WO2015095835A1

WO2015095835A1 - Methods of modulating radioiodine uptake for the treatment of radioiodine-refractory cancers

Info

Publication number: WO2015095835A1
Application number: PCT/US2014/071739
Authority: WO
Inventors: Saurabh Saha; Dean WELSCH; Gary Decrescenzo; Jeffrey James ROIX
Original assignee: Biomed Valley Discoveries, Inc.
Priority date: 2013-12-20
Filing date: 2014-12-19
Publication date: 2015-06-25

Abstract

The present invention provides, inter alia, methods for modulating iodine uptake in a subject in need thereof, which subject is refractory to radioiodine. The methods include administering to the subject an effective amount of an ERK1/2 inhibitor and an effective amount of radioiodine. Also provided are methods and kits for treating or ameliorating the effects of thyroid cancer in a human patient in which the thyroid cancer has an N-RAS mutation and is refractory to radioiodine treatment.

Description

METHODS OF MODULATING RADIOIODINE UPTAKE FOR THE TREATMENT OF RADIOIODINE-REFRACTORY CANCERS

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims benefit of U.S. Patent Application Serial No. 61/918,959, filed on December 20, 2013 which application is incorporated by reference herein in its entirety.

FIELD OF INVENTION

[0002] The present invention provides, inter alia, methods for modulating iodine uptake in a subject who is refractory to radioiodine. Also provided are methods and kits for treating or ameliorating the effects of thyroid cancer in a human patient in which the thyroid cancer has an N-RAS mutation and is refractory to radioiodine treatment.

INCORPORATION BY REFERENCE OF SEQUENCE LISTING

[0003] This application contains references to amino acids and/or nucleic acid sequences that have been filed concurrently herewith as sequence listing text file 0375752.txt, file size of 649 KB, created on December 19, 2014. The aforementioned sequence listing is hereby incorporated by reference in its entirety pursuant to 37 C.F.R. § 1 .52(e)(5).

BACKGROUND OF THE INVENTION

[0004] Radiotherapy is frequently used to treat numerous cancers. However, the efficacy of any radiotherapeutic is dependent on a number of factors, including the uptake of the radiotherapeutic by the target cancer. High uptake by thyroid cancers is associated with a 10-year survival rate of approximately 60%, whereas thyroid cancers refractory to radiotherapy only have a 10% survival rate.

[0005] Thus, there is, inter alia, a need for methods of enhancing the uptake of radio-labeled therapeutics in cancer treatment regimens, particularly for patients who have become refractory to such radiotherapeutics. The present invention is directed to meeting these and other needs.

SUMMARY OF THE INVENTION

[0006] One embodiment of the present invention is a method of modulating iodine uptake in a subject in need thereof, which subject is refractory to radioiodine. The method comprises administering to the subject an effective amount of an ERK1/2 inhibitor and an effective amount of radioiodine.

[0007] Another embodiment of the present invention is a method for treating or ameliorating the effects of thyroid cancer in a human patient in which the thyroid cancer has an N-RAS mutation and is refractory to radioiodine treatment. The method comprises administering to the patient an effective amount of an ERK1/2 inhibitor and an effective amount of radioiodine.

[0008] A further embodiment of the present invention is a method for treating or ameliorating the effects of thyroid cancer in a human patient in which the thyroid cancer has an N-RAS mutation and is refractory to radioiodine treatment. The method comprises administering to the patient an effective amount of BVD-523 and an effective amount of ¹³¹1. [0009] An additional embodiment of the present invention is a kit for modulating iodine uptake in a subject in need thereof, which subject is refractory to radioiodine. The kit comprises an effective amount of an ERK1/2 inhibitor and an effective amount of radioiodine packaged together with instructions for their use.

DETAILED DESCRIPTION OF THE INVENTION

[0010] One embodiment of the present invention is a method of modulating iodine uptake in a subject in need thereof, which subject is refractory to radioiodine. The method comprises administering to the subject an effective amount of an ERK1/2 inhibitor and an effective amount of radioiodine.

[0011] As used herein, "modulating iodine uptake" refers to increasing or decreasing iodine uptake, or initiating iodine uptake where none was previously observed. Preferably, iodine uptake is increased or it is initiated in a patient for which iodine uptake was not previously observed. Preferably, the iodine is in the form of a radiolabeled therapeutic and/or diagnostic.

[0012] As used herein, a "subject" is a mammal, preferably, a human. In addition to humans, categories of mammals within the scope of the present invention include, for example, farm animals, domestic animals, laboratory animals, etc. Some examples of farm animals include cows, pigs, horses, goats, etc. Some examples of domestic animals include dogs, cats, etc. Some examples of laboratory animals include primates, rats, mice, rabbits, guinea pigs, etc. In each such case, the subject suffers from a disease or condition that may be treated, diagnosed, and/or monitored with a radioiodine according to the present invention.

[0013] As used herein, "resistant" and "refractory" are used interchangeably. Being "refractory" to radioiodine means that radioiodine has reduced efficacy in, e.g., treating cancer or killing cancer cells, or as a diagnostic/monitoring mechanism compared to the same subject prior to becoming resistant to radioiodine..

[0014] As used herein, an "ERK1/2 inhibitor" means those substances that (i) directly interact with ERK1 and/or ERK2, e.g., by binding to ERK1/2 and (ii) decrease the expression or the activity of ERK1 and/or ERK2 protein kinases. Therefore, inhibitors that act upstream of ERK1/2, such as MEK inhibitors and RAF inhibitors, are not ERK1/2 inhibitors according to the present invention. Preferred ERK1/2 inhibitors of the present invention do not decrease the amount of phosphorylated ERK1 and/or ERK2 but decrease the activity of phosphorylated ERK1 and/or ERK2. Non-limiting examples of ERK1/2 inhibitors according to the present invention include AEZS-131 (Aeterna Zentaris), AEZS-136 (Aeterna Zentaris), BVD-523 (BioMed Valley Discoveries, Inc.), SCH-722984 (Merck & Co.), SCH-772984 (Merck & Co.), SCH-900353 (MK-8353) (Merck & Co.), pharmaceutically acceptable salts thereof, and combinations thereof. Preferably, the ERK1/2 inhibitor is BVD- 523 or a pharmaceutically acceptable salt thereof. In the present invention, BVD-523, a preferred ERK1/2 inhibitor, corresponds to a compound according to formula (I):

and pharmaceutically acceptable salts thereof. BVD-523 may be synthesized according to the methods disclosed, e.g., in U.S. Patent No. 7,354,939. Enantiomers and racemic mixtures of both enantiomers of BVD-523 are also contemplated within the scope of the present invention.

