CA3215236A1 - Method for treating lung cancer and non-small cell lung cancer - Google Patents

Abstract

A method of treating NSCLC or lung cancer in an individual including administering to the individual an effective amount of a composition comprising nanomolar potent particles comprising acylfulvene. The treatment can be based upon the individual having squamous cellular carcinoma. Another aspect includes compositions (such as pharmaceutical compositions), medicine, kits, and unit dosages useful for the methods described herein.

Description

Method for Treating Lung Cancer and Non-Small Cell Lung Cancer CROSS-REFERENCE TO RELATED APPLICATION
[0001] This application claims the benefit of U.S. Provisional Patent Application No.
63/173,968, filed April 12, 2021, which is incorporated by reference herein in its entirety.
TECHNIC AL FIELD

[0002] The application relates to a cancer treatment, and more particularly to a method for treating lung cancer. This application also relates to methods and compositions for the treatment of non-small-cell lung cancer (NSCLC) and lung cancer by administering compositions comprising nanomolar potent acylfulvenes.
BACKGROUND

[0003] Lung cancer remains the most common cause of cancer-related mortality.
This is true for both men and women. In 2020 in the United States lung cancer accounted for more deaths than breast cancer, prostate cancer, colon cancer, and brain cancer.

[0004] Most lung cancer caused by smoking is non-small cell lung cancer (NSCLC), which represents about 90% of all lung cancers. 'there are three main types of NSCLC: squamous cell carcinoma, large cell carcinoma, and adenocarcinoma. Adenocarcinoma is the most common form of lung cancer and is the lung cancer most frequently found in both smokers and non-smokers. Squamous cell carcinoma is generally found in a proximal bronchus.
Early-stage NSCLC tends to be localized, and if detected early it can often be treated by surgery with a favorable outcome and improved survival. Other treatment options include radiation treatment, drug therapy, and a combination of these methods.

[0005] NSCLC is staged by the size of the tumor and its presence in other tissues including lymph nodes. In the occult stage, cancer cells are found in sputum samples or lavage samples and no tumor is detectable in the lungs. In stage 0, only the innermost lining of the lungs exhibit cancer cells and the tumor has not grown through the lining. In stage IA, the cancer is considered invasive and has grown deep into the lung tissue but the tumor is less than 3 cm across. In this stage, the tumor is not found in the bronchus or lymph nodes.
In stage IB, the tumor is either larger than 3 cm across or has grown into the bronchus or pleura, but has not grown into the lymph nodes. In stage IIA, the tumor is more than 3 cm across and has grown into the lymph nodes. In stage IIB, the tumor has either been found in the lymph nodes and is greater than 3 cm across or grown into the bronchus or pleura; or the cancer is not in the lymph nodes but is found in the chest wall, diaphragm, pleura, bronchus, or tissue that surrounds the heart. In stage IIIA, cancer cells are found in the lymph nodes near the lung and bronchi and in those between the lungs but on the side of the chest where the tumor is located. Stage IIIB, cancer cells are located on the opposite side of the chest from the tumor and in the neck. Other organs near the lungs may also have cancer cells and multiple tumors may be found in one lobe of the lungs. In stage IV, tumors are found in more than one lobe of the same lung or both lungs and cancer cells are found in other parts of the body. In all stages, the cancer must be treated.

[0006] Accordingly, there is always a need for treatments for non-small-cell lung cancer (NSCLC) and lung cancer.
SUMMARY

[0007] This application discloses a method of treating lung cancer and NSCLC
in an individual. The method includes administering to the individual an effective amount of a composition comprising nanomolar potent particles comprising acylfulvene.

[0008] Another aspect includes compositions (such as pharmaceutical compositions), medicine, kits, and unit dosages useful for the methods described herein.

[0009] Another aspect of this application includes a method for treating lung cancer and NSCLC using an effective amount of hydroxyureamethyl-acylfulvene together with an effective amount of one or more of an additional therapeutic agent selected from the group consisting of temozolomide, bevacizumab, everolimus, carmustine, lomustine, procarbazine, vincristine, irinotecan, cisplatin, carboplatin, methotrexate, etoposide, vinblastine, bleomycin, actinomycin, cyclophosphamide, and ifosfamide.

[0010] Another aspect of this application includes a method for treating lung cancer and NSCLC using an effective amount of hydroxyureamethyl-acylfulvene together with radiation therapy. The radiation therapy can be selected from lung irradiation, fractionated radiotherapy, radio surgery, and a combination thereof The radiation may be applied before, during, and after any treatment with an effective amount of hydroxyureamethyl-acylfulvene.

[0011] Another aspect of this application includes a method in which the patient is human or animal.

