Phospholipid analogs as diapeutic agents and methods thereof
09550002 ยท 2017-01-24
Assignee
Inventors
Cpc classification
G01N33/57492
PHYSICS
A61K51/0489
HUMAN NECESSITIES
A61P35/00
HUMAN NECESSITIES
International classification
A61K51/00
HUMAN NECESSITIES
Abstract
The present invention provides methods and uses of phospholipid ether analogs as diagnostic and therapeutic agents for numerous cancers.
Claims
1. A method for the treatment of a cancer in a subject comprising: administering, to a subject previously diagnosed with cancer, an effective amount of .sup.125I-labeled 18-(p-iodophenyl)octadecyl phosphocholine or a salt thereof for treating the cancer.
2. The method of claim 1, wherein the subject is human.
3. The method of claim 1, wherein the phosphocholine is selectively uptaken by cancer cells within the subject.
4. The method of claim 1, wherein the cancer is lung cancer.
5. The method of claim 1, wherein the cancer is adrenal cancer.
6. The method of claim 1, wherein the cancer is melanoma or subcutaneous cancer.
7. The method of claim 1, wherein the cancer is intestinal cancer, colon cancer or colorectal cancer.
8. The method of claim 1, wherein the cancer is ovarian cancer or cervical cancer.
9. The method of claim 1, wherein the cancer is prostate cancer.
10. The method of claim 1, wherein the cancer is liver cancer or hepatocellular carcinoma.
11. The method of claim 1, wherein the cancer is squamous cell carcinoma.
12. The method of claim 1, wherein the cancer is retinoblastoma.
13. The method of claim 1, wherein the cancer is glioma.
14. The method of claim 1, wherein the cancer is breast cancer.
15. The method of claim 1, wherein the cancer is carcinosarcoma.
16. The method of claim 1, wherein the cancer is pancreatic cancer.
17. The method of claim 1, comprising administering between about 0.5 Ci and about 500 mCi of the .sup.125I-labeled 18-(p-iodophenyl)octadecyl phosphocholine to the subject.
18. The method of claim 1, comprising administering less than 0.3 g/kg of body weight of the .sup.125I-labeled 18-(p-iodophenyl)octadecyl phosphocholine to the subject.
19. A method for the treatment of a cancer in a subject comprising: administering, to a subject previously diagnosed with as having cancer, an effective amount of .sup.131I-labeled 18-(p-iodophenyl)octadecyl phosphocholine or a salt thereof for treating the cancer.
20. The method of claim 19, wherein the subject is human.
21. The method of claim 19, wherein the phosphocholine is selectively uptaken by cancer cells within the subject.
22. The method of claim 19, wherein the cancer is lung cancer.
23. The method of claim 19, wherein the cancer is adrenal cancer.
24. The method of claim 19, wherein the cancer is melanoma or subcutaneous cancer.
25. The method of claim 19, wherein the cancer is intestinal cancer, colon cancer or colorectal cancer.
26. The method of claim 19, wherein the cancer is ovarian cancer or cervical cancer.
27. The method of claim 19, wherein the cancer is prostate cancer.
28. The method of claim 19, wherein the cancer is liver cancer or hepatocellular carcinoma.
29. The method of claim 19, wherein the cancer is squamous cell carcinoma.
30. The method of claim 19, wherein the cancer is retinoblastoma.
31. The method of claim 19, wherein the cancer is glioma.
32. The method of claim 19, wherein the cancer is breast cancer.
33. The method of claim 19, wherein the cancer is carcinosarcoma.
34. The method of claim 19, wherein the cancer is pancreatic cancer.
Description
BRIEF DESCRIPTION OF DRAWINGS
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I. GENERAL DESCRIPTION OF THE INVENTION
(33) General Description of the Invention: Before the present methods are described, it is understood that this invention is not limited to the particular methodology, protocols, cell lines, and reagents described, as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention which will be limited only by the appended claims.
(34) It must be noted that as used herein and in the appended claims, the singular forms a, an, and the include plural reference unless the context clearly dictates otherwise. Thus, for example, reference to a cell includes a plurality of such cells and equivalents thereof known to those skilled in the art, and so forth. As well, the terms a (or an), one or more and at least one can be used interchangeably herein. It is also to be noted that the terms comprising, including, and having can be used interchangeably.
(35) Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are now described. All publications mentioned herein are incorporated herein by reference for the purpose of describing and disclosing the chemicals, cell lines, vectors, animals, instruments, statistical analysis and methodologies which are reported in the publications which might be used in connection with the invention. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention.
(36) As defined herein, the term isomer includes, but is not limited to optical isomers and analogs, structural isomers and analogs, conformational isomers and analogs, and the like. In one embodiment, this invention encompasses the use of different optical isomers of an anti-tumor compound of Formula 3A. It will be appreciated by those skilled in the art that the anti-tumor compounds useful in the present invention may contain at least one chiral center. Accordingly, the compounds used in the methods of the present invention may exist in, and be isolated in, optically-active or racemic forms. Some compounds may also exhibit polymorphism.
(37) It is to be understood that the present invention may encompass the use of any racemic, optically-active, polymorphic, or stereroisomeric form, or mixtures thereof, which form possesses properties useful in the treatment of tumor-related conditions described and claimed herein. In one embodiment, the anti-tumor compounds may include pure (R)-isomers. In another embodiment, the anti-tumor compounds may include pure (S)-isomers. In another embodiment, the compounds may include a mixture of the (R) and the (S) isomers. In another embodiment, the compounds may include a racemic mixture comprising both (R) and (S) isomers. It is well known in the art how to prepare optically-active forms (for example, by resolution of the racemic form by recrystallization techniques, by synthesis from optically-active starting materials, by chiral synthesis, or by chromatographic separation using a chiral stationary phase).
(38) The invention includes the use of pharmaceutically acceptable salts of amino-substituted compounds with organic and inorganic acids, for example, citric acid and hydrochloric acid. The invention also includes N-oxides of the amino substituents of the compounds described herein. Pharmaceutically acceptable salts can also be prepared from the phenolic compounds by treatment with inorganic bases, for example, sodium hydroxide. Also, esters of the phenolic compounds can be made with aliphatic and aromatic carboxylic acids, for example, acetic acid and benzoic acid esters. As used herein, the term pharmaceutically acceptable salt refers to a compound formulated from a base compound which achieves substantially the same pharmaceutical effect as the base compound.
(39) This invention further includes method utilizing derivatives of the anti-tumor compounds. The term derivatives includes but is not limited to ether derivatives, acid derivatives, amide derivatives, ester derivatives and the like. In addition, this invention further includes methods utilizing hydrates of the anti-tumor compounds. The term hydrate includes but is not limited to hemihydrate, monohydrate, dihydrate, trihydrate and the like.
(40) This invention further includes methods of utilizing metabolites of the anti-tumor compounds. The term metabolite means any substance produced from another substance by metabolism or a metabolic process.
(41) As defined herein, contacting means that the anti-tumor compound used in the present invention is introduced into a sample containing the receptor in a test tube, flask, tissue culture, chip, array, plate, microplate, capillary, or the like, and incubated at a temperature and time sufficient to permit binding of the anti-tumor compound to a receptor. Methods for contacting the samples with the anti-tumor compound or other specific binding components are known to those skilled in the art and may be selected depending on the type of assay protocol to be run. Incubation methods are also standard and are known to those skilled in the art.
(42) In another embodiment, the term contacting means that the anti-tumor compound used in the present invention is introduced into a patient receiving treatment, and the compound is allowed to come in contact in vivo.
(43) As used herein, the term treating includes preventative as well as disorder remittent treatment. As used herein, the terms reducing, suppressing and inhibiting have their commonly understood meaning of lessening or decreasing. As used herein, the term progression means increasing in scope or severity, advancing, growing or becoming worse. As used herein, the term recurrence means the return of a disease after a remission.
(44) As used herein, the term administering refers to bringing a patient, tissue, organ or cells in contact with an anti-tumor phospholipid ether compound. As used herein, administration can be accomplished in vitro, i.e. in a test tube, or in vivo, i.e. in cells or tissues of living organisms, for example, humans. In certain embodiments, the present invention encompasses administering the compounds useful in the present invention to a patient or subject. A patient or subject, used equivalently herein, refers to a mammal, preferably a human, that either: (1) has a disorder remediable or treatable by administration of the anti-tumor substance using a phospholipid ether compound or (2) is susceptible to a disorder that is preventable by administering the anti-tumor compound using a phospholipid ether compound
(45) As used herein, pharmaceutical composition means therapeutically effective amounts of the anti-tumor compound together with suitable diluents, preservatives, solubilizers, emulsifiers, and adjuvants, collectively pharmaceutically-acceptable carriers. As used herein, the terms effective amount and therapeutically effective amount refer to the quantity of active therapeutic agent sufficient to yield a desired therapeutic response without undue adverse side effects such as toxicity, irritation, or allergic response. The specific effective amount will, obviously, vary with such factors as the particular condition being treated, the physical condition of the patient, the type of animal being treated, the duration of the treatment, the nature of concurrent therapy (if any), and the specific formulations employed and the structure of the compounds or its derivatives. In this case, an amount would be deemed therapeutically effective if it resulted in one or more of the following: (a) the prevention of disease (e.g., pancreatic cancer, breast cancer); and (b) the reversal or stabilization of such disease. The optimum effective amounts can be readily determined by one of ordinary skill in the art using routine experimentation.
(46) Pharmaceutical compositions are liquids or lyophilized or otherwise dried formulations and include diluents of various buffer content (e.g., Tris-HCl, acetate, phosphate), pH and ionic strength, additives such as albumin or gelatin to prevent absorption to surfaces, detergents (e.g., Tween (Polysorbate) 20, Tween 80, Pluronic F68, bile acid salts), solubilizing agents (e.g., glycerol, polyethylene glycerol), anti-oxidants (e.g., ascorbic acid, sodium metabisulfite), preservatives (e.g., Thimerosal, benzyl alcohol, parabens), bulking substances or tonicity modifiers (e.g., lactose, mannitol), covalent attachment of polymers such as polyethylene glycol to the protein, complexation with metal ions, or incorporation of the material into or onto particulate preparations of polymeric compounds such as polylactic acid, polglycolic acid, hydrogels, etc, or onto liposomes, microemulsions, micelles, unilamellar or multilamellar vesicles, erythrocyte ghosts, or spheroplasts. Such compositions will influence the physical state, solubility, stability, rate of in vivo release, and rate of in vivo clearance. Controlled or sustained release compositions include formulation in lipophilic depots (e.g., fatty acids, waxes, oils).
(47) Also encompassed by the invention are methods of administering particulate compositions coated with polymers (e.g., poloxamers or poloxamines). Other embodiments of the compositions incorporate particulate forms protective coatings, protease inhibitors or permeation enhancers for various routes of administration, including topical, parenteral, pulmonary, nasal and oral. In one embodiment the pharmaceutical composition is administered parenterally, paracancerally, transmucosally, tansdermally, intramuscularly, intravenously, intradermally, subcutaneously, intraperitonealy, intraventricularly, intracranially and intratumorally.
(48) Further, as used herein pharmaceutically acceptable carriers are well known to those skilled in the art and include, but are not limited to, 0.01-0.1M and preferably 0.05M phosphate buffer or 0.9% saline. Additionally, such pharmaceutically acceptable carriers may be aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media.