[0015] In one aspect of this embodiment, the radioiodine is a radiolabeled iodine molecule that is safe and effective for use in a subject, such as, e.g., a human. Non-limiting examples of radioiodine according to the present invention may be selected from the group consisting of ¹²³l, ¹²⁴l, ¹²⁵l, ¹³¹1, and combinations thereof. Preferably, the radioiodine is ¹³¹1. Preferably, the effective amount of ¹³¹1 delivers an absorbed radiation dose to a lesion of 2000 cGy or more with 300 mCi or less of ¹³¹1. However, the particular specific activity and dose will be determined by a medical professional and may vary depending on the patient and intended use of the radioiodine.

[0016] In another aspect of this embodiment, the subject has a cancer selected from the group consisting of thyroid cancer, bone cancer, and nodal metastases. In the present invention, other cancers susceptible to radioiodine treatment are also contemplated. [0017] In a further aspect of this embodiment, the ERK1/2 inhibitor is in a first unit dosage form and the radioiodine is in a second unit dosage form, separate from the first.

[0018] The ERK1/2 inhibitor and radioiodine may be co-administered to the subject, either simultaneously or at different times, as deemed most appropriate by a physician. If the ERK1/2 inhibitor and radioiodine are administered at different times, for example, by serial administration, the ERK1/2 inhibitor may be administered to the subject before the radioiodine. Alternatively, the radioiodine may be administered to the subject before the ERK1/2 inhibitor.

[0019] In a further aspect of this embodiment, the subject has at least one mutation in an oncogene. As used herein, a "mutation" means a change, in e.g., a nucleic acid. As used herein, an "oncogene" means a gene whose mutation has the potential to cause cancer. Preferably, the mutation in an oncogene is selected from the group consisting of a BRAF mutation, a RAS mutation, a RET/PTC rearrangement, a PAX8 rearrangement, and combinations thereof. More preferably, the mutation in an oncogene is an N- RAS mutation.

[0020] The following Tables 1 , 2, 3, and 4 show the SEQ ID Nos. of representative nucleic acid and amino acid sequences of wild type B-RAF, N- RAS, RET, and PAX8 from various animal sources, respectively, in the sequence listing. These sequences may be used in methods, such as the methods set forth below, for identifying subjects with a mutation in an oncogene, which information may be used to select subjects who would benefit from the methods according to the present invention. Table 1 - B-RAF sequences

polypeptide

or nucleic

acid

SEQ ID No. sequence Organism

40 polypeptide cow, Bos taurus

41 nucleic acid horse, Equus caballus

42 polypeptide horse, Equus caballus

43 nucleic acid chicken, Gallus gallus

44 polypeptide chicken, Gallus gallus

Table 2 - N-RAS sequences

Table 3 - RET sequences

polypeptide

or nucleic

SEQ ID acid

No. sequence Organism Other Information

66 polypeptide human, Homo sapiens isoform c

67 nucleic acid human, Homo sapiens variant 4

68 polypeptide human, Homo sapiens

69 nucleic acid rat (Rattus norvegicus) variant 1

70 polypeptide rat (Rattus norvegicus) isoform a

71 nucleic acid rat (Rattus norvegicus) variant 2

72 polypeptide rat (Rattus norvegicus) isoform b

73 nucleic acid mouse, Mus musculus

74 polypeptide mouse, Mus musculus

75 nucleic acid mouse, Mus musculus variant 2

76 polypeptide mouse, Mus musculus isoform a

77 nucleic acid mouse, Mus musculus variant 4

78 polypeptide mouse, Mus musculus isoform c rabbit, Oryctolagus

79 nucleic acid cuniculus

rabbit, Oryctolagus

80 polypeptide cuniculus

guinea pig, Cavia

81 nucleic acid porcellus

guinea pig, Cavia

82 polypeptide porcellus

dog, Canis lupus

83 nucleic acid familiaris

dog, Canis lupus

84 polypeptide familiaris

dog, Canis lupus

85 nucleic acid familiaris variant X1 dog, Canis lupus

86 polypeptide familiaris variant X1

87 nucleic acid cat, Felis catus

88 polypeptide cat, Felis catus

89 nucleic acid cow, Bos taurus

90 polypeptide cow, Bos taurus

91 nucleic acid chicken, Gallus gallus

92 polypeptide chicken, Gallus gallus Table 4 - PAX8 sequences

polypeptide

or nucleic

SEQ ID acid

No. sequence Organism Other Information

132 polypeptide cow, Bos taurus variant X9

[0021] Methods for identifying mutations in nucleic acids, such as the above identified oncogenes, are known in the art. Nucleic acids may be obtained from biological samples. In the present invention, biological samples include, but are not limited to, blood, plasma, urine, skin, saliva, and biopsies. Biological samples are obtained from a subject by routine procedures and methods which are known in the art.

[0022] Non-limiting examples of methods for identifying mutations include PCR, sequencing, hybrid capture, in-solution capture, molecular inversion probes, fluorescent in situ hybridization (FISH) assay, and combinations thereof.

[0023] Various sequencing methods are known in the art. These include, but are not limited to, Sanger sequencing (also referred to as dideoxy sequencing) and various sequencing-by-synthesis (SBS) methods as disclosed in, e.g., Metzker, 2005, sequencing by hybridization, by ligation (for example, WO 2005021786), by degradation (for example, U.S. Patent Nos. 5,622,824 and 6,140,053) and nanopore sequencing (which is commercially available from Oxford Nanopore Technologies, UK). In deep sequencing techniques, a given nucleotide in the sequence is read more than once during the sequencing process. Deep sequencing techniques are disclosed in e.g., U.S. Patent Publication No. 20120264632 and International Patent Publication No. WO 2012125848. [0024] PCR-based methods for detecting mutations are known in the art and employ PCR amplification, where each target sequence in the sample has a corresponding pair of unique, sequence-specific primers. For example, the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method allows for rapid detection of mutations after the genomic sequences are amplified by PCR. The mutation is discriminated by digestion with specific restriction endonucleases and is identified by electrophoresis. See, e.g., Ota et al., 2007. Mutations may also be detected using real time PCR. See, e.g., International Application Publication No. WO 2012046981 .

