MX2007007497A - Phospholipid ether analogs for detecting and treating cancer. - Google Patents

Abstract

The present invention provides methods for treating, 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 using phospholipid ether analogs.

Description

ANALOGUES OF PHOSPHOLIPID ETHER FOR THE DETECTION AND TREATMENT OF CANCER FIELD OF THE INVENTION The present invention generally provides methods and techniques for the detection and treatment of various cancers.

BACKGROUND OF THE INVENTION The invention is generally related to phospholipid ether analogues and the use thereof, and is specifically related to the use of phospholipid ether analogues and combinations thereof for the diagnosis of metastasis, treatment, pharmacokinetic studies. , of dosimetry, and toxicity of various types of cancer, such as, giant cell lung cancer, prostate cancer and metastasis thereof.

Giant cell lung cancer (C) Giant cell lung cancer (C) is the leading cause of death from cancer in the United States today. Surgical resection in appropriately selected patients offers the best chance of cure. A pre-operative appraisal praises Jif ubión me -.-, local ethics, Regional and distant in this way is decisive for optimal management. Image formation with FDG PET scanning has recently become a "gold standard" for C imaging, due to improved sensitivity, particularly when compared to CT imaging. However, its sensitivity to identify the involvement of mediastinal lymph nodes is only about 90%, and lacks specificity, particularly in patients with inflammatory or granulomatous disease, is particularly problematic. In addition, its utility to diagnose brain tumors or metastases is limited due to the high metabolic background noise of normal brain tissue. The evaluation of the state of the mediastinal lymph nodes is essential due to the nodal metastasis, which occurs in almost half of all patients with C. It is probably the most frequent barrier to cure. Accurate grading can also save patients the morbidity of unnecessary, non-curative surgical procedures. Therefore, there remains a need for a technique for image formation that is more sensitive, specific, and accurate than any technology currently available. The current conventional modalities have limitations. The formation of anatomical images with computed tomography (CT) and magnetic resonance imaging (RI) are impractical for total body selection, although they are the methods for Non-invasive image formation that is used more widely for the evaluation of a localized regional dissemination. However, CT depends on the size criteria of more than one centimeter to diagnose abnormal nodes. Scanning by positron emission tomography (PET) with 18F-fluorodeoxyglucose (FDG) has generated considerable interest as a technique for oncological imaging. Prospective, a recent study compared to the ability of a standard procedure for graduation using C (bone scan by CT, ultrasound, etc.), and PET scan to detect metastases in mediastinal lymph nodes and distant sites. The mediastinal involvement was confirmed histopathologically, and the distant metastases were confirmed by different tests for imaging. The sensitivity and specificity of PET to detect mediastinal metastases were 91% and 86%. respectively; to detect distant metastases, 82% and 93%, respectively. This is compared with the sensitivity and specificity for CT imaging of the mediastinal involvement of 75% and 66%, respectively. A meta-analysis involving 39 studies and more than 1000 patients, it was also found that PET-FDG was more accurate than CT for mediastinal grading, (sensitivity and specificity of 85% and 90%, respectively, for FDG-PET exploration and 61% and 79% for CT), although FDG-PET was less specific when CT showed elongated mediastinal lymph nodes (78%) . FDG-PET, has been shown to reduce useless thoracotomies in patients. However, due to the false positive and false negative ratio, confirmatory mediastinoscopies are often recommended. For example, a retrospective study involving more than 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 an extra-thoracic disease, particularly in patients with intermediate stages of lung cancer. A study conducted by the American College of Physicians involving more than 300 patients, found that a disease Unsuspected metastatic or second primary malignancies were identified in 18 of 287 patients (6.3%). More studies, although not all, suggest that by correctly identifying advanced disease, PET could avoid unnecessary thoracotomy in 1 of 5 patients. Conventional techniques for anatomical imaging such as CT scanning are also deficient to predict survival after treatment. In a recent study involving 73 NSCLC patients, who received treatment with concurrent chemo / radiotherapy based on cisplatin or radiotherapy alone for advanced disease, the response through conventional CT imaging does not correlate with survival. The response by FDG-PET scans, however, does not correlate significantly with survival (p <0.001). Survival from the date of follow-up examination with PET was 84% and 84% at 1 and 2 years, respectively, for 24 patients who had achieved a complete PET response, although only 43% and 31% of the 32 patients they had not done it (p = 0.010). These results corroborate similar findings recently reported by other authors, which also show an absorption correlation on exploration. with PET, with a biological aggressiveness of the tumor, and that PET imaging later, after finishing the treatment is quite predictive of future survival. It is generally accepted that image formation by FDC-PET is a poor method to identify a metastatic disease to the brain in patients with NSCLC. Under normal conditions, the gray matter of the brain had a high glucose utilization and therefore the absorption of FDC is normally high. While cerebral metastatic disease is often quite metabolic and often does not demonstrate increased absorption of FDG, it is often less than the gray matter of the brain and therefore brain metastases may not be striking. In one series, the sensitivity and specificity for the identification of a metastatic brain disease in patients with NSCLC was 60% and 99% for FDG-PET, and 100% and 100% for conventional imaging. Therefore, imaging using FDG-PET is not considered to be the best method to evaluate a patient with NSCLC for a metastatic disease for the brain. Another disadvantage of FDG is that it is not specific for tumors, although it accumulates in both malignant and non-malignant hypermetabolic tissues. The overwhelming majority of False positive results (positive result when the radiological abnormality is not due to cancer) with lung FDG-PET scans are due to inflammatory and infectious causes. FDG is a non-specific tracer and accumulates in areas of infection or inflammation. In the lung, these areas may be parenchymal nodes localized or more diffuse (subsegmented, segmented or lobular) or in the hilar and mediastinal nodes. In a recent study from Japan, of 116 lung nodules 1-3 cm in diameter, 15 of the 73 malignant nodules were false-negative in FDG-PET and 15 of 43 benign nodules were false-positive in PET-FDG. In focal pneumonias, which cause nodules of ground vitreous opacity, the false positive proportion was as high as 80%. In another study, ten patients with extrapulmonary cancer had positive FDG-PET uptake in the lung; 6 had intense focal or multifocal absorption and four had absorption in a more segmented or lobular pattern. In all 10 patients, the absorption was due to consolidation or atelectasis and the final diagnosis in the follow-up was an inflammation or pulmonary infection. In addition to active bacterial pneumonias, false-positive FDC-PET results may be present in many other infectious and inflammatory conditions in the lung. In the Midwest of the United States, many asymptomatic people have pulmonary nodules and elongated nodes due to a previous infection with histoplasma; Although many of these nodes are at rest, some represent a latent or active infection. Pulmonary sarcoidosis is probably one of the most common active inflammatory granulomatous processes in the lung. Interestingly, when serial FDC-PET scans have been performed in a patient who has been treated with oral corticosteroids for pulmonary sarcoidosis, the FDC absorption decreased and then faded. FDG-PET is often also negative in malignancies with a low metabolic rate, such as carcinoma or bronchoalveolar carcinoid. Therefore, a radiopharmacist who could identify early and accurately a metastatic disease in patients with NSCLC could have a significant impact on patient care, both in terms of graduation and response to therapy. Although PET imaging has improved the diagnostic effectiveness in this area compared to CT, it continues to make a need for a precise imaging technique that is not based on metabolic activity, which is not specific, but which is based on a tumor-specific function that may not Select invasive to the total body, including the brain.

