Companion diagnostic for hydroxyurea methylacylfulvene therapy

Abstract

A companion diagnostic method, kit, and integrated system for quantifying expression of Prostaglandin Reductase 1 (PTGR1) in a patient sample are disclosed. When the measured expression equals or exceeds a predetermined threshold, the patient is selected to receive hydroxyurea methylacylfulvene (LP-184) or a related acylfulvene. The invention enables personalized cancer therapy, improves objective response rates, and minimizes unnecessary exposure to ineffective treatment. Also provided are pharmaceutical combinations, uses, and claims for treating PTGR1 -positive solid tumors and hematological malignancies.

Description

COMPANION DIAGNOSTIC FOR HYDROXYUREA METHYLACYLFULVENETHERAPYPRIOR RELATED APPLICATION DATA

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 669,682, filed July 10, 2024, which is incorporated by reference herein in its entirety.INCORPORATION BY REFERENCE OF SEQUENCE LISTING

[0002] The Sequence Listing titled LP 10040. xml which was created on July 9, 2025 and is 5 kilobytes in size, is hereby incorporated by reference in its entirety.TECHNICAL FIELD

[0003] This application relates to oncology and, more particularly, to a companion in vitro diagnostic (IVD) method, kit, and system for identifying and monitoring patients who will benefit from treatment with hydroxyurea methylacylfulvene (also known as LP-184) and structurally related acylfulvenes.BACKGROUND

[0004] Cancer remains one of the leading causes of morbidity and mortality worldwide. Although many cytotoxic and targeted agents are available, drug resistance and inter-patient heterogeneity continue to limit durable responses. The development of therapeutic strategies for patients with advanced cancer has markedly improved overall survival. However, resistance to anticancer reagents is inevitable, and the prognosis of advanced cancer remains poor. There are several potential sources of cancer drug resistance, including alterations to drug transporters, the suppression of apoptosis, mitochondrial alterations, the promotion of DNA damage repair, autophagy, epithelial-mesenchymal transition, and cancer stem cells (CSCs). Appropriate strategies that consider the mechanisms are necessary to cure cancer.

[0005] Hydroxyurea methylacylfulvene is a semisynthetic derivative of the sesquiterpene illudin S that exerts potent DNA-alkylating activity. Clinical and pre-clinical studies demonstrate marked cytotoxicity in multiple solid and hematologic malignancies; however, clinical benefit correlates with expression of the enzyme Prostaglandin Reductase 1 (PTGR1, Gene ID 10979).

[0006] Companion diagnostics are essential tools in personalized medicine, enabling the identification of patients who are most likely to benefit from a specific therapeutic product.Companion diagnostics — tests that are essential for the safe and effective use of a therapeutic product — allow clinicians to pre-select likely responders, avoid ineffective therapy, and monitor emergent resistance. Hydroxyurea Methylacylfulvene is a potent chemotherapeutic agent used in the treatment of certain cancers and other proliferative diseases. The efficacy and safety of this treatment can vary significantly among patients, necessitating a reliable biomarker to guide its use.

[0007] Accordingly, there is always a need for improved methods to treat cancer.SUMMARY

[0008] This application discloses the discovery that treatment of a cancer in a subject using a companion diagnostic method. This method can include the steps of obtaining a biological sample from a patient, isolating nucleic acids from the biological sample, detecting and quantifying PTGR1 gene expression levels in the isolated nucleic acids, and comparing the detected PTGR1 expression levels to a reference value to predict the patient's response to Hydroxyurea Methylacylfulvene treatment. The method can include monitoring changes in PTGR1 expression levels during the course of treatment to assess efficacy and adjust the therapeutic regimen as needed.

[0009] In one aspect, a method of predicting or monitoring a subject’s response to hydroxyurea methylacylfulvene comprises: obtaining a biological sample containing nucleic acid; isolating the nucleic acid under RNase-inhibiting conditions; detecting and quantifying PTGR1 expression using a closed-cartridge RT-qPCR platform; and comparing the normalized cycle-threshold (ACt) to a pre-validated cutoff to classify the subject as Hydroxyurea-Methylacylfulvene responsive or non-responsive.