[0025] Hybrid capture methods are known in the art and are disclosed in e.g., U.S. Patent Publication No. 20130203632 and U.S. Patent Nos. 8,389,219 and 8,288,520. These methods are based on the selective hybridization of the target genomic regions to user-designed oligonucleotides. The hybridization can be to oligonucleotides immobilized on high or low density microarrays (on-array capture), or solution-phase hybridization to oligonucleotides modified with a ligand (e.g. biotin) which can subsequently be immobilized to a solid surface, such as a bead (in-solution capture).

[0026] Molecular Inversion Probe (MIP) techniques are known in the art and are disclosed in e.g., Absalan et al., 2008. This method uses MIP molecules, which are special "padlock" probes (Nilsson et al., 1994) for genotyping. A MIP molecule is a linear oligonucleotide that contains specific regions, universal sequences, restriction sites and a Tag (index) sequence (16-22 bp). A MIP hybridizes directly around the genetic marker/SNP of interest. The MIP method may also use a number of "padlock" probe sets that hybridize to genomic DNA in parallel (Hardenbol et al., 2003). In case of a perfect match, genomic homology regions are ligated by undergoing an inversion in configuration (as suggested by the name of the technique) and creating a circular molecule. After the first restriction, all molecules are amplified with universal primers. Amplicons are restricted again to ensure short fragments for hybridization on a microarray. Generated short fragments are labeled and, through a Tag sequence, hybridized to a cTag (complementary strand for index) on an array. After the formation of Tag-cTag duplex, a signal is detected.

[0027] In another aspect of this embodiment, the method further comprises administering to the subject an effective amount of thyroid stimulating hormone (TSH) prior to administration of the ERK1/2 inhibitor and the radioiodine. Preferably, the TSH is in a third unit dosage form, separate from the first and second unit dosage forms. The TSH, or similar agent, such as, e.g., thyrotropin alfa (Thyrogen), a highly purified recombinant form of human TSH, is used to stimulate cancer cells to take up radioiodine.

[0028] Another embodiment of the present invention is a method for treating or ameliorating the effects of thyroid cancer in a human patient in which the thyroid cancer has an N-RAS mutation and is refractory to radioiodine treatment. The method comprises administering to the patient an effective amount of an ERK1/2 inhibitor and an effective amount of radioiodine.

[0029] As used herein, the terms "treat," "treating," "treatment" and grammatical variations thereof mean subjecting an individual subject to a protocol, regimen, process or remedy, in which it is desired to obtain a physiologic response or outcome in that subject, e.g., a patient. In particular, the methods of the present invention may be used to slow the development of disease symptoms or delay the onset of the disease or condition, or halt the progression of disease development. However, because every treated subject may not respond to a particular treatment protocol, regimen, process or remedy, treating does not require that the desired physiologic response or outcome be achieved in each and every subject or subject population, e.g., patient population. Accordingly, a given subject or subject population, e.g., patient population, may fail to respond or respond inadequately to treatment.

[0030] As used herein, the terms "ameliorate", "ameliorating" and grammatical variations thereof mean to decrease the severity of the symptoms of a disease in a subject.

[0031] Suitable and preferred ERK1/2 inhibitors, radioiodine, and effective amounts of radioiodine are as disclosed herein. Methods of identifying mutations are also as set forth above.

[0032] In one aspect of this embodiment, the ERK1/2 inhibitor is in a first unit dosage form and the radioiodine is in a second unit dosage form, separate from the first. Methods of co-administration are as disclosed herein.

[0033] In another aspect of this embodiment, the method further comprises administering to the subject an effective amount of thyroid stimulating hormone (TSH) prior to administration of the ERK1/2 inhibitor and the radioiodine. Preferably, the TSH is in a third unit dosage form, separate from the first and second unit dosage forms.

[0034] A further embodiment of the present invention is a method for treating or ameliorating the effects of thyroid cancer in a human patient in which the thyroid cancer has an N-RAS mutation and is refractory to radioiodine treatment. The method comprises administering to the patient an effective amount of BVD-523 and an effective amount of ¹³¹1.

[0035] Effective amounts of radioiodine are as disclosed herein. Methods of identifying mutations are also as set forth above.

[0036] In one aspect of this embodiment, the BVD-523 is in a first unit dosage form and the ¹³¹1 is in a second unit dosage form, separate from the first. Methods of co-administration are as disclosed herein.

[0037] In another aspect of this embodiment, the method further comprises administering to the subject an effective amount of thyroid stimulating hormone (TSH) prior to administration of the BVD-523 and the ¹³¹1. Preferably, the TSH is in a third unit dosage form, separate from the first and second unit dosage forms.

[0038] An additional embodiment of the present invention is a kit for modulating iodine uptake in a subject in need thereof, which subject is refractory to radioiodine. The kit comprises an effective amount of an ERK1/2 inhibitor and an effective amount of radioiodine packaged together with instructions for their use.

[0039] Suitable and preferred subjects, ERK1/2 inhibitors, and radioiodine and amounts thereof are as disclosed herein. The kits of the invention may be used to treat subjects with cancers having various mutational backgrounds and/or that are characterized as disclosed above. Methods of identifying such mutations are also as set forth above. [0040] In one aspect of this embodiment, the ERK1/2 is in a first unit dosage form and the radioiodine is in a second unit dosage form, separate from the first. Methods of co-administration are as disclosed herein.

[0041] In another aspect of this embodiment, the kit further comprises an effective amount of thyroid stimulating hormone (TSH). Preferably, the TSH is in a third unit dosage form, separate from the first and second unit dosage forms.