[0013] FIG. I shows hydroxyureamethyl-acylfulvene exhibits nanomolar potency in a range of NSCLC cell lines;
[0014] FIG. 2 shows hydroxyureamethyl-acylfulvene sensitivity on an X axis and PTGRI
transcript levels across 19 NSCLC cell lines;

[0015] FIG. 3 shows hydroxyureamethyl-acylfulvene potency in terms of nanomolar IC5Os compared among Oxaliplatin, Cisplatin, Pemetrexed, Paclitaxel and Gemcitabine obtained from the GDSC database;
[0016] FIG. 4 shows hydroxyureamethyl-acylfulvene exhibits nanomolar potency in a brain metastasis model LXFE 2478, originating from primary lung cancer, in terms of nanomolar IC50s (11( axis) as compared Erlotinib, Gefitinib and Osimertinib;
[0017] FIG. 5 shows results from H460 nude mouse xenograft models where hydroxyureamethyl-acylfulvene was administered intraperitoneally on the days indicated by the arrows on the X axis (N = 10 in each group).
[0018] FIG. 6 shows the value adjacent to the highly mutated gene is the permutation test p-value of PTGR1 relative gene expression between driver mutated (black) and not-mutated (white) samples.
SEQUENCES
[0019] SEQ ID NO: I amino acid sequence of PTGR1, [0020] SEQ ID NO:2 ___________ amino acid sequence cif KEAP I
[0021] SEQ ID NO:3¨ amino acid sequence of KRAS, [0022] SEQ ID NO:4--amino acid sequence of TP53, and [0023] SEQ ID NO:5--amino acid sequence of STK11.
DEFINITIONS
[0024] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of skill in art to which the subject matter herein belongs. As used herein, the following definitions are supplied in order to facilitate the understanding of the present invention.
100251 Amino acid sequence aligned with the amino acid sequence set out in SEQ
ID NO: X
(when refen-ing to a variant polypeptide) means that the variant amino acid sequence and the amino acid sequence set out in SEQ ID NO: X are aligned by a suitable method which allows comparison of the sequences with each other and identifications of the positions in the amino acid sequence of the variant wherein either the same amino acid is present (identical position), or another amino acid is present (substitution), or one or more extra amino acids are present (insertion or extension) or no amino acid is present (deletion or truncation) if compared with the amino acid sequence set out in SEQ ID NO: X.

[0026] The term "based upon" includes assessing, determining, or measuring the patient characteristics as described herein (and preferably selecting a patient suitable for receiving treatment).
[0027] The term "concurrent administration" means that the administration of the first therapy and that of a second therapy in a combination therapy overlap with each other.
[0028] The term "effective amount" used herein refers to an amount of a compound or composition sufficient to treat a specified disorder, condition or disease such as ameliorate, palliate, lessen, and/or delay one or more of its symptoms. In reference to NSCLC, an effective amount comprises an amount sufficient to cause a tumor to shrink and/or to decrease the growth 113 rate of the tumor (such as to suppress tumor growth) or to prevent or delay other unwanted cell proliferation in NSCLC. In some embodiments, an effective amount is an amount sufficient to delay development of NSCLC. In some embodiments, an effective amount is an amount sufficient to prevent or delay recurrence. An effective amount can be administered in one or more administrations. In the case of NSCLC, the effective amount of the drug or composition may: (i) reduce the number of NSCLC cells; (ii) reduce tumor size; (iii) inhibit, retard, slow to some extent and preferably stop NSCLC cancer cell infiltration into peripheral organs; (iv) inhibit (i.e., slow to some extent and preferably stop) tumor metastasis; (v) inhibit tumor growth; (vi) prevent or delay occurrence and/or recurrence of tumor; and/or (vii) relieve to some extent one or more of the symptoms associated with NSCLC.
[0029] The term "healthy individual" shall be taken to mean an individual who is known not to suffer from cancer (e.g., lung cancer or NSCLC), such knowledge being derived from clinical data on the individual, including, but not limited to, a different diagnostic assay to that described herein.
[0030] A "reference level" means a level of the compound of the present invention or additional biomarker(s) that is indicative of a particular disease state, phenotype, or lack thereof, as well as combinations of disease states, phenotypes, or lack thereof [0031] A "reference sample" refers to a sample containing reference level of a biomarker. For example, a reference sample can be obtained from a subject that does not have a particular disease, disease state or phenotype, such as cancer or acute injury.
100321 "Likely to respond" or "responsiveness" as used herein refers to any kind of improvement or positive response either clinical or non-clinical selected from, but not limited to, measurable reduction in tumor size or evidence of disease or disease progression, complete response, partial response, stable disease, increase or elongation of progression free survival, or increase or elongation of overall survival.

