Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
  • A61K51/02—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by the carrier, i.e. characterised by the agent or material covalently linked or complexing the radioactive nucleus
  • A61K51/04—Organic compounds
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
  • A61K51/02—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by the carrier, i.e. characterised by the agent or material covalently linked or complexing the radioactive nucleus
  • A61K51/04—Organic compounds
  • A61K51/0404—Lipids, e.g. triglycerides; Polycationic carriers
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
  • A61P35/04—Antineoplastic agents specific for metastasis
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer

Definitions

• the invention generally relates to phospholipids ether analogs and use thereof and specifically relates to use of phospholipid ether analogs and combinations thereof for diagnosis of metastasis, treatment, pharmacokinetic, dosimetry, and toxicity studies of various cancer types, such as non-small cell lung cancer, prostate cancer and metastasis thereof.
• NSCLC Non-small cell lung cancer
• NSCLC Non-small cell lung cancer
• Imaging with FDG PET scanning has recently become the "gold standard" for imaging NSCLC, due to improved sensitivity, particularly when compared with CT imaging.
• its sensitivity for identifying mediastinal lymph node involvement is only about 90%, and lack of specificity, particularly in patients with inflammatory or granulomatous disease, is particularly problematic.
• its utility in diagnosing brain tumors or metastases is limited due to high metabolic background of normal brain tissue.
• CT computed tomography
• MRI magnetic resonance imaging
• PET Positron-emission tomography
• FDG F- fluorodeoxyglucose
• FDG-PET has been shown to reduce futile thoracotomies in patients. However, because of the false positive and false negative rate, confirmatory mediastinoscopies are often recommended. For example, a retrospective study involving over 200 patients with NSCLC found that the sensitivity, specificity, positive and negative predictive values, and accuracy for FDG-PET were 64%, 77%, 45%, 88%, and 75%, respectively.
• FDG-PET also plays a role in diagnosing extra-thoracic disease, particularly in patients with intermediate stages of lung cancer. A study done by the American College of Physicians involving over 300 patients found that unsuspected metastatic disease or second primary malignancies was identified in 1 8 of 287 patients (6.3%).
• the sensitivity and specificity for the identification of cerebral metastatic disease in patients with NSCLC was 60% and 99% for FDG-PET, and 100% and 100% for conventional imaging. Therefore, FDG-PET imaging is not considered to be the best method of evaluating a patient with NSCLC for metastatic disease to the brain.
• FDG fluorescence desorption spectroscopy
• Another disadvantage of FDG is that it is not specific for tumors, but accumulates in both malignant and non-malignant hypermetabolic tissues.
• FDG is a nonspecific tracer and accumulates in areas of infection or inflammation. In the lung, these areas can be localized lung parenchymal nodules or more diffuse (subsegmental, segmental or lobar) or in the hilar and mediastinal nodes.
• FDG-PET is also frequently negative in malignancies with a low metabolic rate, such as bronchoalveolar carcinoma or carcinoid.
• a radiopharmaceutical that could accurately identify early metastatic disease in the patients with NSCLC would have a significant impact on patient care, in terms of both staging and response to therapy.
• PET imaging has improved diagnostic efficacy in this area compared to CT, there remains a need for an accurate imaging technique that is not based upon metabolic activity, which is non-specific, but is based upon a tumor-specific function that can non-invasively screen the whole body, including the brain, TPara 181 Prostate Cancer
• Factors increasing the likelihood of systemic recurrence include a high preoperative PSA level as well as pathologic features of the surgical specimen including Cleason score >7, seminal vesicle involvement, and lymph node involvement. In contrast, extracapsular extension, positive surgical margins and Gleason score ⁇ 7 are factors generally associated with local recurrence.
• the velocity of PSA rise following prostatectomy has been utilized to determine whether disease recurrence is local or systemic. For instance, Partin et al reported that a PSA rise of less than 0.75 ng/ml/year was more frequently associated with local recurrence.
• patients with a rising PSA due to local recurrence in whom systemic recurrence cannot be excluded with confidence, may unnecessarily undergo hormonal ablation, which is generally not considered curative and is associated with osteoporosis development, decreased libido, weight gain, menopausal symptoms, and overall malaise, as well as the evolution of hormonally independent prostate cancer.
• CT computed tomography
• MRI magnetic resonance imaging
• Radioimmunoscintigraphy with lndium-1 1 1 capromab pendetide has been utilized in patients following prostatectomy with a rising PSA who have a high clinical suspicion of occult metastatic disease and no clear evidence for metastatic disease in other imaging studies.
• This scan is based on a radiolabeled murine monoclonal antibody which is specific for PSMA (Prostate-specific membrane antigen), a transmembrane protein which is specifically expressed by both normal and malignant prostate epithelial cells.
• PSMA Prostate-specific membrane antigen
• ProstaScint radioimmunoscintigraphy has been shown to be promising in diagnosing locally recurrent disease in the prostate bed in patients with rising PSA
• clinical results for this scan have been somewhat variable, with sensitivities ranging between 44% and 92% and specificities between 36% and 86%.
• false-negative ProstaScint studies have been reported in 10% to 20% of cases.
• ProstaScint has been reported in neurofibromatosis, lymphomas, renal carcinomas, pelvic kidneys, myolipomas, and meningiomas, as well as in the bone marrow of vertebral bodies. Given this data, use of the ProstaScint scan for patients at risk for occult metastases from prostate cancer remains controversial.
• PET positron-emission tomography
• FDC-PET can distinguish between active and quiescent bone metastases in patients with prostate cancer.
• the intensity of FDG uptake is thought to reflect the metabolic and biological activity of these lesions in contrast to the traditional bone scan with technetium-diphosphonate compounds in which nonspecific osteoblast activity may be detected as a false positive signal following treatment.
• a false negative reading may be obtained since early metastases, which initially seed into the bone marrow, will not necessarily produce a signal until an osteoblastic response occurs. Therefore, a persistently positive bone scan does not necessarily indicate the presence of residual viable metastases and a negative bone scan result may not reflect accurately the patient's metastatic tumor burden.
• FDC-PET may therefore prove beneficial in guiding the management of patients with bony metastases and in a retrospective study FDC-PET and helical CT have been shown independently to be more effective than i n In-monoclonal antibody imaging in detecting metastatic disease.
• the present invention provides a method for detecting and locating recurrence of cancer, radiation and chemo insensitive cancer or metastasis of cancer selected from the group consisting of Lung cancer, Adrenal cancer, Melanoma, Colon cancer, Colorectal cancer, Ovarian cancer, Prostate cancer, Liver cancer, Subcutaneous cancer, Squamous cell cancer, Intestinal cancer, Hepatocellular carcinoma, Retinoblastoma, Cervical cancer, Glioma, Breast cancer and Pancreatic cancer in subject that has or is suspected of having cancer.
• the method comprising the steps of: (a) administering a phospholipid ether analog to the subject; and (b) determining whether an organ suspected of having recurrence of cancer, radiation and chemo insensitive cancer or metastasis of cancer in the subject retains a higher level of the analog than surrounding region(s) wherein a higher retention region indicates detection and location of the recurrence of cancer, radiation insensitive and chemo cancer or metastasis of cancer.
• the phospholipid analog is selected from:
• X is selected from the group consisting of radioactive isotopes of halogen; n is an integer between 8 and 30; and Y is selected from the group comprising NH2, NR2, and NR3, wherein R is an alkyl or arylalkyl substituent or
• X is a radioactive isotope of halogen
• n is an integer between 8 and 30
• Y is selected from the group consisting of H, OH 1 COOH, COOR and OR
• 2 is selected from the group consisting of NH2, NR2, and NR3, wherein R is an alkyl or arylalkyl substituent.
• X is selected from the group of radioactive halogen isotopes consisting of 1 SF 1 ⁇ a, 76Br, 77 ⁇ r , 82 B r,i22
• the phospholipid ether isl 8-(p-lodophenyl)octadecyl phospnocboltne, 1 -O-[l 8-(p-lodophenyl)octadecyl]-l ,3-propanediol-3- phosphocholine, or 1 -O-D ⁇ - ⁇ -lodophenyOoctadecylJ-ii.-O-methyl-rac- glycero-3-phosphocholine, wherein iodine is in the form of a radioactive isotope.
• the detection is carried out by a of PET, CT, MRI scanning methods combination thereof.
• Another embodiment of the present invention provides a method for the treatment of recurrence of cancer, radiation and chemo insensitive cancer or metastasis of cancer in a subject.
• the method comprises administering to the subject an effective amount of a compound comprising a phospholipid ether analog, in a preferred embodiment, the recurrence of cancer, radiation and chemo insensitive cancer or metastasis of cancer occurs in the group selected from Lung cancer, Adrenal cancer, Melanoma, Colon cancer, Colorectal cancer, Ovarian cancer, Prostate cancer, Liver cancer, Subcutaneous cancer, Squamous cell cancer, Intestinal cancer, Hepatocellular carcinoma, Retinoblastoma, Cervical cancer, Glioma, Breast cancer, Pancreatic cancer and Carcinosarcoma.