Prostate cancer Only in 2004, approximately 230,110 new cases of prostate cancer were diagnosed in the United States. Despite improved techniques in the definitive local treatment of prostate cancer clinically confined to organs by radical prostatectomy, such that many men have been cured with primary therapy alone, no less than 40% of patients experienced biochemical reappearance with long follow-up term. This reappearance is typically defined as a postoperative PSA level that is greater than or equal to 0.4 ng / ml because patients with PSA levels above this threshold generally develop clinical evidence of recurrence within 6-49 days. months although a PSA level greater than or equal to 0.2 has been proposed more recently. With limited success, clinical and pathological criteria are currently used to determine the likelihood of a reoccurrence of systemic disease. Increasing factors of the likelihood of a systemic reappearance include a high preoperative level of PSA, as well as pathological features of the surgical specimen including the Cleason score > 7, involvement seminal vesicular disease, and involvement of lymph nodes. In contrast, extracapsular extension, positive surgical margins and the Gleason score < 7 are factors associated in general with local reappearance. In addition, the rate of PSA emergence after prostatectomy has been used to determine if the reappearance of the disease is local or systemic. For example, Partin et al. Reported that an increase in PSA less than 0.75 ng / ml / year was more frequently associated with a local reappearance. In addition, Patel et al. Reported that twice the PSA time of more than 12 months correlates with a local reappearance. Despite these clinical and pathological criteria, the inventors have been unable to accurately select patients adequately for local therapy in such a way that many men can receive unnecessary hormonal ablation. One of the greatest challenges in the treatment of patients with prostatic cancer confined clinically organ or patients with biochemical reappearance after a definitive treatment of a presumed disease confined to organs remains to accurately distinguish a localized versus metastatic disease. These diagnostic capabilities are important to identify patients who may benefit from modalities of effective local treatment including surgery, external beam radiation, brachytherapy, and cryotherapy. Because inventors do not currently have an accurate means of graduation, patients with metastatic disease that is hidden unnecessarily may undergo local treatment with associated risks of therapy. In addition, patients with an increase in PSA due to a local reappearance, in whom the systemic reappearance can not be excluded with confidence, can unnecessarily undergo hormonal ablation, in general it is not considered curative and is associated with the development of osteoporosis, libido decreased, weight gain, menopausal symptoms, and malaise, as well as the evolution of prostate cancer and hormonally independent. While conventional studies for imaging, for example, computed tomography (CT) and magnetic resonance imaging (MRI) are useful for assessing a soft tissue metastasis, the vast majority of prostate cancer metastasizes only to bone. In this way, the usefulness of CT and MRI examination to assess the disease is sub-optimal and modalities for more sensitive imaging are needed for prostate cancer, whether it reappears locally or metastatically.

Radioimmunoscintigraphy with indium-111 capromab pendetide (ProstaScint, Cytogen Corp, Princeton, NJ) has been used in patients after a prostatectomy with an increase in PSA who had a high clinical suspicion of hiding a metastatic disease and without clear evidence of a metastatic disease in other imaging studies. This scan is based on a radiolabelled murine monoclonal antibody that is specific for PSMA (prostate-specific membrane antigen), a transmembrane protein that is expressed specifically in both normal and malignant prostatic epithelial cells. While the ProstaScint radioimmunoscintigraphy has shown that it is promising to diagnose a locally recurrent disease in the prostate bed in patients with an increase in PSA, the clinical results for this exploration have been somewhat variable, with sensitivities varying between 44% and 92% and specificities between 36% and 86%. In addition, when posterior biopsy was used as the reference standard for a local reappearance, false-negative ProstaScint studies have been reported in 10% to 20% of cases. In addition, the false-positive absorption of 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 These data, the use of a ProstaScint scan for patients at risk of hiding metastases from prostate cancer remains controversial. In patients with metastatic prostatic cancer, training has now been used. Imaging by positron emission tomography (PET) to measure the metabolic activity of bone metastases. This technique has proven to be effective in distinguishing active bone metastases from osteoblastic activity that occurs as a result of bone healing after successful treatment of a metastatic disease. This issue has been better assessed by PET than by either bone scan or CT. In addition, changes in the findings can be observed by PET exploration before 4 weeks after the start of systemic treatment in patients with metastatic prostate cancer, while, in many cases, no significant change on conventional bone exploration has been observed. Therefore, imaging using PET technology can be useful to monitor the response to treatment in these patients. PET image formation with 18F-FDC has generated considerable interest as a technique for imaging. Recently, it has been shown that FDC-PET can distinguish between active and quiescent bone metastases in patients with prostate cancer. It shows that the intensity of absorption of FDG reflects the metabolic and biological activity of these lesions in contrast to traditional bone exploration with tecténio-diphosphonate compounds in which the non-specific osteoblastic activity can be suppressed as a false positive signal after treatment. In addition, a false negative reading can be obtained because the first metastases, which are initially sown in the bone marrow, will not necessarily produce a signal until an osteoblastic response is present. Therefore, a persistently positive bone scan does not necessarily indicate the presence of residual viable metastases and a negative bone scan result may not accurately reflect the concern for a metastatic tumor in the patient. FDC-PET, therefore, may prove to be beneficial in guiding the management of patients with bone metastases and in a retrospective FDC-PET and helical CT study, they have been independently shown to be more effective than monoclonal antibody 1 imaging: L1In to detect a metastatic disease. Although FDG-PET scanning is a promising technique for imaging prostate cancer patients, most prostate cancers are slow growing and therefore do not accumulate FDC, and thus the image is not well formed with that agent. Therefore, FDG is excreted in 1 to urine and in this way the accumulation of FDG in the bladder will reduce to a minimum the probability to detect local reappearances of prostate cancer. In fact, Morris et al. Reported difficulty in detecting soft tissue metastases by FDC-PET only when the metastatic sites are obscured by anatomical trajectories of tracer excretion. More recently, PET-CT has been found to be more effective than PET alone in identifying metastatic lesions in patients with suspected occult metastases. In a prospective study of patients with various tumor types, the specificity and accuracy with multiple radiological interpretations were significantly higher for PET-CT. Accordingly, there remains a need to develop a more sensitive and specific imaging test, an agent for molecular imaging, such as phospholipid ether (PLE) compounds. It may be convenient to have tumor-selective radiopharmaceuticals, with minimal accumulation in the bladder, that could accurately identify a metastatic disease early in patients with prostate cancer, which could have an important impact on the care of patients, in terms of both graduation as a response to therapy.

SUMMARY OF THE INVENTION The present invention provides a method for detecting and locating the reappearance of cancer, cancer insensitive to radiation and cancer chemo or metastasis 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 a subject who has or is suspected of having cancer. The method comprises the steps of: (a) administering a phospholipid ether analogue to the subject; and (b) determining whether an organ with suspected recurrence of cancer, cancer insensitive to radiation and chemo or cancer metastasis in the subject maintains a higher level of the analogue than the surrounding regions where a region with higher retention indicates detection and location of the reappearance of cancer, cancer insensitive to radiation and chemo or cancer metastasis. In a preferred embodiment, the phospholipid analog is selected from: where 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 consisting of NH2, NR2, and NR3, wherein R is an alkyl or arylalkyl substituent or where 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, and Z is selected from the group consisting of NH2, NR2, and NR3, wherein R is an alkyl or arylalkyl substituent. In addition, in certain embodiments, X is selected from the group of halogen radioactive isotopes consisting of 18F, 36C1, 76Br, 77R tírv, 82R Brv, 122T i, 123t ±, 1 4T i, 125T, ITU I, "and 11 A, t .. From" ma.y ,, or "r Preferably, the phospholipid ether is 18- (p-iodophenyl) octadecylphosphocholine, 1-0- [18- (p-iodophenyl) octadecyl] -1,3-propandiol-3-phosphocholine, or 1-0- [18- (p -iodophenyl) octadecyl] -2-0-methyl-rac-glycero-3-phosphocholine, where the iodine is in the form of a radioactive isotope. In this method, preferably, the detection is carried out by one of the PET, CT, MRI scan methods or combinations thereof. Another embodiment of the present invention provides a method for the treatment of the reappearance of cancer, cancer insensitive to radiation and chemo or cancer metastasis in a subject. The method comprises administering to the subject an effective amount of a compound comprising a phospholipid ether analogue, in a preferred embodiment, the reappearance of cancer, cancer insensitive to radiation and chemo or cancer metastasis occurs in the selected group 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 carcinosarcoma. Also preferably, the phospholipid analogue is selected from: where 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 consisting of NH2, NR2, and NR3, wherein R is an alkyl or arylalkyl substituent or where 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, and Z is selected from the group consisting of NH2, NR2, and NR3, wherein R is an alkyl or arylalkyl substituent. In this method, preferably, X is selected from the group of radioactive halogen isotopes consisting of 18F, 36C1, 76Br, 77r r, 82 Brr, 122-i, 123-i, 124x i, 125-iG, 131 i, and 211 A, t. Higher Preferably, the effective amount of the phospholipid ether analogue is a combination of at least two isotopes one with a path variation of about 0.1A to 1mm and a second with a path variation of about 1mm to 1m. More preferably, the effective amount of the phospholipid ether analogue is a combination of at least two 125 I and 131 I isotopes. Also, preferably, the phospholipid ether is 18- (p-iodophenyl) octadecylphosphocholine, 1-0- [18- (p-iodophenyl) octadecyl] -1,3-propandiol-3-phosphocholine, or 1-0- [18 - (p-iodophenyl) octadecyl] -2-0-methyl-rac-glycero-3-phosphocholine, where iodine is in the form of a radioactive isotope. In certain embodiments, the effective amount of the phospholipid ether analog is fractionated. In still other embodiments, the effective amount of the phospholipid ether analogue is between about 0.5 μCi to 3 Ci, treatable in a linear and dose-dependent manner. In other embodiments, it is provided that the dosage can be adapted to the volume of cancer. Still other embodiments provide that the dosage for a radiation-insensitive tumor is greater than the dosage of a tumor sensitive to radiation and less than 3 Ci and can be adapted to the volume of cancer. Another embodiment of the present invention provides the use of the phospholipid ether analogue for the production of a pharmaceutical composition for the treatment of the reappearance of cancer, cancer insensitive to radiation and chemo or cancer metastasis. In this embodiment, the phospholipid analogue is selected from: where 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 consisting of NH2, NR2, and NR3, wherein R is an alkyl or arylalkyl substituent or where X is a radioactive isotope of halogen; n is an integer between 8 and 30; And it is selected from the group consisting of H, OH, COOH, COOR and OR, and Z is selected of the group consisting of NH2, NR2, and NR3, wherein R is an alkyl or arylalkyl substituent. Also in this embodiment, preferably X is selected from the group of radioactive isotopes of halogen consisting of 18F, "° C, 76Br, 77Br, 82Br, 122I, 123I, 124I, 125I, 131I, 211At and combinations thereof. Preferably, the phospholipid ether is 18- (p-iodophenyl) octadecylphosphocholine, 1-0- [18- (p-iodophenyl) octadecyl] -1,3-propandiol-3-phosphocholine, or 1-0- [18- ( p-iodophenyl) octadecyl] -2-0-methyl-rac-glycero-3-phosphoccline, where the iodine is in the form of a radioactive isotope The additional objects, features and advantages of the invention will become apparent from the following detailed description, the drawings and appended claims.