[0033] The term "lung tissue", and "lung cancer" refer to tissue or cancer, respectively, of the lungs themselves, as well as the tissue adjacent to and/or within the strata underlying the lungs and supporting structures such as the pleura, intercostal muscles, ribs, and other elements of the respiratory system. The respiratory system itself is taken in this context as representing nasal cavity, sinuses, pharynx, larynx, trachea, bronchi, lungs, lung lobes, aveoli, aveolar ducts, aveolar sacs, aveolar capillaries, bronchioles, respiratory bronchioles, visceral pleura, parietal pleura, pleural cavity, diaphragm, epiglottis, adenoids, tonsils, mouth and tongue, and the like.
The tissue or cancer may be from a mammal and is preferably from a human, although monkeys, apes, cats, dogs, cows, horses and rabbits are within the scope of the present invention. The term "lung condition" as used herein refers to a disease, event, or change in health status relating to the lung, including for example lung cancer and various non-cancerous conditions.
100341 As applied to polypeptides, the term "substantial similarity" or "substantially similar"
means that two peptide sequences, when optimally aligned, such as by the programs GAP or BESTFIT using default gap weights, share at least 95% sequence identity, even more preferably at least 98% or 99% sequence identity. Preferably, residue positions which are not identical differ by conservative amino acid substitutions. A "conservative amino acid substitution" is one in which an amino acid residue is substituted by another amino acid residue having a side chain (R group) with similar chemical properties (e.g., charge or hydrophobicity).
In general, a conservative amino acid substitution will not substantially change the functional properties of a protein. In cases where two or more amino acid sequences differ from each other by conservative substitutions, the percent sequence identity or degree of similarity may be adjusted upwards to correct for the conservative nature of the substitution.
Means for making this adjustment are well-known to those of skill in the art. See, e.g., Pearson (1994) Methods Mol. Biol. 24: 307-331 . Examples of groups of amino acids that have side chains with similar chemical properties include (1 ) aliphatic side chains: glycine, alanine, valine, leucine and isoleucine; (2) aliphatic-hydroxyl side chains: serine and threonine; (3) amide-containing side chains: asparagine and glutamine; (4) aromatic side chains: phenylalanine, tyrosine, and tryptophan; (5) basic side chains: lysine, arginine, and histidine; (6) acidic side chains: aspartate and glutamate, and (7) sulfur-containing side chains are cysteine and methionine. Preferred conservative amino acids substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamate-aspartate, and asparagine-glutamine.
Alternatively, a conservative replacement is any change having a positive value in the PAM250 log-likelihood matrix disclosed in Gonnet et al. (1992) Science 256: 1443-1445. A "moderately conservative" replacement is any change having a nonnegative value in the PAM250 log-likelihood matrix.
[0035] The term "treatment" or "treating" is an approach for obtaining beneficial or desired results including clinical results. For purposes of this invention, beneficial or desired clinical results include, but are not limited to, one or more of the following:
alleviating one or more symptoms resulting from the disease, diminishing the extent of the disease, stabilizing the disease (e.g., preventing or delaying the worsening of the disease), preventing or delaying the spread (e.g., metastasis) of the disease, preventing or delaying the recurrence of the disease, delay or slowing the progression of the disease, ameliorating the disease state, providing a remission (partial or total) of the disease, decreasing the dose of one or more other medications required to treat the disease, delaying the progression of the disease, increasing the quality of life, and/or prolonging survival. Also encompassed by "treatment" is a reduction of pathological consequence of NSCLC or lung cancer. The methods of the invention contemplate any one or more of these aspects of treatment.
[0036] The term "therapeutic effect" refers to a beneficial local or systemic effect in animals, particularly mammals, and more particularly humans, caused by administration of a compound or composition of the invention. The phrase -therapeutically-effective amount"
means that amount of a compound or composition of the invention that is effective to treat a disease or condition caused by aben-ant biological activity at a reasonable benefit/risk ratio. In some embodiments, the therapeutically effective amount of hydroxyureamethyl-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, mg/day, 640 mg/day, 680 mg/day. and 720 mg/day.
DETAILED DESCRIPTION
[0037] This application discloses methods of treating non-small-cell lung cancer (NSCLC) and lung cancer in an individual in need thereof, comprising administering to the individual an effective amount of a composition comprising nanomolar potent acylfulvene.
[0038] One embodiment includes a method for treating patients with lung cancer and/or non-small cell lung carcinoma or non-small cell lung carcinoma in which hydroxyureamethyl acylfulvene or salt thereof is administered in a therapeutically effective amount to the patient with lung cancer or non-small cell lung carcinoma.