• the phospholipid analog is selected from:
• X is selected from the group consisting of radioactive isotopes of halogen; n is an integer between 8 and 30; and Y is selected from the group comprising NHh 1 NR2, and NR3, wherein R is an alkyl or arylalkyl substituent or
• X is a radioactive isotope of halogen
• n is an integer between 8 and 30
• Y is selected from the group consisting of H, OH, COOH, COOR and OR
• Z is selected from the group consisting of NH2, NR 2 , and NR3, wherein R is an alkyl or arylalkyl substituent.
• X is selected from the group of radioactive halogen isotopes consisting of i8F, 36Cl, 76Br, 77Br, 8 2 ⁇ r,i22
• the effective amount of phospholipid ether analog is a combination of at least two isotopes, one with one with a path range of about 0.1 A to 1 mm and a second with a path range of about 1 mm to 1 m.
• the effective amount of phospholipid ether analog is a combination of at least two isotopes, 125 I and 131 I.
• the phospholipid ether isl 8-(p-lodophenyl)octadecyl phosphocholine, 1 -O-[l 8-(p-lodophenyl)octadecyl]-l ,3-propanediol-3- phosphocholine, or l -O-[l 8-(p-lodophenyl)octadecyl]-2 ⁇ O-methyl-rac- glycero-3-phosphocholine, wherein iodine is in the form of a radioactive isotope.
• the effective amount of phospholipid ether analog is fractionated. In yet other embodiments, the effective amount of phospholipid ether analog is about O.S ⁇ Ci to about 3Ci treatable in a linear and dose dependent manner. Other embodiments provide that the dosage is adaptable to the cancer-volume. Yet other embodiments provide that the dosage for radiation insensitive tumor is greater than dosage for radiation sensitive tumor and less than 3Ci and is adaptable to the cancer- volume.
• Another embodiment of the present invention provides the use of phospholipid ether analog for the production of a pharmaceutical composition for the treatment of recurrence of cancer, radiation and chemo insensitive cancer or metastasis of cancer.
• the phospholipid analog is selected from:
• X is selected from the group consisting of radioactive isotopes of halogen; n is an integer between 8 and 30; and Y is selected from the group comprising NHz, NR2, and NR3, wherein R is an alkyl or arylalkyl substituent or
• X is a radioactive isotope of halogen
• n is an integer between 8 and 30
• Y is selected from the group consisting of H, OH, COOH, COOR and OR
• Z is selected from the group consisting of NH2, NR2, and NR3, wherein R is an alkyl or arylalkyl substituent.
• X is selected from the group of radioactive halogen isotopes consisting of i»F f 36 CI, 76 Br, 77 Br, sz ⁇ r ⁇ l, '23
• the phospholipid ether isl 8-(p- lodophenyOoctadecyl phosphocholine, 1 -O-D 8-(p-lodophenyl)octadecyl]- 1 ,3-propanediol-3-phosphocholine, or 1 -O-[l 8-(p-lodophenyl)octadecyl]- 2-0-methyl-rac-glycero-3-phosphocholine, wherein iodine is in the form of a radioactive isotope.
• Elastically scattered electrons (which include backscattered electrons) are generally scattered through larger angles than are inelastically scattered electrons, (right)
• An incident electron ionizes the sample atom by ejecting an electron from an inner-shell (the K shell, in this case).
• De- excitation in turn, produces characteristic X-radiation (above) or an Auger electron (below).
• Secondary electrons are ejected with low energy from outer loosely bound electron shells, a process not shown.
• Fig. 3 Scintigraphy of the anterior chest of Patient 03 acquired at 1 , 2, and 6 days after iv administration of 1 mCi 1 31 l-NM-324. Uptake is seen in the left lingular lung cancer (T) with increasing tumor-to-background ratios over time.
• Fig. 4 Time course (days) of NM404 in a SCID mouse with a human RL-251 adrenal tumor (T) xenograft.
• FIG. 5 Tissue distribution 1-1 125-NM404 in RL 251 Adrenal Cancer in SCID mice depicting that while accumulation in the tumor increased, distribution in blood, spleen and kidney reduced by days 1 through 14.
• Fig. 6 Apparent SCCl and SCC6 tumor regression after injection of 125I-NM404. By day 41 , the tumor is significantly reduced.
• Fig. 7. The image shows one of the tumor-bearing animals treated with 250 ⁇ Ci of I-125-NM404 at 4 weeks following injection. The hair above the tumor has fallen off, presumably due to the strong accumulation of radioactivity in the tumor. Additionally, the surface of the tumors appears "caved in” and shows darker areas, presumably from hemorrhage and necrosis. The figure shows the effect of 1-125-NM404 on the tumor. Although tumor size (outer dimensions) may not shrink, 1-125- NM404 causes central necrosis. The measurement method measuring outer tumor dimensions may have underestimated tumor volume response following I-125-NM404.
• FIG. 8 PC3 human prostate cancer model implanted into SCID mouse.
• PC3 is known to be radiation insensitive.
• the curves between control (no radioactivity, cold NM404) and treatment (1-1 25-NM404) only separate about 4-5 weeks after treatment, until then the growth of the tumors looks the same indicating that: 1) NM404 takes a few days to about 1 week to fully accumulate in the tumor, and 2) the isotope 1-125 has a low radiation flow (since it has a long half life). Both factors contribute to a delayed onset of the therapy effect, at a time when all NM404 has cleared out of normal tissues.
• Fig. 9 Scintigraphic comparison of NM404 (bottom panel) and NM324 (top panel) at 1 , 2, and 4 days in a SCID mouse with human prostate PC-3 tumor (arrow) implanted in the flank. Liver and background radioactivity are much improved with NM404.
• Fig. 10 Tumor volume for each group was recorded over the 10-week assessment period depicting the control and dosage of 50, 1 50, 250 and 500 ⁇ Ci.
• control animals show rapidly growing tumors over the 10-week assessment period.
• the 50 uCi dose group did not show any difference to control animals, hence these seem to be ineffective dose levels in this animal model.
• the 1 50, 250 and 500 ⁇ Ci dose groups show a substantial and prolonged treatment effect. Tumor volumes are stable and same tumors appear "collapsed" (the tumor surface has caved in). Additionally, hair above the tumors fell off confirming substantial accumulation of radioactivity in these tumors.
• Fig. 1 A549 Tumor Xenografts (I Xl O 6 cells, s.c.) in Female SCID Mice Fractionated Dose (3 x 50 mCi), having 2 independent controls for each dosage.
• a fractionated dosing of NM404 e.g. 3 x 50 micro-Ci versus a single dose of 150 micro-Ci
• the fractionated dose may be safer since it is eliminated from normal tissues in between fractionated injections.
• FIG. 12 A549 Large Tumors vs Small Tumors 1 50 microcuries
• FIG. 13 Bioscan image (A) obtained 4 days post I Z5
• FIG. 1 Fused 3D surface-rendered MRI image (blue) and 3D , microPET image (A) obtained 24h after iv injection of 124 I-NM4O4 (80 ⁇ Ci) into a rat with a CNS-I glioma brain tumor. Images were fused using Amira
• 124 l-MicroPET images a human A549 lung tumor xenograft in a SCID mouse 48h post injection of 124 I-NM4O4 (80 ⁇ Ci).
• Fig. 1 7. i 2 ⁇ -MicroPET "mages (coronal, sagittal, and axial) 96h after iv injection of 124 I-NM4O4 into a PC-3 (flank) tumor -bearing mouse.
• NM324 top panel at 1 , 2, and 4 days in a SCID mouse with human prostate
• PC-3 tumor implanted in the flank. Liver and background radioactivity are much improved with NM404.
• Fig. 20 Time course of 125 I-NM4O4 in human RL-251 adrenal cancer xenograft in a SCID mouse. Prolonged tumor (1 .5x0.5 cm, arrow) retention is evident even after 20 days.
• Bioscan image obtained 4 days post NM404 injection (B). Positionally matched fused photo/Bioscan image of excised prostate/vesicular gland (C) showing intense uptake of radioactivity in the prostate tumor.
• Nude mouse leg at various times following intratibial injection (arrow in first panel depicts injection site and direction) of 2x10 5 human PC3 prostate tumor cells. Tumor begins protruding through the bone by 28 days (arrow) and by 46 days the tumor has literally destroyed the tibia leaving only the fibula intact.
• FIG. 24 Fused 3D surface-rendered MRI image (blue) and 3D microPET image (A) obtained 24h after iv injection of 124 I-NM4O4 (100 ⁇ Ci) into a rat with a CNS-I glioma brain tumor. Images were fused using Amira (v3.1 ). Right panels show (B) contrast-enhanced coronal MRI slice through the tumor (arrow) and (C) fused coronal MRI and 124 I-NM4O4 microPET images corroborating presence and location of the tumor.
• FIG. 25 Posterior whole body planar nuclear medicine image (A) 4 days after iv administration of 13 'l-NM-404 (0.8 mCi) to a patient with non small cell lung cancer (6 cm dia., arrow). Lung tumor is easily detected in corresponding axial (B) and coronal (C) computed tomography (CT) scans.
