Treating pediatric cancers with hydroxyureamethyl acylfulvene

Abstract

Disclosed are methods, compositions, and kits for treating pediatric cancers using hydroxyureamethyl acylfulvene (LP-184) or pharmaceutically acceptable salts / solvates thereof. The methods include monotherapy and combination regimens for pediatric sarcoma, medulloblastoma, glioma (including DIPG), hepatoblastoma, Wilms tumor, and related malignancies. Combination therapies include LP-184 with vincristine, topoisomerase I poisons (topotecan or irinotecan), PARP inhibitors (e.g., rucaparib), or spironolactone, yielding additive or greater-than-additive reductions in tumor growth or cell viability.

Description

TREATING PEDIATRIC CANCERS WITH HYDROXYUREAMETHYL ACYLFULVENETECHNICAL FIELD

[0001] This application relates to pediatric cancer treatments, and more specifically, this application relates to cancer treatments using an acylfulvene.BACKGROUND

[0002] Pediatric cancers are a heterogeneous set of malignant diseases arising in children and adolescents (typically <18 years of age). In contrast to many adult malignancies that are frequently associated with cumulative environmental exposures and aging biology, pediatric tumors are often driven by germline predisposition syndromes, dysregulation of developmental pathways, and somatic alterations characteristic of embryonal or immature cell lineages. Although pediatric cancers are relatively rare on a population basis, they remain a leading cause of disease-related mortality in children, underscoring the need for specialized research, diagnostic approaches, and therapeutics tailored to the pediatric setting.

[0003] Current standard treatments typically involve multimodal regimens including cytotoxic chemotherapy, surgery, and radiation. While many pediatric cancers are initially chemosensitive, durable control remains challenging for high-risk, relapsed, or refractory disease. Moreover, the acute and long-term toxicities of conventional therapy — cardiotoxicity, neurocognitive effects, growth and endocrine disturbances, infertility, and secondary malignancies — are of particular concern in children with decades of life expectancy.

[0004] Regulatory frameworks encourage dedicated pediatric evaluation, including requirements or incentives for pediatric investigation plans and orphan-drug pathways. Nevertheless, significant gaps persist for high-priority entities such as relapsed acute leukemias, high-risk neuroblastoma, diffuse midline glioma, and metastatic sarcomas, where novel mechanisms of action and biomarker-guided strategies are urgently needed.

[0005] Accordingly, there remains a need for improved treatments for pediatric cancer.SUMMARY

[0006] In one aspect, the disclosure provides a method of treating a pediatric cancer selected from pediatric sarcoma, medulloblastoma, glioma, and hepatoblastoma in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of hydroxyureamethyl acylfulvene (LP-184) or a pharmaceutically acceptable salt or solvate thereof.

[0007] In another aspect, administration of hydroxyureamethyl acylfulvene is intravenous, intraperitoneal, or oral administration.

[0008] In another aspect, the dose of hydroxyureamethyl acylfulvene is about 0.05 mg / kg to about 30 mg / kg. The dose of hydroxyureamethyl acylfulvene can be selected from about 0.5 mg / kg, about 2.0 mg / kg, about 4.0 mg / kg, about 10 mg / kg, or about 30 mg / kg.

[0009] In another aspect, the pediatric cancer is selected from medulloblastoma, diffuse intrinsic pontine glioma (DIPG), Ewing sarcoma, rhabdomyosarcoma, hepatoblastoma, and Wilms tumor.

[0010] In another aspect, the treatment regimen of hydroxyureamethyl acylfulvene results in sustained tumor regression or a tumor-growth delay relative to control.

[0011] In another aspect, the regimen treats a pediatric cancer that is a sarcoma xenograft exhibiting no tumor regrowth at study termination.

[0012] In another aspect, a method of treating a pediatric sarcoma comprises administering an effective amount of hydroxyureamethyl acylfulvene.

[0013] In another aspect, the pediatric sarcoma is selected from Ewing sarcoma and rhabdomyosarcoma.

[0014] In another aspect, a method of treating a pediatric cancer comprises administering hydroxyureamethyl acylfulvene in combination with vincristine.

[0015] In another aspect, hydroxyureamethyl acylfulvene is administered intravenously at about 1 mg / kg on Days 1, 4, 7, and 10, and vincristine is administered intraperitoneally or intravenously on a weekly schedule selected from Days 1, 8, and 15.

[0016] In another aspect, the combination of hydroxyureamethyl acylfulvene and vincristine yields an additive antitumor effect measured as tumor-growth delay relative to either single agent.

[0017] In another aspect, the combination treatment with hydroxyureamethyl acylfulvene and vincristine is employed in a pediatric sarcoma.

[0018] In another aspect, a method of treating a pediatric cancer comprises administering hydroxyureamethyl acylfulvene prior to, concurrently with, or subsequent to a topoisomerase I poison selected from topotecan and irinotecan.

[0019] In another aspect, a method of treating a pediatric cancer characterized by a biomarker selected from (i) increased expression or activity of PTGR1, (ii) homologous recombination deficiency (HRD) positivity, and (iii) nucleotide excision repair (NER) positivity comprises administering an effective amount of hydroxyureamethyl acylfulvene.

[0020] In another aspect, increased PTGR1 expression is determined by an assay demonstrating at least a two-fold elevation relative to a normal reference.

[0021] In another aspect, a method of sensitizing a pediatric cancer to radiotherapy comprises administering hydroxyureamethyl acylfulvene before, during, or after a course of radiation therapy.

[0022] In another aspect, hydroxyureamethyl acylfulvene is administered within about 24 hours of a radiation fraction.

[0023] In another aspect, a kit is provided comprising (a) hydroxyureamethyl acylfulvene or a pharmaceutically acceptable salt or solvate thereof; and (b) a PARP inhibitor, wherein the kit includes instructions for administering the hydroxyureamethyl acylfulvene prior to, concurrently with, or subsequent to the PARP inhibitor to treat a pediatric cancer.

[0024] In another aspect, a method of reducing tumor growth rate in a pediatric subject with a cancer selected from pediatric sarcoma, medulloblastoma, glioma, rhabdoid tumor, and hepatoblastoma comprises administering hydroxyureamethyl acylfulvene in an amount effective to reduce tumor growth rate by at least about 30% relative to a pretreatment rate.

[0025] In another aspect, a method of selecting a pediatric subject for treatment with hydroxyureamethyl acylfulvene comprises: (i) obtaining a tumor cell sample from the subject; (ii) determining an hydroxyureamethyl acylfulvene ICso using a 3-day viability assay; and (iii) selecting the subject when the ICso is < about 100 nM, and thereafter administering hydroxyureamethyl acylfulvene to the selected subject.

[0026] In another aspect, a method of treating a pediatric cancer comprises administering hydroxyureamethyl acylfulvene in combination with spironolactone, wherein the combination produces a statistically significant reduction in tumor cell viability measured at about 24 hours relative to either single agent in cells derived from the subject.

[0027] In another aspect, a method of extending event-free survival (EFS) in a pediatric sarcoma comprises administering LP-184, wherein EFS is defined as the time to 4-fold tumor-volume increase and is extended relative to vehicle control in a confirmatory cohort derived from the subject’s tumor.

[0028] In another aspect, a method of treating Ewing sarcoma in a pediatric subject comprises administering hydroxyureamethyl acylfulvene and vincristine, wherein the combination yields an additive antitumor effect measured as tumor-growth delay relative to either single agent.