[0039] Hy droxy-UreaMethyl Acylfulvene or hydroxyurearnelhyl-acylfulyene (currentiy, termed as LP-184 by Lantern Pharma, Inc.) is a semisynthetic or synthetic antitumor agent derived from the mushroom toxin illudin S. The structure of each isomer is shown below.
.7".'44111 =
OH
=

100401 Specific embodiments are applicable to multiple histological types of NSCLC. The NSCLC may be squamous cell carcinoma (i.e., epidermoid carcinoma), large cell carcinoma, adenocarcinoma, adenosquamous carcinoma, carcinomas with pleomorphic, sarcomatoid, or sarcomatous elements, carcinoid tumor, or salivary gland carcinoma. In some embodiments the NSCLC is squamous cell carcinoma. In some embodiments, the squamous cell carcinoma is papillary, clear cell, small cell, or basaloid. In some embodiments, the NSCLC
is adenocarcinoma. In some embodiments, the adenocarcinoma is acinar, papillary, bronchioloalveolar carcinoma (e.g., nonmucinous, mucinous, mixed mucinous and nonmucinous or indeterminate cell type), solid adenocarcinoma with mucin, adenocarcinoma with mixed subtypes, well-differentiated fetal adenocarcinoma, mucinous (colloid) adenocarcinoma, mucinous cystadenocarcinoma, signet ring adenocarcinoma, or clear cell adenocarcinoma. In some embodiments, the large cell carcinoma is large-cell neuroendocrine carcinoma, combined large-cell neuroendocrine carcinoma, basaloid carcinoma, lymphoepithelioma-like carcinoma, clear cell carcinoma, or large cell carcinoma with rhabdoid phenotype. In some embodiments, the carcinoma with pleomorphic, sarcomatoid, or sarcomatous elements is carcinomas with spindle and/or giant cells, spindle cell carcinoma, giant cell carcinoma, carcinosarcoma, or pulmonary blastoma. In some embodiments, the carcinoma of salivary-gland type is mucoepidermoid carcinoma or adenoid cystic carcinoma.
In one example, hydroxyureamethyl acylfulvene or its salt may be administered either prior to, concomitantly with, or subsequent to the administration of a chemotherapeutic agent or agents.

[0041] The NSCLC of any of the methods herein may be an occult tumor, a stage 0 tumor, a stage I tumor (stage IA (Ti, NO, MO) or stage TB (T2, NO, MO)), a stage II
tumor (stage IIA
(Ti, Ni, MO) and stage JIB (T2, Ni, MO)), a stage IIIA tumor (Ti, N2, MO, T2, N2, MO, T3, Ni, MO, or T3, N2, MO), a stage IIIB tumor (Any T, N3, MO or T4, any N, MO), or a stage IV
tumor (Any T, any N, M1). In some embodiments of any of the methods described herein, the NSCLC is early stage NSCLC, non-metastatic NSCLC, primary NSCLC, advanced NSCLC, locally advanced NSCLC, metastatic NSCLC, NSCLC in remission, or recurrent NSCLC. In some embodiments, the NSCLC is localized resectable, localized unresectable, or unresectable.
In some embodiments, the NSCLC is unresectable stage IV NSCLC. In some embodiments, the NSCLC is inoperable Stage IIIA and/or IIIB NSCLC, PS 0-1, and FEV 1>800 ml.
[0042] In another embodiment, any of the above methods of treatment comprises the further step of co-administering to the patient one or more second therapeutic agents.
The choice of a combination of agent or second therapeutic agent may be made from any second therapeutic agent known to be useful for co-administration with hydroxyureamethyl acylfulvene or salt.
The choice of second therapeutic agent is also dependent upon the particular disease or condition to be treated. Examples of second therapeutic agents that may be employed in the methods of this application are those set forth above for use in combination compositions comprising a compound of this invention and a second therapeutic agent.
[0043] 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, melphalam, dacarbazine, temozolomide, doxorubicin, imatinib, erlotinib, bevacizumab, cetuximab and a Raf kinase inhibitor.
[0044] In another embodiment, the second therapeutic is one or more chemotherapeutic agents selected from paclitaxel or cisplatinum.
[0045] Some embodiments include a treatment for lung cancer or NSCLC that includes (I) conducting a physical exam of the patient; (2) using a magnetic resonance imaging ("MR1") to confirm the presence of a tumor in the patient's lung; (3) conducting a lung biopsy to obtain genetic information about the cancer, which includes molecular subtype and genetic markers;
and (4) prescribing effective treatment of hydroxyureamethyl-acyifulvene to a subject having lung cancer or NSCLC.
[0046] Another embodiment includes a method of treating lung cancer or NSCLC
that includes detecting, in a human subject, the presence of certain genetic information;
administering hydroxyureamethyl-acyifulvene or a pharmaceutically acceptable salt thereof, to the subject, if the human subject overexpress or under expresses certain markers. The marker can be substantially similar to PTGR1 (SEQ ID NO: 1), KEAP1 (SEQ ID NO: 2), KRAS (SEQ
ID NO: 3), TP53 (SEQ ID NO: 4), and STK11 (SEQ ID NO: 5)..
[0047] Another embodiment includes a method of treating lung cancer or NSCLC
in a subject, comprising: (a) obtaining or having obtained an expression level in a sample from a subject for a plurality of targets, wherein the plurality of targets comprises the group consisting of PTGR1 (SEQ ID NO: 1) together with one or more of the following, KEAP1, KRAS, TP53 and STK11;
(b) determining that the subject is sensitive to a treatment with a Hydroxyureamethyl-acylfulvene; and (c) administering a cancer treatment including a hydroxyureamethyl-acylfulvene.
[0048] The administration period can be a multi-week treatment cycle as long as the tumor remains under control and the regimen is clinically tolerated. In some embodiments, a single dosage of hydroxyureamethyl-acyifulvene or other therapeutic agent can be administered once a week, and preferably once on each of day 1 and day 8 of a three- week (21 -day) treatment cycle. In some embodiments, a single dosage of hydroxyureamethyl-acyifulvene or other therapeutic agent can be administered once a week, twice a week, three times per week, four times per week, five times per week, six times per week, or daily during a one-week, two-week, three-week, four-week, or five-week treatment cycle. The administration can be on the same or different day of each week in the treatment cycle.
[0049] Protein sequences of certain proteins identified herein include PTGR1 (SEQ ID NO:
1), KEAP1 (SEQ ID NO: 2), KRAS (SEQ ID NO: 3), TP53 (SEQ ID NO: 4), and STK11 (SEQ
ID NO: 5).
[0050] 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 hydroxyureamethyl acylfulvene or journal discussion of the same.
[0051] Hydroxyureamethyl-acylfulvene for use in accordance with the present invention can be mainly administered by parenteral administration, specifically including subcutaneous administration, intramuscular administration, intravenous administration, transcutaneous administration, intrahecal administration, epidural administration, intra joint administration and local administration, or may also be administered in various dosage forms, for example by oral administration if possible.

