference between Saline and Chemo+PAN).

[0083] PAN-811 blocks MTX/5-FU-elicited damage to neurogenesis in the subgranular zone

[0084] DCX is a protein that expresses in both neural precursor cells and immature neurons, which involves neurogenesis in adult brain. DCX positive cells were labeled with green fluorescence (FIG. 3A), which not only shown in cell body but also in cell processes in the subgranular zone (SGZ) of DG. The density of DCX+ cells in the Saline+PAN group was about same as that in the Saline group. However, the number of DCX+ cells was clearly less in the Chemo group, in comparison with that in the Saline control. There were more DCX+ cells in the Chemo+PAN group than in the Chemo group. The density of cell processes in the PAN and Chemo+PAN groups appeared also higher. DCX+ cells in SGZ were manually quantified in blind way to avoid bias (FIG. 3B). MTX/5-FU in Chemo group reduced number of DCX+ cells to 67% of that in the Saline group (F _{3, 16} = 13.27, p < 0.01 by Tukey HSD Test following ANOVA). PAN-811 did not cause any decrease of DCX+ cells in number. In the Chemo+PAN group, the number of DCX+ cells was significantly higher than that in the Chemo group (F _{3, 16} = 13.27, p < 0.01 by Tukey HSD Test following ANOVA) and 94% of that in the Saline control (no statistically significant difference between two groups). Thus, PAN-811 provided a full preservation of neurogenesis in the SGZ under MTX/5-FU stress.

[0085] Discussion

[0086] In line with previous findings in the rat [Winocur et al., Behav Neurosci. 2016; 130: 428-436; Winocur et al., Psychopharmacology (Berl). 2014; 231 : 2311-2320] and the mouse [Winocur et al., Pharmacol Biochem Behav. 2006; 85: 66-75; Winocur et al., Clin Cancer Res. 2012; 18: 3112-3121], deficits were observed in spatial learning and memory, NMTS rule learning, and discrimination learning following i.p. delivery of TX/5-FU. The impairment was widespread, affecting a range of cognitive processes associated with the hippocampus [Morris et al., Nature. 1982; 297: 681-683], frontal lobes [Winocur, Neuropsychologia. 1992; 30: 769-781], and corpus striatum [McDonald et al., Functional dissociation of bain regions in learning and memory: Evidence for multiple systems. In: Foster JK, Jelicic M, editors. Memory: Systems, Process or Function. New York: Oxford University Press; 1999. p. 66-103], respectively. MTX/5-FU-elicited CNS impairment could also be enduring. The measurement of cognitive functions started from Day 31 and finished by the end of Day 66 (the end of the final test), and cognitive dysfunction manifested through the whole time period. Additionally, histo pathology was examined on Day 97 and MTX/5-FU-induced cellular changes were clearly observed at that time.

[0087] The combination of MTX and 5-FU has dual effects. It can damage both cancer cells and neural stem cells [Winocur et al., Behav Neurosci. 2016; 130: 428-436; Winocur et al., Behav Brain Res. 2015; 281 : 239-244]. The former introduces a therapeutic benefit with respect to the disease while the latter has negative side effects on the nervous system. Second, oxidative stress (OS) is a common factor in cytotoxicity, and both MTX and 5-FU increase in vivo OS and result in damage [Miketova et al., Biol Res Nurs. 2005; 6: 187-195; Caron et al., Pediatr Blood Cancer. 2009; 53: 551 -556; Numazawa et al., Basic Clin Pharmacol Toxicol. 2011 ; 108: 40-45; Lamberti et al., J Exp Clin Cancer Res. 2012; 31 : 60; Baba et al., Exp Ther Med. 2012; 4: 452-458]. To gain insight into underlying mechanisms for MTX/5-FU-elicited cognitive impairment, we examined the effect of chemotherapy on neurogenesis in adult rat brains by labeling neural precursor cells and immature neurons with DCX. In the granular zone of the DG of hippocampus, MTX/5-FU significantly reduced the number of DCX+ cells, indicating a deleterious effect on the neurogenesis of adult brain. The present results indicate that suppression of neurogenesis in the dentate gyrus is an underlying mechanism of chemotherapy-induced impairment on the SM task, a widely accepted test of hippocampal function.

[0088] PAN-811 at a dose of 12 mg/kg does not introduce any neurotoxicity by comparison with control group, as shown with cognitive tests and IHC examination. Furthermore, PAN-81 1 at this dose does not show fatal toxicity to rats, since rat number in Saline+PAN group remained same as that in Saline group by the end of experiment.

[0089] Functionally, systemic delivery of PAN-81 1 via the i.p. route achieved a significant preservation of hippocampus-controlled cognitive function under MTX/5-FU stress, as demonstrated by suppression of MTX/5-FU-elicited error increase in the SM test and by blocking MTX/5-FU-induced percent time reduction in target zone during the PT test of spatial memory. Similarly, PAN-811 also provided effective neuroprotection from MTX/5-FU stress on frontal lobe and striatal function, as reflected in improved performance in the Chemo+PAN group on the NMTS and DL tests. This is the first demonstration that PAN- 81 1 can protect against MTX/5-FU-induced cognitive impairment in an animal model. [0090] All publications, patents and patent applications are herein incorporated by reference in their entirety to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated by reference in its entirety.