[0029] In another aspect, hydroxyureamethyl acylfulvene is administered prior to vincristine by an interval of about 1 to about 72 hours.BRIEF DESCRIPTION OF THE DRAWINGS

[0030] FIG. 1 depicts hydroxyureamethyl acylfulvene ICso values (nM) obtained in a 3 -day viability assay.

[0031] FIG. 2 shows increased cleaved PARP (c-PARP) by Western blot 96 h after exposure to 50 nM LP-184.

[0032] FIG. 3 shows that hydroxyureamethyl acylfulvene induces apoptosis in ATRT cell lines

[0033] FIG. 4A shows hydroxyureamethyl acylfulvene in combination with the PARP inhibitor rucaparib reduces viability in an ATRT cell line.

[0034] FIG. 4B shows hydroxyureamethyl acylfulvene in combination with the PARP inhibitor rucaparib reduces viability in another ATRT cell line.

[0035] FIG. 4C shows hydroxyureamethyl acylfulvene in combination with the PARP inhibitor rucaparib reduces viability in yet another ATRT cell line.

[0036] FIG. 5 shows hydroxyureamethyl acylfulvene plus spironolactone (spir) decreases viability in ATRT cells.

[0037] FIG. 6A shows representative antitumor responses to hydroxyureamethyl acylfulvene in pediatric sarcoma PDX models.

[0038] FIG. 6B shows hydroxyureamethyl acylfulvene plus vincristine shows an additive antitumor effect in the EW-5 Ewing’s sarcoma PDX model.DETAILED DESCRIPTION

[0039] This application provides a therapy for treating pediatric sarcomas, medulloblastomas, gliomas, and hepatoblastoma. In embodiments, the therapy includes administering agents such as an acylfulvene or an illudin. In other embodiments, the therapy includes administering a combination of other therapies. In other embodiments, the therapy includes a therapy that can be used to treat biochemical occurrence and recurrence of these pediatric tumors, in which an acylfulvene (e.g., hydroxyureamethyl acylfulvene) or a salt thereof is administered in a therapeutically effective amount to the patient.Illudin or Acylfulvene

[0040] In one embodiment, this application includes the use of an illudin or illudin analog (e.g., acylfulvene). Acylfulvene is a class of cytotoxic semi-synthetic derivatives of illudin, a natural product that can be extracted from the jack o'lantern mushroom (Omphalotus olearius). Acylfulvene, derived from the sesquiterpene illudin S by treatment with acid (reverse Prins reaction), is far less reactive to thiols than illudin S.

[0041] In one example, the acylfulvene is (-) - hydroxyureamethyl acylfulvene (termed LP-184 by Lantern Pharma Inc.), exhibits negative optical rotation, as shown below:

[0042] In another example, the acylfulvene is (+)-hydroxyureamethyl acylfulvene (termed LP -284 by Lantern Pharma Inc ), exhibits positive optical rotation, as shown below:

[0043] (+) - hydroxyuream ethyl acylfulvene and (-) - hydroxyuream ethyl acylfulvene are enantiomers and are now known publicly.Nucleotide Excision Repair (NER) Pathway

[0044] Nucleotide Excision Repair (NER) is a DNA repair mechanism that has a role in maintaining the integrity of the DNA molecule by identifying and repairing various types of damage, particularly bulky lesions that distort the DNA helix. These lesions can be caused by environmental factors such as UV radiation, chemicals, and other mutagens.

[0045] NER is a versatile pathway that can repair a wide range of DNA lesions, making it crucial for the maintenance of genomic stability. Deficiencies in the NER pathway can lead to a higher susceptibility to cancer. Studying NER has also provided insights into various disease processes.

[0046] NER positivity can be determined through various methods, including genetic testing, genomic profiling, or specific biomarker assays. These tests aim to identify genetic or genomic alterations associated with NER, such as mutations or loss of function in genes involved in the NER pathway.Homologous Recombination Deficiency (HRD) Positive

[0047] Homologous recombination deficiency (HRD) positivity refers to the presence of genetic or genomic alterations in a tumor that indicate a defect or impairment in the homologous recombination DNA repair pathway. HRD positivity is often assessed in the context of cancer, particularly in relation to predicting response to certain treatments.

[0048] When tumors have HRD, they are more likely to have difficulties repairing DNA damage, such as double-stranded breaks. This can make them more susceptible to certain therapies that exploit these repair deficiencies.

[0049] HRD positivity can be determined through various methods, including genetic testing, genomic profding, or specific biomarker assays. These tests aim to identify genetic or genomic alterations associated with HRD, such as mutations or loss of function in genes involved in the homologous recombination pathway.

[0050] Assessing HRD positivity can have implications for treatment decisions. For example, in ovarian cancer, HRD positivity has been used as a predictive biomarker for the response to a drug. Drugs that block an alternative DNA repair pathway relied upon by HRD-positive cancer cells, leading to their selective targeting and cell death.

[0051]

[0021] In one embodiment, acylfulvene or hydroxyureamethyl acylfulvene or its salt may be administered either prior to, concurrently with, or subsequent to the administration of another agent.

[0052] One embodiment includes a method of treating pediatric sarcomas, medulloblastomas, gliomas, rhabdoid tumors, and hepatoblastoma in a subject in need thereof.

[0053] Another embodiment provides a method for treating these cancers by administering hydroxyureamethyl acylfulvene as a monotherapy. This method involves the administration of a therapeutically effective dose of hydroxyureamethyl acylfulvene to patients diagnosed with these tumors. The compound can be administered via oral, intravenous, or intraperitoneal routes, with the dosage adjusted based on the patient's weight, age, and disease severity. The treatment regimen may involve daily, weekly, or bi-weekly administration, depending on the desired therapeutic effect and patient tolerance.

[0054] Another embodiment provides a method for treating pediatric sarcomas, medulloblastomas, gliomas, rhabdoid tumors, and hepatoblastoma in which hydroxyureamethyl acylfulvene is used in combination with one or more conventional chemotherapeutic agents to enhance treatment. Suitable chemotherapeutic agents include, but are not limited to, doxorubicin, cisplatin, and 5-fluorouracil.

[0055] Another embodiment provides a method for treating these cancers in which hydroxyureamethyl acylfulvene is employed as an adjuvant therapy following surgical resection of tumors. After the primary tumor has been surgically removed, patients are administered hydroxyureamethyl acylfulvene to target residual cancer cells and prevent recurrence.

[0056] Another embodiment provides a method for treating pediatric sarcomas, medulloblastomas, gliomas, rhabdoid tumors, and hepatoblastoma in which hydroxyureamethylacylfulvene is encapsulated in nanoparticles for targeted delivery to cancer cells. This approach utilizes nanoparticles that are designed to preferentially accumulate in cancerous cells, thereby increasing the local concentration of the drug while reducing systemic exposure and toxicity.

[0057] Another embodiment provides a method for treating these pediatric tumors in which hydroxyureamethyl acylfulvene is used in conjunction with radiotherapy for the treatment. The compound may be administered before, during, or after radiation treatment to sensitize cancer cells to radiation, thereby enhancing the effectiveness of radiotherapy.