[0007] In another aspect, the disclosure provides: (i) a single-use kit preloaded with lysis buffer, wash solutions, lyophilized primers / probes for PTGR1 and PPIA, and an HL7-FHIR compatible software module; (ii) use of the method as a companion diagnostic essential for safe and effective Hydroxyurea-Methylacylfulvene therapy; and (iii) pharmaceutical combinations of Hydroxyurea-Methylacylfulvene with PARP inhibitors wherein PTGR1 positivity predicts synergistic cytotoxicity.

[0010] Another aspect of this application includes the treatments of cancer that can include solid tumors, and hematological malignancies. Tumors such as, but not limited to, hyperplasticor neoplastic disease, such as a carcinoma, sarcoma, or mixed type cancer, including breast, colon, rectal, endometrial, gastric, prostate or brain, mesothelioma, ovarian, lung or pancreatic cancer can be targeted for therapy.

[0011] Another aspect is a predictive diagnostic method. A biological sample (blood, FFPE tissue, biopsy, or other bodily fluid) is processed under RNase-inhibiting conditions. PTGR1 expression is quantified (e.g., closed-cartridge RT-qPCR, qPCR, RT-PCR, or NGS), normalized to a housekeeping gene, and compared with a pre-validated cut-off (e.g., ACt < 8.5). Expression above the cut-off classifies the patient as “Hydroxyurea-Methylacylfulvene-responsive,” whereas lower expression predicts non-response. The same assay can be repeated during therapy to adjust dosing or detect emerging resistance.

[0012] Another aspect is clinical applications. Kits and tests can guide treatment decisions for a broad range of malignancies, including solid tumors (lung, breast, ovarian, prostate, colorectal, pancreatic, brain, sarcoma, adipose tissue) and hematologic cancers (AML, multiple myeloma, mantle-cell lymphoma, double-hit lymphoma). Hydroxyurea-Methylacylfulvene therapy may be combined with radiotherapy or co-administered with PARP or CHK1 / 2 inhibitors to exploit synthetic lethality.

[0013] Another aspect is therapeutic guidance. Patients classified as responsive receive Hydroxyurea-Methylacylfulvene at -5-300 mg / m2intravenously or 30-120 mg / day orally in 28-day cycles. Serial PTGR1 measurements (at least once per cycle) support real-time regimen adjustments.

[0014] Another aspect is a single-use kit. A disposable cartridge pre-loaded with lysis buffer, wash solutions, lyophilized primers / probes for PTGR1 (plus a control gene), and RT-qPCR reagents is read by an automated instrument that extracts nucleic acid, performs amplification / detection, interprets results, and transmits an HL7-FHIR report to the electronic medical record. Spin-column and magnetic-bead versions are available, together with positive / negative control cartridges and reagents stable for > 12 months at 4 °C.

[0015] Another aspect is an information-processing system. Embedded software (or a cloud service) normalizes Ct values, applies a machine-learning classifier (e.g., XGBoost trained on > 500 cases) with tumor-type-specific coefficients, and displays a binary “6-hydroxymethylacylfulvene -responsive / non-responsive” result plus dosage guidanceadjusted for renal and hepatic function.

[0016] Another aspect is a screening assay for drug discovery. An in-vitro workflow compares cytotoxicity of test agents in paired cell lines that differ by > 5-fold in PTGR1 expression; compounds showing > 10-fold selective killing in PTGRl-high cells are flagged as PTGR1 -dependent.

[0017] Another aspect is a combination product. The diagnostic kit and a hydroxyurea methyl acyl fulvene pharmaceutical formulation are packaged and labeled together as a single FDA / EMA companion-diagnostic combination essential for the safe and effective use of hydroxyurea methylacylfulvene.

[0018] Another aspect is an integrated precision-oncology solution. This aspect includes a validated PTGR1 -based assay, ready -to-use kit and software, treatment algorithms, supportive drug combinations, screening tools, and co-packaged therapeutic products — all focused on matching 6-hydroxymethylacylfulvene to the patients most likely to benefit.BRIEF DESCRIPTION OF THE FIGURES

[0019] FIG 1 shows RT-qPCR amplification curves illustrating detection of PTGR1 in FFPE tissue relative to a housekeeping gene (PPIA).