[0042] The kits may also include suitable storage containers, e.g., ampules, vials, tubes, a radiation shielded container, etc., for each agent of the present invention, which, e.g., may be in the form of pharmaceutical compositions and other reagents, e.g., buffers, balanced salt solutions, etc., for use in administering the agents to subjects. The agents of the invention and other reagents may be present in the kits in any convenient form, such as, e.g., in a solution or in a powder form. The kits may further include a packaging container, optionally having one or more partitions for housing the pharmaceutical composition and other optional reagents.

[0043] In the present invention, an "effective amount" or a "therapeutically effective amount" of an ERK1/2 inhibitor, radioiodine, or thyroid stimulating hormone of the invention, including pharmaceutical compositions containing same that are disclosed herein, is an amount of these agents or compositions that is sufficient to effect beneficial or desired results as described herein when administered to a subject. Effective dosage forms, modes of administration, and dosage amounts may be determined empirically, and making such determinations is within the skill of the art. It is understood by those skilled in the art that the dosage amount will vary with the route of administration, the rate of excretion, the duration of the treatment, the identity of any other drugs being administered, the age, size, and species of mammal, e.g., human patient, and like factors well known in the arts of medicine and veterinary medicine. In general, a suitable dose of an agent or composition, according to the present invention, will be that amount of the agent or composition which is the lowest dose effective to produce the desired effect. The effective dose of an agent or composition of the present invention may be administered as two, three, four, five, six or more sub-doses, administered separately at appropriate intervals throughout the day.

[0044] A suitable, non-limiting example of a dosage of a ERK1/2 inhibitor disclosed herein is from about 1 mg/kg to about 2400 mg/kg per day, such as from about 1 mg/kg to about 1200 mg/kg per day, 75 mg/kg per day to about 300 mg/kg per day, including from about 1 mg/kg to about 100 mg/kg per day. Other representative dosages of such agents include about 1 mg/kg, 5 mg/kg, 10 mg/kg, 15 mg/kg, 20 mg/kg, 25 mg/kg, 30 mg/kg, 35 mg/kg, 40 mg/kg, 45 mg/kg, 50 mg/kg, 60 mg/kg, 70 mg/kg, 75 mg/kg, 80 mg/kg, 90 mg/kg, 100 mg/kg, 125 mg/kg, 150 mg/kg, 175 mg/kg, 200 mg/kg, 250 mg/kg, 300 mg/kg, 400 mg/kg, 500 mg/kg, 600 mg/kg, 700 mg/kg, 800 mg/kg, 900 mg/kg, 1000 mg/kg, 1 100 mg/kg, 1200 mg/kg, 1300 mg/kg, 1400 mg/kg, 1500 mg/kg, 1600 mg/kg, 1700 mg/kg, 1800 mg/kg, 1900 mg/kg, 2000 mg/kg, 2100 mg/kg, 2200 mg/kg, and 2300 mg/kg per day. The effective dose of ERK1/2 inhibitor disclosed herein, e.g., BVD-523, may be administered as two, three, four, five, six or more sub-doses, administered separately at appropriate intervals throughout the day. [0045] A suitable, non-limiting example of a dosage of radioiodine is from about 1 mCi to about 500 mCi, including from about 10 mCi to about 400 mCi. Other representative dosages include about 1 mCi, 2 mCi, 3 mCi, 4 mCi, 5 mCi, 6 mCi, 7 mCi, 8 mCi, 9 mCi, 10 mCi, 20 mCi, 30 mCi, 40 mCi, 50 mCi, 60 mCi, 70 mCi, 80 mCi, 90 mCi, 100 mCi, 125 mCi, 150 mCi, 175 mCi, 200 mCi, 250 mCi, 300 mCi, and 400 mCi.

[0046] A suitable, non-limiting example of a dosage of thyroid stimulating hormone is from about 0.1 mg to about 10 mg, including from about 0.5 mg to about 5 mg. Other representative dosages include about 0.1 mg, 0.2 mg, 0.3 mg, 0.4 mg, 0.5 mg, 0.6 mg, 0.7 mg, 0.8 mg, 0.9 mg, 1 .0 mg, 2 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg, and 10 mg.

[0047] The ERK1/2 inhibitors, TSH, radioiodine, or pharmaceutical compositions containing same of the present invention may be administered in any desired and effective manner: for oral ingestion, or as an ointment or drop for local administration to the eyes, or for parenteral or other administration in any appropriate manner such as intraperitoneal, subcutaneous, topical, intradermal, inhalation, intrapulmonary, rectal, vaginal, sublingual, intramuscular, intravenous, intraarterial, intrathecal, or intralymphatic. Further, the ERK1/2 inhibitors, TSH, radioiodine, or pharmaceutical compositions containing same of the present invention may be administered in conjunction with other treatments. The ERK1/2 inhibitors, TSH, radioiodine, or the pharmaceutical composition containing the same of the present invention may be encapsulated or otherwise protected against gastric or other secretions, if desired. [0048] The pharmaceutical compositions of the invention comprise one or more active ingredients, e.g., ERK inhibitors, TSH, and radioiodine, in admixture with one or more pharmaceutically-acceptable diluents or carriers and, optionally, one or more other compounds, drugs, ingredients and/or materials. Regardless of the route of administration selected, the agents/compounds of the present invention are formulated into pharmaceutically-acceptable dosage forms by conventional methods known to those of skill in the art. See, e.g., Remington, The Science and Practice of Pharmacy (21 ^st Edition, Lippincott Williams and Wilkins, Philadelphia, PA.).