[0058] Another embodiment includes methods for preventing pediatric sarcomas, medulloblastomas, gliomas, rhabdoid tumors, and hepatoblastoma in an individual at risk, comprising the step of administering to the individual an effective amount of hydroxyureamethyl acylfulvene. In some embodiments, the individual has a genetic predisposition or family history of these cancers. In some embodiments, the method further comprises administering at least one additional therapeutic agent. Examples of additional therapeutic agents include growth inhibitory agents, such as cytotoxic agents, peptides, small molecules, and antibodies.

[0059] Another embodiment includes a pharmaceutical composition having a therapeutically effective amount of an illudin or an illudin analog thereof, derivative, or a pharmaceutically acceptable salt thereof. The illudin analog can be hydroxyureamethyl acylfulvene.

[0060] In another embodiment, the second therapeutic is one or more chemotherapeutic agents selected from camptothecin derivatives, paclitaxel, docetaxel, epothilone B, 5-FU, gemcitabine, oxaliplatin, cisplatinum, carboplatin, melphalan, dacarbazine, temozolomide, doxorubicin, imatinib, erlotinib, bevacizumab, cetuximab, and a Raf kinase inhibitor.

[0061] In another embodiment, the second therapeutic is one or more chemotherapeutic agents selected from paclitaxel or cisplatinum.

[0062] The term “combination therapy” can include or includes the administration of the therapeutic agents as described above in further combination with other biologically active ingredients and non-drug therapies (e.g., surgery or radiation treatment). Where the combination therapy further comprises a non-drug treatment, the non-drug treatment may be conducted at any suitable time so long as a beneficial effect from the co-action of the combination of the therapeutic agents and non-drug treatment is achieved. For example, in appropriate cases, the beneficial effect is still achieved when the non-drug treatment is temporally removed from the administration of the therapeutic agents, perhaps by days or even weeks.

[0063] In another aspect, a composition or combination therapy herein, or a pharmaceutically acceptable salt or solvate thereof, may be administered in combination with radiation therapy. Radiation therapy can also be administered in combination with a composition of the present invention and another chemotherapeutic agent described herein as part of a multiple agent therapy.

[0064] In another embodiment, methods are provided for treating or preventing pediatric tumors associated with increased expression or activity of a protein having at least 90% or 95% amino acid sequence identity to PTGR1, comprising administering to an individual in need of such treatment an effective amount of hydroxyureamethyl acylfulvene, thereby effectively treating or preventing the pediatric tumor. In some embodiments, the cell proliferative disorder is a pediatric cancer, such as hepatoblastoma, medulloblastoma, rhabdoid tumor, or glioma. In some embodiments, the individual has a predisposition to these cancers due to genetic mutations, such as those found in the SMARCB1, APC, or TP53 genes.

[0065] Combination therapy can be achieved by administering two or more agents, e.g., an acylfulvene and one or more other therapeutic agents, each of which is formulated and administered separately, or by administering two or more agents in a single formulation. Other combinations are also encompassed by combination therapy. For example, two agents can be formulated together and administered in conjunction with a separate formulation containing a third agent. While the two or more agents in the combination therapy can be administered simultaneously, they need not be. For example, administration of a first agent (or combination of agents) can precede administration of a second agent (or combination of agents) by minutes, hours, days, or weeks. Thus, the two or more agents can be administered within minutes of each other or within 1, 2, 3, 6, 9, 12, 15, 18, or 24 hours of each other or within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14 days of each other or within 2, 3, 4, 5, 6, 7, 8, 9, or 10 weeks of each other. In some cases, even longer intervals are possible. While in many cases it is desirable that the two or more agents used in a combination therapy be present within the patient's body at the same time, this need not be so.

[0066] The methods of combination therapy may or should result in a synergistic effect, wherein the effect of a combination of compounds or other therapeutic agents is greater than the sum of the effects resulting from administration of any of the compounds or other therapeutic agents as single agents. A synergistic effect may also be an effect that cannot be achieved by administration of any of the compounds or other therapeutic agents as single agents. The synergistic effect may include, but is not limited to, an effect of treating pediatric sarcomas, medulloblastomas, gliomas, rhabdoidtumors, or hepatoblastoma by reducing tumor size, inhibiting tumor growth, or increasing survival of the subject. The synergistic effect may also include reducing cancer cell viability, inducing cancer cell death, and inhibiting or delaying cancer cell growth.

[0067] Therapeutically effective doses can vary, as recognized by those skilled in the art, depending on the diseases treated, the severity of the disease, the route of administration, the age and general health condition of the patient, excipient usage, the possibility of co-usage with other therapeutic treatments such as use of other agents, and the judgment of the treating physician. For example, guidance for selecting an effective dose can be determined by reference to the prescribing information for acylfulvene or hydroxyureamethyl acylfulvene or journal discussion of the same.

[0068] The term “effective amount” as used herein refers to the amount of an agent needed to alleviate at least one or more symptoms of the disease or disorder, and relates to a sufficient amount of pharmacological composition to provide the desired effect. The term “therapeutically effective amount” therefore refers to an amount of the agent that is sufficient to provide a particular effect when administered to a typical subject. An effective amount may be an amount sufficient to decrease the symptoms of a disease responsive to inhibition of another agent. For cancer therapy, efficacy in vivo can, for example, be measured by assessing the duration of survival, time to disease progression (TTP), the response rates (RR), duration of response, and / or quality of life. Effective amounts may vary, as recognized by those skilled in the art, depending on route of administration, excipient usage, and co-usage with other agents. An effective amount as used herein, in various contexts, would also include an amount sufficient to delay the development of a symptom of the disease, alter the course of a symptom disease (for example but not limited to, slowing the progression of a symptom of the disease), or reverse a symptom of the disease. Thus, it is not generally practicable to specify an exact “effective amount.” However, for any given case, an appropriate “effective amount” can be determined by one of ordinary skill in the art using only routine experimentation.

[0069] The dosage ranges for the administration of an agent according to the methods described herein depend upon, for example, the form of the agent, its potency, and the extent to which symptoms, markers, or indicators of a condition described herein are desired to be reduced, for example, the percentage reduction desired for tumor growth. The dosage should not be so large as to cause adverse side effects. Generally, the dosage will vary with the age, condition, and sex ofthe patient and can be determined by one of skill in the art. The dosage can also be adjusted by the individual physician in the event of any complication.

[0070] The term “therapeutically effective amount,” as used herein, refers to an amount of a pharmaceutical agent to treat, ameliorate, or prevent an identified disease or condition, or to exhibit a detectable therapeutic or inhibitory effect. The effect can be detected by any assay method known in the art. The precise effective amount for a subject will depend upon the subject's body weight, size, and health; the nature and extent of the condition; and the therapeutic or combination of therapeutics selected for administration. Therapeutically effective amounts for a given situation can be determined by routine experimentation that is within the skill and judgment of the clinician. In a preferred aspect, the disease or condition to be treated is pediatric cancer, such as sarcomas, medulloblastomas, gliomas, rhabdoid tumors, or hepatoblastoma. In another aspect, the disease or condition to be treated is a cell proliferative disorder.