• the term “isomer” includes, but is not limited to optical isomers and analogs, structural isomers and analogs, conformational isomers and analogs, and the like.
• this invention encompasses the use of different optical isomers of an anti-tumor compound of Formula 3A.
• 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.
• 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.
• the anti-tumor compounds may include pure (R)-isomers.
• the anti-tumor compounds may include pure (S)- isomers.
• the compounds may include a mixture of the (R) and the (S) isomers.
• the compounds may include a racemic mixture comprising both (R) and (S) isomers.
• 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.
• 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 he prepared from the phenolic compounds by treatment with inorganic bases, for example, sodium hydroxide.
• esters of the phenolic compounds can be made with aliphatic and aromatic carboxylic acids, for example, acetic acid and benzoic acid esters.
• pharmaceutically acceptable salt refers to a compound formulated from a base compound which achieves substantially the same pharmaceutical effect as the base compound.
• This invention further includes method utilizing derivatives of the anti-tumor compounds.
• derivatives includes but is not limited to ether derivatives, acid derivatives, amide derivatives, ester derivatives and the like.
• this invention further includes methods utilizing hydrates of the anti-tumor compounds.
• hydrate includes but is not limited to hemihydrate, monohydrate, dihydrate, trihydrate and the like.
• 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.
• "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.
• 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.
• the term “treating” includes preventative as well as disorder remittent treatment.
• the terms “reducing”, “suppressing” and “inhibiting” have their commonly understood meaning of lessening or decreasing.
• progression means increasing in scope or severity, advancing, growing or becoming worse.
• recurrence means the return of a disease after a remission.
• administering refers to bringing a patient, tissue, organ or cells in contact with an anti-tumor phospholipid ether compound.
• 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.
• 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
• pharmaceutical composition means therapeutically effective amounts of the anti-tumor compound having radioactivity together with suitable diluents, preservatives, solubilizers, emulsifiers, and adjuvants, collectively “pharmaceutically-acceptable carriers.”
• 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.
• compositions are liquids or lyophilized or otherwise dried formulations and include diluents of various buffer content (e.g., Tris-HCI, 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
• 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.
• the pharmaceutical composition is administered parenterally, paracancerally, transmucosally, tansdermally, intramuscularly, intravenously, intradermally, subcutaneously, intraperitonealy, intraventricular ⁇ , intracraniaJly and intratumorally.
• pharmaceutically acceptable carriers are well known to those skilled in the art and include, but are not limited to, 0,0T-0.1 M 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 nonaqueous 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.
• 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.
• 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.
• 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.
• a pharmaceutical composition can be delivered in a controlled release system.
• the agent may be administered using intravenous infusion, an implantable osmotic pump, a transdermal patch, liposomes, or other modes of administration.
• 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.
• 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. 1 1 5-1 38 (1984). Other controlled release systems are discussed in the review by Langer (Science 249:1 527-1 533 (1990).
• 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.
• 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.
• the pharmaceutical preparations administerable by the invention can be prepared by known dissolving, mixing, granulating, or tablet-forming processes.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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.
• compositions which contain an active component are 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.
• 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.
• auxiliary substances such as wetting or emulsifying agents, pH buffering agents which enhance the effectiveness of the active ingredient.
• 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.
• inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides
• organic bases as isopropylamine, trimethylamine, 2-ethylamino ethanol, histidine, procaine, 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.
• the active compound in another method according to the invention, can be delivered in a vesicle, in particular a liposome (see Langer, Science 249:1 527-1 533 (1 990); 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. 31 7-327; see generally ibid).
• 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.
• 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.
• phospholipids ether compounds and specially 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 27/27 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.
• 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 124 I has a 4 day physical half life it can be shipped anywhere in the world whereas FDG with its 1 10 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 131 I 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 18 F for PET scanning or potentially 19 F (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.
• the PLE compounds may be designed to more accurately estimate the specificity and sensitivity of radiolabeled PLE analogs such as NM404 as an imaging agent in prostate cancer and other cancers. Based upon preclinical models, PLE analogs such as NM404 are likely to exhibit high uptake in tumors giving this agent the significant potential for use in staging, following response to therapy, or potentially as a therapeutic agent when coupled with higher doses of 131 I, 125 !, or 211 At an alpha-emitting halogen with therapeutic efficacy.
• the present invention generally provides methods and techniques for the detection and treatment of various cancers.
• the present invention provides a method for detecting and locating recurrence of cancer, radiation and chemo insensitive cancer or metastasis of cancer selected from the group consisting of Lung cancer, Adrenal cancer, Melanoma, Colon cancer, Colorectal cancer, Ovarian cancer, Prostate cancer, Liver cancer, Subcutaneous cancer, Squamous cell cancer, Intestinal cancer, Hepatocellular carcinoma, Retinoblastoma, Cervical cancer, Glioma, Breast cancer and Pancreatic cancer in subject that has or is suspected of having cancer.
• the method comprising the steps of: (a) administering a phospholipid ether analog to the subject; and .
• the phospholipid analog is selected from:
• X is selected from the group consisting of radioactive halogen isotopes
• n is an integer between 8 and 30
• Y is selected from the group comprising NH2, NR2, and NR3, wherein R is an alkyl or arylalkyl substituent or
• X is a radioactive halogen isotope
• n is an integer between 8 and 30
• Y is selected from the group consisting of H, OH 1 COOH, COOR and OR
• Z is selected from the group consisting of NH2, NR2, and NR3, wherein R is an alkyl or arylalkyl substituent.
• X is selected from the group of radioactive halogen isotopes consisting of 1 8 F, 36 CI 1 76 Br, 77Br, 82Br,i 22
• the phospholipid ether isl 8-(p-lodophenyl)octadecyl phosphocholine, 1 -O-[l ⁇ -t ⁇ -lodophenyOoctadecylj-l ,3-propanediol-3- phosphocholine, or 1 -O-[18-(p-lodophenyl)octadecyl]-2-0 ⁇ methyl-rac- glycero-3-phosphocholine, wherein iodine is in the form of a radioactive isotope.
• the detection is carried out by PET CT MRI scanning methods and combination thereof.
• alkyl refers to a straight chain, branched or cyclic, saturated or unsaturated aliphatic hydrocarbons.
• the alkyl group has 1 -16 carbons, and may be unsubstituted or substituted by one or more groups selected from halogen, hydroxy, alkoxy carbonyl, amido, alkylamido, dialkylamido, nitro, amino, alkylamino, dialkylamino, carboxyl, thio and thioalkyl.
• a "hydroxy” group refers to an OH group.
• alkoxy refers to an — O-alkyl group wherein alkyl is as defined above.
• a “thio” group refers to an — SH group.
• a “thioalkyl” group refers to an -SR group wherein R is alkyl as defined above.
• An “amino” group refers to an -NH2 group.
• An “alkylamino” group refers to an — NHR group wherein R is alkyl is as defined above.
• a “dialkylamino” group refers to an — NRR 1 group wherein R and R 1 are all as defined above.
• An “amido” group refers to an — CONH2.
• An “alkylamido” group refers to an — CONHR group wherein R is alkyl is as defined above.
• a “dialkylamido” group refers to an — CONRR' group wherein R and R 1 are alkyl as defined above.
• a "nitro” group refers to an NO2 group.
• a “carboxyl” group refers to a COOH group.
• aryl includes both carbocyclic and heterocyclic aromatic rings, both monocyclic and fused polycyclic, where the aromatic rings can be 5- or 6-membered rings.
• Representative monocyclic aryl groups include, but are not limited to, phenyl, furanyl, pyrrolyl, thienyl, pyridinyl, pyrimidinyl, oxazolyl, isoxazolyl, pyrazolyl, imidazolyl, thiazolyl, isothiazolyl and the like.
• Fused polycyclic aryl groups are those aromatic groups that include a 5- or 6-membered aromatic or heteroaromatic ring as one or more rings in a fused ring system.
• fused polycyclic aryl groups include naphthalene, anthracene, indolizine, indole, isoindole, benzofuranr benzothiophene, indazole, benzimidazole, benzthiazole, purine, quinoline, isoquinoline, ci ⁇ noline, phthalazine, quinazoline, quinoxaline, 1 ,8- naphthyridine, pteridine, carbazole, acridine, phenazine, phenothiazine, phenoxazine, and azulene.
• arylalkyl refers to moieties, such as benzyl, wherein an aromatic is linked to an alkyl group which is linked to the indicated position in the PLE compound.
• the recurrence of cancer, radiation and chemo insensitive cancer or metastasis of cancer occurs in the group selected from Lung cancer, Adrenal cancer, Melanoma, Colon cancer, Colorectal cancer, Ovarian cancer, Prostate cancer, Liver cancer, Subcutaneous cancer, Squamous cell cancer, Intestinal cancer, Hepatocellular carcinoma, Retinoblastoma, Cervical cancer, Glioma, Breast cancer, Pancreatic cancer and Carcinosarcoma.