[0020] FIG. 2 shows PTGR1 -dependent cytotoxicity of LP-184.

[0021] FIG. 3 shows scatter-plot demonstrating dependence of LP-184 cytotoxicity on PTGRl expression in isogenic cell lines.SEQUENCESEQ ID NO 1 :MVRTKTWTLKKHFVGYPTNSDFELKTAELPPLKNGEVLLEALFLTVDPYMRVAAKR LKEGDTMMGQQVAKVVESKNVALPKGTIVLASPGWTTHSISDGKDLEKLLTEWPDT IPLSLALGTVGMPGLTAYFGLLEICGVKGGETVMVNAAAGAVGSVVGQIAKLKGCK VVGAVGSDEKVAYLQKLGFDVVFNYKTVESLEETLKKASPDGYDCYFDNVGGEFS NTVIGQMKKFGRIAICGAISTYNRTGPLPPGPPPEIVIYQELRMEAFVVYRWQGDARQ KALKDLLKWVLEGKIQYKEYIIEGFENMPAAFMGMLKGDNLGKTIVKASEQ ID NO 2:MDDDIAALVVDNGSGMCKAGFAGDDAPRAVFPSIVGRPRHQGVMVGMGQKDSYV GDEAQSKRGILTLKYPIEHGIVTNWDDMEKIWHHTFYNELRVAPEEHPVLLTEAPLN PKANREKMTQIMFETFNTPAMYVAIQAVLSLYASGRTTGIVMDSGDGVTHTVPIYE GYALPHAILRLDLAGRDLTDYLMKILTERGYSFTTTAEREIVRDIKEKLCYVALDFEQ EMATAASSSSLEKSYELPDGQVITIGNERFRCPEALFQPSFLGMESCGIHETTFNSIMK CDVDIRKDLYANTVLSGGTTMYPGIADRMQKEITALAPSTMKIKIIAPPERKYSVWIG GSILASLSTFQQMWISKQEYDESGPSIVHRKCFSEQ ID NO 3:MGKVKVGVNGFGRIGRLVTRAAFNSGKVDIVAINDPFIDLNYMVYMFQYDSTHGKF HGTVKAENGKLVINGNPITIFQERDPSKIKWGDAGAEYVVESTGVFTTMEKAGAHL QGGAKRVIISAPSADAPMFVMGVNHEKYDNSLKIISNASCTTNCLAPLAKVIHDNFGI VEGLMTTVHAITATQKTVDGPSGKLWRDGRGALQNIIPASTGAAKAVGKVIPELNG KLTGMAFRVPTANVSVVDLTCRLEKPAKYDDIKKVVKQASEGPLKGILGYTEHQVV S SDFNSDTHS STFDAGAGIALNDHF VKLISW YDNEFGYSNRVVDLMAHMASKEDETAILED DESCRIPTION

[0022] This application provides a companion diagnostic method having the steps of obtaining a biological sample from a subject, isolating nucleic acids from the biological sample; detecting and quantifying PTGR1 gene expression levels in the isolated nucleic acids; and comparing the detected PTGR1 expression levels to a reference value to predict the subject's response to Hydroxyurea Methylacylfulvene treatment. “PTGR1” (Prostaglandin Reductase 1; RefSeq NM_004763.4) includes naturally occurring splice variants, polymorphic or allelic variants, and functional orthologues in non-human species. Historical symbols “PTGR1,” “PGDH,” and “ZADH2” are equivalent. SEQ ID NO: 1.

[0023] “Companion diagnostic” means an in vitro diagnostic device or test that is essential for the safe and effective use of a specific therapeutic product, consistent with FDA 21 CFR § 809.10 and EMA guideline EMA / CHMP / 579146 / 2021.