[0049] Pharmaceutically acceptable diluents or carriers are well known in the art (see, e.g., Remington, The Science and Practice of Pharmacy (21 ^st Edition, Lippincott Williams and Wilkins, Philadelphia, PA.) and The National Formulary (American Pharmaceutical Association, Washington, D.C.)) and include sugars (e.g., lactose, sucrose, mannitol, and sorbitol), starches, cellulose preparations, calcium phosphates (e.g., dicalcium phosphate, tricalcium phosphate and calcium hydrogen phosphate), sodium citrate, water, aqueous solutions (e.g., saline, sodium chloride injection, Ringer's injection, dextrose injection, dextrose and sodium chloride injection, lactated Ringer's injection), alcohols (e.g., ethyl alcohol, propyl alcohol, and benzyl alcohol), polyols (e.g., glycerol, propylene glycol, and polyethylene glycol), organic esters (e.g., ethyl oleate and tryglycerides), biodegradable polymers (e.g., polylactide-polyglycolide, poly(orthoesters), and poly(anhydrides)), elastomeric matrices, liposomes, microspheres, oils (e.g., corn, germ, olive, castor, sesame, cottonseed, and groundnut), cocoa butter, waxes (e.g., suppository waxes), paraffins, silicones, talc, silicylate, etc. Each pharmaceutically acceptable diluent or carrier used in a pharmaceutical composition of the invention must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not injurious to the subject. Diluents or carriers suitable for a selected dosage form and intended route of administration are well known in the art, and acceptable diluents or carriers for a chosen dosage form and method of administration can be determined using ordinary skill in the art.

[0050] The pharmaceutical compositions of the invention may, optionally, contain additional ingredients and/or materials commonly used in pharmaceutical compositions. These ingredients and materials are well known in the art and include (1 ) fillers or extenders, such as starches, lactose, sucrose, glucose, mannitol, and silicic acid; (2) binders, such as carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone, hydroxypropylmethyl cellulose, sucrose and acacia; (3) humectants, such as glycerol; (4) disintegrating agents, such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, sodium starch glycolate, cross-linked sodium carboxymethyl cellulose and sodium carbonate; (5) solution retarding agents, such as paraffin; (6) absorption accelerators, such as quaternary ammonium compounds; (7) wetting agents, such as cetyl alcohol and glycerol monostearate; (8) absorbents, such as kaolin and bentonite clay; (9) lubricants, such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, and sodium lauryl sulfate; (10) suspending agents, such as ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth; (1 1 ) buffering agents; (12) excipients, such as lactose, milk sugars, polyethylene glycols, animal and vegetable fats, oils, waxes, paraffins, cocoa butter, starches, tragacanth, cellulose derivatives, polyethylene glycol, silicones, bentonites, silicic acid, talc, salicylate, zinc oxide, aluminum hydroxide, calcium silicates, and polyamide powder; (13) inert diluents, such as water or other solvents; (14) preservatives; (15) surface-active agents; (16) dispersing agents; (17) control-release or absorption-delaying agents, such as hydroxypropylmethyl cellulose, other polymer matrices, biodegradable polymers, liposomes, microspheres, aluminum monostearate, gelatin, and waxes; (18) opacifying agents; (19) adjuvants; (20) wetting agents; (21 ) emulsifying and suspending agents; (22), solubilizing agents and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1 ,3-butylene glycol, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor and sesame oils), glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan; (23) propellants, such as chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons, such as butane and propane; (24) antioxidants; (25) agents which render the formulation isotonic with the blood of the intended recipient, such as sugars and sodium chloride; (26) thickening agents; (27) coating materials, such as lecithin; and (28) sweetening, flavoring, coloring, perfuming and preservative agents. Each such ingredient or material must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not injurious to the subject. Ingredients and materials suitable for a selected dosage form and intended route of administration are well known in the art, and acceptable ingredients and materials for a chosen dosage form and method of administration may be determined using ordinary skill in the art.

[0051] The pharmaceutical compositions of the present invention suitable for oral administration may be in the form of capsules, cachets, pills, tablets, powders, granules, a solution or a suspension in an aqueous or nonaqueous liquid, an oil-in-water or water-in-oil liquid emulsion, an elixir or syrup, a pastille, a bolus, an electuary or a paste. These formulations may be prepared by methods known in the art, e.g., by means of conventional pan- coating, mixing, granulation or lyophilization processes.

[0052] Solid dosage forms for oral administration (capsules, tablets, pills, dragees, powders, granules and the like) may be prepared, e.g., by mixing the active ingredient(s) with one or more pharmaceutically-acceptable diluents or carriers and, optionally, one or more fillers, extenders, binders, humectants, disintegrating agents, solution retarding agents, absorption accelerators, wetting agents, absorbents, lubricants, and/or coloring agents. Solid compositions of a similar type maybe employed as fillers in soft and hard-filled gelatin capsules using a suitable excipient. A tablet may be made by compression or molding, optionally with one or more accessory ingredients. Compressed tablets may be prepared using a suitable binder, lubricant, inert diluent, preservative, disintegrant, surface-active or dispersing agent. Molded tablets may be made by molding in a suitable machine. The tablets, and other solid dosage forms, such as dragees, capsules, pills and granules, may optionally be scored or prepared with coatings and shells, such as enteric coatings and other coatings well known in the pharmaceutical- formulating art. They may also be formulated so as to provide slow or controlled release of the active ingredient therein. They may be sterilized by, for example, filtration through a bacteria-retaining filter. These compositions may also optionally contain opacifying agents and may be of a composition such that they release the active ingredient only, or preferentially, in a certain portion of the gastrointestinal tract, optionally, in a delayed manner. The active ingredient can also be in microencapsulated form.

[0053] Liquid dosage forms for oral administration include pharmaceutically-acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs. The liquid dosage forms may contain suitable inert diluents commonly used in the art. Besides inert diluents, the oral compositions may also include adjuvants, such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, coloring, perfuming and preservative agents. Suspensions may contain suspending agents.

[0054] The compositions of the present invention for rectal or vaginal administration may be presented as a suppository, which may be prepared by mixing one or more active ingredient(s) with one or more suitable nonirritating diluents or carriers which are solid at room temperature, but liquid at body temperature and, therefore, will melt in the rectum or vaginal cavity and release the active compound. The compositions of the present invention which are suitable for vaginal administration also include pessaries, tampons, creams, gels, pastes, foams or spray formulations containing such pharmaceutically-acceptable diluents or carriers as are known in the art to be appropriate.

[0055] Dosage forms for the topical or transdermal administration include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches, drops and inhalants. The active agent(s)/compound(s) may be mixed under sterile conditions with a suitable pharmaceutically-acceptable diluent or carrier. The ointments, pastes, creams and gels may contain excipients. Powders and sprays may contain excipients and propellants.