• the phospholipid analog is selected from:
• X is selected from the group consisting of radioactive halogen isotopes halogen; n is an integer between 8 and 30; and Y is selected from the group comprising NHa, NR 2 , and NR3, wherein R is an alkyl or arylalkyl substituent or
• X is a radioactive halogen isotope
• n is an integer between 8 and 30
• Y is selected from the group consisting of H, OH, COOH, COOR and OR
• Z is selected from the group consisting of NH2, NR2, and NR3, wherein R is an alkyl or arylalkyl substituent.
• X is selected from the group of radioactive halogen isotopes consisting Of 18 F, 36Cl 1 76Br, 77Br, 82Br 1 ⁇ i 1 i23
• the effective amount of phospholipid ether analog is a combination of at least two isotopes, one with a path range of about 0.1 ⁇ to 1 mm and a second with a path range of about 1 mm to 1 m, also as discussed in Fig. 2.
• the effective amount of phospholipid ether analog is a combination of at least two isotopes, 125 I and 131 I.
• the phospholipid ether is18-(p-lodophenyl)octadecyl phosphocholine, 1 -O-[1 8-(p-lodophenyl)octadecyl] ⁇ l ,3 ⁇ propanediol-3- phosphocholine, or l -O-[l 8-(p-lodophenyl)octadecyl]-2-0-methyl-rac- glycero-3-phosphocholine, wherein iodine is in the form of a radioactive isotope.
• the effective amount of phospholipid ether analog is fractionated.
• the advantage of using fractionated dosage is that it allows for the PLE analog to be removed from normal tissues.
• fractionated dosing of NM404 e.g. 3 x 50 micro-Ci versus a single dose of 150 micro-Ci
• the effective amount of phospholipid ether analog is about 0.5 ⁇ Ci to about 50OmCi, and as shown in Fig. 10, this is treatable in a linear and dose dependent manner.
• the dosage is adaptable to the cancer-volume as shown in Fig. 12.
• the graph in Fig. 12 shows the difference in tumor growth for the same dose of 1-125-NM404 (1 50 microCi) when injecting in animals with small ( ⁇ 1 cm) and large tumors (> l cm).
• Fig. 1 1 1 The results show that a small tumor showed stunned tumor growth with that dose, however a large tumor population did not show any effects for the same dose, basically behaving like non-radiated control.
• the graph in Fig. 1 2 further illustrates is that the effective tumor dose should be adjusted to tumor volume and that there is a tumor dose per volume of tumor volume that has to be achieved in order to show efficacy.
• the dosage for radiation and chemo insensitive tumor is greater than dosage for radiation sensitive tumor and less than 50OmCi and is adaptable to the cancer-volume, as ascertainable by comparing PC3 (Fig. 8) and A549 cancer models (Fig. 10, 1 1 and 1 2), in which PC3 model is radiation insensitive.
• Another embodiment of the present invention provides the use of phospholipid ether analog for the production of a pharmaceutical composition for the treatment of recurrence of cancer, radiation and chemo insensitive cancer or metastasis of cancer.
• the phospholipid analog is selected from:
• X is selected from the group consisting of radioactive halogen isotopes
• n is an integer between 8 and 30
• Y is selected from the group comprising NHa, NR2, and NR3, wherein R is an alkyl or arylalkyl substituent or
• X is a radioactive halogen isotope
• n is an integer between 8 and 30
• Y is selected from the group consisting of H 1 OH, COOH, COOR and OR
• Z is selected from the group consisting of NH2, NR2, and NR3, wherein R is an alkyl or arylalkyl substituent.
• X is selected from the group of radioactive halogen isotopes
• the phospholipid ether is18-(p- IodophenyOoctadecyl phosphocholine, 1 -O-ll 8-(p-lodophenyl)octadecyl]- 1 ,3 ⁇ propanediol-3-phosphocholine, or 1 -0-[I 8-(p-lodophenyl)octadecyl]- 2-O-methyl-rac-glycero-3-phosphocholine, wherein iodine is in the form of a radioactive isotope.
• Radioiodinated phospholipid ether analogs have shown a remarkable ability to selectively accumulate in a variety of human and animal tumors in xenograft and spontaneous tumor rodent models. It is believed that this tumor avidity arises as a consequence of metabolic differences between tumor and corresponding normal tissues. The results of this study indicate that one factor in the tumor retention of these compounds in tumors is the length of the alkyl chain that determines their hydrophobic properties.
• NM-404 A direct comparison between NM-404 and its predecessor, NM-324, in human PC-3 tumor bearing immune-compromised mice, revealed a dramatic enhancement in both tumor uptake and total body elimination of NM-404 relative to NM-324.
• Cl 8 analog, NM-404 was chosen for follow-up evaluation in human lung cancer patients. Preliminary results have been extremely promising in that selective uptake and retention of the agent in tumors is accompanied by rapid clearance of background radioactivity from normal tissues, especially those in the abdomen.
• Scheme 1 Reagents and conditions: (a) MesSiBr, CH2CI2; (b) BrMg(CH 2 )I iOTHP, Li 2 CuCU (cat), THF, -78 to 20 0 C; (c) PPTS 1 EtOH 7 40 0 C; (d) TsCI, DMAP, CH 2 CI 2 ; (e) BrMg(CHz) 3 OTHP, Li 2 CuCI 4 (cat), THF, -78 to 20 0 C; (g) BrMg(CH 2 ) 6 OTHP, U 2 CuCI 4 (cat), THF, -78 to 20 0 C.
• FPara 1 211 The synthesis was initiated by the conversion of , ⁇ -iodobenzyl alcohol 1 1 to ysHodobenzyl bromide 1 2 as shown in Scheme 1. /7-lodobenzyl bromide was further coupled with Grignard reagent derived from THP protected 1 1 -bromoundecanol 1 3 in the presence of 0.5-0.7 mol% of Li 2 CuCU. 12-( / 0-lodophenyl)dodecanol 1 7 obtained after deprotection of the first coupling product 16 was used for the synthesis of Cl 2 iodinated phospholipids as described earlier.
• Alcohol 1 7 also served as a starting material for the synthesis of ⁇ -iodophenyl alkanols with longer chains.
• tosylate 1 8 with Grignard reagents made from bromides 14 and 1 5 followed by THP deprotection furnished the Cl 5 (20) and Cl 8 (22) alcohols, respectively.
• Cl 5 (20) and Cl 8 (22) alcohols were converted into corresponding alkylphosphocholines 5 (NM-397) and 6 (NM-404) according to published procedures.
• the avidity of PLE analogs to localize in tumors was evaluated in several animal models.
• the PC-3 model represents a human tumor cell line that was used to determine the target (tumor) to non-target ratio of NM404 and NM412 in head to head comparison in order to select a candidate for an initial human pharmacokinetic trial in prostate cancer patients.
• the MatLyLu (Dunning R3327 rat) model a rat prostate tumor line, was used specifically to screen 9 specific analogs prior to entering them into control for dosimetry and tumor-bearing animals for determining tumor/background ratios.
• the Walker-256 carcinosarcoma model was used for quantitative tissue distribution purposes.
• blood levels for 6 were 0.6 ⁇ 0.1 % of ID/g as compared to levels of 0.07 ⁇ 0.00% of ID/g for the Cl 5 (5) analog.
• Total radioactivity levels in the thyroid were relatively low in both 5 and 6 when the extremely small mass of the gland is considered.
• thyroid levels ranged from 26 to 54% injected dose per gram of tissue, these levels are actually quite low and represent an extremely small percentage of the injected dose when the exceedingly small mass of the organ is considered and the data are presented on a percent administered dose per organ basis.
• Non-target tissue uptake can decrease the efficacy of radiodiagnostic imaging by creating high background activity or by causing excessive exposure of radiosensitive tissues to the injected radioactivity.
• the Cl 8 analog 6 (NM-404) displayed a propensity to remain in the circulation much longer than the Cl 2 (4) and Cl 5 (5) analogs.
• a longer plasma half-life may be expected to result in additional opportunities for uptake of the Cl 8 compound 6 by the tumor as it continually circulated through the vasculature.
• This extended plasma half-life may be a result of strong binding of the probe to albumin.
• Uptake and transport of radiolabeled PLE by plasma components may also be an important factor related to the tumor retention of these compounds.
• increase of the chain length from C7 to Cl S results in an increase in the lipophilicity of the PLE analogs. Greater lipophilicity may increase the affinity of these compounds for the cell membrane, and may alter their binding to plasma components.
• NM404 Uptake and transport in the circulation by endogenous lipoproteins such as LDL and HDL may also impact the biological distribution into the tumor.
• unlabeled NM404 was subjected to independent (University of Buffalo Toxicology Research Center) acute toxicity evaluation at 1200 times the anticipated imaging mass dose in rats and rabbits. The agent was well tolerated and no acute toxicities were found at this dose level. Due to its selective tumor uptake and retention properties in a variety of rodent tumor models and subsequent excellent safety profile in rats and rabbits, NM-404 was selected to undergo initial human pharmacokinetic evaluation in non-small cell lung cancer (NSCLC) patients.