[0024] The method can include monitoring changes in PTGR1 expression levels during the course of treatment to assess efficacy and adjust the therapeutic regimen as needed. In otherembodiments, the therapy can includes administering a combination of other therapies. In other embodiments, the combination therapy can be used to treat biochemical occurrence or recurrence of solid cancers (e.g., lung cancer, breast cancer, ovarian cancer, prostate cancer, colon cancer, rectum cancer, and bladder cancer), glioblastoma and atypical teratoid rhabdoid, and renal cell carcinoma). In other embodiments, the therapy can be used to treat biochemical occurrence and recurrence of blood cancers in which an acylfulvene (e.g., hydroxyureamethyl acylfulvene) or salt thereof and a PARP inhibitor administered in a therapeutically effective amount to the subject. In certain embodiments, the combination can provide a treatment for lymphoma, such as mantle cell lymphoma (MCL) and double-hit lymphoma (DHL). In multiple myeloma (MM), the overgrowth of plasma cells in the bone marrow can crowd out normal blood-forming cells.Hydroxyureamethyl Acylfulvene

[0025] 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.

[0026] In one example, the acylfulvene is (-) - hydroxyureamethyl acylfulvene (termed LP- 184 by Lantern Pharma Inc ), which shifts light negatively, is shown below:

[0027] A biological sample, such as blood, tissue, or other bodily fluids, can be obtained from the patient. The sample should be collected and processed using standard clinical procedures to ensure the integrity of the nucleic acids. One example can include the use a vacutainer tube containing EDTA as an anticoagulant, the collection of peripheral blood, and the mixture of the blood with the anticoagulant.

[0028] Tissue sample collection can be a standard procedure known in the art. For example, such a method may include aseptic techniques to collect a tissue biopsy.

[0029] Nucleic acids can be isolated from the biological sample using conventional methods, such as spin columns, magnetic beads, or other suitable techniques. The isolated nucleic acids are then used as templates for detecting PTGR1 expression. The spin column method can include lysing the cells in the biological sample using a lysis buffer, binding the nucleic acids to the spin column membrane, wash the membrane to remove contaminants, and elute the purified nucleic acids using an elution buffer. The magnetic bead method can including lysing the cells in the biological sample using a lysis buffer, binding the nucleic acids to magnetic beads, wash the beads to remove contaminants, and eluting the purified nucleic acids by resuspending the beads in an elution buffer and using a magnetic separator to collect the eluted nucleic acids. RNA or DNA is isolated using spin-column kits (e.g., Qiagen RNeasy), magnetic beads, or automated extraction robots following the manufacturer’s protocols.

[0030] PTGR1 expression levels can be detected and quantified using methods such as quantitative polymerase chain reaction (qPCR), reverse transcription PCR (RT-PCR), or nextgeneration sequencing (NGS). Specific primers and probes targeting PTGR1 are designed and utilized in these assays to ensure accurate and sensitive detection. Quantitative polymerase chain reaction (qPCR) can include designing primers and probes specific to the PTGR1 gene, performing reverse transcription of RNA to cDNA, setting up the qPCR reaction using cDNA, primers, probes, and a master mix containing DNA polymerase, running the qPCR reaction on a thermal cycler with real-time detection, and analyzing the amplification curves to quantify PTGR1 expression levels. Reverse transcription PCR (RT-PCR) can include design primers specific to the PTGR1 gene; perform reverse transcription of RNA to cDNA, setting up the PCR reaction using cDNA, primers, and a master mix containing DNA polymerase, runing thePCR reaction on a thermal cycler and analyzing the PCR products by gel electrophoresis or using a fluorescence-based detection method.

[0031] The reference values for PTGR1 expression are established based on data from a cohort of patients who have been treated with Hydroxyurea Methylacylfulvene. These values serve as a benchmark for comparing the expression levels detected in the patient's sample. Higher or lower expression levels relative to reference values indicate a likelihood of positive or negative response to the treatment, respectively. In some embodiments the diagnostic is executed on a closed-cartridge instrument comprising a sample inlet, pre-aliquoted reagents, real-time optical detection, and a processor. Firmware normalizes PTGR1 ACt values to a housekeeping gene, applies the validated cut-off, generates a PDF report, and transmits results to the electronic medical record via HL7® FHIR.

[0032] In the disclosed methods and diagnostic kits, any portion of the PTGR1 gene — ranging from a short signature segment to the full-length coding sequence — may be employed as the analytical target. For example, primers or probes can be designed against as little as about 10 %, 20 %, 30 %, 40 %, 50 %, or more of the PTGR1 sequence, provided the selected region uniquely identifies PTGR1 and yields sufficient analytical sensitivity and specificity for classifying a subject’s likely response to hydroxyurea methylacylfulvene therapy.