[0071] The efficacy of an agent described herein in, e.g., the treatment of a condition described herein, or to induce a response as described herein (e.g., pediatric tumors) can be determined by the skilled clinician. However, a treatment is considered “effective treatment,” as the term is used herein, if one or more of the signs or symptoms of a condition described herein are altered in a beneficial manner, other clinically accepted symptoms are improved, or even ameliorated, or a desired response is induced e.g., by at least 10% following treatment according to the methods described herein. Efficacy can be assessed, for example, by measuring a marker, indicator, symptom, and / or the incidence of a condition treated according to the methods described herein or any other measurable parameter appropriate, e.g., tumor size and / or growth rate. Efficacy can also be measured by a failure of an individual to worsen as assessed by hospitalization, or need for medical interventions (i.e., progression of the disease is halted). Methods of measuring these indicators are known to those of skill in the art and / or are described herein. Treatment includes any treatment of a disease in an individual or an animal (some non-limiting examples include a human or an animal) and includes: (1) inhibiting the disease, e.g., preventing a worsening of symptoms (e.g., pain or inflammation); or (2) relieving the severity of the disease, e g., causing regression of symptoms. An effective amount for the treatment of a disease means that amount which, when administered to a subject in need thereof, is sufficient to result in effective treatment as that term is defined herein, for that disease. Efficacy of an agent can be determined by assessing physical indicators of a condition or desired response. It is well within the ability of one skilled inthe art to monitor efficacy of administration and / or treatment by measuring any one of such parameters, or any combination of parameters. Efficacy can be assessed in animal models of a condition described herein, for example, treatment of pediatric tumors in a mouse model. When using an experimental animal model, efficacy of treatment is evidenced when a statistically significant change in a marker is observed, e.g., tumor size and / or growth rate.

[0072] In some embodiments, the therapeutically effective amount of hydroxyureamethyl - acylfulvene, acylfulvene, or a pharmaceutically acceptable salt thereof is selected from the group consisting of 0.5 mg / day, 1 mg / day, 2.5 mg / day, 5 mg / day, 10 mg / day, 20 mg / day, 30 mg / day, 60 mg / day, 90 mg / day, 120 mg / day, 150 mg / day, 180 mg / day, 210 mg / day, 240 mg / day, 270 mg / day, 300 mg / day, 360 mg / day, 400 mg / day, 440 mg / day, 480 mg / day, 520 mg / day, 580 mg / day, 600 mg / day, 620 mg / day, 640 mg / day, 680 mg / day, and 720 mg / day.

[0073] The administration dose should be adjusted for the requirement of the individual in need. For example, the administered dosage of hydroxyureamethyl -acylfulvene will be in the range from about 0.05 mg / kg to about 30 mg / kg, preferably 1 mg / kg to 30 mg / kg; or 500 mg-3000 mg flat dose. Thus, one or more doses of about 0.5 mg / kg, 2.0 mg / kg, 4.0 mg / kg, 10 mg / kg, or 30 mg / kg or 500 mg-3000 mg flat dose (or any combination thereof) may be co-administered to the patient. The preferred dosage of acylfulvene will be in the range from 20 mg / kg to about 150 mg / kg, preferably 1 mg / kg to 10 mg / kg. An initial higher loading dose, followed by one or more lower doses may also be administered. In some cases, it is more suitable to apply the lower end of the above described dosage ranges, while in other cases the higher dosages may be used without causing harmful side effects. The goal is often to give the maximum tolerated dosage.

[0074] The term “treat” is used and includes both therapeutic treatment and prophylactic treatment (reducing the likelihood of development). Both terms mean to decrease, suppress, attenuate, diminish, arrest, or stabilize the development or progression of a disease (e.g., a disease or disorder delineated herein), lessen the severity of the disease, or improve the symptoms associated with the disease.

[0075] The pharmaceutical compositions can be included in a container, pack, or dispenser together with instructions for administration.

[0076] The composition of the present invention is capable of further forming salts. The composition of the present invention can form more than one salt per molecule, e.g., mono-, di-, tri-. All of these forms are also contemplated within the scope of the claimed invention.

[0077] As used herein, “pharmaceutically acceptable salts” refer to derivatives of the compounds of the present invention wherein the parent compound is modified by making acid or base salts thereof. Examples of pharmaceutically acceptable salts include, but are not limited to, mineral or organic acid salts of basic residues such as amines, alkali or organic salts of acidic residues such as carboxylic acids, and the like. The pharmaceutically acceptable salts include the conventional non-toxic salts or the quaternary ammonium salts of the parent compound formed, for example, from non-toxic inorganic or organic acids. For example, such conventional non-toxic salts include, but are not limited to, those derived from inorganic and organic acids selected from 2- acetoxybenzoic, 2-hydroxy ethane sulfonic, acetic, ascorbic, benzene sulfonic, benzoic, bicarbonic, carbonic, citric, edetic, ethane disulfonic, 1,2-ethane sulfonic, fumaric, glucoheptonic, gluconic, glutamic, glycolic, glycollyarsanilic, hexylresorcinic, hydrabamic, hydrobromic, hydrochloric, hydroiodic, hydroxymaleic, hydroxynaphthoic, isethionic, lactic, lactobionic, lauryl sulfonic, maleic, malic, mandelic, methane sulfonic, napsylic, nitric, oxalic, pamoic, pantothenic, phenylacetic, phosphoric, polygalacturonic, propionic, salicylic, stearic, subacetic, succinic, sulfamic, sulfanilic, sulfuric, tannic, tartaric, toluene sulfonic, and the commonly occurring amino acids, e g., glycine, alanine, phenylalanine, arginine, etc.

[0078] One embodiment includes the combination of hydroxyureamethyl acylfulvene with topoisomerase I poisons such as topotecan and irinotecan, along with vincristine as a cancer therapy. This combination therapy effectively targets cancer cells through multiple pathways, potentially enhancing treatment efficacy and overcoming resistance mechanisms, particularly in aggressive pediatric malignancies such as neuroblastomas, medulloblastomas, and certain sarcomas.

[0079] Another embodiment includes the combination of hydroxyureamethyl acylfulvene and parp inhibitors.

[0080] Another embodiment includes the combination of hydroxyureamethyl acylfulvene and spironolactone, a diuretic commonly used to manage fluid retention and hypertension.

[0081] It should be understood that all references to pharmaceutically acceptable salts include solvent addition forms (solvates), of the same salt.

[0082] As used herein, the term “selectively” means tending to occur at a higher frequency in one population than in another population. The compared populations can be cell populations. Preferably, an event occurs selectively in population A relative to population B if it occurs greaterthan two times more frequently in population A as compared to population B. An event occurs selectively if it occurs greater than five times more frequently in population A. An event occurs selectively if it occurs greater than ten times more frequently in population A; more preferably, greater than fifty times; even more preferably, greater than 100 times; and most preferably, greater than 1000 times more frequently in population A as compared to population B. For example, cell death would be said to occur selectively in cancer cells if it occurred greater than twice as frequently in cancer cells as compared to normal cells.

[0083] The composition, or pharmaceutically acceptable salts or solvates thereof, are administered orally, nasally, transdermally, pulmonary, inhalationally, buccally, sublingually, intraperitoneally, subcutaneously, intramuscularly, intravenously, rectally, intrapleurally, intrathecally, and parenterally. In one embodiment, the compound is administered orally. One skilled in the art will recognize the advantages of certain routes of administration.

[0084] The dosage regimen utilizing the compounds is selected in accordance with a variety of factors including type, species, age, weight, sex, and medical condition of the patient; the severity of the condition to be treated; the route of administration; and the particular compound or salt thereof employed. An ordinarily skilled physician or veterinarian can readily determine and prescribe the effective amount of the drug required to prevent, counter, or arrest the progress of the condition.