• NSCLC non-small cell lung cancer
• NM-404 displayed significantly lower liver, kidney and abdominal background radioactivity levels, which in addition to providing promise for lung tumor imaging, suggests further evaluation of this agent in human colorectal, pancreatic and prostate cancer patients is warranted. Moreover, a lack of urinary bladder radioactivity, suggests little renal clearance of the agent or its metabolites over the time points examined. This represents a significant advantage over 1 ⁇ F-fluorodeoxyglucose (FDG), a PET agent used routinely for tumor imaging today, which undergoes significant renal elimination, thus prohibiting imaging in the area of the prostate.
• FDG F-fluorodeoxyglucose
• PET imaging with ' 24 I affords over 40 times the sensitivity of planar 131 l-gamma imaging
• PET unlike traditional gamma-camera imaging, also offers significant resolution enhancement and 3-dimensional imaging capabilities, as welj as superior quantitation properties relative to planar scintigraphic imaging.
• the long, 4-day physical half-life of this PET isotope is well suited to the tumor uptake and retention kinetics of NM-404 and the inventors are in the process of extending the imaging studies with NM-404 to include PET.
• NSCLC Non-small cell lung cancer
• PLE Phospholipid ether analogs
• phospholipids ethers are characterized by the presence of an ether-linked long chain alkyl or alkenyl alcohol connected to a glycerophosphocholine molecule ordinarily found in mammalian cells as a minor component of the total phospholipid content.
• PLE phospholipids ethers
• NM404 is (a) selectively retained in 27111 tumor models, including lung and brain tumors as well as PC-3 prostate bone metastases, (b) is not retained in normal, premalignant, or hyperplastic tissues and, (c) is not retained in inflammatory tissues.
• first-in-human pharmacokinetic studies with 131 I-NM4O4 in patients with NSCLC the inventors have found that 131 I-NM4O4 is safe, and that from 24-48 hours is the optimal scintigraphic imaging time point for tumor detection. These studies also revealed significantly lower liver and background activity levels relative to earlier promising analogs and confirmed the agent doesn't cross the intact blood brain barrier.
• NM404 be further developed for PET imaging.
• the inventors have recently radioiodinated NM404 in excellent yield with commercial iodine-124, a relatively new, long-lived PET isotope, the half-life of which appears ideally matched to the pharmacokinetic profile of NM404.
• Initial microPET scans obtained with 124 I-NM4O4 in xenograft and spontaneous mouse and rat tumor models confirmed universal tumor avidity and prolonged retention. Extending these results to PET scanning is now necessary in order to accurately characterize and quantitate the in vivo distribution properties of this agent.
• the primary objective of this proposal is to further develop NM404 for PET/CT imaging in NSCLC patients with this radioisotope.
• the present invention studies (a) the efficacy of imaging primary NSCLC tumors with 124 I-NM4O4 PET/CT in patients with NSCLC undergoing resection, by comparing pre-operative images with pathological findings, (b) the specific tumor accumulation and metabolic fate of NM404 in NSCLC patients undergoing resection, and correlate tumor retention with decreased phospholipase-D activity; (c) preliminary data regarding the sensitivity of imaging locoregional, and metastatic tumors with 124 I-NM4O4 PET/CT in patients with NSCLC, by comparing these results with FDG PET/CT scanning; and (d) preliminary data regarding the specificity of imaging with 124 I-NM4O4 PET/CT, by imaging patients who present with solitary pulmonary nodules, or have a diagnosis of pulmonary sarcoidosis or granulomatous infections such as fungal or mycobacterial infections, or bacterial pneumonias.
• NM404 Due to its prolonged tumor retention properties, 125 l-labeled NM404 has recently afforded significant tumor regression (vide infra) in SCID mice bearing A549 human lung tumor xenografts. Exhibiting both diagnostic and therapeutic utility, NM404 is being developed as a true, potentially universal, diagnostic and therapeutic agent. [Para 145] NM404 has now been evaluated in 27 animal tumor models, including several lung, and it is clear that once the agent enters tumor cells, it reaches a metabolic dead end and becomes trapped. Prolonged tumor retention of this agent is demonstrated in a human adrenal tumor xenograft implanted into SCID mice (Fig. 14). NM404 is also retained in spontaneous murine lung tumors (Fig. 13).
• FPara 1491 Successful Radiolabelinq of NM404 with iodine-124 (Para 1 50]
• the inventors have obtained high specific activity sodium- lodide-124 in 0.1 N NaOH from Eastern Isotopes (Sterling, VA). Radiolabeling of NM404 is achieved in greater than 60% isolated radiochemical yield by modification of an isotope exchange method. Briefly, a 2-ml glass vial is charged with 10 mg of ammonium sulfate dissolved in 50 ⁇ l of deionized water. Glass beads are added, a Teflon lined septum and screw cap are added and the vial gently swirled.
• a solution of 10 ⁇ g (in 10 ⁇ l of ethanol) of stock NM404 is added followed by aqueous sodium iodide-124 (1 -5 mCi) in less than 30 ⁇ l aqueous 0.01 N sodium hydroxide.
• the reaction vile is swirled gently.
• a 5-ml disposable syringe containing glass wool in tandem with another 5-ml charcoal nugget filled syringe with needle outlet are attached.
• the glass wool syringe acts as a condensation chamber to catch evaporating solvents and the charcoal syringe traps free iodide/iodine.
• the reaction vessel is heated in a heating block apparatus for 45 minutes at 1 50°C after which Four 20 ml volumes of air are injected into the reaction vial with a 25-ml disposable syringe and allowed to vent through the dual trap attachment. The temperature is raised to 160°C and the reaction vial is heated another 30 minutes. After cooling to room temperature, ethanol (200 ⁇ l) is added and the vial swirled. The ethanolic solution is passed through a pre-equilibrated Amberlite IRA 400-OH resin column to remove unreacted iodide.
• the eluent volume is reduced to 50 ⁇ l via a nitrogen stream (use charcoal syringe trap) and the remaining volume injected onto a silica gel column (Perkin Elmer, 3 ⁇ m X 3cm disposable cartridge column eluted at 1 ml/ min with hexane/isopropanol/water (52:40:8)) for purification.
• Final purity is determined by TLC (plastic backed silica gel-60 eluted with chloroform-methanol-water (65:35:4, Rf -0.1).
• the HPLC solvents are removed by rotary evaporation and the resulting radioiodinated NM404 is solubilized in aqueous 2% Polysorbate-20 and passed through a 0.22 ⁇ m filter into a sterile vial. Radiochemical purity is typically greater than 99%.
• I-NM4O4 pilot therapy study in SCID mice with human A549 lung tumor xenografts.
• 125 I-NM4O4 was administered as either a single dose or alternatively in 3 doses (once a week for 3 weeks) to groups of 6 mice and a separate cohort received an equivalent mass dose of unlabeled NM404 for comparison.
• Single doses were 50 or 500 ⁇ Ci and the repeat dose group received a total of 3 weekly 50 ⁇ Ci doses. Tumor growth was monitored for 10 weeks following final injection.
• NM404 has been administered in a tracer dose for imaging (0.3 ⁇ g/kg body mass) in the Phase 1 NSCLC trial at the University of Wisconsin. A 70 kg subject would thus receive approximately 21 ⁇ g of NM404, although recent improvements in the exchange labeling procedure have resulted in much smaller mass doses being injected. Given the recent improvements in labeling and specific activity, the inventors were able to already reach a mass dose in the range of 142,9 ng/kg BW which corresponds to 0.22 nmol/kg BW or a total mass dose of 0.010 mg per 70kg patient. Further improvements are anticipated based on a new labeling methodology as described below, which will likely result in a greater than 50-fold reduction in the mass of compound required. Taken those future improvements into account, inventors believe that the intended clinical mass dose of 1-125-NM404 to be given in the clinical trials performed would be roughly 3-5 ng/kg BW or about 250 ng per 70kg patient.
• test article was a solution of C-NM404 (active ingredient) containing inactive ingredients (vehicle).
• the control solution for this study was the vehicle without the active ingredient.
• test article was formulated as follows:
• Inactive Ingredient 2% Polysorbate20 in sterile water (injection grade)
• Inactive Ingredient 2% Polysorbate20 in sterile water
• the test article (C-NM-404) was administered at greater than 200 times the anticipated clinical dose.
• the control and test rats were injected intravenously in the lateral tail vein.
• the rats were injected with the test or control article intravenously at 2 ml/kg of body weight using a 25 gauge needle and a 1 -ml syringe.
• the injections were given by alternating rats from the control group with rats from the test group.
• the injections were given cautiously over a 30 second to one minute time interval.
• the injection to control rat #27-01 was given at 9:03 am and last injection to test rat #27-16 was given at 1 1 :01 am.
• the thymus, heart, lungs, spleen, kidneys, liver, brain, and testes were collected, examined grossly, weighed and sectioned for pathology.
• the tissue samples (except thymus) were placed in jars of "Z-fix" fixative for histology.
• the unique numbers were also applied to each cage indicating which rats were housed within that cage. There were four cages of control rats and four cages of test rats. The rats were weighed and the mean weight of the control group was 0.234 kilograms and 0.238 kilograms for the test group. The two groups of eight (8) rats were established by random assignment. The rats were weighed daily until the termination of the study on Day 14.