[0052] The injections for administration include for example sterile, aqueous or non-aqueous solutions, suspensions and emulsions. The aqueous solutions and suspensions include for example distilled water for injections and physiological saline. The non-aqueous solutions and suspensions include for example propylene glycol, polyethylene glycol, vegetable oils such as olive oil, alcohols such as ethanol, and Polysorbate 80 (under trade name).
Such composition may contain auxiliary agents such as preservatives, moistening agents, emulsifying agents, dispersing agents, stabilizers (for example, lactose) and dissolution auxiliary agents (for example, meglumine). These are sterilized by filtering through bacteria-retaining filters, blending sterilizing agents, or irradiation. Alternatively, these may be produced once into a sterile solid composition and then dissolved or suspended in sterile water or sterile solvents for injections, prior to use.
[0053] 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 of the 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.
[0054] 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. lung cancer) 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 in the 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 lung cancer 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.
In some embodiments, the therapeutically effective amount of hydroxyureamethyl-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.
[0055] The methods described herein are useful for various aspects of lung cancer or NSCLC
treatment. In some embodiments of any of the methods, the method comprises a method of inhibiting NSCLC cell proliferation (such as NSCLC tumor growth) in an individual, comprising administering to the individual a) an effective amount of a composition comprising particles comprising hydroxyureamethyl-acylfulvene. In some embodiments, at least about 10% (including for example at least about any of 20%, 30%, 40%, 60%, 70%, 80%, 90%, or 100%) cell proliferation is inhibited.
[0056] In some embodiments of any of the methods described herein, the method of treatment results in an objective response (such as a partial response or complete response).
[0057] In some embodiments of any of the methods described herein, the method of treatment results in improved quality of life.
100581 It should also be understood that a specific dosage and treatment regimen for any particular patient will depend upon a variety of factors, including the activity of the specific compound employed, the age, body weight, general health, sex, diet, time of administration, rate of excretion, drug combination, and the judgment of the treating physician and the severity of the particular disease being treated. The amount of a compound of the present invention in the composition will also depend upon the particular compound in the composition. It may be preferable to give the maximum tolerable dose.
[0059] The treatment may include the use of one or more biomarkers. The biomarker may be less than, greater than, or equal to the level of those markers in healthy people. In one example, upregulation of the following genes could be implicated in increased sensitivity to hydroxyureamethyl-acylfulvene: CD55, GLIS3, PRKCDBP, EGF, GLRX, SLC16A7, ABLIM3, DUSP4, ABCG2, HSPB8, TSPAN8, FKBP7, RGS2, CCPG1, DDIT4L, CTSL, PMP22, ACS33, HYAL1, KRT83, SLC16A14, AKR1B10, CA12, SDC2, HHIPL2, MCTP1 [0060] In some embodiments, expression level is determined by measuring the expression level of a gene of interest for a given patient population, determining the median expression level of that gene for the population, and comparing the expression level of the same gene for a single patient to the median expression level for the given patient population. For example, if the expression level of a gene of interest for the single patient is determined to be above the median expression level of the patient population, that patient is determined to have high expression of the gene of interest. Alternatively, if the expression level of a gene of interest for the single patient is determined to be below the median expression level of the patient population, that patient is determined to have low expression of the gene of interest. In some embodiments, the single patient has NSCLC and the patient population does not have cancer (i.e., normal). In some embodiments, the single patient has one histological type of NSCLC
(e.g., squamous cell carcinoma) and the patient population has a second histological type of NSCLC (e.g., adenocarcinoma). In some embodiments, the single patient and the patient population have the same histological type of NSCLC (e.g., squamous cell carcinoma).
[0061] These methods to identify expression levels are not limited by the technique that is used to identify the expression level of the gene of interest. Nucleic acid (e.g., RNA or DNA) or protein levels of the gene of interest can be measured. Methods for measuring gene expression and/or determining sequence for detection of polymorphism are well known in the art and include, but are not limited to, immunological assays, nuclease protection assays, northern blots, in situ hybridization, ELISA, reverse transcriptase Polymerase Chain Reaction (RT-PCR), Real-Time Polymerase Chain Reaction, expressed sequence tag (EST) sequencing, cDNA microarray hybridization or gene chip analysis, subtractive cloning, Serial Analysis of Gene Expression (SAGE), Massively Parallel Signature Sequencing (MPSS), and Sequencing-By-Synthesis (SBS). Diagnostic procedures can also be performed in situ directly upon tissue sections (fixed and/or frozen) of patient tissue obtained from biopsies or resections.
[0062] Examples and Results [0063] Example 1