[0085] All percentages and ratios used herein, unless otherwise indicated, are by weight. Other features and advantages of the present invention are apparent from the different examples. The provided examples illustrate different components and methodology useful in practicing the present invention. The examples do not limit the claimed invention. Based on the present disclosure, the skilled artisan can identify and employ other components and methodology useful for practicing the present invention.

[0086] As used herein, a “subject in need thereof’ is a subject having a pediatric sarcoma, medulloblastoma, glioma, rhabdoid tumor, hepatoblastoma, or a precancerous condition of these cancers. Preferably, a subject in need thereof has a diagnosis of these pediatric tumors or a genetic predisposition indicating a high risk. A “subject” includes a mammal. The mammal can be e.g., any mammal, e.g., a human, primate, bird, mouse, rat, dog, cat, cow, horse, goat, camel, sheep, or pig. Preferably, the mammal is a human. The subject of the present invention includes any humansubject who has been diagnosed with, has symptoms of, or is at risk of developing a pediatric tumor or a precancerous condition.

[0087] A subject in need thereof may have refractory or resistant cancer. “Refractory or resistant cancer” means cancer that does not respond to treatment. The cancer may be resistant at the beginning of treatment, or it may become resistant during treatment. In some embodiments, the subject in need thereof has cancer recurrence following remission on most recent therapy. In some embodiments, the subject in need thereof received and failed all known effective therapies for pediatric tumor treatment. In some embodiments, the subject in need thereof received at least one prior therapy. In certain embodiments, the prior therapy is monotherapy. In certain embodiments, the prior therapy is combination therapy.

[0088] In some embodiments, a subject in need thereof may have a secondary cancer as a result of a previous therapy. “Secondary cancer” means cancer that arises due to or as a result of previous carcinogenic therapies, such as chemotherapy or radiation therapy.

[0089] Cancer is a group of diseases that may cause almost any sign or symptom. The signs and symptoms will depend on where the cancer is, the size of the cancer, and how much it affects the nearby organs or structures. If a cancer spreads (metastasizes), then symptoms may appear in different parts of the body.

[0090] Treating pediatric sarcomas, medulloblastomas, gliomas, rhabdoid tumors, and hepatoblastoma can result in a reduction in the size of a tumor. A reduction in the size of a tumor may also be referred to as “tumor regression.” Preferably, after treatment, tumor size is reduced by 5% or greater relative to its size prior to treatment; more preferably, tumor size is reduced by 10% or greater; more preferably, reduced by 20% or greater; more preferably, reduced by 30% or greater; more preferably, reduced by 40% or greater; even more preferably, reduced by 50% or greater; and most preferably, reduced by greater than 75%. Size of a tumor may be measured by any reproducible means of measurement. The size of a tumor may be measured as a diameter of the tumor.

[0091] Treating these pediatric tumors results in a decrease in the number and size of tumors. Preferably, after treatment, tumor number or size is reduced by 5% or greater relative to the number prior to treatment; more preferably, tumor number or size is reduced by 10% or greater; more preferably, reduced by 20% or greater; more preferably, reduced by 30% or greater; more preferably, reduced by 40% or greater; even more preferably, reduced by 50% or greater; and mostpreferably, reduced by greater than 75%. Number of tumors may be measured by any reproducible means of measurement. The number of tumors may be measured by counting tumors visible to the naked eye or at a specified magnification. Preferably, the specified magnification is 2x, 3x, 4x, 5x, lOx, or 50x.

[0092] Treating these pediatric tumors can result in a decrease in the number of metastatic lesions in other tissues or organs distant from the primary tumor site. Preferably, after treatment, the number of metastatic lesions is reduced by 5% or greater relative to number prior to treatment; more preferably, the number of metastatic lesions is reduced by 10% or greater; more preferably, reduced by 20% or greater; more preferably, reduced by 30% or greater; more preferably, reduced by 40% or greater; even more preferably, reduced by 50% or greater; and most preferably, reduced by greater than 75%. The number of metastatic lesions may be measured by any reproducible means of measurement. The number of metastatic lesions may be measured by counting metastatic lesions visible to the naked eye or at a specified magnification. Preferably, the specified magnification is 2x, 3x, 4x, 5x, lOx, or 50x.

[0093] Treating these pediatric tumors can result in an increase in average survival time of a population of treated subjects in comparison to a population receiving carrier alone. Preferably, the average survival time is increased by more than 30 days; more preferably, by more than 60 days; more preferably, by more than 90 days; and most preferably, by more than 120 days. An increase in average survival time of a population may be measured by any reproducible means. An increase in average survival time of a population may be measured, for example, by calculating for a population the average length of survival following initiation of treatment with an active compound. An increase in average survival time of a population may also be measured, for example, by calculating for a population the average length of survival following completion of a first round of treatment with an active compound.

[0094] Treating these pediatric tumors can result in an increase in average survival time of a population of treated subjects in comparison to a population of untreated subjects. Preferably, the average survival time is increased by more than 30 days; more preferably, by more than 60 days; more preferably, by more than 90 days; and most preferably, by more than 120 days. An increase in average survival time of a population may be measured by any reproducible means. An increase in average survival time of a population may be measured, for example, by calculating for a population the average length of survival following initiation of treatment with an activecompound. An increase in average survival time of a population may also be measured, for example, by calculating for a population the average length of survival following completion of a first round of treatment with an active compound.

[0095] Treating these pediatric tumors can result in an increase in average survival time of a population of treated subjects in comparison to a population receiving monotherapy with a drug that is not a compound of the present invention, or a pharmaceutically acceptable salt or solvate thereof. Preferably, the average survival time is increased by more than 30 days; more preferably, by more than 60 days; more preferably, by more than 90 days; and most preferably, by more than 120 days. An increase in average survival time of a population may be measured by any reproducible means. An increase in average survival time of a population may be measured, for example, by calculating for a population the average length of survival following initiation of treatment with an active compound. An increase in average survival time of a population may also be measured, for example, by calculating for a population the average length of survival following completion of a first round of treatment with an active compound.

[0096] Treating these pediatric tumors can result in a decrease in the mortality rate of a population of treated subjects in comparison to a population receiving carriers alone. Treating these pediatric tumors can result in a decrease in the mortality rate of a population of treated subjects in comparison to an untreated population. Treating these pediatric tumors can result in a decrease in the mortality rate of a population of treated subjects in comparison to a population receiving monotherapy with a drug that is not a compound of the present invention, or a pharmaceutically acceptable salt or solvate thereof. Preferably, the mortality rate is decreased by more than 2%; more preferably, by more than 5%; more preferably, by more than 10%; and most preferably, by more than 25%. A decrease in the mortality rate of a population of treated subjects may be measured by any reproducible means. A decrease in the mortality rate of a population may be measured, for example, by calculating for a population the average number of disease-related deaths per unit time following initiation of treatment with an active compound. A decrease in the mortality rate of a population may also be measured, for example, by calculating for a population the average number of disease-related deaths per unit time following completion of a first round of treatment with an active compound.

[0097] Treating these pediatric tumors can result in a decrease in tumor growth rate. Preferably, after treatment, tumor growth rate is reduced by at least 5% relative to the rate prior to treatment;more preferably, tumor growth rate is reduced by at least 10%; more preferably, reduced by at least 20%; more preferably, reduced by at least 30%; more preferably, reduced by at least 40%; more preferably, reduced by at least 50%; even more preferably, reduced by at least 75%. Tumor growth rate may be measured by any reproducible means of measurement. Tumor growth rate can be measured according to a change in tumor diameter per unit time.