• Duration of Treatment Single dose Reference therapy, dose and mode of administration, batch number: 2% Polysorbate20 in sterile water, 2 ml/kg of body weight, given via tail vein injection over 30-60 seconds.
• Inventors approach to the development of safe and effective cancer therapies is to design small-molecule carrier molecules which are capable of being selectively retained in cancer tissue, but not or minimally in non-cancerous tissues.
• An extension of this approach to radiotherapy would exploit the selective delivery of the radiopharmaceutical to deposit therapeutic levels of radiation within the tumor mass while minimizing radiation-induced damage to normal tissues.
• This technology is based on the unique biochemical and pharmacological properties of phospholipid ethers (PLE's) and especially its sub-group alkyl phosphocholine analogues, such as NM404, which displays a high degree of tumor selectivity.
• Phospholipids are an essential component of cellular membranes where they impart structural integrity and are intimately associated with a variety of cell signaling processes.
• Phosphatidylcholine commonly known as lecithin
• Phospholipid ethers represent a minor subclass of phospholipids that also reside in membranes. As the name implies, these lipids contain an ether rather than an ester linkage at the C-I position.
• Platelet-activating factor (PAF) represents one of the better known phospholipid ethers.
• the iodinated APC analogues were readily labeled with all iodine radioisotopes using an isotope exchange method.
• These PLE analogs are specifically designed to incorporate aromatic radioiodine in order to render the molecule stable towards in vivo deiodination.
• the low level of thyroid activity in all prior preclinical imaging and tissue distribution studies (on both a % injected dose/g and % injected dose/organ basis) has confirmed the in vivo stability of the radioiodinated PLE analogs.
• NM- 324 [ ⁇ -O-iodophenyO-dodecylphosphocholine], initially showed the most promise in animal tumor localization studies.
• NM324 During initial human pharmacokinetic studies with the prototype agent, NM324, an unacceptable accumulation in liver tissue was observed and additional experiments to identify PLE compounds with superior tumor localization and background clearance properties were performed.
• NM404 [18-(4-iodophenyl)-octadecylphosphocholine] emerged due to its enhanced ability to localize in tumor, its increased metabolic clearance from the liver, and its longer plasma half-life.
• the lead compound NM404 has now been evaluated in over 25 animal tumor models and in every tumor model and tumor type studied so far, NM404 has shown tumor-selective retention. Prolonged tumor retention of 12S I-NM4O4 has been demonstrated in mice for periods of 20-60 days post-injection. Such very extensive and protracted tumor retention characteristics may significantly enhance the radiotherapeutic efficacy of the agent, especially for isotopes with a slow radioactive decay like e.g. iodine- 1 25.
• NM324, the predecessor of NM404 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. Three major enzymatic pathways are involved in the metabolism of PLE. O-Alkyl glycerol monooxygenase (AGMO) is responsible for cleavage of the alkyl ether linkage at C-I to form either the long chain fatty alcohol or subsequently, the corresponding fatty acid. Phospholipases C (PLC) and D (PLD), on the other hand, give rise to the glycerol or phosphatidic acid products, respectively.
• AMO O-Alkyl glycerol monooxygenase
• Phospholipases C (PLC) and D (PLD) give rise to the glycerol or phosphatidic acid products, respectively.
• NM324 was not a substrate for this enzyme when compared to [3H]-lyso-PAF (platelet activating factor), which was extensively metabolized.
• [3H]-lyso-PAF platelet activating factor
• NM324 was analyzed as a substrate for PLC isolated from Bacillus cereus and was not hydrolyzed relative to l - ⁇ almitoyl-2-pH]-palmitoyl-L-3-phosphatidylcholine (DPPC), which underwent significant hydrolysis.
• DPPC l - ⁇ almitoyl-2-pH]-palmitoyl-L-3-phosphatidylcholine
• PLE analogs were subjected to a phospholipase D (PLD) assay.
• the PLD which was isolated from cabbage, is similar to mammalian PLD 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 PLD.
• NM404 is a metabolic substrate to human Phospholipase D, and that the relative absence of Phospholipase D in cancer cell membranes is the underlying mechanism for tumor-selective retention of NM404. Although known from the literature (reference???), it is still unclear why cancers lack PLD in their membranes.
• NM404 precursors were also subjected to in vitro metabolism studies in various cell lines including Walker tumor cells, rat muscle (H9c2), and rat hepatocytes.
• 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 48h time period studied.
• NM404 may be due to a decrease in the membrane levels of PLD, thus precluding metabolism and clearance of NM404 from the cell.
• PLD membrane levels of PLD
• PLE analogs were sequestered by and subsequently metabolized by normal cells (which contained normal levels of PLD). If malignant tumor cells would possess a normal complement of PLD, that the agent would have been metabolized and eliminated from the tumor cells as well. Conversely it could be deduced that the lack of metabolism and clearance of the agent from malignant cells would support the hypothesis that these neoplastic cells lack PLD relative to surrounding normal host cells.
• NM324 and NM404 are similar in structure to miltefosine (hexadecylphosphocholine), an antitumor ether lipid studied most extensively in Europe.
• miltefosine hexadecylphosphocholine
• 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 (Lohmeyer M, Bittman R. Drugs of the Future 1994; 1 9: 1021 -1037).
• 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 uncertainty remains regarding the mode of PLE uptake into cells, most evidence now supports the idea that these ether lipids are directly absorbed into cell membranes where they accumulate.
• iodine-125 is the most appropriate radioisotope for combination with the NM404 targeting backbone, since: [Para 205] The long isotope half-life of iodine-125 matches perfectly with the long and stable tumor retention of NM404, delivering therapeutic radiation doses for an extended period of time. [Para 206] The effect of iodine-125 is caused by both, low-energy gamma/X-ray irradiation and by Auger electrons, all of which are of very limited treatment distance. Since NM404 is taken up into the tumor, iodine- 125 can effectively deliver a tumor dose but it is sparing surrounding healthy tissue.
• Iodine-125 has Auger electrons as one of its radio-decay byproducts (Figure 2). Auger electrons cause a pronounced biological effect, but have a very short treatment distance. Since NM404 is taken up directly into all cell membranes of cancer cells (including the nuclear membrane), the treatment distance to the DNA is very low. It may effectively allow Auger electrons to be a major contributor to I-125-NM404 treatment effects.
• the iodine-125 isotope is used for formulation in this application because it exhibits favorable characteristics for cancer radiotherapy, with properties listed below: [Para 209] Gamma Irradiation
• 1-131 -NM404 could potentially be used for radiotherapy, the inventors believe 1-125 to be the more optimal isotope because its lower energy radiation has a shorter radiation haif distance than 1-1 31 and thus hypothesize that it will produce less damage to healthy tissue. Therefore, it was decided that 1-125-NM404 would be used for all clinical radiotherapy studies performed in order to reduce the potential for collateral damage to healthy tissues.
• iodine-125 Due to its 60-day physical half-life and low energy 35 keV photon emission, iodine-125 is suitable for imaging experiments in mice and rats. Iodine-125 also provides therapeutic efficacy when used in permanent prostate brachytherapy implants ("brachytherapy seeds").
• the major advantage of 1251 is that all the photons are of low energy, insuring very limited exposure of normal tissues surrounding the tumor.
• the major difference between brachytherapy seeds containing iodine-125 and 1-125- NM404 are the effect of Auger electrons. Since brachytherapy seeds have a metal capsule around the iodine-125, only the low-energy gamma and X-ray are of therapeutic value, and Auger electrons are eliminated by the metal capsule.
• I-125-NM404 is taken up into the cancer cell membranes (including into the nuclear membrane), so that Auger electrons can have a major contribution to the therapeutic effect.
• iodine-131 has been used with great efficacy in the treatment of thyroid cancer, a significant disadvantage of iodine-131 is that there is a higher energy gamma emission which could actually expose adjacent surrounding tissues to more radiation than would occur with - iodine-125.
• Units of EDE and ED are mSv/MBq or rem/mCi .
• Phospholipid ethers can easily be labeled with iodine radioisotopes using radiolabeling methods developed in the labs.
• the iodophenyl phospholipid ether analogs are specifically designed so that the radioiodine affixed to each molecule is stable to facile in wV ⁇ deiodination. It was found that any chemical modification of the phosphocholine moiety or shortening the chain length of the iodophenylalkyl moiety to less than 8 methylenes resulted in little or no tumor uptake.
• the inventors have now synthesized over 20 radiolabeled PLE compounds and tested in vitro and in vivo.
• NM-324 Clinical Evaluation of NM324: Although first generation compounds NM-324 and NM-294 displayed similar animal tumor localization characteristics, NM-324 was easier to chemically synthesize and was thus selected as the lead compound for initial clinical studies. Although images obtained in several human lung cancer patients detected tumors, images were complicated by high liver radioactivity ( Figure 3).
• Second Generation PLE Analogs In order to decrease liver uptake and prolong the plasma phase, the inventors examined 9 structural analogs of NM-324 to identify agents that would display improved tumor- to-background tissue ratios with decreased liver uptake. The new PLE analogs were synthesized and radiolabeled with 125 I for initial image analysis in Copenhagen rats bearing Dunning R3327 prostate tumors.