12 [0064] Nineteen human NSCLC cell lines representing diverse molecular, demographic, and histological features were treated with hydroxyureamethyl-acylfulvene in a 96 well format in triplicate wells, across concentrations ranging from 14 nM to 10 M. The treatments were carried out for 72 hours and cell viability was assayed using Promega's CellTiter Fluor reagent.
Drug sensitivity was measured in terms of 1050 value generated from the dose response curve plotted in GraphPad Prism, as shown in FIG. 1. Representative dose response curves are not shown. Overall, LP 184 exhibited strong nanomolar potency in the majority of NSCLC cell lines tested, indicating broad anti-tumor cytotoxicity in this panel. In these 19 NSCLC cell lines, the 1050 range was 45 to 1805 nM, with median 1050 of 371 nM and mean 1050 of 571 nM.
[0065] Example 2 [0066] This study of hydroxyureamethyl-acylfulvene responses in primary NSCLC
cell lines was extended to in vitro models of brain metastases originating from primary lung cancers.
Two such models LXFA 983 and LXFE 2478 were tested for their sensitivity to hydroxyureamethyl-acylfulvene both in 2D and 3D culture systems. For 2D
cultures, the CellTiter-Glok assay provided a cell viability readout whereas for 3D
cultures, the 3D
Clonogenic assay provided a vital stain-based colony formation readout. As shown in Figure 4, hydroxyureamethyl-acylfulvene retained efficacy in these models, ranging between IC50 of 88 nM and 3209 nM.
[0067] The translational relevance of these results is underscored by the blood brain barrier crossing property of hydroxyureamethyl-acylfulvene.
[0068] As depicted in FIG. 2, hydroxyureamethyl-acylfulvene sensitivity was found in the NSCLC cell lines tested to correlate with PTGR1 transcript levels (Pearson Correlation Coefficient, r = ¨0.603, p value 6.076E-05). The mean hydroxyureamethyl-acylfulvene IC50 value of 571 nM was used to divide the NSCLC cell lines in high and low sensitivity groups. PTGR1 expression was compared among two groups of NSCLC cell lines tested: 11 cell lines with hydroxyureamethyl-acylfulvene IC50 <571 nM and 8 cell lines with IC50 >
571 nM.
100691 To investigate the influence of KEAP1 mutations on PTGR1 transcript levels in the panel of 19 NSCLC cell lines tested, PTGR1 expression was compared, among two groups of cell lines: 7 cell lines with KEAP1 mutation and 12 cell lines without KEAP1 mutation.
We found that the difference in PTGR1 expression between KEAP1 mutant and wild type cell lines is significant upon one-tailed t-test analysis (p value 0.0253). In comparison,