(49) Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's and fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers such as those based on Ringer's dextrose, and the like. Preservatives and other additives may also be present, such as, for example, antimicrobials, antioxidants, collating agents, inert gases and the like.
(50) Controlled or sustained release compositions administerable according to the invention include formulation in lipophilic depots (e.g. fatty acids, waxes, oils). Also comprehended by the invention are particulate compositions coated with polymers (e.g. poloxamers or poloxamines) and the compound coupled to antibodies directed against tissue-specific receptors, ligands or antigens or coupled to ligands of tissue-specific receptors.
(51) Other embodiments of the compositions administered according to the invention incorporate particulate forms, protective coatings, protease inhibitors or permeation enhancers for various routes of administration, including parenteral, pulmonary, nasal and oral.
(52) Compounds modified by the covalent attachment of water-soluble polymers such as polyethylene glycol, copolymers of polyethylene glycol and polypropylene glycol, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone or polyproline are known to exhibit substantially longer half-lives in blood following intravenous injection than do the corresponding unmodified compounds (Abuchowski et al., 1981; Newmark et al., 1982; and Katre et al., 1987). Such modifications may also increase the compound's solubility in aqueous solution, eliminate aggregation, enhance the physical and chemical stability of the compound, and greatly reduce the immunogenicity and reactivity of the compound. As a result, the desired in vivo biological activity may be achieved by the administration of such polymer-compound abducts less frequently or in lower doses than with the unmodified compound.
(53) In yet another method according to the invention, a pharmaceutical composition can be delivered in a controlled release system. For example, the agent may be administered using intravenous infusion, an implantable osmotic pump, a transdermal patch, liposomes, or other modes of administration. In one embodiment, a pump may be used (see Langer, supra; Sefton, CRC Crit. Ref. Biomed. Eng. 14:201 (1987); Buchwald et al., Surgery 88:507 (1980); Saudek et al., N. Engl. J. Med. 321:574 (1989). In another embodiment, polymeric materials can be used. In yet another embodiment, a controlled release system can be placed in proximity to the therapeutic target, for example liver, thus requiring only a fraction of the systemic dose (see, e.g., Goodson, in Medical Applications of Controlled Release, supra, vol. 2, pp. 115-138 (1984). Other controlled release systems are discussed in the review by Langer (Science 249:1527-1533 (1990).
(54) The pharmaceutical preparation can comprise the anti-tumor compound alone, or can further include a pharmaceutically acceptable carrier, and can be in solid or liquid form such as tablets, powders, capsules, pellets, solutions, suspensions, elixirs, emulsions, gels, creams, or suppositories, including rectal and urethral suppositories. Pharmaceutically acceptable carriers include gums, starches, sugars, cellulosic materials, and mixtures thereof. The pharmaceutical preparation containing the anti-tumor compound can be administered to a patient by, for example, subcutaneous implantation of a pellet. In a further embodiment, a pellet provides for controlled release of anti-tumor compound over a period of time. The preparation can also be administered by intravenous, intra-arterial, or intramuscular injection of a liquid preparation oral administration of a liquid or solid preparation, or by topical application. Administration can also be accomplished by use of a rectal suppository or a urethral suppository.
(55) The pharmaceutical preparations administerable by the invention can be prepared by known dissolving, mixing, granulating, or tablet-forming processes. For oral administration, the anti-tumor compounds or their physiologically tolerated derivatives such as salts, esters, N-oxides, and the like are mixed with additives customary for this purpose, such as vehicles, stabilizers, or inert diluents, and converted by customary methods into suitable forms for administration, such as tablets, coated tablets, hard or soft gelatin capsules, aqueous, alcoholic or oily solutions. Examples of suitable inert vehicles are conventional tablet bases such as lactose, sucrose, or cornstarch in combination with binders such as acacia, cornstarch, gelatin, with disintegrating agents such as cornstarch, potato starch, alginic acid, or with a lubricant such as stearic acid or magnesium stearate.
(56) Examples of suitable oily vehicles or solvents are vegetable or animal oils such as sunflower oil or fish-liver oil. Preparations can be effected both as dry and as wet granules. For parenteral administration (subcutaneous, intravenous, intra-arterial, or intramuscular injection), the anti-tumor compounds or their physiologically tolerated derivatives such as salts, esters, N-oxides, and the like are converted into a solution, suspension, or expulsion, if desired with the substances customary and suitable for this purpose, for example, solubilizers or other auxiliaries. Examples are sterile liquids such as water and oils, with or without the addition of a surfactant and other pharmaceutically acceptable adjuvants. Illustrative oils are those of petroleum, animal, vegetable, or synthetic origin, for example, peanut oil, soybean oil, or mineral oil. In general, water, saline, aqueous dextrose and related sugar solutions, and glycols such as propylene glycols or polyethylene glycol are preferred liquid carriers, particularly for injectable solutions.
(57) The preparation of pharmaceutical compositions which contain an active component is well understood in the art. Such compositions may be prepared as aerosols delivered to the nasopharynx or as injectables, either as liquid solutions or suspensions; however, solid forms suitable for solution in, or suspension in, liquid prior to injection can also be prepared. The preparation can also be emulsified. The active therapeutic ingredient is often mixed with excipients which are pharmaceutically acceptable and compatible with the active ingredient. Suitable excipients are, for example, water, saline, dextrose, glycerol, ethanol, or the like or any combination thereof.
(58) In addition, the composition can contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents which enhance the effectiveness of the active ingredient.
(59) An active component can be formulated into the composition as neutralized pharmaceutically acceptable salt forms. Pharmaceutically acceptable salts include the acid addition salts, which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed from the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, 2-ethylamino ethanol, histidine, procaine, and the like.
(60) For topical administration to body surfaces using, for example, creams, gels, drops, and the like, the anti-tumor compounds or their physiologically tolerated derivatives such as salts, esters, N-oxides, and the like are prepared and applied as solutions, suspensions, or emulsions in a physiologically acceptable diluent with or without a pharmaceutical carrier.
(61) In another method according to the invention, the active compound can be delivered in a vesicle, in particular a liposome (see Langer, Science 249:1527-1533 (1990); Treat et al., in Liposomes in the Therapy of Infectious Disease and Cancer, Lopez-Berestein and Fidler (eds.), Liss, N.Y., pp. 353-365 (1989); Lopez-Berestein ibid., pp. 317-327; see generally ibid).
(62) For use in medicine, the salts of the anti-tumor compound may be pharmaceutically acceptable salts. Other salts may, however, be useful in the preparation of the compounds according to the invention or of their pharmaceutically acceptable salts. Suitable pharmaceutically acceptable salts of the compounds include acid addition salts which may, for example, be formed by mixing a solution of the compound according to the invention with a solution of a pharmaceutically acceptable acid such as hydrochloric acid, sulphuric acid, methanesulphonic acid, fumaric acid, maleic acid, succinic acid, acetic acid, benzoic acid, oxalic acid, citric acid, tartaric acid, carbonic acid or phosphoric acid.
(63) Generally, NM404 is a promising new tumor-selective diagnostic imaging agent to monitor the treatment response of several tumor treatment modalities. Radioiodinated NM404, a second-generation phospholipid ether analog, had displayed remarkable tumor selectivity in 10/10 xenograft tumor models and more recently in another 14/14 spontaneous rodent tumor models. Due to a lack of metabolic phospholipase enzymes in the membranes of tumor cells, the prevailing hypothesis of this approach is that phospholipid ether analogs become trapped exclusively in tumor cell membranes because of their inability to become metabolized and eliminated. Thus, the differential clearance rates of phospholipid ethers from normal cells versus viable tumor cells form the basis of this concept. Results obtained in a variety tumor models indicate that NM404 is sequestered and selectively retained by viable tumor cells and localizes in both primary and metastatic lesions regardless of anatomic location including those found in lymph nodes. Unlike FDG, this agent does not localize in infectious sites. Other advantages of NM404 over FDG include the following: NM404 is selective for and retained indefinitely by malignant tumor cells whereas FDG in not selective for tumor cells and goes to infectious sites and hyperplasias (Barret's Esophagus). Further, since .sup.124I has a 4 day physical half life it can be shipped anywhere in the world whereas FDG with its 110 min half-life, may have limited distribution within 200 miles of the production site. NM404 undergoes prolonged retention (not metabolized) and therefore affords a significant therapeutic potential when mated with an appropriate radioisotope like .sup.131I whereas FDG does not possess any therapeutic potential. NM404 can be labeled with a variety of iodine isotopes expanding it versatility (diagnosis and therapy as well as a tool for experimental animal studies) whereas FDG is limited to .sup.18F for PET scanning or potentially .sup.19F (stable) for magnetic resonance imaging albeit at very low sensitivity levels. Regardless of its tumor targeting ability, due to its rapid metabolism in tumor cells, it has not potential for therapy. NM404 affords the potential to not only accurately predict local tumor response to various treatment modalities, but also allows detection of distant metastatic lesions in cases of sub-therapeutic primary tumor treatment.
II. THE INVENTION
(64) The present invention generally provides methods and techniques for the detection and treatment of various cancers. In one preferred embodiment, the present invention provides a method for detecting and locating Lung cancer, Adrenal cancer, Melanoma, Colon cancer, Colorectal cancer, Ovarian cancer, Prostate cancer, Liver cancer, Subcutaneous cancer, Intestinal cancer, Hepatocellular carcinoma, Retinoblastoma, Cervical cancer in subject that has or is suspected of having cancer. The method comprises the steps of:
(65) (a) administering a phospholipid ether analog to the subject; and
(66) (b) determining whether an organ suspected of having cancer of the subject retains a higher level of the analog than surrounding region(s) wherein a higher retention region indicates detection and location of the cancer. In this method, the phospholipid analog is selected from:
(67) ##STR00003##
where X is selected from the group consisting of radioactive isotopes of iodine; n is an integer between 16 and 30; and Y is selected from the group comprising NH.sub.2, NR.sub.2, and NR.sub.3, wherein R is an alkyl or arylalkyl substituent or
(68) ##STR00004##
where X is a radioactive isotope of iodine; n is an integer between 16 and 30; Y is selected from the group consisting of H, OH, COOH, COOR and OR, and Z is selected from the group consisting of NH.sub.2, NR.sub.2, and NR.sub.3, wherein R is an alkyl or aralkyl substituent. In this method, X is selected from the group of radioactive isotopes of iodine consisting of .sup.122I, .sup.123I, .sup.124I, .sup.125I, and .sup.131I. Preferably, in this method, the phospholipid ether is 18-(p-Iodophenyl)octadecyl phosphocholine, 1-O-[18-(p-Iodophenyl)octadecyl]-1,3-propanediol-3-phosphocholine, or 1-O-[18-(p-Iodophenyl)octadecyl]-2-O-methyl-rac-glycero-3-phosphocholine, wherein iodine is in the form of a radioactive isotope. Various phospholipid ethers and related methodologies for the manufacture and use of the phospholipid ether compounds are described in U.S. Pat. Nos. 4,925,649; 4,965,391; 5,087,721; 5,347,030; 6,255,519 and 6,417,384 and all of which are herein incorporated by reference.