• NM-404 not only exhibited much lower liver activity than its predecessor NM324 but also maintained prolonged tumor retention (Figure 4. NM404 was therefore selected to undergo further imaging and biodistribution analysis in a variety of animal-tumor models. [Para 228] Accordingly, NM404 has now been evaluated in over 20 xenograft and spontaneous animal tumor models as shown in Table 2. In all tumor models the agent displayed significant tumor uptake and retention regardless of location. Although tumor retention can be explained by a lack of metabolic phospholipase enzymes in the tumor cell membranes, the exact mechanism of tumor cell uptake is unknown.
• the inventors know the agent does not localize in benign intestinal adenomas (polyps) so it was desirable to further evaluate the propensity of the agent to localize in an intermediate stage of tumorigenesis, namely hyperplasia.
• the inventors are currently evaluating the selectivity of NM404 in a unique mouse tumor model developed at Wisconsin, wherein both preneoplastic hyperplasias and malignant adenocarcinomas form spontaneously in the mammary gland. Preliminary results in this model indicate that the agent is not taken up and retained in preneoplastic lesions, and thus, appears to be exclusively retained by malignant tumor cells.
• NM404 has been found to be retained in tumor tissue for long and extended periods of time. Tumor concentrations are almost stable for many weeks following administration of NM404, showing slow elimination from cancerous tissue over time. In contrast, NM404 is eliminated from normal tissue within a few days reaching very low levels. [Para 233] Additionally, NM404 was designed to have a long blood half life. This ensures prolonged exposure of NM404 to the tumor tissue and ensures uptake of up to 10-25% of the injected dose into the tumor tissue. The inventors believe that it is very important for a radiotherapy compound to have a large portion of the injected dose accumulating in the tissue of interest.
• NM404 will continuously accumulate in tumor tissue over time.
• An example of this pattern is provided in Figure 5. This may lead to a delayed onset of the therapeutic effect until the accumulation of NM404 into the tumor tissue has been continuing for several days or weeks.
• the first-generation prototype compound NM324 was found to have a elimination half life time in plasma of 2.43 hours in rats.
• the lead compound NM404 has a elimination half life time of roughly 209 hours in rats (distribution phase half life is 4.86 hours).
• iodine-125 Due to its 60-day physical half-life and low energy 35 KeV photon emission, iodine-125 is suitable for imaging experiments in mice and rats. Iodine-125 also affords therapeutic characteristics.
• Figure 6 2 nude mice were each inoculated with subcutaneous squamous cell lines SCCl and SCC6 tumor cell implants on opposing flanks. SCCI and SSC6 cells were used because one is radiosensitive relative to the other. After 14 days when the average tumor size was approaching 0.5 cm in diameter, one of the mice received 20 ⁇ Ci of I-125-NM-404 and the other one receive unlabeled NM404 in an equal mass dose.
• A549 human non-small cell lung cancer, NSCLC, from ATCC
• NSCLC human non-small cell lung cancer
• Tumor cell suspension (1 xl O 6 cells in phosphate-buffered saline) is injected s.c. into the right flank of female SCID mice (6-8 weeks, CB-
• mice 5mm in diameter, mice will be divided into groups of 6 for therapy study. fPara 2451 Dosing:
• Tumor size measured once a week.
• mice [Para 251 ] The tumor model used for this study was A549, a human non- small cell lung cancer, (NSCLC) obtained from ATCC (Manassas, VA). The tumor cells are maintained in Ham's F-I 2K media supplemented with 10% fetal bovine serum. Tumor cell suspension (1 x10 6 cells in phosphate- buffered saline) is injected s.c. into the right flank of female SCID mice (6-8 weeks, CB-I 7 /IcrHsd- Prkcd scld , Harlan). Animals are given free access to food and water. Upon reaching 5-10 mm in tumor diameter, mice will be enrolled in the study.
• NSCLC human non- small cell lung cancer
• A549 human non-small cell lung cancer, NSCLC, from ATCC
• Ham's F- 2K media supplemented with 10% fetal bovine serum.
• Tumor cell suspension (1 x10 6 cells in phosphate-buffered saline) is injected s.c. into the right flank of female SCID mice (6-8 weeks, CB-
• mice 5mm in diameter, mice will be divided into groups of 6 for therapy study.
• PC-3 human prostate cancer, from ATCC
• Tumor cell suspension (I xI O 6 cells in phosphate-buffered saline) is injected s.c. into the right flank of male SCID mice (6-8 weeks, CB-I 7 / ⁇ crHsd- Prkcd sc/tf , Harlan),
• mice Animals are given free access to food and water, tumor growth and animal weight are monitored. Upon reaching 4-5mm in diameter, mice will be divided into groups of 6 for therapy study. fPara 2781 Dosing:
• Tumor size (caliper) measured once a week.
• the present invention provides preliminary data regarding the use of the second-generation PLE analog, NM404, in imaging patients with prostate cancer.
• This agent currently under investigation at the University of Wisconsin, is selectively retained in tumors in high levels, and has high sensitivity and specificity in preclinical models. It has passed acute toxicology testing in both rats and rabbits at >1000 times the anticipated human imaging dose, and the unlabeled agent was administered at 10 times the anticipated imaging mass dose to 10 normal volunteers at the University of Michigan and University of Wisconsin to document safety.
• the inventors hypothesize that imaging with NM404 will ultimately prove as sensitive as imaging with FDC, will also be more specific, may afford therapeutically utility and due to its relatively long half-life be available in virtually every PET facility regardless of location.
• NM404 has high tumor uptake
• this agent also has the potential to be developed as a therapeutic agent when coupled with higher doses of 131 I 1 125 I, another halogen astatine, lodine-125 would be especially desirable in prostate cancer patients due to its short Auger electron path length in tissue, which would theoretically minimize radiation effects in neighboring normal tissues like the rectum.
• the 60 day half-life of iodine- 125 matches exceedingly well with the prolonged tumor retention properties of NM404.
• Phospholipids are an essential component of cellular membranes where they impart structural integrity and are heavily associated with a variety of cell signaling processes. Phosphatidylcholine, commonly known as lecithin, is such an example. Phospholipid ethers, on the other hand, represent a minor subclass of phospholipids that also reside in membranes.
• NM404 12-(4-iodophenyl)- octadecylphosphocholine was selected due to its enhanced ability to localize in tumor, its increased metabolic clearance from the liver, and its longer plasma half-life.
• lymph node metastases were clearly delineated by scintigraphy in a metastatic prostate tumor model following intravenous administration of NM404, but the tracer was not retained by uninvolved lymph nodes.
• iodine-1 23 may also prove suitable for this agent when scanned in a 3-dimensional SPECT mode. Imaging with iodine-1 23 will require further investigation.
• planar 2-dimensional imaging like that currently being performed with iodine-1 31 in humans, requires a delay period to allow background activity to clear from neighboring normal tissues and blood. It is likely that earlier imaging may be possible when scanning with PET and 3D SPECT where neighboring radioactivity is less interfering due to the 3- dimensional nature of these modalities. In organs where background radioactivity remains inherently low (brain for example), it may be possible to use gamma emitting isotopes like iodine-123 which in addition to providing beautiful images, would permit image acquisition later on the same day of injection.
• PET imaging with 124 I affords over 40 times the sensitivity of planar 131 l-gamma scintigraphy.
• PET unlike traditional gamma camera imaging, also offers significant resolution enhancement, image quantification, and 3-dimensional capabilities. Due to the preliminary success of 131 I-NM4O4 in the current lung cancer imaging trial, it is now imperative to label NM404 with iodine-1 24 and evaluate its tumor detection efficacy by PET in order to overcome the limitations inherently associated with planar scintigraphy
• FDG-PET has paved the way for hybrid imaging, its lack of tumor cell specificity will always limit its diagnostic efficacy.
• New molecularly targeted agents like NM404, which display universal tumor uptake and selective retention regardless of location, as well as selectivity for malignant tumor cells and not inflammatory or hyperplastic lesions, will represent a significant improvement in the detection and characterization of cancer.
• NM404 for malignant tumors seen in mouse models
• FDG tumor selectivity
• the agent could be manufactured in one facility and shipped to virtually any location in the world due to its 4-day half life.
• MicroPET images acquired 24h after iv injection of 124 I-NM4O4 were corroborated with contrast-enhanced MRI images and showed intense uptake of the tracer in the brain tumor accompanied by little or no uptake in surrounding intact brain tissue.
• This study represents the first PET image obtained with NM404 and demonstrates the ability to efficiently radiolabel, purify, and formulate NM404 for PET imaging. These compounds may then be used for extending NM404 PET utility into human cancer patients.
• fPara 3061 Determine tumor uptake and retention characteristics of 124 I NM404 by PET-CT in patients with radio ⁇ raphically evident metastatic prostate cancer.