BRIEF DESCRIPTION OF THE FIGURES Figure 1. Structures of certain PLE analogues. Figure 2. Classical models showing the production of photonic radiation from electromagnetic braking, characteristic X-rays and electrons Auger The electrons (on the left) are dispersed elastically and inelastically by the positively charged core. The dispersed electron inelastically loses energy, which appears as photonic braking radiation electromagnetic. The electrons dispersed elastically (which include, backscattered electrons) in general are dispersed through greater angles of the scattered electrons inelastically, (to the right). An incidental electron ionizes the sample atom by ejecting an electron from an inner layer (the K layer, in this case). In turn, the lack of excitation produces characteristic X radiation (superior) or an Auger electron (inferior). Secondary electrons are expelled without low energy from the loose outer layers of electrons, a process not shown. Figure 3. Scintigraphy of the anterior thorax of patient 03 obtained on days 1, 2, and 6 after i.v. administration. of 1 mCi of 1311-NM-324. Absorption is observed in lung cancer Ungular left (t) with increasing proportions of tumor in the background over time. Figure 4. Time Course (days) of NM404 in a SCID mouse with a human adrenal tumor (T) RL-251 xenograft. Figure 5. Distribution of tissues I-1125-NM404 in adrenal cancer RL 251 in SCID mice that represents that while the accumulation in the tumor increased, the distribution in blood, spleen and kidney was reduced in days 1 to 14.

Figure 6. Evident regression of the SCC1 and SCC6 tumor after the injection of 125I-NM404. On day 41, the tumor was significantly reduced. Figure 7. The image shows one of the animals carrying tumors treated with 250 μCi of I-125-NM404 at 4 weeks after injection. The upper hair of the tumor fell off, probably due to the significant accumulation of radioactivity in the tumor. Additionally, the surface of the tumors appears "sunken" and shows dark areas, probably due to hemorrhage and necrosis. The Figure shows the effect of I-125-NM404 on the tumor. Although the size of the tumor (external dimensions) may not decrease, I-125-NM404 causes central necrosis. The measurement method for measuring the external dimensions of the tumor could have an underestimated tumor volume response after I-125-NM404. Figure 8. PC3 human prostate cancer model implanted in a SCTD mouse. It is known that PC3 is insensitive to radiation. The curves between control (without radioactivity, cold NM404) and treatment (I-125-NM404) are only separated approximately 4-5 weeks after treatment; until then the growth of the tumors seems to be the same, which indicates that: 1) NM404 takes a few days to approximately 1 week, accumulate totally in the tumor, and 2) isotope 1-125 has a Low radiation flow (because it has a long half-life). Both factors contribute to a delayed appearance of the therapeutic effect, at the time when all of NM404 had cleared from normal tissues. Figure 9, Scintigraphic comparison of NM404 (lower panel) and NM324 (upper panel) on days 1, 2, and 4 in a SCID mouse with a human prostate PC-3 tumor (arrow) implanted on the flank. The liver and antecedent radioactivity were greatly improved with NM404. Figure 10. The tumor volume of each group was recorded through a 10-week titration period representing the control and dosage of 50, 150, 250 and 500 μCi. In this figure, the control animals showed rapid growth tumors through a measurement period of 10 weeks. This confirms that the compound itself C-NM404 did not have a substantial effect on the growth of the tumors. The group with a dose of 50 μCi did not show any difference with respect to the control animals, therefore, these seem to be inefficient levels of dosage in this animal model. However, the control groups with a dosage of 150, 250 and 500 μCi showed a substantial and prolonged treatment effect. Tumor volumes were stable and the same tumors appeared to "collapse" (the surface of the tumor collapsed). Additionally, the hair on the tumors fell off, confirming a substantial accumulation of radioactivity in these tumors. The results show a linear dose effect of I-125-NM404 on tumor volume. Figure 11. A549 tumor xenografts (1 x 106 cells, s.c.) in fractionated doses in female SCID mice (3 x 50 mCi), having 2 independent controls for each dosage. A fractional dosage of NM404 (eg, 3 x 50 micro-Ci against an individual dosage of 150 micro-Ci) produced the same therapeutic effect. The fractionated dose may be safer because it is removed from normal tissues between activated injections. Figure 12. Large A549 tumors against small tumors of 150 micrccuries. Figure 13. Bioexploration image (A) obtained 4 days after injection with 125I-NM404 in an Apc ^ mouse. Digital photo (B) and fused image that coincides positionally (C) of a lung excised from the mouse that contains a spontaneous lung tumor (2 mm dia, arrow) that shows an intense NM404 uptake in the tumor. Figure 14. Time course of 125I-NM404 in human RL-251 adrenal cancer xenograft in a mouse SCID. Prolonged tumor (1.5 x 0.5 cm, arrow) retention is evident even after 20 days. Figure 15. Image by MRI that provided a 3D surface fused (blue) and 3D microPET image (A) obtained 24 hours after injection i.v. of 12 I-NM404 (80 μCi) in a rat with a glioma brain tumor of CNS-1. The images were merged using Amira (v3.1). The lower panels show (B) a plate for MRI and coronal enhanced contrast through the tumor (arrow) and (C) Fused coronal MRI and microPET imaging 124I-NM404 that corroborate the presence and location of the tumor. Figure 16. Images of 124I-MicroPET of a human lung tumor xenograft - 549 in a SCID mouse 48 hours after injection of 124I-NM404 (80 μCi).