13 PTGR1 expression is entirely independent of mutations in KRAS, TP53 and STK11 which are commonly altered in NSCLC but currently lack effective targeted therapy options [0070] Example 3 [0071] A broad comparison in selected NSCLC cell lines was performed using hydroxyureamethyl-acylfulvene responses with previously published responses of commonly prescribed standard chemotherapeutics obtained from the GDSC
database.
Among those were Oxaliplatin and Cisplatin that also act as DNA alkylating agents but likely via a mechanism not overlapping with that perceived for hydroxyureamethyl-acylfulvene. Known responses were obtained to the antimetabolites Pemetrexed and Gemcitabine, also currently considered as a standard therapy option for NSCLC.
Lung adenocarcinomas can also be treated with taxanes. Publicly available IC50 data on Oxaliplatin, Cisplatin, Pemetrexed, Paclitaxel and Gemcitabine gathered after a 72-hour treatment as with hydroxyureamethyl-acylfulvene , and typically reported as mean values without standard error were obtained from the GDSC database. In this analysis of relative cytotoxicity across selected NSCLC cell lines, as shown in FIG. 3, hydroxyureamethyl-acylfulvene turned out to be up to 3800 times more potent than some of these chemotherapeutics approved for medical use in NSCLC.
[0072] Example 4 [0073] The brain metastasis model LXFE 2478 harbors a heterozygous EGFR-activating mutation, i.e., EGFR exon 20 insertion (M766 A767insASV). The patient from whom this model was derived was reported to be resistant to radiotherapy, cisplatin/erlotinib/pemetrexed combination and PD-Li antibody treatments. The published in vitro 2D efficacy of various EGFR inhibitors in this model were compared with that of hydroxyureamethyl-acylfulvene under similar conditions, and found that hydroxyureamethyl-acylfulvene is about 6 times more potent than earlier generation EGFR
inhibitors Erlotinib and Gefitinib whereas about 2.4 times less potent than the latest generation EGFR inhibitor Osimertinib, thereby placing hydroxyureamethyl-acylfulvene within this spectrum (FIG. 4)..
[0074] Example 5 [0075] Hydroxyureamethyl-acylfulvene anti-tumor response was evaluated in vivo in the NCI-H460 lung tumor model as a subcutaneous xenograft in nude mice. Ten mice were included in the vehicle control and treatment groups. As shown in FIG. 5,

14 hydroxyureamethyl-acylfulvene treatment was conducted using a regimen of five 5 mg/kg injections administered intraperitoneally on days 1, 3, 6, 9 and 12. The individual mouse tumor volumes and body weights on the sampling days are not shown. This treatment yielded statistically significant differences in mean tumor volumes of vehicle control and treatment groups on days 8, 12 and 15. hydroxyureamethyl-acylfulvene thus demonstrated anti-tumor efficacy in a lung cancer model.
[0076] Example 6 [0077] PTGR1 expression status in relation to multiple relevant genes' mutation status in clinical datasets of NSCLC was investigated. In an analysis of 533 NSCLC
adenocarcinoma patient records from the TCGA portal, PTGR1 was found to be highly expressed in KEAP1 mutated samples. In the plot in FIG. 6, the value adjacent to the highly mutated gene is the permutation test p-value of PTGR1 relative gene expression between driver mutated (black) and not-mutated (white) samples. This result is the most statistically significant for KEAP1 (p value 0.00126), with PTGR1 being highly expressed in KEAP1 mutated samples.
Within this PTGR1 high subset, there were distinct brackets enriched in KEAP1, KRAS, BRAF, EGFR, NRF2, MET, and AKT1 mutations. Populations harboring such mutations co-occurring with elevated PTGR1 levels potentially represent molecularly defined NSCLC
patient subgroups that benefit from an hydroxyureamethyl-acylfulvene based regimen.
[0078] SEQ ID NO.: 1: (PTGR1) [0079] MVRTKTWTLKKHFVGYPTNSDFELKTAELPPLKNGEVLLEALFLTVDPYMR
VAAKRLKEGDTMMGQQVAKVVESKNVALPKGTIVLASPGWTTHSISDGKDLEKLL
TEWPDTIPLSLALGTVGMPGLTAYFGLLEICGVKGGETVMVNAAAGAVGSVVGQIA
KLKGCKVVGAVGSDEKVAYLQKLGFDVVFNYKTVESLEETLKKASPDGYDCYFDN
VGGEFSNTVIGQMKKFGRIAICGAISTYNRTGPLPPGPPPEIVIYQELRMEAFVVYRW
QGDARQKALKDLLKWVLEGKIQYK_EYIIEGFENMPAAFMGMLKGDNLGKTIVKA
[0080] SEQ ID NO.: 2 (KEAP1) [0081] MQPDPRPSGAGACCRFLPLQ SQCPEGAGDAVMYASTECKAEVTPSQHGNRT