[0056] The compositions of the present invention suitable for parenteral administrations may comprise one or more agent(s)/compound(s) in combination with one or more pharmaceutically-acceptable sterile isotonic aqueous or non-aqueous solutions, dispersions, suspensions or emulsions, or sterile powders which may be reconstituted into sterile injectable solutions or dispersions just prior to use, which may contain suitable antioxidants, buffers, solutes which render the formulation isotonic with the blood of the intended recipient, or suspending or thickening agents. Proper fluidity can be maintained, for example, by the use of coating materials, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. These compositions may also contain suitable adjuvants, such as wetting agents, emulsifying agents and dispersing agents. It may also be desirable to include isotonic agents. In addition, prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents which delay absorption.

[0057] In some cases, in order to prolong the effect of a drug (e.g., pharmaceutical formulation), it is desirable to slow its absorption from subcutaneous or intramuscular injection. This may be accomplished by the use of a liquid suspension of crystalline or amorphous material having poor water solubility. [0058] The rate of absorption of the active agent/drug then depends upon its rate of dissolution which, in turn, may depend upon crystal size and crystalline form. Alternatively, delayed absorption of a parenterally- administered agent/drug may be accomplished by dissolving or suspending the active agent/drug in an oil vehicle. Injectable depot forms may be made by forming microencapsule matrices of the active ingredient in biodegradable polymers. Depending on the ratio of the active ingredient to polymer, and the nature of the particular polymer employed, the rate of active ingredient release can be controlled. Depot injectable formulations are also prepared by entrapping the drug in liposomes or microemulsions which are compatible with body tissue. The injectable materials can be sterilized for example, by filtration through a bacterial-retaining filter.

[0059] The formulations may be presented in unit-dose or multi-dose sealed containers, for example, ampules and vials, and may be stored in a lyophilized condition requiring only the addition of the sterile liquid diluent or carrier, for example water for injection, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the type described above.

[0060] The following examples are provided to further illustrate the methods of the present invention. These examples are illustrative only and are not intended to limit the scope of the invention in any way. EXAMPLES

Example 1

Methods

[0061] In general, the procedures set forth in Ho et al., 2013 are followed Specifically, the experiments are performed as follows.

Patients

[0062] Patients will have differentiated thyroid carcinoma of follicular cell origin, or a variant thereof, determined histologically. Patients are also required to meet one of the following criteria for radioiodine-refractory disease: 1 ) an index metastatic lesion that was not radioiodine-avid on diagnostic radioiodine scanning performed up to 2 years before enrollment, 2) a radioiodine-avid metastatic lesion that remained stable in size or progressed despite radioiodine treatment 6 months or more before entry into the study, and 3) ¹⁸F-fluorodeoxyglucose (FDG)-avid lesions on PET scanning.

Study Design

[0063] Patients are to adhere to a low-iodine diet for 5 days before the study. Then, patients undergo a thyrotropin alfa-stimulated iodine-124 PET- CT study, followed by treatment with BVD-523 at a dose of 75 mg given orally twice daily for 4 weeks. In the fourth week of BVD-523 treatment, patients undergo a second iodine-124 PET-CT study. Spot urinary iodine measurements may be performed before each study to rule out iodine contamination effects. Patients are discontinued from the study if the second iodine-124 PET study does not show an increase in iodine uptake to a pre- specified dosimetry threshold. If this threshold is met, maximum tolerable activity can be determined in thyrotropin alfa-stimulated whole-body and blood dosimetry studies while the patient continues to receive BVD-523 and a low- iodine diet. A therapeutic dose of iodine-131 can then be administered after stimulation with thyrotropin alfa with BVD-523 treatment continuing until 2 days after iodine-131 exposure. Toxic effects are monitored for at least 30 days following discontinuation of BVD-523 and patients receive suppressive thyroid hormone treatment throughout the study.

[0064] Those patients who receive iodine-131 treatment are subjected to CT imaging, MRI, or both 2 and 6 months after radioiodine therapy. A radiologist can assess radiologic response in these patients according to the Response Evaluation Criteria in Solid Tumors (RECIST). Additional measurements taken 1 , 2, and 6 months after iodine-131 administration include levels of serum thyrotropin, free thyroxine, thyroglobulin and thyroglobulin antibodies.

Iodine-124 PET-CT Studies

[0065] For 2 consecutive days, patients receive 0.9 mg of thyrotropin alfa intramuscularly once daily. The next day, 6 mCi of iodine-124 is administered orally. Two days later, PET-CT imaging is performed without contrast agents. A patient may be scanned from the canthomeatal line to the midthigh with the use of seven to nine bed positions. Following the scan, the number, size, and maximal standardized uptake value of lesions in each body region are recorded.

[0066] If the second iodine-124 PET-CT study indicates increased iodine uptake in the index lesion(s), the administered activity of iodine-131 necessary to deliver a projected absorbed dose of 2000 cGy or more to the lesion can be estimated. The estimate is based on the lesion activity concentration measured via PET multiplied by a recovery coefficient based on the lesion dimensions measured via CT and on an assumed biologic half-life of at least 2 days. Patients are eligible for the standard iodine-131 dosimetry protocol to determine the actual activity to be administered if it appears that one or more lesions could be treated with a dose of 2000 cGy or more with an iodine-131 administered activity of up to 300 mCi. Localization of individual iodine-124 uptake to a specific, corresponding lesion on CT may be determined using image analysis tools and PET volume computer-assisted reading systems, as has been described. (Fox et al., 201 1 ).