[0098] Treating these pediatric tumors can result in a decrease in tumor regrowth. Preferably, after treatment, tumor regrowth is less than 5%; more preferably, tumor regrowth is less than 10%; more preferably, less than 20%; more preferably, less than 30%; more preferably, less than 40%; more preferably, less than 50%; even more preferably, less than 75%. Tumor regrowth may be measured by any reproducible means of measurement. Tumor regrowth is measured, for example, by measuring an increase in the diameter of a tumor after a prior tumor shrinkage that followed treatment. A decrease in tumor regrowth is indicated by the failure of tumors to reoccur after treatment has stopped.

[0099] Treating or preventing a pediatric cell proliferative disorder can result in a reduction in the rate of cellular proliferation. Preferably, after treatment, the rate of cellular proliferation is reduced by at least 5%; more preferably, by at least 10%; more preferably, by at least 20%; more preferably, by at least 30%; more preferably, by at least 40%; more preferably, by at least 50%; even more preferably, by at least 75%. The rate of cellular proliferation may be measured by any reproducible means of measurement. The rate of cellular proliferation is measured, for example, by measuring the number of dividing cells in a tissue sample per unit time.

[0100] Treating or preventing a pediatric cell proliferative disorder can result in a reduction in the proportion of proliferating cells. Preferably, after treatment, the proportion of proliferating cells is reduced by at least 5%; more preferably, by at least 10%; more preferably, by at least 20%; more preferably, by at least 30%; more preferably, by at least 40%; more preferably, by at least 50%; even more preferably, by at least 75%. The proportion of proliferating cells may be measured by any reproducible means of measurement. Preferably, the proportion of proliferating cells is measured, for example, by quantifying the number of dividing cells relative to the number of nondividing cells in a tissue sample. The proportion of proliferating cells can be equivalent to the mitotic index.

[0101] Treating or preventing a pediatric cell proliferative disorder can result in a decrease in the size of an area or zone of cellular proliferation. Preferably, after treatment, the size of an area orzone of cellular proliferation is reduced by at least 5% relative to its size prior to treatment; more preferably, reduced by at least 10%; more preferably, reduced by at least 20%; more preferably, reduced by at least 30%; more preferably, reduced by at least 40%; more preferably, reduced by at least 50%; even more preferably, reduced by at least 75%. The size of an area or zone of cellular proliferation may be measured by any reproducible means of measurement. The size of an area or zone of cellular proliferation may be measured as a diameter or width of an area or zone of cellular proliferation.

[0102] Treating or preventing a pediatric cell proliferative disorder can result in a decrease in the number or proportion of cells having an abnormal appearance or morphology. Preferably, after treatment, the number of cells having an abnormal morphology is reduced by at least 5% relative to its size prior to treatment; more preferably, reduced by at least 10%; more preferably, reduced by at least 20%; more preferably, reduced by at least 30%; more preferably, reduced by at least 40%; more preferably, reduced by at least 50%; even more preferably, reduced by at least 75%. An abnormal cellular appearance or morphology may be measured by any reproducible means of measurement. An abnormal cellular morphology can be measured by microscopy, e.g., using an inverted tissue culture microscope. An abnormal cellular morphology can take the form of nuclear pleomorphism, increased mitotic activity, or abnormal cell shapes typical of pediatric cancers.

[0103] Administering a composition of the present invention to a cell or a subject in need thereof can result in modulation (i.e., stimulation or inhibition) of the activity of a protein methyltransferase of interest. This modulation can influence gene expression and may play a role in the regulation of pediatric cancer progression, particularly in tumors such as hepatoblastomas, medulloblastomas, and rhabdoid tumors.

[0104] Treating pediatric sarcomas, medulloblastomas, gliomas, rhabdoid tumors, or hepatoblastomas can result in cell death, and preferably, cell death results in a decrease of at least 10% in the number of cells in a population. More preferably, cell death means a decrease of at least 20%; more preferably, a decrease of at least 30%; more preferably, a decrease of at least 40%; more preferably, a decrease of at least 50%; most preferably, a decrease of at least 75%. The number of cells in a population may be measured by any reproducible means. The number of cells in a population can be measured by fluorescence-activated cell sorting (FACS), immunofluorescence microscopy, and light microscopy. Methods of measuring cell death are asshown in Li et al., Proc. Natl. Acad. Sci. USA. 100(5): 2674-8, 2003. In an aspect, cell death occurs by apoptosis, necrosis, or other forms of programmed cell death specific to pediatric tumors.

[0105] Preferably, an effective amount of a composition of the present invention, or a pharmaceutically acceptable salt or solvate thereof, is not significantly cytotoxic to normal cells. A therapeutically effective amount of a compound is not significantly cytotoxic to normal cells if the administration of the compound in a therapeutically effective amount does not induce cell death in greater than 10% of normal cells. A therapeutically effective amount of a compound does not significantly affect the viability of normal cells if the administration of the compound in a therapeutically effective amount does not induce cell death in greater than 10% of normal cells. In an aspect, cell death occurs selectively in cancer cells, with minimal impact on healthy, nonproliferating cells.

[0106] Contacting a cell with a composition of the present invention, or a pharmaceutically acceptable salt or solvate thereof, can induce or activate cell death selectively in pediatric cancer cells. Administering to a subject in need thereof a compound of the present invention, or a pharmaceutically acceptable salt or solvate thereof, can induce or activate cell death selectively in pediatric cancer cells. Contacting a cell with a composition of the present invention, or a pharmaceutically acceptable salt or solvate thereof, can induce cell death selectively in one or more cells affected by a pediatric cell proliferative disorder. Preferably, administering to a subject in need thereof a composition of the present invention, or a pharmaceutically acceptable salt or solvate thereof, induces cell death selectively in one or more cells affected by a pediatric cell proliferative disorder.

[0107] The present invention relates to a method of treating or preventing pediatric cancers such as sarcomas, medulloblastomas, gliomas, rhabdoid tumors, or hepatoblastomas by administering a composition of the present invention, or a pharmaceutically acceptable salt or solvate thereof, to a subject in need thereof, where administration of the composition results in one or more of the following: prevention of cancer cell proliferation by accumulation of cells in one or more phases of the cell cycle (e.g., Gl, Gl / S, G2 / M), induction of cell senescence, promotion of tumor cell differentiation, or promotion of cell death in cancer cells via cytotoxicity, necrosis, or apoptosis, without a significant amount of cell death in normal cells. Antitumor activity in animals with a therapeutic index of at least 2 has been observed. As used herein, “therapeutic index” is defined as the maximum tolerated dose divided by the efficacious dose.

[0108] The terms “cancer” and “cancerous” refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth / proliferation. Examples of pediatric cancers include, but are not limited to, sarcomas (e.g., osteosarcoma, Ewing sarcoma), medulloblastoma, glioma (e.g., diffuse intrinsic pontine glioma), rhabdoid tumors, and hepatoblastomas. These cancers may originate in different tissues or organs and may exhibit distinct patterns of growth and metastasis.

[0109] The term “pediatric subject” refers to a human subject less than 18 years of age unless otherwise specified; “adolescent and young adult (AYA)” may refer to subj ects approximately 15- 39 years of age; and “therapeutically effective amount” refers to an amount sufficient to produce a measurable clinical benefit, including tumor response, disease stabilization, or survival improvement, with an acceptable safety profile for the intended pediatric population.