(69) In another embodiment, the present invention provides a method for the treatment of cancer in a subject. The method comprises administering to the subject an effective amount of a molecule comprising a phospholipid ether analog, as described above. In this method, the cancer is selected from a group consisting of Lung cancer, Adrenal cancer, Melanoma, Colon cancer, Colorectal cancer, Ovarian cancer, Prostate cancer, Liver cancer, Subcutaneous cancer, Intestinal cancer, Hepatocellular carcinoma, Retinoblastoma, Cervical cancer, Glioma, Breast cancer, Pancreatic cancer, carcinosarcoma and Prostrate cancer.
(70) The present invention also contemplates the use of a phospholipid ether analog for the production of a pharmaceutical composition for the treatment of cancer. These phospholipid analogs are selected from the group discussed above.
(71) Yet another embodiment of the present invention provides a method of differentiating inflammation, adenoma, hyperplasia from neoplasia in a subject. The method comprises the steps of:
(72) (a) administering a phospholipid ether analog to the subject; and
(73) (b) determining whether an organ suspected of having inflammation, adenoma, hyperplasia or neoplasia of the subject retains a higher level of the analog than surrounding region(s). When the subject exhibits a higher retention region, it indicates detection and location of the neoplasia and when the subject exhibits a lower retention region, it indicates the presence of an organ suspected of having the adenoma, hyperplasia or inflammation.
(74) Another embodiment of the present invention provides a method of detecting neoplasia in a tissue sample having a phospholipase D (PLD). The method comprises the step of:
(75) (a) quantifying the PLD protein activity level or the PLD mRNA level in the tissue sample; and
(76) (b) determining whether the tissue sample has a lower level of protein activity than surrounding tissue region(s) wherein a lower activity region indicates detection and location of the neoplasia, or
(77) (c) determining whether the tissue sample has a lower level of mRNA than surrounding tissue region(s) wherein a lower mRNA level region indicates detection and location of the neoplasia.
(78) In this method, the PLD protein activity or the mRNA level may be quantified by contacting the tissue sample with a PLE analog, as described above.
(79) Yet another embodiment of the present invention provides an anti-tumor agent selected by a method of screening a tissue sample having a PLD, comprising the step of: (a) quantifying the PLD protein activity or PLD mRNA level, wherein reduced PLD protein activity or reduced mRNA level compared to the surrounding tissue region(s) is indicative of neoplasia. The PLD protein activity or the mRNA level may be quantified by contacting the tissue sample with a PLE analog, as described above.
(80) The following sections discuss the use and methods related to only certain phospholipid ether compounds, however, such uses are exemplary and should not be deemed to narrow the scope of the present invention.
(81) For example, NM404 a phospholipid ether has demonstrated marked specificity for neoplastic tissue but not in preneoplastic tissue in many experimental tumor models. The high tumor to background avidity and tumor selectivity of NM404 suggests it may be potentially superior to .sup.18F-FDG PET scanning for intra-treatment tumor imaging. The precise mechanism of tumor specificity of NM404 is under investigation, and currently is not as well described as the glucose utilization mechanism for .sup.18F-FDG uptake. It is not well established whether NM404 uptake in neoplastic tissue depends on the viability of that tissue, or if this uptake phenomenon is related to some membrane or matrix component that is independent of tissue viability. If this uptake and specificity are linked to tumor viability, it would follow that NM404 uptake in tumors recently sterilized by radiation would be non existent or poor, whereas tumors resistant to radiation would show continued uptake. Recently, Inventors demonstrated NM404 uptake and killing in both radio sensitive and radio resistant squamous cancer cells (SCC1 and 6) in nude mice. Such an assay would be invaluable in managing patients treated with radiation therapy since patients manifesting no post-treatment NM404 localization would indicate cure, whereas those with resistant tumors (continued uptake of NM404) could be offered other non-radiation options (surgery, chemotherapy, etc).
(82) One approach to the development of sensitive, more available imaging exams is to design carrier molecules which are capable of selectively delivering a radiopharmaceutical probe to the desired target tissue. The inventors approach has been to capitalize on unique biochemical or pharmacological properties of molecules displaying a high degree of tissue or tumor selectivity.
(83) Snyder and coworkers.sup.16,17 observed that a variety of animal and human tumor cells contain much higher concentrations of naturally occurring ether lipids in the cell membranes than normal tissue. He proposed that the accumulation of ether lipids in tumors was a result of a lower capacity of tumor cells to metabolize these lipids due to a lack of key metabolic enzymes. The inventors have capitalized on this observation by synthesizing a number of radioiodinated phospholipid ether (PLE) analogs as potential tumor-selective imaging agents. Several of these PLE analogs have exhibited a striking and apparently universal ability to localize in and be selectively retained by a wide variety of spontaneous and transplanted rat, murine, and human tumor models (24/24).
(84) The inventors prevailing hypothesis (
(85) Following examples depict preferred embodiments of the present invention and are for illustrative purposes only. These examples should not be deemed to narrow the scope of the present invention.
III. EXAMPLES
A. Example I
Synthesis, Radiolabeling, and Formulation of NM404
(86) The inventors' synthetic approach was based on the copper-catalyzed cross-coupling reaction of Grignard reagents with alkyl tosylates or halides for the alkyl chain elongation (see the scheme below). The synthesis was started from p-iodobenzyl alcohol 1 which was converted into p-iodobenzyl bromide 2 by reaction with trimethylsilyl bromide. p-Iodobenzyl bromide 2 was further coupled with Grignard reagent 3 in the presence of Li.sub.2CuCl.sub.4 as a catalyst. 12-(p-Iodophenyl)dodecanol 5 obtained after deprotection of the first coupling product 4 was converted into tosylate 6. In the next step, tosylate 6 was coupled with Grignard reagent 7 containing 6 carbon atoms and this completed the chain elongation process. THP deprotection of 8 gave 18-(p-iodophenyl)octadecanol 9 which was converted into 10 (NM-404) by two-step procedure as shown in the scheme.
(87) ##STR00005##
(88) Further, rapid high yield synthesis process for labeling NM404 with any isotope if iodine, including .sup.124I, .sup.125I and .sup.131I was carried out by the following process: First, an aluminum heating block apparatus was preheated to 145 C. and a condenser was prepared using a 5 ml disposable syringe barrel fitted with a bent 1.5 inch 18 ga disposable needle and a rubber septum at the top.
(89) Second, the HPLC system was initiated and the reservoir was filled with filtered degassed solvent (hexane/isopropanol/water (40:52:8). The system was equilibrated followed by a systematic check-up of the ancillary systems such as the pump, detectors, chart recorders and computer integrators.
(90) Third, a 3-ml disposable syringe charcoal trap as prepared by using a glass wool plug in bottom, filling the syringe with 2.5 mL with granulated charcoal, adding another glass wool plug and inserting a septum on top. A short tubing adaptor needle was placed on the syringe and an 18-ga needle was inserted through the septum on the top. The charcoal trap was connected to the top-of the condenser and vented to the atmosphere through a sodium thiosulfate trap.
(91) Fourth, 5 mg of ammonium sulfate was added in 20 l of deionized water in 2 ml borosilicate glass v-vial followed by 20 g of unlabeled NM404 in 20-l of absolute ethanol to the vial. The vial was gently swirled or flicked to ensure mixing and 6 borosilicate glass beads (3 mm) were also added to the vial. The vial was then sealed with a Teflon-coated butyl rubber septum and an aluminum crimp cap. The septum was punctured with an 18-ga needle and the desired amount of aqueous sodium iodide-131 (in 0.1 N NaOH, typically 5 mCi in 15 l) was added via a Hamilton microsyringe through the septum. The vial was again gently swirled or flicked to ensure mixing. The vial was assayed in a dose calibrator.
(92) Fifth, the charcoal trap syringe was inserted into the reaction vial and the reaction vial was lowered into the heating block well (filled half way with sand). The reaction vial was heated at 145 C. for 40 min during which most of the solvent distilled off and condensed in the condenser. A stream of air (425 ml) was slowly inserted through the reaction vial with a 25-ml syringe. The temperature of the reaction vile was increased to 155 C. and heating was continued for an additional 30 minutes. The reaction vial was removed from the block heater and the condenser/trap assembly was disconnected and discarded and vial was allowed to cool to room temperature.
(93) Sixth, 0.5 ml of absolute ethanol was added into the reaction vial. The vial was gently swirled and assayed in the dose calibrator.
(94) Seventh, a radio-TLC analysis of the crude labeled product mixture was conducted on silica gel (chloroform/methanol/water (65/35/4).
(95) Eighth, Amberlite IRA 400-OH resin column was prepared by presoaking 1.0 g of resin in 5 ml of abs. ethanol for 30 minutes. Ethanol was decanted and the resin was rinsed with two additional 5 ml portions of ethanol. The wet resin was added into a 3 ml disposable syringe barrel with a glass wool plug at the bottom and fitted with an Acrodisc filter and a 1-way stopcock. The ethanolic solution of the crude radioiodinated product was gradually eluted through the resin column into a 5 ml vial.
(96) Ninth, a septum was inserted and the solvent was blown off with a stream of nitrogen. A charcoal syringe was attached on the outlet of the vial prior to initiating nitrogen flow. Once dry, 50 l of ethanol was used to dilute and transfer contents to a 300 l v-vial. The source vial was rinsed with a second 50 l ethanol wash and transferred to the v-vial.
(97) Tenth, HPLC pump was stabilized and a solvent flow of 1.0 ml/min was established. The reaction mixture was purified by HPLC on a Perkin-Elmer cartridge silica column (4.333 mm, 3 m silica) eluted with hexane/isopropanol/water (40:52:8) at 1.0 ml/min. Peak detection was performed by UV at 230 and 254 nm and by radioactivity. Once the appropriate peak was collected in a sterile vial, a small sample for radio-TLC analysis was removed and the remaining solvent was evaporated with a stream of nitrogen to give the desired compound as a dry residue. Specific activity was calculated as necessary.
(98) Eleventh, Polysorbate 20 was added at a ratio of 0.1 l/1.0 g of NM-404 to the flask from a stock solution of 5% Polysorbate 20 in absolute ethanol. Polysorbate 20 is the pharmaceutical grade of Tween 20 that is now used in both human and animal studies with NM404. The solvent was removed by rotary evaporation for 10 min at <30 C. The residue was dissolved with mixing in sufficient sterile water to yield a 2% Polysorbate 20 solution. The formulated product was passed through a sterile 0.2 m Pall-Gelman Acrodisc filter (13 mm) into a dry, sterile, multidose vial (Hollister-Stier) vented with another sterile 0.2 m filter. 100 l of product solution was diverted into a vial for QC analysis.
(99) Twelfth, radioactivity was measured in the dose calibrator and quality control tests (sterility, apyrogenicity) were performed.