Tissue Genotypinq

[0067] Tumor genotypes are determined by a number of methods known by those of skill in the art, including mass-spectrometry genotyping and Sanger sequencing. Genes of interest in this context include, but are not limited to, BRAF, N-RAS, K-RAS, PIK3CA, and AKT1 . If oncogenic point mutations in these genes cannot be found in a given tumor, samples may be screened for RET and PAX8-peroxisome proliferator-activated receptor gamma (PPARG) rearrangements. Tumor cDNA is used as a template for qPCR assays to identify altered expression of exons 10 and 1 1 relative to 12 and 13 of RET. Samples with greater expression of exons 12 and 13 are considered to be positive for a RET/PTC translocation. Positive controls for this assay may include cell line cDNA from medullary thyroid cancers and papillary thyroid cancers for testing expression of full-length RET and RET fusion mRNA, respectively. Statistical Analysis

[0068] The primary endpoint for the study is the percentage of patients with BVD-523-induced increases in iodine uptake in the index tumor(s) as quantified by iodine-124 PET at baseline and after 4 weeks of BVD-523. A second primary endpoint may be the tumor response at approximately 2 and 6 months after iodine-131 treatment, according to Response Evaluation Criteria In Solid Tumors (RECIST), version 1 .1 .

[0069] A secondary endpoint may be an assessment of whether treatment with iodine-131 after BVD-523 is associated with decreased serum thyroglobulin levels at 2 and 6 months. An additional, exploratory endpoint may be an assessment of differences in BVD-523 efficacy for enhancing radioiodine uptake between patients with BRAF mutations and patients with wild-type BRAF.

Example 2

BVD-523 Enhances Radioiodine Uptake in Thyroid Cancer

Study Population

[0070] Patients are referred and screened for the study. Baseline characteristics such as age, sex, tumor type, tumor genotype, exposure to prior radioiodine treatments, and exposure to other thyroid cancer treatments are assessed.

[0071] It is expected that a significant number of patients will exhibit iodine-124 uptake that is new, increased, or both. Further, it is expected that a significant number of these patients will show that an absorbed radiation dose in their lesion(s) would equal or exceed 2000 cGy with 300 mCi of radioiodine or less, will continue to receive BVD-523, and will receive therapeutic radioiodine (¹³¹ l). Additionally, it is expected that patients receiving radiotherapy will show at least partial response if not stable disease or tumor regression. Thyrotropin-suppressed serum thyroglobulin levels should decrease as well. It is further expected that responses will be durable for at least 6 months after radioiodine administration.

Safety

[0072] Toxic effects due to BVD-523 are expected to be grade 1 or 2 and consistent with previous studies of BVD-523.

DOCUMENTS

ABSALAN, Farnaz; Mostafa Ronaghi (2008). Molecular Inversion Probe Assay. Methods in Molecular Biology 396. Humana Press, pp. 315- 330.

FOX, J.J. et al. Practical approach for comparative analysis of multilesion molecular imaging using a semiautomated program for PET/CT. J

Nucl Med 201 1 ;52:1727-32.

HARDENBOL, P. et al. Multiplexed genotyping with sequence-tagged molecular inversion probes. Nat. Biotechnol. 2003, no. 21 , pp. 673-678. HO, A.L. et al. Selumetinib-enhanced radioiodine uptake in advanced thyroid cancer. NEJM 2013;368(7):623-32.

METZKER, Emerging technologies in DNA sequencing Genome Res. 2005.

15: 1767-1776.

NILSSON, M. et al. Padlock probes: circularizing oligonucleotides for localized DNA detection. Science. 1994, no.265, p.2085-2088.

OTA et al., Single nucleotide polymorphism detection by polymerase chain reaction-restriction fragment length polymorphism. Nat Protoc. 2007; 2(1 1 ):2857-64.

[0073] All documents cited in this application are hereby incorporated by reference as if recited in full herein.

[0074] Although illustrative embodiments of the present invention have been described herein, it should be understood that the invention is not limited to those described, and that various other changes or modifications may be made by one skilled in the art without departing from the scope or spirit of the invention.

Claims

WHAT IS CLAIMED IS:

1 . A method of modulating iodine uptake in a subject in need thereof, which subject is refractory to radioiodine, the method comprising administering to the subject an effective amount of an ERK1/2 inhibitor and an effective amount of radioiodine.

2. The method according to claim 1 , wherein the subject is a mammal.

3. The method according to claim 2, wherein the mammal is selected from the group consisting of humans, primates, farm animals, and domestic animals.

4. The method according to claim 2, wherein the mammal is a human.

5. The method according to claim 1 , wherein the ERK1/2 inhibitor is selected from the group consisting of AEZS-131 (Aeterna Zentaris), AEZS- 136 (Aeterna Zentaris), BVD-523 (BioMed Valley Discoveries, Inc.), SCH- 722984 (Merck & Co.), SCH-772984 (Merck & Co.), SCH-900353 (MK-8353) (Merck & Co.), pharmaceutically acceptable salts thereof, and combinations thereof.

6. The method according to claim 1 , wherein the ERK1/2 inhibitor is BVD- 523.

7. The method according to claim 1 , wherein the radioiodine is selected from the group consisting of ¹²³l, ¹²⁴l, ¹²⁵l, ¹³¹1, and combinations thereof.

8. The method according to claim 7, wherein the radioiodine is ¹³¹1.

9. The method according to claim 7, wherein the effective amount of ¹³¹1 delivers an absorbed radiation dose to a lesion of 2000 cGy or more with 300 mCi or less of ¹³¹1.

10. The method according to claim 1 , wherein subject has a cancer selected from the group consisting of thyroid cancer, bone cancer, and nodal metastases.

1 1 . The method according to claim 1 in which the ERK1/2 inhibitor is in a first unit dosage form and the radioiodine is in a second unit dosage form, separate from the first.

12. The method according to claim 1 , wherein the ERK1/2 inhibitor and the radioiodine are co-administered to the subject.

13. The method according to claim 1 , wherein the ERK1/2 inhibitor and the radioiodine are administered to the subject serially.

14. The method according to claim 13, wherein the ERK1/2 inhibitor is administered to the subject before the radioiodine.

15. The method according to claim 1 , wherein the subject has at least one mutation in an oncogene.

16. The method according to claim 15, wherein the mutation in an oncogene is selected from the group consisting of a BRAF mutation, a RAS mutation, a RET/PTC rearrangement, a PAX8 rearrangement, and combinations thereof.

17. The method according to claim 15, wherein the mutation in an oncogene is an N-RAS mutation.

18. The method according to claim 1 1 further comprising administering to the subject an effective amount of thyroid stimulating hormone (TSH) prior to administration of the ERK1/2 inhibitor and the radioiodine.