[0033] During the course of Hydroxyurea Methylacylfulvene treatment, PTGR1 expression levels are periodically monitored to assess therapeutic efficacy. Changes in expression levels can indicate the success of the treatment or the need for adjustments in the therapeutic regimen. For example, a decrease in PTGR1 expression may suggest effective tumor suppression, while an increase could indicate resistance or relapse.

[0034] 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 the same.

[0035] 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 sufficientamount 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 PARP. 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.

[0036] 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.

[0037] 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 cancer. In another aspect, the disease or condition to be treated is a cell proliferative disorder.

[0038] 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., solid cancers or blood cancers) 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 blood cancers 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, acylfulvene, Irofulven 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.

[0039] The term “treat” is used and includes both therapeutic treatment and prophylactic treatment (reducing the likelihood of development). Both terms mean 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.

[0040] The pharmaceutical compositions can be included in a container, pack, or dispenser together with instructions for administration.

[0041] 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.

[0042] 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-hydroxyethane 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, salicyclic, stearic, subacetic, succinic, sulfamic, sulfanilic, sulfuric, tannic, tartaric, toluene sulfonic, and the commonly occurring amine acids, e.g., glycine, alanine, phenylalanine, arginine, etc.

[0043] Other examples of pharmaceutically acceptable salts include hexanoic acid, cyclopentane propionic acid, pyruvic acid, malonic acid, 3-(4-hydroxybenzoyl)benzoic acid, cinnamic acid, 4-chlorobenzenesulfonic acid, 2-naphthalenesulfonic acid, 4-toluenesulfonic acid, camphorsulfonic acid, 4-methylbicyclo-[2.2.2]-oct-2-ene-l-carboxylic acid, 3-phenylpropionic acid, trimethylacetic acid, tertiary butylacetic acid, muconic acid, and the like. The present invention also encompasses salts formed when an acidic proton in the parent compound either is replaced by a metal ion, e.g., an alkali metal ion, an alkaline earth ion, or an aluminum ion; or coordinates with an organic base such as ethanolamine, diethanolamine, triethanolamine, tromethamine, N-methylglucamine, and the like.

[0044] It should be understood that all references to pharmaceutically acceptable salts include solvent addition forms (solvates), of the same salt.

[0045] 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 greater than 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.

[0046] The composition, or pharmaceutically acceptable salts or solvates thereof, are administered orally, nasally, transdermally, pulmonary, inhalationally, buccally, sublingually, intraperintoneally, 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.

[0047] 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; the renal and hepatic function of the patient; 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.

[0048] Techniques for formulation and administration of the disclosed compounds of the invention can be found in Remington: the Science and Practice of Pharmacy, 19.sup.th edition, Mack Publishing Co., Easton, Pa. (1995). In an embodiment, the compounds described herein,and the pharmaceutically acceptable salts thereof, are used in pharmaceutical preparations in combination with a pharmaceutically acceptable carrier or diluent. Suitable pharmaceutically acceptable carriers include inert solid fdlers or diluents and sterile aqueous or organic solutions. The compounds will be present in such pharmaceutical compositions in amounts sufficient to provide the desired dosage amount in the range described herein.

[0049] 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.

[0050] As used herein, a “subject in need thereof’ is a subject having a precancerous condition. Preferably, a subject in need thereof has cancer. 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 a pig. Preferably, the mammal is a human. The subject of the present invention includes any human subject who has been diagnosed with, has symptoms of, or is at risk of developing a cancer or a precancerous condition.

[0051] 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 cancer 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.

[0052] 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 from previous carcinogenic therapies, such as chemotherapy.

[0053] 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 itaffects the nearby organs or structures. If a cancer spreads (metastasizes), then symptoms may appear in different parts of the body.

[0054] Treating cancer can result in a reduction in size of a tumor. A reduction in 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% or greater. 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.

[0055] Treating cancer results in a decrease in number and size of tumors. Preferably, after treatment, tumor number or size is reduced by 5% or greater relative to 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 most preferably, 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.

[0056] Treating cancer can result in a decrease in 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.

[0057] Treating cancer 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 averagesurvival 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.

[0058] Treating cancer 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 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.