The planes of the corresponding image formation are indicated by a green dotted line in coronal view. Figure 17. Images 12 I-MicroPET (coronal, sagittal, and axial) 96 hours after injection i.v. of 124I-NM404 in a mouse bearing a PC-3 tumor (flank). At any point of time, an activity that is not directed will be evident. Figure 18. l24I-MicroPET images of a transgenic mouse with a spontaneous pancreatic c-myc adenocarcinoma (5 mm) 18 hours after injection of 12? NM404 (80 μCi). Figure 19. Scintigraphic comparison of NM404 (lower panel) and NM324 (upper panel) on days 1, 2, and 4 in a SCID mouse with a human prostate PC-3 tumor (arrow) implanted in the flank. The radioactivity in liver and background was greatly improved with NM404. Figure 20. Time course of 125I-NM404 in human RL-251 adrenal cancer xenograft in a SCID mouse. After 20 days, prolonged retention of the tumor is evident (1.5 x 0.5 cm, arrow). Figure 21. Extinct prostate complex / vesicular gland (A). Bioexploration image obtained 4 days after the injection of NM404 (B). The fused photo / bioexploration image coincided positionally with the excised prostate / vesicular gland (C) showing intense radioactivity absorption in the prostate tumor. Figure 22. MicroCT images with high-density 3D proportional surface of a naked mouse leg at various times after an intratibial injection (the arrow on the first panel represents the site and direction of the injection) of 2 x 10 5 cells Human PC3 prostatic tumor. The tumor begins to protrude through bone around 28 days (arrow) and at 46 days the tumor has literally destroyed the tibia leaving intact only the fibula. Figure 23. MicroCT image of surfaces provided co-registered and coronal plate fused with exploration with positionally coincident biexploration radionuclides (color) 4 days after the injection of NM404 labeled with 125I. The focal NM404 activity correlates well with the PC3 tumor in the tibia (arrow). Figure 24. Surface MRI image provided 3D fused (blue) and 3D microPET image (A) obtained 24 hours after the iv injection of 12 I-NM404 (100 μCi) in a rat with a glioma brain tumor of CNS-1 . The images were merged using Amira (v3.1). The panels on the right show (B) a coronal MRI plate of improved contrast through the tumor (arrow) and (C) images by fused coronal MRI and 12? -NM404 microPET that corroborate the presence and location of the tumor. Figure 25. Image by flat nuclear medicine of total posterior body (A) 4 days after i.v. of 131I-NM-404 (0.8 mCi) to a patient with giant cell lung cancer (6 cm dia, arrow). The lung tumor is easily detected in the corresponding axial (B) and coronal (C) computed topographic (CT) scans.

DETAILED DESCRIPTION OF THE INVENTION General description of the invention: Before describing the methods of the present invention, it should be understood that this invention is not limited to the methodology, protocols, cell lines, and particular reagents described, since these may vary. It should also be understood that the terminology used herein is for the purpose of describing only the particular embodiments, and is not intended to limit the scope of the present invention which will be limited only by the appended claims. It should be noted that in the sense in which it is used herein and in the appended claims, the singular forms "a", "one", and "the" include, the plural reference, unless the context clearly dictates otherwise. Thus, for example, the reference to "a cell" includes a plurality of these cells and the equivalents thereof known to those skilled in the art, etc. As well as, the terms "a" (or "one"), "one or more" and "at least one" may be used interchangeably herein. It should also be noted that the terms "comprising", "including", and "having" can be used interchangeably.

Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art to which this invention pertains. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, preferred methods and materials are not currently described. All publications mentioned herein are incorporated herein by reference for the purpose of describing and exposing chemical products, cell lines, vectors, animals, instruments, statistical analysis and methodologies reported in publications that could be used in conjunction with the invention. Nothing herein should be construed as an admission that the invention is not authorized to precede this disclosure by virtue of the prior invention. In the sense in which the present is defined, the term "isomer" includes, but is not limited to, optical isomers and analogues, isomers and structural analogues, isomers and conformational analogues, and the like. In one embodiment, this invention encompasses the use of different optical isomers of an anti-tumor compound of the formula 3A. It will be appreciated by those skilled in the art that 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 can exist and be isolated in optically active or racemic forms. Some compounds may also exhibit polymorphism. It should be understood that the present invention may encompass the use of any racemic, optically active, polymorphic, or stereoisomeric form, or mixtures thereof, that form possesses properties useful in the treatment of conditions related to tumors described and claimed herein. . In one embodiment, the anti-tumor compounds may include pure (R) isomers. In another embodiment, the anti-tumor compounds may include pure (S) isomers. In another embodiment, the compounds may include a mixture of (R) and (S) isomers. In another embodiment, the compounds may include a racemic mixture comprising both (R) and (S) isomers. It is well known in the art, how to prepare the 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 be prepared from phenolic compounds by treatment with inorganic bases, for example, sodium hydroxide. Also, the esters of the phenolic compounds can be prepared with aliphatic and aromatic carboxylic acids, for example, esters of acetic acid and benzoic acid. As used herein, the term "pharmaceutically acceptable salt" refers to a compound formulated from a base compound that achieves substantially the same pharmaceutical effect as the base compound. This invention further includes a method for using derivatives of anti-tumor compounds. The term "derivatives" includes, without limitation, ether derivatives, acid derivatives, amide derivatives, ester derivatives and the like. In addition, this invention also includes methods for utilizing hydrates of anti-tumor compounds. The term "hydrate", includes, without limitation, hemihydrate, monohydrate, dihydrate, trihydrate and the like. This invention further includes methods for utilizing the metabolites of anti-tumor compounds. The term "metabolite" means any substrate produced from another substance by metabolism or a metabolic process. In the sense in which it defines herein, "contacting" means that the anti-tumor compound used in the present invention is introduced into a sample containing the receptor in a test tube, flask, tissue culture, chip, matrix, plate, microplate, capillary, or the like, and incubated at a temperature and time sufficient to allow the binding of the anti-tumor compound to a receptor. The methods for contacting the samples with the anti-tumor compound or other specific binding components are known to those skilled in the art and can be selected depending on the type of analysis protocol to be performed. Incubation methods are also standard and are known to those skilled in the art. In another embodiment, the term "contacting" means that the anti-tumor compound used in the present invention is introduced into a patient receiving the treatment, and the compound is allowed to come in contact in vivo. In the sense in which it is used herein, the term "treatment" includes a preventive treatment, as well as a sender of a disorder. In the sense in which it is used in the present, the terms "reduce", "suppress" and "inhibit", have their commonly understood meaning to lessen or diminish. In the sense in which it is used in the present, the term "progression" means an increase in the scope or severity, advancement, growth or worsening. In the sense in which it is used in the present, the term "recurrence" means the return of a disease after a remission. As used herein, the term "administer" refers to contacting a patient, tissue, organ or cells with an anti-tumor phospholipid ether compound. In the sense in which it is used herein, the administration can be carried out in vi tro, ie, in a tube for testing, or in vivo, ie, in cells or tissues of living organisms, for example, Humans. In certain embodiments, the present invention encompasses administering the compounds useful in the present invention to a patient or subject. A "patient" or "subject", used equivalently herein, refers to a mammal, preferably a human being, which either: (1) has a disorder that can be remedied or treated by administering the anti-tumor substance using a phospholipid ether compound or (2) is susceptible to a disorder that is can be prevented by administering the anti-tumor compound using a phospholipid ether compound. As used herein, "pharmaceutical composition" means therapeutically effective amounts of the anti-tumor compound having radioactivity, together with diluents, preservatives, solubilizers, emulsifiers, and adjuvants, collectively "pharmaceutically-acceptable carriers". "In the sense in which it is used herein, the terms" effective amount "and" therapeutically effective amount ", refer to the amount of the active therapeutic agent sufficient to provide a desired therapeutic response without undue adverse side effects such as , toxicity, irritation, or allergic response. The "effective amount" specifies, will obviously vary with factors such as, the particular condition that will be treated, the physical condition of the patient, the type of animal that will be treated, the duration of the treatment, the nature of the concurrent therapy (in its case ), and the specific formulations used and the structure of the compounds or their derivatives. In this case, it could be determining a therapeutically effective amount, if it resulted in one or more of the following: (a) disease prevention (eg, pancreatic cancer, breast cancer); and (b) the investment or stabilization of this disease. The effective optimum amounts can be easily determined by someone skilled in the art using routine experimentation. The pharmaceutical compositions are liquids or formulations lyophilized or otherwise dried and include diluents of various buffer contents (eg, Tris-HCl, acetate, phosphate), pH and ionic strength, additives such as albumin or gelatin to prevent absorption to surfaces, detergents (e.g., Tween (Polysorbate) 20, Tween 80, Pluronic F68, salts of bile acid), solubilizing agents (e.g., glycerol, polyethylene glycol), antioxidants (e.g., ascorbic acid, sodium metabisulfite) , preservatives (eg, thimerosal, benzyl alcohol, parabens), bulking agents or tonicity modifiers (eg, lactose, mannitol), covalent binding of polymers, such as, polyethylene glycol to protein, complexation with metal ions, or the incorporation of the material in or on preparations in macroparticles of polymeric compounds such as, acid polylactic, polyglycolic acid, hydrogels, etc., or on liposomes, microemulsions, micelles, unilamellar or multilamellar vesicles, leftovers of erythrocytes, or spheroplasts. These compositions will influence the physical state, solubility, stability, release rate in vivo, and rate of clearance in vivo. Controlled or sustained release compositions include a formulation in lipophilic deposits (eg, fatty acids, waxes, oils). Also encompassed by the invention are methods for administering compositions in macroparticles coated with polymers (eg, poloxamers or poloxamines). Other embodiments of the compositions incorporate coatings in macro particulate forms, protease inhibitors or permeation enhancers for the various routes of administration, including topical, parenteral, pulmonary, nasal and oral. In one embodiment, the pharmaceutical composition is administered parenterally, paracancerally, transmucosally, tansmerically, intramuscularly, intravenously, intradermally, subcutaneously, intraperitoneally, intraventricularly, intracranially, and intratumorally. In addition, in the sense in which it is used herein, "pharmaceutically acceptable carriers" are well known to those skilled in the art, and they include, but are not limited to: 0.01-0 phosphate buffer. IM and preferably 0.05M or 0.9% saline. Additionally, these pharmaceutically acceptable carriers can be aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic / aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer and fatty oils. Intravenous vehicles include fluid and nutrient regenerators, electrolyte regenerators such as those based on Ringer's dextrose, and the like. Also, preservatives and other additives may be present, such as, for example, antimicrobials, antioxidants, intercalating agents, inert gases and the like. Sustained or controlled release compositions that can be administered according to the invention include, a formulation in lipophilic deposits (eg, fatty acids, waxes, oils).