[0110] An “individual at risk of having a pediatric cancer” refers to an individual having a higher than average propensity of acquiring these cancers. Examples of individuals at risk include, without limitation, individuals with genetic predispositions such as mutations in the TP53, RBI, SMARCB1, APC, or NF1 genes, family history of pediatric cancers, or previous exposure to carcinogenic treatments or environmental factors.

[0111] The term “kit” means a combination partner as defined above can be dosed independently or by use of different fixed combinations with distinguished amounts of the combination partners, i.e., simultaneously or at different time points. The parts of the kit of parts can then, e.g., be administered simultaneously or chronologically staggered, that is, at different time points and with equal or different time intervals for any part of the kit of parts. The ratio of the total amounts of the combination partners to be administered in the combined preparation can be varied. The combination partners can be administered by the same route or by different routes.

[0112] The term “level of expression” or “expression level” as used herein refers to the amount of a polynucleotide, mRNA, or an amino acid product or protein in a biological sample. Expression levels can be used as biomarkers to determine the activity or effect of therapeutic agents, particularly in pediatric cancers where certain genetic expressions may be indicative of treatment response.

[0113] One skilled in the art may refer to general reference texts for detailed descriptions of known techniques discussed herein or equivalent techniques. These texts can, of course, also be referred to in making or using an aspect of the invention.EXAMPLES

[0114] In order that the disclosure disclosed herein may be more efficiently understood, examples are provided below. It should be understood that these examples are for illustrative purposes only and are not to be construed as limiting the disclosure in any manner.Methods

[0115] Pediatric brain tumor cells were maintained in DMEM supplemented with 10% FBS and 1% penicillin-streptomycin (10,000 U / mL). All cell lines used in experiments were within 10-15 passages after being thawed. Cell line authentication was performed, and all cell lines were regularly tested for mycoplasma, as determined by PCR detection methods, and all lines were tested negative (date of last test: 7-12-23). Cell viability was measured using the Cell- Titer Gio assay conducted over 3 or 4 days. Apoptosis was measured using immunofluorescence for cleaved caspase 3 or western blot analysis for cleaved PARP.Tumor xenografts

[0116] All animal procedures were approved by the UT Health San Antonio ethics committee. For in vivo determination of tumor line sensitivity to LP-184, a single mouse testing (SMT) approach was adopted. 26 pediatric CDX / PDX models were tested including Ewing sarcoma (9), malignant rhabdoid tumor (MRT, 4), rhabdomyosarcoma (RMS, 6), hepatoblastoma (HB, 6) and a single Wilms tumor. Details of the tumor lines are given in Table 1. The SMT design is valuable for determining high-level activity against a wide variety of tumor histologies. To determine the dose response relationship for hydroxyureamethyl acylfulvene using a conventional design (5 mice / group), we selected two models, one that demonstrated sensitivity in the SMT screen (EW-5) and one that was not tested in the SMT screen (ES-1) to validate the SMT data. For combination studies, hydroxyureamethyl acylfulvene was combined with vincristine to determine whether there was additive or greater than additive activity (5 mice / group). Study endpoints were tumor regression and event-free survival (EFS). EFS was defined as the time after initiation of treatment for the tumor volume to increase 4- fold. Mice were entered onto study when tumor volume was 200-400mm3. Tumor diameters were measured weekly, and studies were terminated 14+ weeks after the initiating treatmentif tumor volumes did not reach the EFS endpoint.

[0117] LP-184 demonstrated nanomolar potency across pediatric CNS tumor cell lines. FIG. 1 depicts hydroxyureamethyl acylfulvene ICso values (nM) obtained in a 3 -day viability assay, with the x-axis listing cell lines and the y-axis showing corresponding ICso values (log scale). The distribution evidences differential sensitivity (lower ICso indicating greater sensitivity), supporting broad activity and the rationale for biomarker-guided development. Table shows the specific data shown in FIG. 1.Table 1Cell line Pediatric cancer type LP-184 IC50 [nM]CHLA02 ATRT 1770CHLA05 ATRT 162CH1.A06 ATRT 37CHLA266 ATRT 24BT37 ATRT 22DAOY Medulloblastoma 99;D283MED Medulloblastoma 13D341MED Medulloblastoma 541CHLA01RMED Medulloblastoma 1000: SF8628 DIPG 71^ SF7761 DIPG 105

[0118] LP-184 induces apoptosis in atypical teratoid / rhabdoid tumor (AT / RT) cells. FIG. 2 shows increased cleaved PARP (c-PARP) by Western blot 96 h after exposure to 50 nM LP-184, and FIG. 3 shows increased cleaved caspase-3 (c-CASP3) by immunofluorescence under the same conditions. A subset ofpediatric brain tumor cell lines were highly sensitive to LP-184, as reflectedby low IC50 values; in AT / RT cells, apoptosis at 96 h was confirmed by C-CASP3 immunofluorescence and c-PARP Western blot.Example 2

[0119] LP-184 synergized with PARP inhibitor Rucaparib in vitro in ATRT cell lines. FIGS. 4A, 4B, and 4C show hydroxyureamethyl acylfulvene in combination with the PARP inhibitor rucaparib reduces viability in ATRT cell lines. Cells were treated with hydroxyureamethyl acylfulvene (25 nM) ± rucaparib (2 pM), and viability was measured at 72 h (CellTiter-Glo). The combination produced a statistically significant reduction in cell viability relative to single-agent treatments across the ATRT panel: BT37 (FIG. 4 A), CHLA-06 (FIG. 4B), and CHLA-05 (FIG. 4C).Example 3

[0120] Combination of hydroxyureamethyl acylfulvene and Spironolactone was additive in vitro in an ATRT cell line. FIG. 5 shows hydroxyureamethyl acylfulvene plus spironolactone (spir) decreases viability in ATRT cells. CHLA-06 cells were treated for 24 h with hydroxyureamethyl acylfulvene (40 or 80 nM) ± spironolactone (10 pM), and viability was measured (CellTiter-Glo). The combinations produced a statistically significant reduction in viability relative to single-agent treatments.Example 4

[0121] Table 2 shows antitumor activity of hydroxyureamethyl acylfulvene in pediatric sarcoma xenograft models. Mice bearing pediatric sarcoma xenografts received hydroxyureamethyl acylfulvene (4 mg / kg, i.v.) on Days 1, 4, 7, and 10, with subsequent follow-up for survival. Tumor response outcomes are summarized for the tested models. The SMT screen was conducted against 26 pediatric sarcoma xenograft models to identify therapeutically important antitumor activity (tumor regression and biologically meaningful extension of EFS over the control EFS). hydroxyureamethyl acylfulvene was administered intravenously at 4 mg / kg twice weekly for 2 weeks, hydroxyuream ethyl acylfulvene was highly active in 19 of 26 models that showed no tumor regrowth at the termination of study (day 98).TABLE 2

[0122] FIG. 6 A shows representative antitumor responses to hydroxyureamethyl acylfulvene in pediatric sarcoma PDX models. Single-agent hydroxyureamethyl acylfulvene (4 mg / kg, i.v.) administered on Days 1, , 7, and 10 produced sustained tumor regression in multiple subcutaneous xenografts (N=l per model; SMT design). The y-axis shows tumor volume; the x-axis shows weeks post-treatment initiation.