(100) All unlabeled NM404 were taken from the original stock batch that recently underwent acute toxicology testing in order to minimize potential synthetic differences between studies. Radioiodination of NM404 was routinely achieved by an isotope exchange reaction in a melt of pivalic acid developed by the inventors.sup.19 or by the new method described herein and prepared for injection according to standard methods described by the inventors..sup.22 This procedure was used effectively for preparing sterile material for the initial human trials with NM324, the predecessor of NM404 and has been used over 40 times to prepare .sup.125I- and .sup.131I-labeled NM404. Generally, following purification and accurate mass quantification by HPLC, the radiopharmaceutical was dissolved in absolute ethanol (50-500 l) and Polysorbate 20 (0.1 l/g of compound). The ethanol is removed under vacuum and the residue dissolved in sterile water to give a final solution containing no more than 2-3% Polysorbate 20. Sterilization was achieved by filtration through a sterile 0.2 m filter unit. Final radiochemical purity must exceed 97% before using in animals. Quantification and calculation of final specific activity were achieved by HPLC analysis using known mass standards, and quantification of radioactivity (.sup.125I) was accomplished by dilution and counting in a PE Wallac gamma-counter in order to avoid attenuation concerns. Quantification of higher energy isotopes including .sup.131I were done with a dose calibrator with built in settings for these isotopes. Specific activities of 1 mCi per 100 g of radioiodinated NM404 were typically achieved. Injection volumes were typically around 100 l per mouse. Tissue distribution data were expressed as a percent injected dose (+SEM) per gram of tissue and also as percent injected dose per organ when whole organs were weighed according to published procedures established by the inventors..sup.22 At each time point, tumor-to-tissue-ratios were calculated on a percent injected dose per gram of tissue basis.
(101) General tissue distribution (TD) analysis: Biodistribution studies were performed in female mice according to the standard procedure developed by the inventors..sup.27 Radioiodinated NM404 (5 Ci in 100 l) was administered via tail vein injection. At the predetermined time points animals (3/time point) were euthanized by exsanguination while under pentobarbital anesthesia. A total of 16 tissues including blood, plasma, adrenal glands, bladder, bone marrow, fat, heart, kidney, liver, lung, muscle, spleen, ovaries, skin, thyroid, and tumor were excised, rinsed, and dissected free of extraneous tissue. Large organs were minced and duplicate tissue samples will be weighed and placed in plastic tubes for isotope counting. Injection site and residual carcass radioactivity were also determined in a well counter. These standard procedures have been utilized for many years in the inventor's laboratory under appropriate animal care and radiation safety approval. Tissue distribution tables were generated by a computer program which produces decay-corrected tissue radioactivity concentration data on a percent injected dose/g, % kg dose, and percent injected dose/organ+SEM basis. At each time point, tumor to tissue ratios were calculated based on a percent injected dose per gram of tissue basis. A control TD study (3 mice/time point, 15 total mice) were performed on tumor bearing mice at 4, 7, 14, 21, and 28 days most NM404 injection in order to establish comparative TD tables for all of the therapeutic regimens. General imaging protocols: Animals received .sup.125I-NM404 (10 Ci) via tail vein injection and at predetermined timepoints thereafter were anesthetized (sodium pentobarbital anesthesia, 0.06 mg/g bw) and underwent radionuclide scanning using a Bioscan AR2000 radio-TLC scanner modified for mouse imaging (1 mm high resolution collimator/1 min acquisition time per lane/1 mm lane increments). Data were quantitated and presented using Winscan 2D software from Bioscan. Once excised, control and treated tumors were also scanned ex vivo on the Bioscan unit in order to allow for more accurate ROI analysis by eliminating whole body radionuclide attenuation. Animals (sodium pentobarbital anesthesia, 0.06 mg/g bw) underwent microCT scanning (Imtek MicroCAT I, 390 step acquisition/43Kvp/410 A) using medium resolution acquisition parameters. Data sets were reconstructed 3-dimensionally and are visualized with AMIRA 3D-visualization software. The software allows for ROI density analysis and convenient on-screen measuring.
B. Example II
Preclinical Studies with First Generation PLE Analogs
(102) Phospholipid ethers can easily be labeled with iodine radioisotopes using an isotope exchange method developed by the inventors..sup.19 The iodophenyl phospholipid ether analogs are specifically designed so that the radioiodine affixed to each molecule is stable to facile in vivo deiodination. Over 20 radiolabeled PLE compounds were synthesized and tested in vitro and in vivo..sup.20-22 Two of these, namely NM294 and NM324 [12-(3-iodophenyl)-dodecyl-phosphocholine], initially showed the most promise in animal tumor localization studies. These prototype compounds, labeled with iodine-125, selectively localized in tumors over time in the following animal tumor models; 1) Sprague-Dawley rat bearing Walker 256 carcinosarcoma; 2) Lewis rat bearing mammary tumor; 3) Copenhagen rat bearing Dunning R3327 prostate tumors; 4) Rabbits bearing Vx2 tumors; and 5) athymic mice bearing human breast (HT39), small cell lung (NCI-69), colorectal (LS174T), ovarian (HTB77IP3), and melanoma tumors. Optimal tumor localization of these agents takes from one to several days.
(103) Mechanistic Studies with PLE Analogs: NM324 and NM404 are similar in structure to miltefosine (hexadecylphosphocholine), an antitumor ether lipid studied most extensively in Europe. The antitumor properties of miltefosine and several other antitumor phospholipid ether analogs have been demonstrated in a wide range of tumor cell lines including prostate-, bladder-, and terato-carcinomas, murine and human leukemias, as well as lung, colon, ovarian, brain and breast cancers..sup.23 In contrast to many anticancer drugs, these phospholipid ether analogs do not bind directly to DNA and are not mutagenic. Although the precise antiproliferative mechanism of action has not been determined, they apparently act at several tumor cell sites. These compounds have been associated with a variety of cellular effects including transport, promotion of cytokine formation, apoptosis induction, and interference with a variety of key lipid metabolism and cell signaling enzymes most of which are located in the cellular membrane. Although a debate exists regarding the mode of uptake into cells, the majority of reports now support the idea that these ether lipids are directly absorbed into cell membranes where they accumulate. A widespread belief is that these agents act by perturbing membrane phospholipid metabolism; however, cellular distribution studies with these agents have been limited by spontaneous cellular compartmental redistribution during homogenization and subcellular fractionation procedures. In contrast to the tracer imaging doses (several g) the inventors have employed, antitumor effects are seen only at doses generally exceeding 300-1000 mg per day..sup.23
(104) Formal metabolism studies were conducted on several PLE analogs including NM324, the predecessor of NM404. In these studies, each agent was examined to determine their ability to serve as substrates for enzymes associated with PLE metabolism. As shown in
(105) Finally, several PLE analogs were subjected to a PL.sub.D assay. The PL.sub.D, which was isolated from cabbage, is similar to mammalian PL.sub.D in that the cabbage form affords phosphatidylethanol-type products in addition to phosphatidic acid when the enzymatic reaction is performed in the presence of ethanol. Several of the PLE analogs subjected to these assay conditions did give rise to the phosphatidylethanol adduct, indicating possible interaction with PL.sub.D.
(106) Several NM404 precursors were also subjected to in vitro metabolism studies in various cell lines including Walker tumor cells, rat muscle (H9c2), and rat hepatocytes. In these studies, the extent of metabolism was determined on the basis of radiolabeled products formed after incubation for various time periods and the results normalized to cell number or the amount of cellular protein. Subsequent lipid extraction of the incubation medium and cell suspension demonstrated little generation of PLE metabolites in the Walker tumor cells whereas a significant production of metabolites was seen in both the muscle cells and hepatocytes over the 48 h time period studied. These results correlate nicely with in vivo biodistribution studies completed on all analogs. Although several studies have been completed, the role of metabolic trapping in the uptake and retention of radiolabeled PLE analogs in tumor cells is not well defined and currently remains an active area of examination.
(107) Clinical Evaluation of NM324: Of several promising first generation PLE analogs, NM324 was more readily synthesized and was thus selected as the lead compound for initial clinical studies. Although images obtained in 5 human lung cancer patients detected tumors, images were complicated by high liver radioactivity (
(108) Second Generation PLE Analogs: In order to decrease liver uptake and prolong the plasma phase, 9 structural analogs of NM324 were synthesized and radiolabeled with .sup.125I for initial image analysis in Copenhagen rats bearing Dunning R3327 prostate tumors. Based upon this initial screen, NM347, NM404 [18-(4-iodophenyl)-octadecylphosphocholine] and NM412 (
(109) More recent imaging studies with NM404 and NM412 in animal models showed that both were superior to NM324 in visualizing a variety of tumors. Significantly, lymph node metastases were clearly delineated in a metastatic prostate tumor model following intravenous administration of either NM404 or NM412. Most importantly, the tracer was not retained by uninvolved lymph nodes..sup.24 (
(110) Animal imaging studies performed in mouse models aimed at determining the uptake characteristics in a wide variety of tumor models are summarized in Table 1. Preliminary results in B6 Apc.sup.Min/+ mice indicate that NM404 is not taken up by adenomatous polyps but is taken up and retained by mammary adenocarcinomas in this model, thus indicating a possible specificity for malignant tumor cells. These studies are aimed at determining the potential of NM404 to noninvasively characterize tumors. NM404 has displayed significant tumor uptake and retention in every adenocarcinoma model studied.