QID S VV RAC SDFLVQQLDP SNAIGIANFAEQIGCVELHQRAREYIYMHFGEVAKQEE
FFNLSHC QLVTLIS RDDLNVRCESEVFHACINWVKYDCEQRRFYV QALLRAVRCHS
LTPNFLQMQLQKCEILQSDSRCKDYLVKIFEELTLHKPTQVMPCRAPKVGRLIYTAG
GYFRQSL SYLEAYNPSDGTWLRLADLQVPRSGLAGCVVGGLLYAVGGRNNSPDGN
TDS S AL D CYNPMTNQW S P CAP MSVPRNRI GVGVID GHIYAV GGS HGCIHHN S VERY
EPERDEWHLVAPMLTRRIGVGVAVLNRLLYAVGGFDGTNRLNSAECYYPERNEWR
MITAMNTIRSGAGVCVLHNCIYAAGGYDGQDQLNSVERYDVETETWTFVAPMKHR
RSALGITVHQGRIYVLGGYDGHTFLDSVECYDPDTDTWSEVTRMTSGRS GVGVAV
TMEPCRKQIDQQNCTC
[0082] SEQ ID NO.: 3 (KRAS) [0083] MTEYKLVVVGAGGVGKSALTIQLIQNHFVDEYDPTIEDSYRKQVVIDGETCL
LDILDTAGQEEY SAMRDQYMRTGEGFL C VFAINN TKSFEDIHHYREQIKRVKD S ED
VPMVLVGNKCDLPSRTVDTKQAQDLARSYGIPFIETSAKTRQRVEDAFYTLVREIRQ
YRLKKISKEEKTPGCVKIKKCIIM
100841 SEQ ID NO.: 4 (TP53) WFTEDPGPDEAPRMPEAAPPVAPAPAAPTPAAPAPAPSWPLS S SVPSQKTYQGSYGF
RLGFLHS GT AK SVTCTYS P ALNKMFC QL AKTCPVQLWVDSTPPPGTRVRAMAIYKQ
SQHMTEV VRRCPHHERC SD SDGLAPPQHLIRVEGN LRVEYLDDRN TFRH S V V VPYE

GRDRRTEEENLRKKGEPHHELPP GS TKRALPNNT S S SP QP KKKPLD GEYF TL QIRGR
ERFEMFRELNEALELKDAQAGKEPGGSRAHS SHLKSKK GQ ST SRHKK LMF KTEGPD
SD
[0086] SEQ ID NO.: 5 (STK11) [0087] MEVVDPQQLGMFTEGELMSVGMDTFIHRIDSTEVIYQPRRKRAKLIGKYLM
GDLLGEGSYGKVKEVLDSETLCRRAVKILKKKKLRRIPNGEANVKKEIQLLRRLRH
KNVIQLVDVLYNEEKQKMYMVMEYCVCGMQEMLDSVPEKRFPVCQAHGYFCQLI
DGLEYLHSQGIVHKDIKPGNLLLTTGGTLKISDLGVAEALHPFAADDTCRTSQGSPA
FQPPEIANGLDTFSGFKVDIWSAGVTLYNITTGLYPFEGDNIYKLFENIGKGSYAIPG
DCGPPLSDLLKGMLEYEPAKRFSIRQIRQHSWFRKKHPPAEAPVPIPPSPDTKDRWRS
MTVVPYLEDLHGADEDEDLFDIEDDIIYTQDFTVPGQVPEEEASHNGQRRGLPKAV
CMNGTEAAQLSTKSRAEGRAPNPARKACSASSKIRRLSACKQQ
[0088] Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it is apparent to those skilled in the art that certain minor changes and modifications will be practiced. Therefore, the description and examples should not be construed as limiting the scope of the invention.

Claims (9)

Claims

1. A method of treating NSCLC in an individual comprising administering to the individual an effective amount of a composition comprising nanomolar potent particles comprising acylfulvene, wherein treatment is based upon the individual having squamous cellular carcinoma.

2. The method of claim 1, wherein the composition comprising nanoparticles comprising paclitaxel and albumin and the platinum-based agent are administered intravenously.

3. The method of claim 1, wherein the individual is human.

4. The method of claim 1, wherein the method further comprises the administration of radiation.

5. The method of claim 1, wherein the NSCLC is Stage IIIB NSCLC or Stage IV

NSCLC.

The method of claim, wherein the additional therapeutic agent is selected from the group consisting of cisplatin, paclitaxel, and other available therapies.

7.
The method of claim 1, further comprising measuring expression of genetic information of brain cancer to determine if the expression of the genetic information is greater than or less than a reference level of the genetic information.

8. The method of claim 7, wherein the genetic information is PTGR1, KEAP1, KRAS, TP53, or STK11.

9.
A method of treating lung cancer in a human subject with a targeted drug therapy comprising:
extracting a biological sample from the human subject;
measuring the level of expression of PTGRI, KEAN, KRAS, 1P53 and STKI I or combination thereof in the biological sam.ple;
identifying a patient having cancer sensitive to treatment with an illudin-based anti-cancer agent;
treating the human subject with the illudin-based anti-cancer agent having the following struc ture:

(1, er r¨ACIii.
A , ....1,, 4,..,,, ,k .-" ........k \N> __________________________ ticD,"' ... õ.,==== /
H
wheretn the expression level in the human suhject of PTGR1 and one or more KEAP1, KRAS, TP53 and STK1 1 mutation is greater than the level in human without the sd id tumor cancer.