[0123] Combination of hydroxyureamethyl acylfulvene and Vincristine was additive in vivo in EW-5 Ewing’s sarcoma PDX model. Combination of certain DNA damaging agents, such as topoisomerase I poisons (topotecan and irinotecan) with vincristine have greater than anticipated antitumor activity based on single agent activity. To determine whether combination of hydroxyureamethyl acylfulvene with vincristine had greater than anticipated activity, hydroxyureamethyl acylfulvene was administered at 1 mg / kg twice weekly for 2 weeks IV, andvincristine was administered TP at 0.5 mg / kg every 7 days x 3 starting on the same day as the initial dose of LP-184. There was an additive effect on antitumor activity when vincristine was combined with hydroxyureamethyl acylfulvene in the EW-5 xenograft model with some tumor growth delay.

[0124] FIG 6B shows hydroxyureamethyl acylfulvene plus vincristine showed an additive antitumor effect in the EW-5 Ewing’s sarcoma PDX model. Treatment with 1 mg / kg hydroxyureamethyl acylfulvene i.v. on Days 1, 4, 7, and 10 in combination with 0.5 mg / kg Vincristine i.p. on Days 1, 8, and 15 produced a tumor growth delay compared to single agents in the EW-5 subcutaneous xenograft model (N=5 per arm). The y-axis shows tumor volume and the x-axis shows weeks post-treatment initiation.

[0125] While several exemplary aspects and embodiments have been provided, those skilled in the art will recognize possible modifications, combinations, permutations, and variations. Therefore, the appended claims and any claims subsequently introduced are to be interpreted to encompass all such modifications, combinations, permutations, and variations within their true spirit and scope.

Claims

CLAIMS1. A method of treating a pediatric cancer selected from pediatric sarcoma, medulloblastoma, glioma, and hepatoblastoma in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of hydroxyureamethyl acylfulvene or a pharmaceutically acceptable salt or solvate thereof.

2. The method of claim 1, wherein the hydroxyureamethyl acylfulvene is (-)- hydroxyureamethyl acylfulvene (LP-184).

3. The method of claim 1, wherein administration is intravenous, intraperitoneal, or oral.

4. The method of claim 1, wherein the dose is about 0.05 mg / kg to about 30 mg / kg.

5. The method of claim 1, wherein the dose is selected from about 0.5 mg / kg, about 2.0 mg / kg, about 4.0 mg / kg, about 10 mg / kg, or about 30 mg / kg.

6. The method of claim 1, wherein the pediatric cancer is selected from medulloblastoma, diffuse intrinsic pontine glioma, Ewing sarcoma, rhabdomyosarcoma, hepatoblastoma, and Wilms tumor.

7. A method of treating a pediatric cancer in a subject in need thereof, comprising intravenously administering hydroxyureamethyl acylfulvene at about 4 mg / kg on Days 1, 4, 7, and 10.

8. The method of claim 7, wherein the treatment results in sustained tumor regression or a tumor-growth delay relative to control.

9. The method of claim 7, wherein the pediatric cancer is a sarcoma xenograft exhibiting no tumor regrowth at study termination.

10. A method of treating a pediatric sarcoma in a subject in need thereof, comprising administering an effective amount of hydroxyureamethyl acylfulvene.

11. The method of claim 10, wherein the pediatric sarcoma is selected from Ewing sarcoma and rhabdomyosarcoma.

12. A method of treating a pediatric cancer, comprising administering hydroxyureamethyl acylfulvene in combination with vincristine.

13. The method of claim 12, wherein hydroxyureamethyl acylfulvene is administered intravenously at about 1 mg / kg on Days 1, 4, 7, and 10, and vincristine is administered intraperitoneally or intravenously on a weekly schedule selected from Days 1, 8, and 15.

14. The method of claim 12, wherein the combination yields an additive antitumor effect measured as tumor-growth delay relative to either single agent.

15. The method of claim 12, wherein the pediatric cancer is a pediatric sarcoma.

16. A method of treating a pediatric cancer in a subject in need thereof, comprising administering hydroxyureamethyl acylfulvene prior to, concurrently with, or subsequent to a topoisomerase I poison selected from topotecan and irinotecan.

17. The method of claim 16, wherein the regimen further comprises vincristine administered on a weekly schedule.

18. A method of treating a pediatric cancer characterized by a biomarker selected from (i) increased expression or activity of PTGR1, (ii) homologous recombination deficiency (HRD) positivity, and (iii) nucleotide excision repair (NER) positivity, comprising administering an effective amount of hydroxyureamethyl acylfulvene.

19. The method of claim 18, wherein increased PTGR1 expression is determined by an assay demonstrating at least a two-fold elevation relative to a normal reference.

20. A method of sensitizing a pediatric cancer to radiotherapy, comprising administering hydroxyureamethyl acylfulvene to the subject before, during, or after a course of radiation therapy.

21. The method of claim 20, wherein the hydroxyureamethyl acylfulvene is administered within about 24 hours of a radiation fraction.

22. A kit comprising: (a) hydroxyureamethyl acylfulvene or a pharmaceutically acceptable salt or solvate thereof; and (b) a PARP inhibitor; wherein the kit includes instructions for administering the hydroxyureamethyl acylfulvene prior to, concurrently with, or subsequent to the PARP inhibitor to treat a pediatric cancer.

23. A method of reducing tumor growth rate in a pediatric subject with a cancer selected from pediatric sarcoma, medulloblastoma, glioma, rhabdoid tumor, and hepatoblastoma, comprising administering hydroxyureamethyl acylfulvene in an amount effective to reduce tumor growth rate by at least about 30% relative to a pretreatment rate.

24. A method of selecting a pediatric subject for treatment with hydroxyureamethyl acylfulvene, comprising: (i) obtaining a tumor cell sample from the subject; (ii) determining an hydroxyureamethyl acylfulvene ICso using a 3 -day viability assay; and (iii) selecting the subject when the ICso is < about 100 nM, and thereafter administering hydroxyureamethyl acylfulvene to the selected subject.

25. A method of treating a pediatric cancer, comprising administering hydroxyureamethyl acylfulvene in combination with a PARP inhibitor, wherein the combination produces a statistically significant reduction in tumor cell viability relative to either single agent in an ex vivo assay of cells derived from the subject.

26. The method of claim 25, wherein the PARP inhibitor is rucaparib and the hydroxyureamethyl acylfulvene exposure corresponds to about 25 nM in the ex vivo assay used to guide dosing.

27. A method of treating a pediatric cancer, comprising administering hydroxyureamethyl acylfulvene in combination with spironolactone, wherein the combination produces a statistically significant reduction in tumor cell viability measured at about 24 hours relative to either single agent in cells derived from the subject.

28. A method of extending event-free survival (EFS) in a pediatric sarcoma, comprising administering LP-184, wherein EFS is defined as the time to 4-fold tumor-volume increase and is extended relative to vehicle control in a confirmatory cohort derived from the subject’s tumor.

29. A method of treating Ewing sarcoma in a pediatric subject, comprising administering hydroxyuream ethyl acylfulvene and administering vincristine, wherein the combination yields an additive antitumor effect measured as tumor-growth delay relative to either single agent.

30. The method of claim 12 or claim 29, wherein hydroxyureamethyl acylfulvene is administered prior to vincristine by an interval of about 1 to about 72 hours.

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