Also encompassed by the invention are compositions in macroparticles coated with polymers (eg, poloxamers or poloxamines) and the compound coupled with antibodies directed against specific-tissue receptors, ligands, antigens or coupled with ligands of tissue-specific receptors. . Other embodiments of the compositions administered according to the invention incorporate macroparticulate forms, protective coatings, protease inhibitors or permeation enhancers for the various routes of administration, including parenteral, pulmonary, nasal and oral. Compounds modified by the covalent attachment of water-soluble polymers such as polyethylene glycol, polyethylene glycol and propylene glycol copolymers, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone or polyproline, are known to exhibit virtually longer half-lives in the blood after an injection intravenously than the corresponding unmodified compounds (Abuchowski et al., 1981; Newmark et al., 1982; and Katre et al., 1987). These modifications can also increase the solubility of the compound in aqueous solution, eliminate aggregation, improve the physical and chemical stability of the compound, greatly reduce the immunogenicity and reactivity of the compound. As a result, the desired in vivo biological activity can be achieved by administering these sequestrants of the polymeric compound less frequently or at lower doses than with the unmodified compound. Still in another method according to the invention, a pharmaceutical composition can be delivered in a controlled release system. For example, the agent can be administered using intravenous infusion, an implantable osmotic pump, a transdermal patch, liposomes, or other modes of administration. In one embodiment, a pump can be used (see Langer, supra; Sefton, CRC Crit. Ref. Biomed. Eng. 14: 201 (1987); Buchwaid et al., Surgery 88: 507 (1980); Saudek et al., N. Engl. J. Med. 321: 574 (1989). In another embodiment, polymeric materials can be used. In yet another embodiment, a controlled release system can be placed in close proximity to the therapeutic target, e.g., the liver, thus requiring only a fraction of the systemic dose (see, e.g., Goodson, in Medical Applications of Controlled Relay, supra, vol. 2, pp. 115-138 (1984). Other controlled release systems by Langer are analyzed in the review (Science 249: 1 527-1533 (1990) . The pharmaceutical preparation may comprise anti-tumor compound alone, or may further include a pharmaceutically acceptable carrier, and may be in solid or liquid form such as tablets, powders, capsules, granules, 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 granule. In yet another embodiment, a granule provides controlled release of the anti-tumor compound over a period of time. The preparation can also be administered by intravenous, intra-arterial, or intramuscular injection, of an oral administration in liquid preparation of a liquid or solid preparation, by topical application, administration can also be carried out by the use of a rectal suppository or a urethral suppository. The pharmaceutical preparations that can be administered by the invention can be prepared by known processes of dissolution, mixing, granulating, or tabletting. For oral administration, anti-tumor compounds or their derivatives physiologically tolerated such as salts, esters, N-oxides, and the like are mixed with the usual additives for this purpose, such as vehicles, stabilizers, or inert diluents, and are converted by the usual methods into suitable forms for administration, such as, tablets, coated tablets, hard or soft gelatin capsules, aqueous or alcoholic aqueous solutions. Examples of suitable inert carriers are conventional tablet bases such as lactose, sucrose or corn starch in combination with binders, such as acacia, corn starch, gelatin, with disintegrating agents such as corn starch, potato starch. , alginic acid, or with a lubricant such as, stearic acid or magnesium stearate. Examples of suitable oily vehicles or solvents are vegetable or animal oils such as sunflower oil or fish liver oil. The preparations can be made in both dry and wet granules. For parenteral administration (subcutaneous, intravenous, intra-arterial, or intramuscular injection), the anti-tumor compounds or their physiologically tolerated derivatives such as salts, esters, N-oxides, and the like become a solution, suspension, or expulsion, if desired with the usual substances and suitable for this purpose, for example, solubilizers or other auxiliaries. Examples are sterile liquids such as water and oils, with or without the addition of a surfactant or other pharmaceutically acceptable adjuvants. The illustrative oils are those of origin, petroleum, animal, vegetable or synthetic, for example, peanut oil, soybean oil, or mineral oil. In general, water, saline, dextrose, aqueous and related sugar solutions, and glycols such as propylene glycols or polyethylene glycol are the preferred liquid carriers, particularly for injectable solutions. The preparation of pharmaceutical compositions containing an active component is well understood in the art. These compositions can be prepared as aerosols delivered to the nasopharynx or as injectables, either as liquid solutions or suspensions; however, solid forms suitable for solution, or suspension in liquids prior to injection can also be prepared. The preparation can also be emulsified. The active therapeutic ingredient is often mixed with excipients that 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.

In addition, the composition may contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents that improve the effectiveness of the active ingredient. An active component can be formulated into the composition as neutralized pharmaceutically acceptable salt forms. The pharmaceutically acceptable salts include, the acid addition salts, which are formed with organic acids such as hydrochloric or phosphoric acids, or organic acids such 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 organic bases such as, isopropylamine, trimethylamine, 2-ethylaminoethanol, histidine, procaine, and the like. For topical administration to the body surfaces using, for example, creams, gels, drops, and the like, the anti-tumor compounds or their physiologically tolerated derivatives such as salts, esters, N-oxides, and the like are prepared and applied as solutions, suspensions, or emulsions in a physiologically acceptable diluent with or without a pharmaceutical carrier.

In another method according to the invention, the active compound can be delivered in a vesicle, in particular a liposome (see, Langer, Science 249: 1 527-1533 (1990); Treat et al., In Liposomes in the Therapy of Infectious Disease and Cancer, Lopez-Berestein and Fidler (eds.), Liss, NY, pp. 353-365 (1989), Lopez-Berestein ibid., Pp. 31 7-327, see ibid in general). For use in medicine, the salts of the anti-tumor compound may be pharmaceutically acceptable salts. However, other salts may be useful in the preparation of the compounds according to the invention or their pharmaceutically acceptable salts. Suitable pharmaceutically acceptable salts of the compounds include acid addition salts which, for example, can 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, sulfuric acid , methanesulfonic acid, fumaric acid, maleic acid, succinic acid, acetic acid, benzoic acid, oxalic acid, citric acid, tartaric acid, carbonic acid or phosphoric acid. In general, the compounds of phospholipid ether and especially NM404, is an imaging agent for promising diagnosis, selective of new tumors to monitor the response to the treatment of various modalities of tumor treatment. Radioiodinated NM404, a second generation phospholipid ether analogue, exhibited a remarkable tumor selectivity 27/27 tumor models. Due to the lack of metabolic phospholipase enzymes in the membranes of tumor cells, the prevailing hypothesis of this procedure is that the phospholipid ether analogues are trapped exclusively in the membranes of the tumor cell due to their inability to be metabolized and eliminated. In this way, the differential clearance rates of the phospholipids ethers of normal cells against tumor cell lines form the basis of this concept. The results obtained in a variety of tumor models indicate that NM404 is sequestered and selectively maintained by tumor tumor cells and located in both primary and metastatic lesions, regardless of the anatomic location including those found in lymph nodes. Unlike FDG, this agent is not located in infectious sites. Other advantages of NM404 with respect to FDG include the following: NM404 is selective and is maintained indefinitely by malignant tumor cells, whereas FDG is not selective for tumor cells and does not go to infectious sites and hyperplasias (Barret's esophagus). Also, since 124I has a physical half-life of 4 days, you can board anywhere in the world while FDG with its half-life of 110 min, may have a limited distribution within 321.86 Km (200 miles) of the production site. NM404 undergoes prolonged retention (without metabolism) and therefore produces a significant therapeutic potential when it is matched with a suitable radioisotope similar to 'I whereas FDG does not possess any therapeutic potential. NM404 can be labeled with a variety of iodine isotopes that expand its versatility (diagnosis and therapy as well as a tool for studies in experimental animals) while FDG is limited to 18F for PET scanning or potentially 19F (stable) for formation of magnetic resonance imaging, however at very low sensitivity levels. Regardless of its ability to target tumors, due to its rapid metabolism in tumor cells, it has no potential for therapy. NM404 produces the local tumor response for potential prediction or not only accurate to various treatment modalities, but also allows the detection of distant metastatic lesions in cases of primary subtherapeutic tumor treatment. PLE compounds can be designed for a more accurate estimate of the specificity and sensitivity of radiolabelled PLE analogues such as, NM404 as an agent for imaging prostate cancer and other cancers. Based on preclinical models, PLE analogs such as, NM404 probably exhibit high tumor uptake giving this agent significant potential to be used at titration, after response to therapy, or potentially as a therapeutic agent when coupled with higher doses of 131I, 1 5I, or 211At an alpha-emitting halogen with therapeutic efficacy.