(111) TABLE-US-00001 Tumor Model Species Category Uptake Human Tumor Xenografts Prostate PC-3 SCID Mouse Adenocarcinoma Yes Lung A-549 (NSCLC) SCID Mouse Adenocarcinoma Yes Lung NCI H-69 (Oat Nude Mouse Adenocarcinoma Yes Adrenal H-295 SCID Mouse Adenocarcinoma Yes Adrenal RL-251 SCID Mouse Adenocarcinoma Yes Melanoma A-375 Nude Mouse Adenocarcinoma Yes Colon LS-180 Nude Mouse Adenocarcinoma Yes Ovarian HTB-77 Nude Mouse Adenocarcinoma Yes Animal Tumor Xenografts Mammary MCF-7 Rat Adenocarcinoma Yes Prostate MatLyLu Rat Adenocarcinoma Yes Walker-256 Rat Carcinosarcoma Yes Recent Rodent Models TRAMP prostate Spontaneous mouse Adenocarcinoma Yes Liver CT-26 Mouse xenograft Colorectal Yes adenocarcinoma Subcutaneous CT-26 Mouse xenograft Colorectal Yes adenocarcinoma Min Mouse Intestinal Endogenous Mouse Adenocarcinoma Yes Melanoma Mouse xenograft Adenocarcinoma Yes SCC1 and 6 Nude mouse Squamous cell Yes carcinoma Mammary SCC and Apc.sup.Min/+mouse SCC and Yes AC Adenocarcinoma Hepatocellular Spontaneous mouse Adenocarcinoma Yes Carcinoma Retinoblastoma Spontaneous mouse Blastoma Yes Cervical Spontaneous mouse Adenocarcinoma Yes Adenocarcinoma Pancreatic c-myc and Spontaneous mouse Adenocarcinoma Yes kras Glioma L9 Rat Xenograft Glioma Yes Intestinal polyp Apc.sup.Min/+mouse Adenoma No.sup.1 Mammary Hyperplasia Apc.sup.Min/+mouse Alveolar No.sup.1 Hyperplasia .sup.2Tumor uptake was <<1% injected dose/gram
(112) Relevance of CT26 Murine Tumor Model: Inventors explored NM-404 as a predictor of tumor response in a murine (BALB/c mice) model with subcutaneous CT26 cell inoculation into the flanks of the mice. The CT26 cell line is a poorly differentiated murine adenocarcinoma that was induced by rectal injection of N-nitroso-N-methylurethane in BALB/c mice. The cell line is simple to grow in vitro and results in a predictable growth pattern when injected in the vasculature (tail vein injection, metastatic model), or into the skin (
(113) Preliminary Imaging Results with NM404 in CT26 Tumors: In a preliminary experiment to show that NM404 localizes in subcutaneous CT26 xenografts, two animals were injected (IV tail vein) with .sup.125I-NM404 (10 Ci) and subsequently imaged on a modified Bioscan AR2000 radioTLC scanner (equipped with high resolution 1 mm collimator and 2-D acquisition and analysis software) at 1, 4, and 7 days post injection. On day 7, the animal was euthanized and the tumor removed, photographed, and scanned ex vivo on the Bioscan (
(114) Initial RF ablation of a subcutaneous mouse CT26 tumor was successful and resulted in severe cellular damage (
C. Example III
Non-Small Cell Lung Cancer
(115) Imaging and biodistribution studies were performed in SCID (severe combined immune deficiency mutation) mice bearing the human NSCLC adenocarcinoma A549 cell line (adenocarcinoma has become the most frequent human lung cancer histologic type). Preliminary pilot results in five animals were encouraging and demonstrated that the new agent NM404 overcomes a limitation of the NM-324 compound. While there is reasonably good tumor uptake with NM324, imaging is compromised by a high first pass clearance by the liver. However, NM404 shows excellent tumor visualization, especially striking in the delayed images, with minimal liver and kidney uptake. Moreover, tissue biodistribution studies further confirmed the high levels of radioactivity residing in the tumors. A comparison of NM324 and NM404 images in a SCID mouse-human NSCLC model are shown in
(116) Extensive biodistribution data for the prototype agent .sup.125I-NM324 in several tumor models have previously been compiled. Counsell R E, Schwendner S W, Meyer K L, Haradahira T, Gross M D. Tumor visualization with a radioiodinated phospholipid ether. J Nucl Med 31(3):332-336, 1990; Plotzke K P, Fisher S J, Wahl R L, Olken N M, Skinner S, Gross M D, Counsell R E. Selective localization of a radioiodinated phospholipid ether analog in human tumor xenografts. J Nucl Med 34(5):787-792, 1993; Rampy M A, Brown R S, Pinchuk A N, Weichert J P, Skinner R W, Fisher S J, Wahl R L, Gross M D, Etheir S P, Counsell R E. Biological disposition and imaging of a radioiodinated alkylphosphocholine in two rodent models of breast cancer. J Nucl Med 37(9):1540-1545, 1996. Tumor-to-blood ratios exceeding 8:1 were seen at delayed times post-injection. For example, in a rat mammary tumor model, tumor-to-normal tissue ratios reached a maximum at 96 hours with a tumor-to-blood ratio of 8.6 and tumor-to-muscle ratio of 20:1. Rampy M A, Brown R S, Pinchuk A N, Weichert J P, Skinner R W, Fisher S J, Wahl R L, Gross M D, Etheir S P, Counsell R E. Biological disposition and imaging of a radioiodinated alkylphosphocholine in two rodent models of breast cancer. J Nucl Med 37(9):1540-1545, 1996. Moreover, the biodistribution of PLE-associated radioactivity is heterogeneous in tumor, as demonstrated by microautoradiography studies showing that the PLE radioactivity resides exclusively in viable tumor cells located toward the outer regions rather than the central necrotic regions. Rampy M A, Brown R S, Pinchuk A N, Weichert J P, Skinner R W, Fisher S J, Wahl R L, Gross M D, Etheir S P, Counsell R E. Biological disposition and imaging of a radioiodinated alkylphosphocholine in two rodent models of breast cancer. J Nucl Med 37(9):1540-1545, 1996. Comparative biodistribution data for NM324 and NM404 in SCID mice thus far have only been performed in prostate and A549 lung cancer tumor models. These studies have revealed high tumor to normal tissue ratios and tumor uptake exceeding 25% of the injected dose with NM404, thus supporting our desire to study the biodistribution of PLE analogs in more spontaneous tumor models and humans.
(117) One additional study addressing the relative sensitivity of NM404 to NM324 was performed in a SCID mouse A549 lung cancer model. The lungs of each animal were excised 10 days after administration of equal doses of each agent and imaged ex vivo for one hour in order to enhance resolution. The low resolution and highly amplified images shown in
(118) It is likely that because the tumor-targeting strategy appears to involve selective tumor retention over time, relatively short-lived nuclides such as .sup.18F or even .sup.99mTc are not practical for labeling at the current time. However, as with the early use of monoclonal antibodies, which were labeled exclusively with radioisotopes of iodine, it may be possible in the future to label PLE analogs with alternative labels, such as iodine-124, wherein the physical half-life matches well with PLE tumor uptake and retention kinetics. In fact, the utility of .sup.124I-labeled NM404 as a tumor selective PET agent is the subject of a pilot project for our microPET acquisition. The aim of that project is to evaluate the feasibility of labeling NM404 with iodine-124, a relatively new positron isotope with a 4-day physical half-life, and to evaluate its promise for PET imaging of tumors in small animal models. In addition to capitalizing on the resolution enhancement and 3-dimensional capabilities PET imaging affords relative to traditional gamma camera imaging, this approach would compliment the use of fluorine-18 FDG in that its uptake into tumor cells occurs via a different biochemical mechanism than glucose utilization.
(119) As has been discussed above, the utility of currently available tracers (e.g. .sup.67Ga and .sup.18F-FDG) is limited by lack of specificity to distinguish neoplasm from inflammation. However, preliminary studies with PLE agents has offered promise in overcoming this clinically significant limitation wherein carrageenan-induced granulomas in rats failed to visualize above background activity and showed no tissue retention. Counsell R E, Schwendner S W, Meyer K L, Haradahira T, Gross M D. Tumor visualization with a radioiodinated phospholipid ether. J Nucl Med 31(3):332-336, 1990. Gallium citrate, however, utilized as a control in that study, did concentrate significantly in the granuloma. Such findings further justify extending the inventors studies with PLE analog agents as potentially useful tumor-selective imaging agents.
(120) Human Studies: Based upon the very promising pharmacokinetic and imaging data in animals, the inventors were encouraged to move studies of radiolabeled phospholipid ethers into the clinical arena. Unlabeled NM404 was initially assessed for its acute toxic effects on rats and rabbits in studies conducted at the Toxicology Research Center, State University of New (SUNY) at Buffalo. No toxic effects were seen at a dose level of 3.2 mg/kg (>150 times the highest anticipated human dose) in these acute dose toxicology studies. Moreover, no platelet activating properties were demonstrated at this high dose level.
(121) Unlabeled NM324 was administered to five normal, disease-free, humans in order to gain approval of the radiolabeled agent for human administration by the Radioactive Drug Research Committee (RDRC). These subjects had no evidence of toxicity, as manifested by symptoms, clinical examination, vital signs and sequential blood chemistries.
(122) As a pilot feasibility project, 4 lung cancer patients were studied under RDRC approval with .sup.131I-labeled NM324 at the Ann Arbor, Mich. VA hospital. Lung tumors were clearly visualized in all three of the patients with lung cancer (two with NSCLC and one with small cell lung cancer), described in detail below. Degree of tumor uptake, at various time points, varied from 1+(barely perceptible above background) to 3+(intense uptake, much greater than normal structures). Note that the patients selected for these initial studies were those with known, relatively large cancers. It was not intended at this stage to study patients in whom problems of tumor staging existed.
(123) Case Histories:
(124) Patient 01 was a 55 year old woman with a right middle lobe lung mass eroding into the right ribs, histologically a mucin-producing adenocarcinoma of probable lung origin. Initial .sup.131I-NM324 scintigraphic images at 6 hours showed a focus of uptake in the right lateral mid-lung. For reasons unrelated to the scintigraphic study, the patient was unable to return to the hospital beyond 6 hours for further imaging session.
(125) Patient 02 was a 62 year old man with a large (977.5 cm), lobulated mediastinal mass extending from the aortopulmonary window and left hilum. Tissue type was a small cell undifferentiated (oat cell) carcinoma. .sup.131I-NM324 scintigraphic images revealed a focus of uptake in the left upper lung, which increased in intensity over time relative to the normal background activity.
(126) Patient 03, a 74 year old man with a right upper lobe NSCLC (adenocarcinoma) treated 5 months previously with radiation therapy. Disease recurred in the left lingula (2.523 cm mass), lower thoracic spine (approx. T8) and right lobe of the liver. .sup.131I-NM324 scintigraphy showed well defined uptake in the lung mass and thoracic spine lesion, which demonstrated increasing target to background ratios over time (
(127) These studies provided an early glimpse of the clinical promise of radiolabeled PLE analogs. Although .sup.131I is a suboptimal agent for imaging purposes, uptake in all three lung tumors was clearly depicted. As expected, based upon prior animal biodistribution experiments, activity in the tumors increased over time, as clearly demonstrated in patients 02 and 03. In patient 03 tumor-to-normal tissue ratios increased from 2.74 at 2 days to 4.23 at 7 days. Patient 01 did not return for later imaging sessions beyond 6 hours. The increasing target-to-background ratios constitute strong evidence that the mechanism for tumor visualization is not one based merely upon abnormal blood flow or tumor hypervascularity. Indeed, animal studies using .sup.99mTc human serum albumin confirmed this..sup.1
(128) Clinical Trial Evaluating Patients with Non-Small Cell Lung Carcinoma (NSCLC) Using NM404
(129) Although NM404 has displayed selective and prolonged tumor retention in 25/25 xenograft and spontaneous rodent models, a physician sponsored IND recently initiated clinical evaluation of the agent in Stage 4 human non-small cell lung cancer patients in order to determine whether or not it would exhibit similar tumor uptake and retention properties in humans. To date, two patients with advanced NSCLC were imaged after an injection of <1 mCi of .sup.131I-NM404. Blood and urine samples were collected at predetermined times, and gamma imaging performed at several time points following administration. In both patients, significant tumor uptake and retention of NM404 was demonstrated in the primary lung tumor, as seen in
(130) Materials and Methods:
(131) Following intravenous injection of iodine-131 labeled NM404 (1mCi/20 g), patients with advanced NSCLC where scanned at 3, 6, 24, 48, 96 h and at 7 and 11 days on a GE Maxxus dual Head SPECT scanner. Blood and urine samples were collected for pharmacokinetic analysis as well as clinical hematologic, renal, and hepatic bioanalysis.
(132) Results:
(133) Initial qualitative imaging results indicate that iodine-131 labeled NM404 clearly localizes in bilateral pulmonary masses as early as 24 h after injection and is selectively retained in these tumors in excess of 11 days. Moreover, background radioactivity in the liver and lower abdominal region including urinary bladder, kidneys, and intestines was significantly less than was observed previously with its predecessor, NM324. No adverse reactions were observed in any of the patients.
(134) Conclusions:
(135) These preliminary findings suggest that NM404 exhibits similar tumor uptake and retention properties in human NSCLC as was seen previously in rodent models.
(136) Although based on only two patients at this point, it appears that NM404 does indeed localize in and undergo selective and prolonged tumor retention in human non-small cell lung cancer.