[0059] Treating cancer can result in 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.

[0060] Treating cancer can result in a decrease in the mortality rate of a population of treated subjects in comparison to a population receiving carrier alone. Treating cancer can result in adecrease in the mortality rate of a population of treated subjects in comparison to an untreated population. Treating cancer 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.

[0061] Treating cancer can result in a decrease in tumor growth rate. Preferably, after treatment, tumor growth rate is reduced by at least 5% relative to number 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 50%; and most 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.

[0062] Treating cancer 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 50%; and most 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 failure of tumors to reoccur after treatment has stopped.

[0063] Treating or preventing a cell proliferative disorder can result in a reduction in the rate of cellular proliferation. Preferably, after treatment, the rate of cellular proliferation is reducedby 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 50%; and most 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.

[0064] Treating or preventing a 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 50%; and most 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.

[0065] Treating or preventing a cell proliferative disorder can result in a decrease in size of an area or zone of cellular proliferation. Preferably, after treatment, size of an area or zone 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 50%; and most preferably, reduced by at least 75%. 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.

[0066] Treating or preventing a 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 50%; and most preferably, reduced by at least 75%. An abnormal cellularappearance 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 pl eiom orphism.

[0067] 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 an activity of a protein methyltransferase of interest.

[0068] Treating cancer or a cell proliferative disorder can result in cell death, and preferably, cell death results in a decrease of at least 10% in 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%. Number of cells in a population may be measured by any reproducible means. A 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 as shown in Li et al., Proc. Natl. Acad. Sci. USA. 100(5): 2674-8, 2003. In an aspect, cell death occurs by apoptosis.

[0069] 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

[0070] 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.EXAMPLE 1

[0071] This example shows the results from an assay utilizing RNA extracted from formalin- fixed, paraffin-embedded (FFPE) tissue samples. FFPE tissues represent a significant source of clinically relevant material, despite the inherent challenges associated with RNA degradation during the fixation and embedding processes. The present methods overcome these challenges by employing sensitive and specific RNA isolation techniques compatible with FFPE-derived nucleic acids, followed by highly efficient amplification.

[0072] The core of the assay involves real-time quantitative polymerase chain reaction (qPCR) measurements. Specifically, primers and probes are designed to target unique sequences within the PTGR1 messenger RNA (mRNA) and selected housekeeping gene mRNA(s) (e.g., GAPDH, ACTB, B2M). The selection of appropriate housekeeping genes ensures consistent and reliable normalization across diverse tissue types and disease states. A standard curve may be generated for both PTGR1 and the housekeeping gene(s) using known RNA concentrations to allow for absolute or relative quantification.

[0073] FIG. 1 graphically demonstrates the analytical validity of this assay. As shown in FIG. 1, the assay consistently and accurately detects and quantifies PTGR1 RNA levels relative to the selected housekeeping gene in cancer cells. This analytical validation confirms the assay's precision, accuracy, specificity, and limit of detection / quantification, establishing its suitability for clinical application. FIG. 1 demonstrates that the assay can detect the abundance ofPTGRl RNA relative to the abundance of a housekeeping gene. The assay utilizes RNA extracted from cancer cells to perform real-time PCR measurements of both PTGR1 and housekeeping gene RNA levels. The data demonstrates that the cartridge assay analytically valid for use as a companion diagnostic.EXAMPLE 2

[0074] A human cancer panel was cultured under standard conditions. Baseline PTGR1 expression in each line was quantified with the cartridge assay described in Example 1 using total RNA. LP-184 (> 98 % purity; Lantern Pharma) was prepared as a 10 mM DMSO stock and diluted in medium such that final DMSO never exceeded 0.25 %. Cells were exposed to nine LP-184 concentrations for 72 h, after which viability was read and ICso values calculated by four-parameter logistic regression.

[0075] FIG.2 demonstrates that the cytotoxic activity of LP-184 in killing tumor cells is strictly dependent on the presence ofPTGRl. To establish this dependency, tumor cells with varying levels of PTGR1 expression were treated with LP-184. The results showed a significant correlation between PTGR1 expression and cell death, indicating that PTGR1 is a factor for LP-184's efficacy. Control experiments with PTGR1 knockdown or inhibition further confirmed that reduced PTGR1 levels diminish LP-184's ability to induce tumor cell death.These findings underscore the potential of PTGR1 as a biomarker for predicting the responsiveness of tumors to LP-184 treatment.