PREFERRED MODALITIES 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 the reappearance of cancer, cancer insensitive to radiation and chemo or metastasis of the cancer selected from the group consisting of lung cancer, adrenal cancer, melanoma, colon cancer, colorectal cancer, ovarian cancer, cancer prostate, liver cancer, subcutaneous cancer, squamous cell cancer, intestinal cancer, hepatocellular carcinoma, retinoblastoma, cervical cancer, glioma, breast cancer and pancreatic cancer in a subject who has or is suspected of having cancer. The method comprises the steps of: (a) administering a phospholipid ether analogue to the subject; and (b) determine whether a suspect organ or one that has a recurrence of cancer, Cancer insensitive to radiation or cancer metastasis in the subject maintains a higher level of the analogue than the surrounding regions, where a region with higher retention-indicates the detection and location of the reappearance of the cancer, cancer insensitive to radiation and chemo or metastasis of the Cancer. In a preferred embodiment, the phospholipid analog is selected from: where 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 consisting of NH2, NR2, and NR3, wherein R is an alkyl or arylalkyl substituent or where 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, COOP and OR, and Z is selected from the group consisting of NH2, NR2, and NR3, wherein R is an alkyl or arylalkyl substituent. In addition, in certain embodiments, X is selected from the group of radioactive halogen isotopes consisting of 18F, 3dCl, 76Br, 77Br, 82Br, 122I, 123I, 124I, 125I, 131I, and 211At. More preferably, the phospholipid ether is 18- (p-iodophenyl) octadecylphosphocholine, 1-0- [18- (p-iodophenyl) octadecyl] -1,3-propandiol-3-phosphocholine, or 1-0- [18- (p-iodophenyl) octadecyl] -2-0-methyl-rac-glycero-3-phosphocholine, where the iodine is in the form of a radioactive isotope. In this method, preferably, the detection is carried out by PET, CT, MRI scan methods or combinations thereof. In the sense in which it is used in the present group "alkyl", it refers to straight chain, branched or cyclic hydrocarbons, saturated or unsaturated. The alkyl group has 1-16 carbon atoms, and may be unsubstituted or substituted by one or more groups selected from halogen, hydroxy, alkoxycarbonyl, amido, alkylamido, dialkylamido, nitro, amino, alkylamino, dialkylamino, carboxyl, thio, and thioalkyl. . A "hydroxy" group refers to an OH group.

An "alkoxy" group refers to an -O-alkyl group wherein alkyl is as defined above. A group "uncle" refers to an SH group. A "thioalkyl" group refers to a -SR group, wherein R is alkyl as defined above. An "amino" group refers to a group -NH2. An "alkylamino" group refers to a group -NHR, wherein R is alkyl and is as defined above. A "dialkylamino" group refers to a group of -NRR ', wherein R and R' are all as defined above. An "amido" group refers to a -CONH2. An "alkylamido" group refers to a group -CONHR wherein R is alkyl as defined above. A "dialkylamido" group refers to a group -CONRR ', wherein R and R' are alkyl as defined above. A "nitro" group refers to a group N02. A "carboxyl" group refers to a COOH group. In the sense in which it is used herein, "aryl" includes both carbocyclic and heterocyclic aromatic rings, both monocyclic and fused polycyclic, wherein the aromatic rings may be 5- or 6-membered rings. Representative monocyclic aryl groups include, by way of example, phenyl, furanyl, pyrrolyl, thienyl, pyridinyl, pyrimidinyl, oxazolyl. isoxazolyl, pyrazolyl, imidazolyl, thiazolyl, isothiazolyl and similar. The 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. Representative fused polycyclic aryl groups include naphthalene, anthracene, indolizine, indole, isoindole, benzofuran benzothiophene, indazole, benzimidazole, benzthiazole, purine, quinoline, isoquinoline, noline, phthalafin, quinazoline, quinoxaline, 1,8-naphthyridine, pteridine, carbazole , acridine, phenazine, phenothiazine, phenoxazine, and azulene. In addition, in the sense in which it is used herein, "arylalkyl" refers to entities, such as benzyl, wherein an aromatic is linked to an alkyl group which is linked to the position indicated in the PLE compound. Another embodiment of the present invention provides a method for the treatment of the reappearance of cancer, cancer insensitive to radiation and chemo or cancer metastasis 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 reappearance of the cancer, cancer insensitive to radiation and chemo or cancer metastasis occurs in the selected group of lung cancer, adrenal cancer, melanoma, colon cancer, colorectal cancer, ovarian cancer, prostate cancer, cancer hepatic, subcutaneous cancer, squamous cell cancer, intestinal cancer, hepatocellular carcinoma, retinoblastoma, cervical cancer, glioma, breast cancer and pancreatic cancer carcinosarcoma. preferably, the phospholipid analogue is selected from: where 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 consisting of NH2, NR2, and NR3, wherein R is an alkyl or arylalkyl substituent or where X is a radioactive isotope of halogen; n is an integer between 8 and 30; And it is selected from the group consisting of H, OH, COOH, COOR and OR, and Z is selected of the group consisting of NH2, NR2, and NR3, wherein R is an alkyl or arylalkyl substituent. In this method, preferably, X is selected from the group of radioactive halogen isotopes consisting of 18F, 36C1, 'cBr, 77Br, 82Br, 122I, i23I, 12 I, I5I, 131I, and 211At and combinations thereof. . More preferably, the effective amount of the phospholipid ether analogue is a combination of at least two isotopes one with a path variation of about 0.1 Á up to 1 vmm and a second with a path variation of about 1 mm up to 1 m, also as discussed in Figure 2. Most preferably, the effective amount of the phospholipid ether analogue is a combination of at least two 125 I and 131 I isotopes. Also, preferably, the phospholipid ether is 18- (p-iodophenyl) octadecylphosphocholine, 1-0- [18- (p-iodophenyl) octadecyl] -1,3-propandiol-3-phosphocholine, or 1-0- [18 - (p-iodophenyl) octadecyl] -2-0-methyl-rac-glycero-3-phosphoccline, where the iodine is in the form of a radioactive isotope. As shown in Figure 11, in certain embodiments, the effective amount of the phospholipid ether analog is fractionated. The advantage of using a fractional dosage is that it allows the PLE analog to be removed from normal tissues. As an example, the fractional dosage of NM404 (for example, 3 x 50 micro-Ci against a single dose of 150 micro-Ci) produces the same therapeutic effect, while still providing low doses of the compound that is then removed from normal tissues between fractionated injections. In still other embodiments, the effective amount of the phospholipid ether analogue is between about 0.5 μCi to 500 mCi, and as shown in Figure 10, this can be treated in a linear manner independent of the dose. Other modalities provide that the dosage can be adapted to the cancer volume as shown in Figure 12. The graph of Figure 12 shows the difference in tumor growth for the same dosage of I-125-NM404 (150 microCi) when It is injected into animals with small (<1 cm) and large (> 1 cm) tumors. The results show that a small tumor showed an impacted tumor growth that is the dose, however, a population of large tumors, showed no effect for the same dosage, behaving basically as a non-irradiated control. The graph of Figure 12 also illustrates that the effective tumor dosage must be adjusted to the tumor volume and that there is a tumor dosage per volume of tumor volume that will have to be reached in order to show efficacy. Still other modalities provide that the Dosage for a tumor insensitive to radiation and chemo is greater than the dosage for a tumor sensitive to radiation and less than 500mCi and can be adapted to the cancer volume, as can be ascertained when comparing PC3 (Figure 8) and models with cancer A549 ( Figure 10, 11 and 12), in which the PC3 model is insensitive to radiation. Another embodiment of the present invention provides the use of the phospholipid ether analogue for the production of a pharmaceutical composition for the treatment or reappearance of cancer, cancer insensitive to radiation and chemo or cancer metastasis. In this embodiment, the phospholipid analogue is selected from: where 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 consisting of NH2, NR2, and NR3, wherein R is an alkyl or arylalkyl substituent or where 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, and Z is selected from the group consisting of NH2, NR2, and NR3, wherein R is an alkyl or arylalkyl substituent. Also in this embodiment, preferably X is selected from the group of halogen radioactive isotopes consisting of 18F, 36C1, 76Br, 77Br, 82Br, 122I, 123I, 124I, 1251, 131I, 211At and combinations thereof. Also preferably, the phospholipid ether is 18- (p-iodophenyl) octadecylphosphocholine, 1-0- [18- (p-iodophenyl) octadecyl] -1,3-propanediol-3-phosphocholine, or 1-0- [18-] (p-iodophenyl) octadecyl] -2-0-methyl-rac-glycero-3-phosphocholine, where the iodine is in the form of a radioactive isotope.