(137) Patient 1:
(138) 55 year old male with bilateral 3 cm left lobe and infiltrative right lobe NSCLC and a brain metastasis and a small right adrenal mass. He has participated in numerous standard and experimental treatment regimens. Images are included in
(139) Patient 2:
(140) 70 year old male recently diagnosed with 6 cm upper lobe non small cell mass, a 5 mm liver mass, an ilial bone met and a very small brain metastasis. He had recently completed low dose carboplatin/taxol chemotherapy and palliative radiotherapy to the ilial and brain metastases the week prior to initiating the NM404 trial. Images are shown in
D. Example IV
Mouse Pancreatic Adenocarcinoma Models
(141) Inventors also studied tumor avidity of NM404, a second-generation PLE analog, in the c-myc mouse pancreatic adenocarcinoma model which is known to produce invasive tumors with mixed acinar/ductal phenotype.
(142) Materials and Methods: Two murine strains that are endogenous for either c-myc, or k-ras, well-known oncogenes, have been developed at the University of Wisconsin. Sandgren E P, Quaife C J, et al., Proc Natl Acad Sci USA. 1991; 88:93-97; Grippo P J. Nowlin P S. Et al., Cancer Research. 63(9):2016-9, 2003.
(143) Expression of c-myc is targeted to pancreatic acinar cells because it is linked to an elastase promoter, which is only expressed in the pancreas. These ela-1-myc endogenous mice develop acinar and ductal neoplasia, which results in death between 2 and 7 months of age. By one month of age, the pancreas appears thickened and firm. Thus mice between the ages of 1-3 months serve as excellent models for the study of pancreatic cancer. Most human pancreatic neoplasms have a ductal morphology and Dr. Sandgren's transgene targeting strategies are aimed at developing tumors that are specific for pancreatic ductal epithelium.
(144) The c-myc model produces tumors that are invasive adenocarcinomas, with mixed acinar/ductal phenotype. The biology of the k-ras model is markedly different. The k-ras tumors have been classified as carcinoma in situ, meaning that they have features of neoplasia, but they do not invade and generally stay small (<2 mm). Their cellular appearance is far more like the early human tumors so from a histological perspective they are a more relevant model of human disease. Further, they resemble the very early stages of the human disease. The ability to detect the early development of the k-ras tumors versus the large and more advanced tumors in the c-myc mice would be an exceptionally important step toward identifying early (perhaps curable) lesions in humans. The fact that k-ras mutations are the cause of over 90% of human pancreatic adenocarcinomas lends further support towards the validity of this model for the evaluation of new tumor imaging agents.
(145) Imaging Studies: In order to determine if NM404 localizes in mouse pancreatic tumors, six c-myc endogenous mice were scanned on a Bioscan AR-2000 radioTLC scanner (modified in the inventors lab for mouse imaging) from 2-21 days after tail vein injection of .sup.125I-NM404 (15 Ci/20 g bw). On the last day, mice also underwent microCT scanning (42 kvp, 410 A, 390 steps, MicroCAT-I, ImTek, Inc., Knoxville, Tenn.). Following in vivo imaging of anesthetized mice, the pancreatic tumors were excised and scanned ex vivo on the same scanner (equipped with high resolution 1 mm collimator and 2-D acquisition and analysis software) in order to avoid tissue attenuation associated with the low energy of iodine-125 (
(146) Results and Discussion: Initial imaging results with NM404 in the c-myc model indicated striking uptake and prolonged retention (>21 days) in all adenocarcinomas ranging from 5-12 mm in diameter. As has been observed in previous cell culture and in vivo animal model studies, NM404 is apparently metabolized and eliminated from normal cells but becomes metabolically trapped in tumor cell membranes. Previous autoradiography experiments in other tumor models have suggested that only viable tumor cells, and not normal tissue or necrotic tissues, are capable of accumulating NM404. The inventors were also able to detect pancreatic tumors in live mice with microCT despite the ubiquitous nature of the pancreas in mice (
(147) Conclusions: NM404 displayed selective and prolonged retention in spontaneous pancreatic adenocarcinomas examined in this study, thus further extending the tumor selectivity of this agent.
E. Example V
Rat Glioma Model
(148) Materials and Methods: All animals were housed and handled in accordance with the University of Wisconsin Research Animal Resources Center guidelines. Rat C6 glioma cells were propagated in DMEM medium (Life Technologies, Gaithersburg, Md.) supplemented with 10% heat-inactivated FBS (BioWhittaker, Walkersville, Md.), 100 U/ml penicillin G, 100 mg/ml streptomycin, and 0.01 M HEPES (Life Technologies, Gaithersburg, Md.). Intracranial tumor implantation was performed as described previously (ref). Briefly, 110.sup.6 C6 cells were resuspended in 5 ml 1.2% methylcellulose and injected into the frontal lobes of anesthetized female Wistar rats (Harlan, Indianapolis, Ind.). Sham-operated animals received intracranial injections of an equal volume of methylcellulose without tumor cells.
(149) Imaging Studies: Ten days after implantation, the presence of intracranial tumors was confirmed with MRI. Briefly, anesthetized rats (6) received 2 ml of Gadodiamide (Gd, Omniscan 287 mg/ml, Nycomed, Princeton, N.J.) intraperitoneally and imaged 10 min later using a 1.5 Tesla clinical MR system (GE Signa LX) and a GE phased array extremity coil. The T1-weighted (TR=500 ms, TE=16.5 ms) multislice sequences covering the entire brain of each rat were inspected to select tumor-bearing rats with varying tumor sizes, and sham-operated rats for NM404 injections.
(150) NM404 [18-(4-iodophenyl)-octadecylphosphocholine] (
(151) Results and Discussion: Initial imaging results with NM404 indicated striking uptake and prolonged retention in all gliomas ranging from 3-5 mm in diameter. Radioactivity in normal brain tissue was minimal in sham operated control animals (
(152) Conclusion: As has been the case in all tumor models examined previously, NM404 displayed selective and prolonged retention by rat C6-gliomas evaluated in this study.
F. Example VI
Murine Liver Tumor
(153) Preliminary results obtained in over 14 xenograft and spontaneous tumor models have universally shown NM404 to undergo selective uptake and prolonged retention in tumors. Further, because NM404 affords lower liver background levels than its predecessors, the inventors expanded evaluation into liver tumors in light of the fact that imaging patients with HCC has been problematic. Many patients have underlying cirrhosis and therefore it is difficult to distinguish regenerating nodules from HCC on cross sectional imaging. Moreover, preliminary studies evaluating PET scanning with FDG have shown only 20-50% sensitivity in detecting the disease. Verhoef C, Valkema R. et al., Liver (2002) 22:51-56.
(154) Materials and Methods: Endogenous Mouse HCC Model. The development of spontaneous hepatocellular cancer in endogenous mice over expressing the TGF gene has been extensively evaluated and is an extremely promising animal model for study of this disease. Lee G H, Merlino G, Fausto N. Cancer Research (1992) 52:5162-5170. TGF is a mitogen for epithelial cells and binds to the EGF receptor; unregulated expression of TGF results in tumor formation. In male CD1 mice expressing the transgene TGF under the control of the zinc-inducible metallothionine 1 (MT1) promoter, 75-80% develop HCC after 12 months of age. However, when the alkylating agent diethylnitrosamine (DEN), a chemical carcinogen, is used to induce tumor growth at 15 days of life, 90% of mice develop HCC by 6 months of age. On histologic examination, these tumors consist of well differentiated hepatocellular carcinomas of a solid pattern. Because the tumors arise spontaneously, the inventors utilize these animals as a suitable model for preclinical studies.
(155) CT26 Colon Adenocarcinoma Xenograft Model: In addition to the spontaneous HCC model, NM404 was also evaluated in a xenograft colon adenocarcinoma tumor model whereby CT26 cells (510.sup.5 cells/50 l) were previously injected directly into the liver parenchyma of female BALB/c mice for creation of focal liver tumors.
(156) Imaging Studies: NM404 (
(157) Results and Discussions: Initial imaging results with NM404 (
(158) Conclusions: As has been the case in all prior tumor models examined, NM404 displayed selective and prolonged retention by both spontaneous and xenograft murine liver tumor models evaluated in this study.
G. Example VII
ApcMin/+ Spontaneous Mammary Carcinoma Model
(159) Materials and Methods: Apc.sup.Min/+ Mouse Model: This model is comprised of mice carrying the Min allele of Apc (Apc.sup.Min/+ mice). This model offers specific advantages over xenograft models in that female Apc.sup.Min/+ mice are predisposed to develop mammary hyperplasias and carcinomas and intestinal adenomas. On the C57BL/6J genetic background, about 5% of untreated females will develop a mammary tumor by 100 days of age. Moser A R, Dove, et al. Proc Natl Acad Sci USA (1993) 90:8977-81. The incidence and multiplicity of the mammary lesions can be increased by a single dose of ethylnitrosourea (ENU), a direct acting alkylating agent. Treatment with ENU results in 90% of B6 Apc.sup.Min/+ females developing an average of 3 mammary squamous cell carcinomas (SCC), but few hyperplasic lesions within 60 days after treatment.