[0076] FIG. 3 illustrates a strong correlation between the abundance of PTGR1 expression and the sensitivity of tumor cells to LP-184. In this study, tumor cells with varying levels ofPTGRl expression were treated with LP-184. The data revealed a positive correlation, where cells with higher PTGR1 expression exhibited increased sensitivity to LP-184, leading to enhanced cell death. Conversely, cells with lower PTGR1 expression showed reduced sensitivity to the drug. Statistical analysis, including correlation coefficients and regression analysis, confirmed the significance of this relationship. These results suggest that PTGR1 expression levels could serve as a predictive biomarker for the effectiveness of LP-184 in targeting specific tumor cells.

[0077] These results establish that LP-184’s cytotoxicity is contingent upon cellular PTGR1; modulating PTGR1 expression alters drug sensitivity by more than 50-fold. The finding corroborates PTGR1 as the requisite bio-activating enzyme and a predictive biomarker for clinical response to hydroxyurea methylacylfulvene.Example 3

[0078] Formalin-fixed paraffin-embedded (FFPE) or fresh-frozen tumor specimens were obtained from subjects enrolled in a Phase I, open-label, dose-escalation study of LP-184. Total RNA (> 100 ng) was extracted with the RNeasy Mini kit (Qiagen) and reverse-transcribed using Superscript IV (Thermo). PTGR1 transcript copy number was quantified by TaqMan™ qRT-PCR on an Applied Biosystems 7500 instrument with the following primers / probe (SEQ ID NOs: 1-3). Expression was normalized to the geometric mean Ct of ACTB and GAPDH, yielding a ACt value for each sample:

[0079] A PTGR1 -negative control tissue (placental trophoblast) produced a ACt of 12.6 ± 0.3, establishing the assay background.

[0080] Subjects received intravenous LP-184 every 21 days according to a 3 + 3 dose-escalation design (1.0-11.0 mg m2). Tumor measurements were performed at baseline and every two cycles and evaluated per RECIST 1.1.TABLE 1

[0081] Across the study cohort (n = 19), PTGR1 ACt values ranged from 2.5 to 9.2 in tumor samples that subsequently exhibited clinical benefit (stable disease > 6 months or partial response). All objective responses clustered below the empirically determined threshold of 9.2; no clinical benefit was observed when ACt exceeded this value. The data demonstrate that a ACt cut-off of < 9.2 reliably predicts achievement of at least stable disease in solid-tumor patients treated with LP-184. As such, Patients selected by this method are expected to derive clinical benefit, as illustrated by Patient 10101-2, who achieved a 15 % reduction in target-lesion burden and durable stable disease.

[0082] While a number of exemplary aspects and embodiments have been discussed above, those of skill in the art will recognize certain modifications, permutations, additions, and subcombinations thereof. It is therefore intended that the following appended claims and claims hereafter are interpreted to include all such modifications, permutations, additions, and subcombinations as are within their true spirit and scope.

Claims

CLAIMS1. A method of identifying a subject who is likely to benefit from treatment with hydroxyurea methylacylfulvene, comprising: a. obtaining a biological sample from the subject; b. isolating nucleic acids from the biological sample; c. detecting and quantifying PTGR1 gene expression levels in the isolated nucleic acids; and d. comparing the detected PTGR1 expression levels to a reference value to predict the subject's response.

2. The method of claim 1, further comprising monitoring changes in PTGR1 expression levels during the course of treatment to assess therapeutic efficacy and adjust the therapeutic regimen.

3. The method of claim 1, wherein the detection and quantification of PTGR1 expression levels are performed using qPCR, RT-PCR, or NGS.

4. The method of claim 1, wherein the biological sample is blood, tissue, or other bodily fluid.

5. The method of claim 1, wherein the biological sample is formalin-fixed, paraffin-embedded (FFPE) tissue, freshly-collected tumor biopsy, whole blood, plasma, serum, bone-marrow aspirate, or a circulating-tumor-cell preparation.