19. The method according to claim 18 in which the TSH is in a third unit dosage form, separate from the first and second unit dosage forms.

20. A method for treating or ameliorating the effects of thyroid cancer in a human patient in which the thyroid cancer has an N-RAS mutation and is refractory to radioiodine treatment, the method comprising administering to the patient an effective amount of an ERK1/2 inhibitor and an effective amount of radioiodine.

21 . The method according to claim 20, wherein the ERK1/2 inhibitor is selected from the group consisting of AEZS-131 (Aeterna Zentaris), AEZS- 136 (Aeterna Zentaris), BVD-523 (BioMed Valley Discoveries, Inc.), SCH- 722984 (Merck & Co.), SCH-772984 (Merck & Co.), SCH-900353 (MK-8353) (Merck & Co.), pharmaceutically acceptable salts thereof, and combinations thereof.

22. The method according to claim 21 , wherein the ERK1/2 inhibitor is BVD-523.

23. The method according to claim 20, wherein the radioiodine is selected from the group consisting of ¹²³l, ¹²⁴l, ¹²⁵l, ¹³¹1, and combinations thereof.

24. The method according to claim 23, wherein the radioiodine is ¹³¹1.

25. The method according to claim 24, wherein the effective amount of ¹³¹1 delivers an absorbed radiation dose to a lesion of 2000 cGy or more with 300 mCi or less of ¹³¹1.

26. The method according to claim 20 in which the ERK1/2 inhibitor is in a first unit dosage form and the radioiodine is in a second unit dosage form, separate from the first.

27. The method according to claim 20, wherein the ERK1/2 inhibitor and the radioiodine are co-administered to the subject.

28. The method according to claim 20, wherein the ERK1/2 inhibitor and the radioiodine are administered to the subject serially.

29. The method according to claim 28, wherein the ERK1/2 inhibitor is administered to the subject before the radioiodine.

30. The method according to claim 26 further comprising administering to the subject an effective amount of thyroid stimulating hormone (TSH) prior to administration of the ERK1/2 and radioiodine.

31 . The method according to claim 30 in which the TSH is in a third unit dosage form, separate from the first and second unit dosage forms.

32. A method for treating or ameliorating the effects of thyroid cancer in a human patient in which the thyroid cancer has an N-RAS mutation and is refractory to radioiodine treatment, the method comprising administering to the patient an effective amount of BVD-523 and an effective amount of ¹³¹1.

33. The method according to claim 32, wherein the effective amount of ¹³¹1 delivers an absorbed radiation dose to a lesion of 2000 cGy or more with 300 mCi or less of ¹³¹1.

34. The method according to claim 32 in which the BVD-523 is in a first unit dosage form and the ¹³¹1 is in a second unit dosage form, separate from the first.

35. The method according to claim 32, wherein the BVD-523 and the ¹³¹1 are co-administered to the subject.

36. The method according to claim 32, wherein the BVD-523 and the ¹³¹1 are administered to the subject serially.

37. The method according to claim 36, wherein the BVD-523 is administered to the subject before the ¹³¹1.

38. The method according to claim 34 further comprising administering to the subject an effective amount of thyroid stimulating hormone (TSH) prior to administration of the BVD-523 and the ¹³¹1.

39. The method according to claim 38 in which the TSH is in a third unit dosage form, separate from the first and second unit dosage forms.

40. A kit for modulating iodine uptake in a subject in need thereof, which subject is refractory to radioiodine, the kit comprising an effective amount of an ERK1/2 inhibitor and an effective amount of radioiodine packaged together with instructions for their use.

41 . The kit according to claim 40, wherein the subject is a mammal.

42. The kit according to claim 40, wherein the mammal is selected from the group consisting of humans, primates, farm animals, and domestic animals.

43. The kit according to claim 41 , wherein the mammal is a human.

44. The kit according to claim 40, wherein the ERK1/2 inhibitor is selected from the group consisting of AEZS-131 (Aeterna Zentaris), AEZS-136 (Aeterna Zentaris), BVD-523 (BioMed Valley Discoveries, Inc.), SCH-722984 (Merck & Co.), SCH-772984 (Merck & Co.), SCH-900353 (MK-8353) (Merck & Co.), pharmaceutically acceptable salts thereof, and combinations thereof.

45. The kit according to claim 40, wherein the ERK1/2 inhibitor is BVD-523.

46. The kit according to claim 40, wherein the radioiodine is selected from the group consisting of ¹²³l, ¹²⁴l, ¹²⁵l, ¹³¹1, and combinations thereof.

47. The kit according to claim 46, wherein the radioiodine is ¹³¹1.

48. The kit according to claim 47, wherein the effective amount of ¹³¹1 delivers an absorbed radiation dose to a lesion of 2000 cGy or more with 300 mCi or less of ¹³¹1.

49. The kit according to claim 40, the subject has a cancer selected from the group consisting of thyroid cancer, bone cancer, and nodal metastases.

50. The kit according to claim 40, wherein the subject has at least one mutation in an oncogene.

51 . The kit according to claim 50, wherein the mutation in an oncogene is selected from the group consisting of a BRAF mutation, a RAS mutation, a RET/PTC rearrangement, a PAX8 rearrangement, and combinations thereof.

52. The kit according to claim 51 , wherein the mutation in an oncogene is an N-RAS mutation.

53. The kit according to claim 40 in which the ERK1/2 inhibitor is in a first unit dosage form and the radioiodine is in a second unit dosage form, separate from the first.

54. The kit according to claim 40, wherein the ERK1/2 inhibitor and the radioiodine are co-administered to the subject.

55. The kit according to claim 40, wherein the ERK1/2 inhibitor and the radioiodine are administered to the subject serially.

56. The kit according to claim 55, wherein the ERK1/2 inhibitor is administered to the subject before the radioiodine.

57. The kit according to claim 53, further comprising an effective amount of thyroid stimulating hormone (TSH).

58. The kit according to claim 57 in which TSH is in a third unit dosage form, separate from the first and second unit dosage forms.

59. The kit according to claim 40, wherein the radioiodine is packaged in a radiation shielded container.