Synthesis and effects of structural activity ratios on tumor avidity of radioiodinated phospholipid ether analogs 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 models with rodents . It is believed that this tumor avidity arises as a consequence of metabolic differences between the tumor and the corresponding normal tissues. The results of this study indicate that a factor in the tumor retention of these compounds in tumors is the length of the alkyl chain that determines their hydrophobic properties. The decrease in chain length from Ci to C7 results in little or no accumulation of tumors and rapid clearance of the compound in rats bearing tumors within 24 hours of administration. Increasing the chain length has the opposite effect, with C15 and C18 analogs exhibiting delayed plasma clearance and improved tumor absorption and retention in tumor bearing rats. Tumor absorption exhibited by the propandiol analogs NM-412 and NM-413 was accompanied by high levels of radioactivity in liver and abdominal 24 hours after injection to tumor bearing rats. In addition to a 2-O-methyl entity for the structure of propandiol also significantly delayed the absorption of tumors. A direct comparison between NM-404 and its predecessor, NM-324, in immuno-compromised mice carrying human PC-3 tumors, revealed a dramatic improvement in both tumor uptake and total body clearance of NM-404 with relation to NM-324. Based on the imaging and studies for tissue distribution in various tumor models in rodents, the C18 analog, NM-404 was selected for a follow-up evaluation in patients with human lung cancer. The preliminary results have been extremely promising since the absorption and retention of the agent in tumors is accompanied by a rapid clearance of a radioactivity from graduation from normal tissues, especially those in the abdomen. These results strongly suggest that extension of human trials to include other cancers is warranted, especially when NM-404 is radiolabelled with iodine-124, a novel commercially available positronic isotope emitter. The relatively long average physical life of 4 days provided by this isotope seems to be adequate for the pharmacodynamic profile of NM-404. Chemical synthesis In the course of the synthesis of the iodinated phospholipid ether analogues, the inventors they seek to develop a general synthetic scheme that could allow altering the chain length in white compounds. The synthetic process is based on a copper-catalyzed cross coupling reaction of Grignard reagent with alkyl tosylates or halides15 (Schemes 1, 2). The choice of the constituent blocks for the elongation of the alkyl chain was determined by the commercial availability of the starting materials. Scheme 1: Reagent and conditions: (a) Me3SiBr, CH2C12; (b) BrMg (CH2) nOTHP, Li2CuCl4 (cat), THF, -78 to 20 ° C; (c) PPTS, EtOH, 40 ° C; (d) TsCl, DMAP, CH2C12; (e) BrMg (CH2) 3OTHP, Li2CuCl4 (cat), THF, -78 to 20 ° C; (g) BrMg (CH2) 6OTHP, Li2CuCl4 (cat), THF, -78 to 20 ° C.

Scheme 1 ~ ~ CH2OH • - /) - CH2B ~~? _j- (CH2) 12OTHP 11 12 16 19 21 I- ^ - (CH2) 15OH I- < j ^ > - (CH2), eOH 20 22 The synthesis was initiated by the conversion of p-iodobenzyl alcohol 11 to p-iodobenzyl bromide 12 as shown in Scheme 1. The p-iodobenzyl bromide was further coupled with a Grignard reagent derived from THP-protected 11-bromoundecanol 13 in the presence of 0.5-0.7 molar% Li2CuCl. The 12- (p-iodophenyl) dodecanol 17 obtained after deprotection of the first coupling product 16 was used for the synthesis of the iodinated C12 phospholipids as described above. The 12 # 13 alcohol 17 also served as a starting material for the synthesis of the alkanoids? -iodophenyl with longer chains. For example, coupling of tosylate 18 with Grignard reagent produced from bromides 14 and 15. Followed by deprotection with THP provided C15 (20) and C18 (22) alcohols, respectively. These alcohols were converted to the corresponding alkylphosphocholines 5 (NM-397) and 6 (NM-404) according to published procedures. The phospholipids ethers of 12,13 propanediol 7 (NM-413) and 8 (NM-412) were synthesized from 3-benzyloxypropanol 25, and the phospholipid ethers 2-O-methyl-rac-glycerol 9 (NM-414) and 10 (NM-410) were obtained from 10-benzyl-2-O-methyl-rac-glycerol 26 using a sequence of reactions that the inventors have reported previously. Radiolabelling with iodine-125 was carried out by a method of Isotopic exchange routinely used in the laboratory.