(160) Apc.sup.Min/+ mice carry a single base pair change in the Apc (adenomatous polyposis coli) gene. The APC/Apc gene encodes a large protein with several potential functional domains. Groden, J., Thliveris, A., Samowitz, W., Carlson, M., Gelbert, L., Albertsen, H., Joslyn, G., Stevens, J., Spirio, L., Robertson, M. and et al. Identification and characterization of the familial adenomatous polyposis coli gene. Cell, (1991) 66, 589-600; Kinzler, K. W., Nilbert, M. C., Vogelstein, B., Bryan, T. M., Levy, D. B., Smith, K. J., Preisinger, A. C., Hamilton, S. R., Hedge, P., Markham, A. and et al. Identification of a gene located at chromosome 5q21 that is mutated in colorectal cancers. Science, (1991) 251, 1366-70. The mouse and human APC proteins are 90% identical and all potential functional domains are conserved. APC regulates -catenin levels. -catenin has multiple roles in the cell, including stabilization of E-cadherin and regulation of transcription through the LEF and TCF family of transcription factors. Aberle, H., Schwartz, H. and Kemler, R. Cadherin-Catenin ComplexProtein Interactions and Their Implications For Cadherin Function. Journal of Cellular Biochemistry, (1996) 61, 514-523; Huber, O., Korn, R., McLaughlin, J., Ohsugi, M., Herrmann, B. G. and Kemler, R. Nuclear localization of beta-catenin by interaction with transcription factor LEF-1. Mechanisms of Development, (1996) 59, 3-10; Behrens, J., Vonkries, J. P., Kuhl, M., Bruhn, L., Wedlich, D., Grosschedl, R. and Birchmeier, W. Functional Interaction of Beta-Catenin With the Transcription Factor Lef-1. Nature, (1996) 382, 638-642. The regulation of -catenin levels involves the interaction of APC, axin or conductin, and glycogen synthase kinase 3 (GSK3) with -catenin. Behrens, J., Jerchow, B. A., Wurtele, M., Grimm, J., Asbrand, C., Wirtz, R., Kuhl, M., Wedlich, D. and Birchmeier, W. Functional interaction of an axin homolog, conductin, with beta-catenin, APC, and GSK3beta. Science, (1998) 280, 596-9; Ikeda, S., Kishida, S., Yamamoto, H., Murai, H., Koyama, S. and Kikuchi, A. Axin, a negative regulator of the Wnt signaling pathway, forms a complex with GSK-3beta and beta-catenin and promotes GSK-3beta-dependent phosphorylation of beta-catenin. EMBO Journal, (1998) 17, 1371-84; Kishida, S., Yamamoto, H., Ikeda, S., Kishida, M., Sakamoto, I., Koyama, S. and Kikuchi, A. Axin, a negative regulator of the wnt signaling pathway, directly interacts with adenomatous polyposis coli and regulates the stabilization of beta-catenin. Journal of Biological Chemistry, (1998) 273, 10823-6; Sakanaka, C., Weiss, J. B. and Williams, L. T. Bridging of beta-catenin and glycogen synthase kinase-3beta by axin and inhibition of beta-catenin-mediated transcription. Proceedings of the National Academy of Sciences of the United States of America, (1998) 95, 3020-3; Rubinfeld, B., Albert, I., Porfiri, E., Fiol, C., Munemitsu, S. and Polakis, P. Binding of GSK3beta to the APC-beta-catenin complex and regulation of complex assembly. Science, (1996) 272, 1023-6; Rubinfeld, B., Souza, B., Albert, I., Muller, O., Chamberlain, S. H., Masiarz, F. R., Munemitsu, S. and Polakis, P. Association of the APC gene product with beta-catenin. Science, (1993) 262, 1731-4; Polakis, P. The adenomatous polyposis coli (APC) tumor suppressor. Biochimica et Biophysica Acta, (1997) 1332, F127-47. This interaction results in the phosphorylation of -catenin, which targets it for degradation by the ubiquitin-proteasome pathway. Rubinfeld, B., Souza, B., Albert, I., Muller, O., Chamberlain, S. H., Masiarz, F. R., Munemitsu, S. and Polakis, P. Association of the APC gene product with beta-catenin. Science, (1993) 262, 1731-4; Su, L. K., Vogelstein, B. and Kinzler, K. W. Association of the APC tumor suppressor protein with catenins. Science, (1993) 262, 1734-7; Polakis, P. Mutations in the APC gene and their implications for protein structure and function. Current Opinion in Genetics & Development, (1995) 5, 66-71; Aberle, H., Bauer, A., Stappert, J., Kispert, A. and Kemler, R. beta-catenin is a target for the ubiquitin-proteasome pathway. EMBO Journal, (1997) 16, 3797-804. Most germline and somatic mutations in APC result in proteins missing some or all of the -catenin binding sites..sup.26,28,29 Polakis, P. Mutations in the APC gene and their implications for protein structure and function. Current Opinion in Genetics & Development, (1995) 5, 66-71; Nagase, H. and Nakamura, YMutations of the APC (adenomatous polyposis coli) gene. Human Mutation. (1993) 2, 425-34; Beroud, C. and Soussi, T. APC gene: database of germline and somatic mutations in human tumors and cell lines. Nucleic Acids Research, (1996) 24, 121-4. Two regions of APC are required for this interaction; the truncated protein encoded by the Min allele lacks both of these regions; Polakis, P. Mutations in the APC gene and their implications for protein structure and function. Current Opinion in Genetics & Development, (1995) 5, 66-71; Su, L. K., Kinzler, K. W., Vogelstein, B., Preisinger, A. C., Moser, A. R., Luongo, C., Gould, K. A. and Dove, W. F. Multiple intestinal neoplasia caused by a mutation in the murine homolog of the APC gene. Science, (1992) 256, 668-70. APC also has a role in the transport of -catenin out of the nucleus. Thus, in the absence of APC function, -catenin would accumulate in the cytoplasm and nucleus, possibly affected both transcription of target genes and cell-cell interaction through E-cadherin. APC mutations are frequent in several tumor types in humans including intestinal tumors and other epithelial tumors. Loss of heterozygosity at the APC locus or increased levels of -catenin have been found in more than 25% of breast cancers. Furuuchi, K., Tada, M., Yamada, H., Kataoka, A., Furuuchi, N., Hamada, J., Takahashi, M., Todo, S., and Moriuchi, T. Somatic mutations of the APC gene in primary breast cancers. Somatic mutations of the APC gene in primary breast cancers, American Journal of Pathology. (2000) 156: 1997-2005; Jonsson, M., Borg, A., Nilbert, M., and Andersson, T. Involvement of adenomatous polyposis coli (APC)/beta-catenin signaling in human breast cancer. Involvement of adenomatous polyposis coli (APC)/beta-catenin signaling in human breast cancer, European Journalof Cancer. (2000) 36: 242-248. Thus, the types of lesions that appear in these mice will be molecularly and histologically similar to breast cancers in humans.
(161) Genetic background can affect the incidence, latency, and type of mammary lesions that develop. For example, FVBxB6 Apc.sup.Min/+ female mice develop an average of 0.2 mammary tumors per mouse, but 4 hyperplasias per mouse within 120 days of treatment. BALB/xB6 Apc.sup.Min/+ develop an average of 1.8 mammary tumors and 0.6 hyperplasias per mouse. Moser A R, Hegge L F, Cardiff R D. Cancer Research (2001) 61:3480-3485. FVBxB6 and BALBxB6 Apc.sup.Min/+ mice develop both mammary SCC and adenocarcinomas (AC).
(162) The hyperplastic lesions in the FVBxB6 Apc.sup.Min/+ mice can be classified as either alveolar hyperplasias or squamous nodules. Moser, A. R., Hegge, L. F., and Cardiff, R. D. Genetic background affects susceptibility to mammary tumors and hyperplasias in Apc.sup.Min/+ mice, Genetic background affects susceptibility to mammary tumors and hyperplasias in Apc.sup.Min/+ mice. Cancer Research (2001) 61:3480-3485. Alveolar hyperplasias are precursors to the adenocarcinomas and the squamous nodules are precursor lesions to the SCC. Thus, by manipulation of the genetic background, mice that develop multiple types of hyperplasias and carcinomas may be generated, often within the same animal. The alveolar hyperplasias resemble atypical lobules (type A) commonly found in samples from human breasts. Cardiff, R. D. and Wellings, S. R. The comparative pathology of human and mouse mammary glands. The comparative pathology of human and mouse mammary glands, Journal of Mammary Gland Biology& Neoplasia. (1999) 4: 105-22. These atypical lobules are more common in cancerous breasts or in the contralateral breast in women with breast cancer. While SCC is not a frequent type of breast tumor, the AC resembles a common type of human breast tumor. In addition, tumors with alterations in the APC pathway are common in human breast cancers. Loss of heterozygosity at the APC locus or increased levels of -catenin have been found in more than 25% of breast cancers. Furuuchi, K., Tada, M., Yamada, H., Kataoka, A., Furuuchi, N., Hamada, J., Takahashi, M., Todo, S., and Moriuchi, T. Somatic mutations of the APC gene in primary breast cancers. Somatic mutations of the APC gene in primary breast cancers, American Journal of Pathology. (2000) 156: 1997-2005. 35. Jonsson, M., Borg, A., Nilbert, M., and Andersson, T. Involvement of adenomatous polyposis coli (APC)/beta-catenin signaling in human breast cancer. Involvement of adenomatous polyposis coli (APC)/beta-catenin signaling in human breast cancer, Eur Journal of Cancer. (2000) 36: 242-248. Thus, the types of lesions that appear in these mice will be molecularly and histologically similar to breast cancers in humans. One of the unique aspects and strengths of this model is the ability to generate mice that develop multiple types of mammary hyperplasias and carcinomas, often within the same animal. In this way we can test the uptake and retention of NM404 in multiple types of hyperplasias and tumors within the same animal.
(163) Polyoma virus infection of mice leads to the development of numerous tumor types including mammary tumors. Endogenous mice expressing the polyoma middle T antigen (PyVT) under the control of the mouse mammary tumor virus LTR (MMTV) develop multifocal mammary dysplasias and tumors rapidly. Amy Moser; Guy, C. T., Cardiff, R. D., and Muller, W. J. Induction of mammary tumors by expression of polyomavirus middle T oncogene: a endogenous mouse model for metastatic disease. Induction of mammary tumors by expression of polyomavirus middle T oncogene: a transgenic mouse model for metastatic disease, Molecular & Cellular Biology. (1992) 12: 954-61. Evidence for in situ carcinoma can be seen as early as three weeks of age, with 100% incidence of mammary tumors by as early as 5 weeks of age. The tumors are primarily classified as AC and/or adenoacanthomas. The mice develop multiple metastatic lesions in the lung within 50 days of the appearance of the primary tumor Lifsted, T., Le Voyer, T., Williams, M., Muller, W., Klein-Szanto, A A., Buetow, K. H., and Hunter, K. W. Identification of inbred mouse strains harboring genetic modifiers of mammary tumor age of onset and metastatic progression. Identification of inbred mouse strains harboring genetic modifiers of mammary tumor age of onset and metastatic progression, Int J of Cancer. (1998) 77: 640-4. Thus, these mice provide a rapid model for metastatic mammary cancer. As with the Apc.sup.Min/+ mice, genetic background affects the time course of tumor development and metastatic spread. Lifsted, T., Le Voyer, T., Williams, M., Muller, W., Klein-Szanto, A A., Buetow, K. H., and Hunter, K. W. Identification of inbred mouse strains harboring genetic modifiers of mammary tumor age of onset and metastatic progression. Identification of inbred mouse strains harboring genetic modifiers of mammary tumor age of onset and metastatic progression, Int J of Cancer. (1998) 77: 640-4. Thus, the inventors use crosses to generate mice with a slower course of tumor development. PyVT can associate with members of the SRC kinase family, phosphatidylinositol-3kinase, the SHC adapter protein and protein phosphatase 2A. Dankort, D. L. and Muller, W. J. Transgenic models of breast cancer metastasis. Transgenic models of breast cancer metastasis, Cancer Treatment & Research. (1996) 83: 71-88. Activation of SRC family kinases is frequently observed in human breast tumors. Amy Moser; Muthuswamy, S. K. and Muller, W. J. Activation of the Src family of tyrosine kinases in mammary tumorigenesis. Activation of the Src family of tyrosine kinases in mammary tumorigenesis, Advances in Cancer Research (1994) 64: 111-23.
(164) Imaging Studies: NM404 (
(165) Results and Discussion: This model is unique in that hyperplastic mammary lesions, mammary carcinomas, and intestinal adenomas develop in the same mouse. Initial imaging results with NM404 (
(166) Conclusions: NM404 has displayed striking tumor avidity in animal and human xenograft tumor models examined to date. Moreover, while it displayed selective and prolonged retention by mammary tumors in this spontaneous tumor model it did not localize in associated intestinal adenomatous polyps.
H. Example VIII
Specificity for Hyperplasia Versus Neoplasia in the ApcMin/+ Endogenous Mammary Adenocarcinoma Model
(167) Materials and Methods: Apc.sup.Min/+ Mouse Model: This model is comprised of mice carrying the Min allele of Apc (Apc.sup.Min/+ mice). This model offers specific advantages over xenograft models in that female Apc.sup.Min/+ mice are predisposed to develop mammary hyperplasias and carcinomas and intestinal adenomas. On the C57BL/6J genetic background, about 5% of untreated females will develop a mammary tumor by 100 days of age. Moser A R, Dove, et al. Proc Natl Acad Sci USA (1993) 90:8977-81. The incidence and multiplicity of the mammary lesions can be increased by a single dose of ethylnitrosourea (ENU), a direct acting alkylating agent. Treatment with ENU results in 90% of B6 Apc.sup.Min/+ females developing an average of 3 mammary squamous cell carcinomas (SCC), but few hyperplasic lesions within 60 days after treatment.