• Inclusion criteria for this study will consist of fifteen patients with metastatic prostate cancer, with at least 5 patients having soft-tissue prostate cancer metastases and at least 5 of the patients with bony metastases identifiable by conventional radiologic studies which include CT scan and bone scan. Following patient enrollment, uptake of radiolabeled NM404 will be measured by 1-1 24 isotope PET-CT scan and correlated with the radiographically evident lesions detected by the patient's other conventional staging studies.
• Radioiodination of stable NM404 with 124 l-sodium iodide is routinely achieved by modification of an ammonium sulfate-mediated isotope exchange reaction reported by Mangner and recently optimized for NM404 in our lab.
• Exchange reaction methodology has been used effectively for initial human trials with NM324, the predecessor of NM404 and is currently being used for preclinical studies and the human lung cancer trial. Briefly, a 2-ml glass vial is charged with 10 mg of ammonium sulfate dissolved in 50 ⁇ l of deionized water.
• the glass wool syringe acts as a condensation chamber to catch evaporating solvents and the charcoal syringe traps free iodide/iodine.
• the reaction vessel is heated in a heating block apparatus for 45 minutes at 1 50° C.
• Four 20 ml volumes of air are injected into the reaction vial with a 25-ml disposable syringe and allowed to vent through the dual trap attachment.
• the temperature is raised to 160° C and the reaction vial is heated another 30 minutes.
• ethanol 200 ⁇ l
• the ethanolic solution is passed through a pre-equilibrated Amberlite IRA 400-OH resin column to remove unreacted iodide.
• the eluent volume is reduced to 50 ⁇ l via a nitrogen stream (use charcoal syringe trap) and the remaining volume injected onto an HPLC silica gel column (Perkin Elmer, 3 ⁇ m X 3cm disposable cartridge column eluted at I mI/ min with hexane/isopropanol/water (52:40:8)) for purification.
• HPLC silica gel column Perkin Elmer, 3 ⁇ m X 3cm disposable cartridge column eluted at I mI/ min with hexane/isopropanol/water (52:40:8)
• the HPLC solvents are removed by rotary evaporation and the resulting radioiodinated NM404 is solubilized in aqueous 2% pharmaceutical grade Polysorbate-20 (0.1 ⁇ l/mg 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 will be achieved by filtration through a sterile 0.2 ⁇ m filter unit.
• Each of the solutions will be tested for pyrogens using the Limulus Amebocyte Lysate test kit. This is the same procedure currently employed for the preparation, purification and sterile formulation of 1-131 -labeled NM404 for lung cancer patient studies.
• the drug Master Formulation Card and Product Preparation Checklist are included in the supplemental section of this proposal.
• This method has afforded sufficient pure injectable 131 I-NM4O4 for our ongoing preclinical and human trials. We have not had a labeling failure to date.
• the 131 I-NM4O4 dose is currently calculated as follows: Animal biodistribution data is used to determine the percentage of injected dose/organ at varying time points. These animal data are extrapolated to man by means of MIRD formalism (MIRDOSE PC v3.1) using standard conversion factors for differences in organ mass and anatomy between rat and standard man, providing predicted human organ doses; Based on these predicted doses, the permissible mCi dose to be injected into humans is determined using the maximal doses legally permitted by RDRC regulations for specific human tissue as defined in the Federal Register (21 CFR Part 361.1) e.g. whole body, blood, blood forming tissues, eye lens, gonads - 3 rem/dose; any other tissue - 5 rem/dose.
• MIRDOSE PC v3.1 MIRD formalism
• PET-CT images (whole body and selected regional conjugate views) for the first five patients will be obtained at 48 and 96' hours post injection. PET images will be obtained using a CE Clinical CE Discovery LS PET-CT scanner utilizing appropriate attenuation correction and optimized to iodine-124. The 48 and 96 hour time points are based on preliminary data regarding optimal imaging times for 131 I-NM4O4 in lung cancer, however, the optimum tumor-targeting time for 124 I-NM4O4 in human prostate cancer is yet to be determined.
• 124 I-NM4O4 tracer accumulation and washout will be recorded on the GE Discovery LS PET/CT scanner or on CE Advance PET scanner available at our clinic.
• a CT scan will be performed first for patient localization and attenuation correction on the Discovery LS PET/CT scanner; a transmission scan will be performed on the Advance PET scanner.
• Each PET scan will be corrected for random, dead time, attenuation, and scatter.
• First scanning will be performed immediately at 48 and 96 h after the 124 I-NM4O4 injection. Whole body scans will be performed with increased scanning time over the tumor site (up to 30 min). Images will be reconstructed using an iterative OSEM reconstruction method with attenuation correction, and smoothed with a Gaussian filter.
• ROIs will be outlined to represent various organs in the field of view (typically heart, lung, muscle, liver, stomach, spleen, intestine, kidney, and bladder). For each ROI and for each time frame, the average radioactivity concentration will be calculated and standardized uptake values (SUVs) calculated.
• SUVs uptake values
• the uptake rate classified as positive for lesion identification will be at most 50% versus the alternative hypothesis that the rate is greater than 50%.
• We anticipate the uptake rate classified as positive for lesion identification will be at least 80%.
• the one-sample binomial test with the null hypothesis that the uptake rate classified as positive is at most 50% has 85% power to detect a rate of 80% at the (onesided) 10% significance level.
• a rate of 90% will be detected with 95% power.
• the proportion of tumors classified as positive for lesion identification will have a standard error of at most 13% and the 90% confidence interval for the proportion will be no wider than 39%.
• FPara 31 81 Determine the specific tumor accumulation and metabolic fate of 124 I NM404 in patients with clinically organ-confined prostate cancer who are candidates for a radical prostatectomy with bilateral pelvic lymph node dissection.
• the prostate biopsy cores will be obtained in sextant distribution (right and left apex, mid-gland and base regions) from the prostate specimen in the OR immediately following surgical removal. These six biopsy cores will be evaluated by frozen section pathology to ensure a representative sampling of both malignant and benign prostate tissue.
• a second set of 6 biopsy cores obtained simultaneously from the same corresponding locations in the prostate will be analyzed for uptake of 124 I-NM4O4. Accordingly, they will be photographed and undergo high-resolution scanning on a Bioscan AR2000 radioscanner and also weighed and radioactivity quantitated in a well counter. Radioactivity concentration will be determined on a %dose/g tissue sample basis for comparison. These results will be compared to histology results in order to confirm localization in tumor relative to surrounding uninvolved tissue.
• the core biopsies will be labeled with the patient's study identification number and location in the prostate from which it was obtained.
• the one-sided t-test with a one-sided 10% significance level has 86%, 90% and 95% power to detect the effect size of 0.8, 0.90, and 1.00, respectively.
• the standard deviation of the tog ratio is 2.0, the 5:1 ratio in the accumulation of NM404 in the tumor to normal tissues will be detected with 86% power.
• the standard deviation of the log ratio is 1.6, a ratio of 5: 1 in the accumulation of NM404 in tumor to normal tissues will be detected with 95% power.
• Imaging procedures in this study will be identical to the first study, except that patients will be imaged only once, based upon the optimal imaging time as identified in the first protocol. Patients will receive an injection of 124 I-NM4O4 (0.3 ⁇ g/kg body weight, 1 mCi or the limit established by dosimetry calculations in the first set of patients). Patients will also have a PET-CT scan 48 hours after infusion. Vital signs will be obtained post 124 I-NM4O4 infusion.
• the null hypothesis that the sensitivity of NM404 in this patient population is at most 30% will be tested against the alternative that the sensitivity is greater than 30%.
• the one-sample binomial test has 83% power to detect a sensitivity of 75% at the one-sided 10% significance level. A sensitivity of 80% will be detected with 90% power.
• patients with occult metastatic disease may unnecessarily undergo local treatment with associated risks of therapy.
• patients with a rising PSA due to local recurrence in whom systemic recurrence cannot be excluded with confidence, may unnecessarily undergo hormonal ablation, which is generally not considered curative and is associated with osteoporosis development, decreased libido, weight gain, menopausal symptoms, and overall malaise, as well as the evolution of hormonally independent prostate cancer.
• CT computed tomography
• MRI magnetic resonance imaging
• Radioimmunoscintigraphy with lndium-1 1 1 capromab pendetide has been utilized in patients following prostatectomy with a rising PSA who have a high clinical suspicion of occult metastatic disease and no clear evidence for metastatic disease in other imaging studies.
• NM404 second-generation PLE analog
• FIG. 331 Certain embodiments of the present invnetion provides preliminary data regarding the use of the second-generation PLE analog, NM404, in imaging patients with prostate cancer. It has been shown that NM404 is (a) selectively retained in a wide variety of tumor types in preclinical models, with a high degree of sensitivity, (b) is safe in humans, (c) can be radiolabeled with 1-1 24, and (d) has appropriate dosimetry characteristics labeled with 1-1 31 .
• Radiolabeled monoclonal antibodies specific to the extracellular domain of prostate-specific membrane antigen preclinical studies in nude mice bearing LNCaP human prostate tumor, J Nucl Med.
• Buhler KR Vessella RL. Establishment and characterization of osseous prostate cancer modelsMntra-tibial injection of human prostate cancer cells.

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