6. The method of claim 1, wherein the subject has a solid tumor selected from lung, breast, ovarian, prostate, colorectal, brain, or pancreatic cancer.

7. The method of claim 1-7, wherein the Hydroxyurea Methyl acylfulvene has the following structure:

8. The method of claim 1, wherein the subject has cancer.

9. The method of claim 1, wherein the cancer is colorectal cancer, pancreatic cancer, primary liver cancers, kidney cancer, ovarian cancer, uterine cancer, lung cancer, breast cancer, prostate cancer, sarcomas, or adipose tissue cancer.

10. The method of claims 1, wherein the subject is an animal.

11. The method of claim 1, wherein the subject or mammal is a human.

12. The method of claims 2 or 3, further comprising subjecting the subject to radiation therapy before, after, or during treatment with HydroxyUreaMethyl Acylfulvene.

13. The method of claim 1, wherein the cancer comprises a solid tumor.

14. A kit for predicting a subject's response to Hydroxyurea Methyl acylfulvene treatment, comprising: a. peagents for isolating nucleic acids from a biological sample; b. primers and probes for detecting and quantifying PTGR1 gene expression levels; and c. instructions for comparing the detected PTGR1 expression levels to a reference value to predict the subject's response.

15. The kit of claim 14, wherein the reagents for isolating nucleic acids include spin columns.

16. The kit of claim 15, wherein the reagents for isolating nucleic acids include magnetic beads.

17. The kit of claim 14, wherein the primers and probes are specific for qPCR detection of PTGR1.

18. The kit of claim 14, wherein the primers and probes are specific for RT-PCR detection ofPTGRl .

19. The kit of claim 14, wherein the primers and probes are specific for NGS detection ofPTGRl.

20. A single-use companion-diagnostic kit for performing the method of claim 1, comprising: a disposable closed cartridge pre-loaded with lysis buffer, wash solutions, reverse-transcription and PCR reagents, lyophilized PTGR1 primers and probe, and lyophilized PPIA primers and probe;an automated cartridge reader configured to perform nucleic-acid extraction, RT-qPCR amplification, and real-time fluorescence detection; and machine-executable instructions stored on a non-transitory computer-readable medium that normalize PTGR1 ACt to PPIA ACt apply the pre-validated reference value, and generate a patient report.

21. The kit of claim 12, further comprising external positive and negative control cartridges containing, respectively, (i) synthetic PTGR1 RNA at the reference-value concentration and (ii) a no-template control.

22. The kit of claim 12, wherein the reagents for isolating nucleic acid comprise spin-column materials.

23. The kit of claim 12, wherein the reagents for isolating nucleic acid comprise magnetic-bead materials.

24. The kit of claim 12, wherein the primers and probes are configured for qPCR detection of PTGR1 / PTGR1.

25. The kit of claim 12, wherein the primers and probes are configured for RT-PCR detection of PTGR1 / PTGR1.

26. The kit of claim 12, wherein the primers and probes are configured for NGS-based detection of PTGR1 / PTGR1 transcripts.

27. A kit of claims 12, further comprising printed or electronic instructions for comparing the detected PTGR1 / PTGR1 expression level to a reference value to predict therapeutic response.

28. A method for determining suitability of LP-184 therapy in a patient with cancer, comprising:(a) obtaining a tumor sample from the patient;(b) measuring PTGR1 transcript level in the sample by quantitative reverse-transcription PCR;(c) determining a normalized delta-Ct (ACt) value for PTGR1 expression; and(d) identifying the patient as suitable for LP- 184 treatment if the ACt value is below 9.2.

29. The method of claim 28, wherein the cancer is a solid tumor.

30. The method of claim 28, further comprising administering LP-184 to the patient identified as suitable for treatment.

31. The method of claim 28, wherein a PTGR1 -negative control included in the assay yields a ACt of approximately 12.6.

32. A kit for predicting anticancer efficacy of LP-184, comprising reagents for measuring PTGR1 expression by qRT-PCR and instructions specifying that ACt values below 9.2 are predictive of at least stable disease on LP-184 therapy.

33. A method of treating a patient with cancer, comprising:(a) identifying the patient as suitable for LP-184 treatment by the method of claim 32; and(b) administering LP-184 to the patient.

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