(168) Genetic background can affect the incidence, latency, and type of mammary lesions that develop. For example, FVBxB6 Apc.sup.Min/+ female mice develop an average of 0.2 mammary tumors per mouse, but 4 hyperplasias per mouse within 120 days of treatment. BALB/xB6 Apc.sup.Min/+ develop an average of 1.8 mammary tumors and 0.6 hyperplasias per mouse. Moser A R, Hegge L F, Cardiff R D. Cancer Research (2001) 61:3480-3485. FVBxB6 and BALBxB6 Apc.sup.Min/+ mice develop both mammary SCC and adenocarcinomas (AC).
(169) Imaging Studies: NM404 (
(170) Results and Discussion: This model is unique in that hyperplastic mammary lesions, mammary carcinomas, and intestinal adenomas develop in the same mouse. Initial imaging results with NM404 (
(171) Conclusions: NM404 has displayed striking tumor avidity in 20/20 animal and human xenograft tumor models examined to date. Moreover, while it displayed selective and prolonged retention by mammary adeno- and squamous cell carcinomas in this spontaneous tumor model, it did not localize in associated focal alveolar hyperplasias or intestinal adenomatous polyps and thus appears to be selective for malignant tumor cells.
I. Example IX
Mechanism of Selective Retention of NM404
(172) Introduction: Certain phospholipid ether analogs, such as NM404, are selectively retained within many types of tumor cells for a prolonged time. The inventors sought to evaluate the mechanism of selective retention of NM404 in tumor cells using both an enzymatic assay to evaluate the activity of phospholipase D (PLD) protein and quantitative PCR. The inventors hypothesized that reduced levels of PLD in tumor cells results in a decrease in the ability to metabolize and excrete NM404.
(173) Methods: Single cell suspensions of murine tumor cell lines including hepa-1 (hepatoma), CT26 (colorectal adenocarcinoma), and TS/A (breast adenocarcinoma) were analyzed with two assays: (1) Amplex Red assay, using a commercially available kit (Molecular Probes) that evaluates PLD protein activity using a fluorescence microplate reader, and (2) quantitative PCR to determine the level of PLD mRNA. Tumor cell lines were compared to normal liver tissue, which exhibits higher levels of uptake and elimination of NM404 and thus likely has lower PLD levels than other normal tissues. For the Amplex Red assay, total protein was extracted using a detergent solution (Triton-X-100) and quantity of PLD compared to a standard positive control. For PCR, mRNA was purified and converted to cDNA using reverse transcriptase (Promega). Conditions for amplification of cDNA for real-time PCR included: (94 C., 30 sec; 65 C., 30 sec; and 72 C., 30 sec) for 50 cycles (iCycler, iQmix, Bio-Rad). The primer for PLD1, (sense) 5-TCTGGTTTCACCCCGTCAGAA-3 (SEQ ID NO: 1), (antisense) 5-TTGCTCATATCTGCGGCGAT-3 (SEQ ID NO:2), was used. Product was compared to a standard cDNA (GAPDH, Biosource) diluted from 1 g to 10.sup.7 g. All assays were performed in duplicate.
(174) Results: PLD was quantitated as shown in the Table 3. Both PLD protein activity and mRNA levels were significantly lower than normal liver tissue (p<0.05, T-test) in all cell lines.
(175) Conclusion: Both reduced PLD protein activity and a decrease in PLD mRNA were observed in murine tumor cell lines. Thus, the mechanism of selective retention of NM404 may be due to a decrease in the breakdown of NM404 by PLD. Decreased PLD activity in tumor may serve as a potential molecular target for anti-tumor agents.
(176) TABLE-US-00002 TABLE 3 PLD protein activity mRNA (g 10.sup.5/ (mU/fluorescence/g 0.01 g of Cell/tissue protein/ml) total cDNA) Hepa-1 3.3 6.2 CT26 7.8 2.4 TS/A 2.8 4.0 Normal liver 14.1 12.2
J. Example X
Therapeutic Attributes in Endogenous Murine Mammary Tumor Model
(177) Models for NM404 Therapy Study: Although long-term survival is not essential for imaging studies, it is advantageous for the proposed therapy studies. The models used for imaging studies suffer from concomitant intestinal tumors which usually lead to the death of the animal. In order to increase the number of tumors developing per mouse and decrease the number of intestinal tumors in hopes of increasing the lifespan of the tumor bearing mice, Dr. Moser has recently crossed male B6 Min/+ mice with female C57BR/cdJ (BR) mice. The resulting BRB6 F1 Min/+ female mice developed significantly more mammary tumors than did the B6 Min/+ mice (P=0.016), an average of nearly 5. The number of mice with tumors and the time to first tumor were not different between these two strains (P=1 and P=0.06, respectively) (
(178) B6 and BRB6 F1 Min/+ mice were very similar with respect to the mammary gland phenotype, but quite different in susceptibility to intestinal tumors. The B6 and BR strains can be considered sensitive backgrounds for Min-induced mammary tumorigenesis as the mice developed a large number of tumors within a short time after ENU treatment. However, the BR strain carries dominant resistance alleles at modifier loci affecting intestinal tumor development, which may prove relevant to the proposed therapy study. A comparison of these strains is presented in Table 2.
(179) TABLE-US-00003 TABLE 2 Genetic background affects mammary and intestinal tumor development in Min/+ mice. Average % with Average # of # with Average # of Average # of survival in # of mammary mammary mammary mammary intestinal days after ENU Strain mice tumor ( ) tumors/mouse lesions (%) lesions/mouse tumors/mouse (range) B6 45 93 3.3 2.0 17 (38) 0.6 0.9 34 10 64 (43-78) BRxB6 18 94 4.9 2.6 7 (39) 0.5 0.7 14 4 91 (58-118) FVBxB6 18 17 0.2 0.5 18 (100) 4.1 2.4 12 6.sup.a 127 (93-178) .sup.aInformation on 16 mice as the intestines of 2 mice were lost in processing. Mice were generated by crossing females of each strain with B6 Min/+ males. Female mice were treated with ENU when between 30-45 days of age and sacrificed when moribund. Only the results from the Min/+ mice are shown. Mammary tumors are defined as those tumors identified at necropsy, while mammary lesions are the small focal lesions noted in the whole mounts of the 1.sup.st, 4.sup.th and 5.sup.th mammary glands. Intestinal tumors were counted in three 4 cm sections from the small intestine (duodenum, jejunum, and ileum), and the entire colon. All Min/+ mice developed intestinal tumors. Values are the means SD
(180) Preliminary Imaging Results with NM404 in Min Mice: In a preliminary experiment to show that NM404 localizes in endogenous FVBxB6 Apc.sup.Min/+ mouse breast tumors, two animals were injected (IV tail vein) with .sup.125I-NM404 (15 Ci) and imaged on a modified Bioscan AR2000 radioTLC scanner (equipped with high resolution 1 mm collimator and 2-D acquisition and analysis software) at 1, 4, and 7 days post injection (
(181) Radiotherapeutic Potential of NM404: During the course of recent mouse tumor uptake and retention studies with imaging doses (15-20 Ci/20 g mouse) of .sup.125I-labeled NM404, several apparent therapeutic responses have been observed (unpublished results). In an Apc.sup.Min/+ mouse mammary tumor model it has generally been noted that tumor growth remains static following a single intravenous injection of NM404. Some of these animals also lost all hair above larger mammary tumors at around 8 days after injection. Moreover, these mice also get intestinal tumors and usually suffer from intestinal bleeding resulting in severe anemia, which renders their feet white. Dr. Moser noted that the feet of these mice had reverted to a pink color around 5 days after a single injection of NM404. Upon eventual dissection of these animals, it was noted that only a very few, if any, of the expected 20 or so intestinal tumors usually found at this age actually remained. The white to pink feet phenomenon was also observed in a separate, but more aggressive, mouse intestinal adenocarcinoma model, wherein dissection at 12 days following NM404 administration, again revealed that most, if not all, of the expected intestinal tumors were gone. In both intestinal models, animals that received NM404 easily outlived their untreated litter mates. These coincidental findings were reconfirmed in two separate age-matched groups each involving more than 6 mice. These observations with .sup.125I-NM404 indicate potential for radiotherapy applications particularly if labeled with iodine-131. Quantitative tumor uptake and retention studies outlined in this proposed mammary tumor model will also provide sufficient data to initiate a comprehensive dosimetry analysis for this agent in order to estimate its true radiotherapeutic potential.
(182) Choice of Isotope: Due to its 60-day physical half-life and low energy 28 KeV photon emission, iodine-125 is suitable for imaging experiments in mice and rats. Iodine-125 also affords therapeutic characteristics as well and is currently used in permanent prostate brachytherapy implants. In one imaging experiment, 2 nude mice were each inoculated with subcutaneous squamous cell 1 and 6 tumor cell implants on opposing flanks. SCC 1 and 6 cells were used because one is radiosensitive relative to the other. After 14 days when the average tumor size (4 total) was approaching 0.5 cm in diameter, one of the mice received 20 Ci of .sup.125I-labeled NM404 and the other one receive unlabeled NM404 in an equal mass dose. The mouse that had received only the unlabeled compound had to be euthanized 20 days after injection due to both tumors reaching the termination size limit as defined in our animal use protocol. Both tumors in the .sup.125I-NM404 mouse regressed dramatically and unexpectedly over the course of several weeks (
(183) Iodine-131 has been used with great efficacy in the treatment of thyroid cancer. Very safe doses of .sup.131I can control subclinical deposits of well-differentiated thyroid cancer, which concentrates iodine very avidly as does the normal thyroid. This active uptake process helps limit the dose to normal tissues. Iodine-131 has both beta and several gamma emissions, but the predominant tissue dose arises from the beta emissions. The inventors have selected .sup.131I-labeled NM404 based upon the clinical success with thyroid cancer coupled with results obtained with Bexxar (an iodine-131-labeled antibody-based agent) in low-grade lymphoma patients. The predominant beta emissions and mostly low energy gamma emissions optimize dose homogeneity within the tumor nodule itself. Also, the shorter half-life (8-days) provides more clinically relevant dose-intensity compared to the 60-day half-life of .sup.125I. These factors will permit the inventors to make the best assessment of the anti-tumor efficacy of this agent. A potential disadvantage of .sup.131I is that there is a higher energy gamma emission as well which could actually expose adjacent surrounding tissues to more radiation than would occur with .sup.125I. The tumors in the endogenous model proposed herein are peripherally located in the mammary glands and thus should not represent an immediate threat to the overall well-being of the animal. Since organ toxicity is also one of the study endpoints, the reaction of the surrounding tissue and key organ systems (marrow, liver, kidneys, bowel, brain, etc) is assessed. Tissue distribution data and actual dosimetry of radiolabeled NM404 will determine its optimal therapeutic potential. It is possible that different isotopes will complement each other in the therapeutic setting.
(184) It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims. All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes.
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