Scheme 2 23, n = 15 25, R = H 27, n = 15, R = H 24, n = 1 8 26, R = 0CH2 28, n = 15, R = OCH 3 29, n = 18, R = H 30, n = 18 R = OCH3 31, n = 15, R = H 7, NM-413. n = 15, R = H 32, n = 15, R = OCH3 8, NM-412, n = 18, R = H 33, n = 18, R = H 9, NM-414, p = 15, R = OCHj 34, n = 18. R = OCH3 10. NM-410, n = 18, R = OCHj Biology. The avidity of PLE analogs to localize in tumors was evaluated in various animal models. The PC-3 model represents a human tumor cell line that was used to determine the target (tumor) at the non-target proportion of NM404 and NM412 in head-to-head comparisons in order to select a candidate for an initial human pharmacokinetic test in patients with prostate cancer. The MatLylu model (rat Dunning R3327), a line with rat prostatic tumor, was used specifically to select 9 specific analogs before entering them in the control for dosimetry and tumor bearing animals or to determine tumor / gradient ratios. Finally, the model of carcinosarcoma Walker-256 was used to quantify the purposes for tissue distribution. To accelerate the selection process and minimize the number of tumor-bearing animals used in studies for tissue distribution with multiple time points, images of new radioiodinated homologs were formed by scintigraphy with gamma camera in the prostate cancer model in rat Dunning R3337 (strains' MAT Lylu). In this way, the male Copenhagen rats received a subcutaneous injection of MAT Lylu cells (1 x 106 cells) in the thigh 10-14 days before the injection of the radioiodinated PLE analogs (30-40 μCi) in 2% solution. Tween-20. The gamma camera images were obtained at multiple time points including 24 and 48 hours after the injection. Homologs (NM-410, NM-413, and NM-414) exhibiting high liver uptake, significant abdominal accumulation and retention or poor tumor absorption and retention did not undergo a subsequent biodistribution analysis. The distribution of radioactivity tissues in rats carrying carcinosarcoma Walker-256 were assessed at various time intervals after intravenous administration of radioiodinated chain length homologs. The first group of compounds that was tested included three alkylphosphocholines: a shorter chain analog with seven carbon atoms 3 (NM-396) and two analogs with a longer chain length, 5 (NM-397, chain length of C15 alkyl) and 6 (NM-404, C18 alkyl chain length). Initial biodistribution experiments performed with 3 (NM-396, analog C7) indicated rapid clearance in tissues accompanied by significant in vivo deiodination. About 24 ñoras, the amount of radioactivity in the thyroid was 213% ID / g, while the radioactivity levels in all the organs examined was < 0.10% ID / g. After these studies, the reduction in the number of methylene groups to seven apparently gave a much more hydrophilic molecule that was rapidly excreted by the kidneys. In contrast to compound 4 (NM-346, C12 analog), analog 3 C7 cleared rapidly from the rat and was not localized to the tumor tissue at any of the time points examined. This observation directs future efforts to assess the effect of increasing the length of the chain aliphatic at the time of tumor absorption and retention. The tissue distribution of homologue 5 C15 (NM-397) was assessed using the same rat tumor model Waiker 256. Radioactivity in the tumor increased with time and peaked at 48 hours after administration (1.65 ± 0.23% ID / g) as opposed to most normal tissues that exhibited their highest levels of radioactivity 6 hours after administration. With the exception of the thyroid, the tumor had its highest concentrations of radioactivity at 24, 48, and 120 hours than any of the other tissues examined. A faster failure of radioactivity occurred in normal tissues compared to the tumor probably due to metabolism and elimination by normal tissues. The accumulation of radioactivity in the thyroid increased throughout the course of study, which suggests the presence of a low level of in vivo deodorization. The levels of radioactivity in the duodenum were similar to those of the tumor with maximum levels that were observed at 48 hours after administration (1.38 ± 0.24% ID / g). Although limited results were obtained with the analog 4 C12 (NM-346) in this model, the results suggest that the profile for tissue distribution of the analogue C15 (NM-397) was similar to that observed with the Analogue 4 C12 with the exception of a 2-fold increase in tumor absorption at 24 hours. The remaining absorption and clearance in other organs and tissues was similar between the two compounds. The effect of further extending the aliphatic chain for the 6 C18 analog (NM-404) represented a dynamic profile of this compound that was similar to the C15 analogue (NM-397) at maximum levels of radioactivity in the tumor 48 hours after the administration (1.14 ± 0.01% ID / g), albeit at slightly lower levels. The amounts of radioactivity detected in liver, kidney and duodenum were significantly lower after the administration of compound 6 C18 compared to the same organs in the studies of analogue C15. In addition, the 6 C18 analog was maintained in the circulation to a much greater degree than the other chain length homologs studied. For example, at 120 hours, blood levels for 6 were 0.6 ± 0.1% ID / g compared to levels of 0.07 ± 0.00% ID / g for analog C15 (5). The levels of total radioactivity in the thyroid were relatively low in both 5 and 6 when the extremely small mass of the gland was considered. In order to examine the transport properties of the PLE analogs, the plasma was isolated from the tumor-carrying rats Walker-256, 7 days after the administration of 6-iodine-125 labeled. We studied the distribution of radioactivity in the plasma compartment of a rat that was receiving 6 (NM-404). The PAGE analysis revealed that the highest radioactivity in circulation (88%) was associated with the albumin fraction after administration of the 6 C18 analogue. This finding is similar to the results reported by Eibl who studied the binding of the phospholipid ether prototype, ET-18-OCH3, with the serum proteins and found that the majority of the lipid ether (71%) bound to the albumin and approximately the 6% to HDL.

Comparative studies for imaging The images of gamma camera scintigraphy, shown in Figure 19, are directly compared to the absorption of the tumor and the body clearance of the analogue I-NM-404 second generation (6) against its shortest chain, the first generation predecessor, 125I-NM-324 (2) on days 1, 2, and 4 after administration of prostate tumor xenografts PC- 3 humans carrying immunocompromised SCID mice. Qualitative scintigraphic comparisons of these two PLE analogs demonstrated an impressive difference in tumor uptake and total body clearance. The agent of Long chain, NM-404, exhibits rapid tumor uptake and prolonged retention accompanied by rapid total elimination of the entire body from radioactivity, while tumor uptake and body clearance were substantially delayed with NM-324, even at 4 days after administration. Significant tumor uptake and retention of the C18 NM-404 analogue accompanied by rapid total elimination from the body clearly defined the superior NM-404 imaging properties in this model. The results of the distribution of extensive quantitative tissues obtained on days 1, 3, 5, and 8 after the administration of NM-404 radioiodinated in this model indicates the rapid elimination of radioactivity of all normal tissues during the 8 days of the period. of evaluation. However, tumor uptake continued to increase until day 5 when 18% dose injected per gram of tumor was reached. The proportions of tumor to antecedent tissue increased steadily throughout the course of the experiment due to prolonged retention in tumors coupled with stable elimination from normal tissues. The proportions of tumor to antecedent tissue exceeded 4, 6.8, 23, and 9 in blood, liver, muscle, and prostate, respectively, 3 days after injection and continued to improve after 5 and 8 days.

Again, although levels in the thyroid ranged from 26 to 54% of dose injected per gram of tissue, these levels are actually very low and represent an extremely small percentage of the injected dose, when exceedingly small of the organ was considered and the data they are represented in a percentage administered dose per organ base. Kotting et al., Have investigated the effects of the alkyl chain length of the biodistribution of three alkylphosphocholine analogues (APC). The Kotting study differed from the experiments since 1) the compounds were administered orally at concentrations that were believed to be cytotoxic to the tumor, and 2) the C22 compound contained a double bond in the alkyl chain. Therefore, direct comparisons with the work described herein can not be performed due to the large dose differences and the unknown bioavailability of the oral agents. In the Kotting study, analogs C16, C18 and C22 were administered orally to rats carrying a mammary carcinoma induced by methylnitrosourea in daily doses of 50-120 o mol / kg. As the length of the alkyl chain increased, the observed levels of the compound in kidney, liver, and lung decreased. In contrast to the results of Tracing obtained with the radiolabelled PLE analogs, Kotting and colleagues, found that APC levels in blood decreased with increasing chain length, while tumor levels increased with increasing chain length.19 Can be expected that oral administration could result in a substantial amount of degradation of the lipid ethers in the Gl tract before absorption. Tumors were rapidly visualized with homologs of alkylphosphocholine C12 (4), C15 (5) and C18 (6) via scintigraphy with gamma camera at both 24 and 48 hours after injection. The results for rat imaging obtained with the propaneol analogues C15 (7, NM-413) and C18 (8, NM-412), on the other hand, exhibited tumor absorption accompanied by high levels of radioactivity in liver and liver. ABS. The imaging results obtained with the analogues of 2-O-Me glycerol C15 (9, NM-414) and C18 (10, NM-410) in the prostate model MAT lylu, indicated high levels of radioactivity in the liver and abdomen with little or no absorption of the agent in the tumors. Differences in clearance and amount of radioactivity from non-target tissues that include the liver and intestinal tract will have a significant impact at the time of application of the phospholipid ether analogues radioiodinated as image forming agents in humans. Absorption in non-target tissue may decrease the effectiveness of radiodiagnostic imaging by creating a high background activity or by causing excessive exposure of radiosensitive tissues to the injected radioactivity. A preliminary clinical trial with 2 (NM-324, meta-iodine isomer of NM-346) in cancer patients, while providing excellent tumor uptake, was limited by radiation dosimetry associated with accumulation in non-target tissues including liver, kidneys, and bladder. The strategy was to examine the alkyl portion of the phospholipid ether analogue structure and determine its function in tumor localization and retention. Qualitative scans for selection of whole rat bodies were obtained in rats carrying MAT-Lylu tumors with radioiodinated PLE analogs with long chain lengths revealed sufficient tumor uptake to allow detection. However, follow-up studies for tissue distribution have shown that sequential increases in chain length from C12 to C15 to C18 resulted in a rapid decline in the amount of radioactivity detected in non-target organs. This substantial decrease in non-target tissue activity was accompanied by a reduction relatively small in the levels of radioactivity present in the tumor. In addition, analog 6 C18 (NM-404) exhibited a propensity to remain in circulation for much longer than the C12 (4) and C15 (5) analogs. A longer average plasma life can be expected, resulting in additional opportunities for the 6 C18 compound to be absorbed into the tumor as they are continuously circulated through the vasculature. This extended plasma half-life may be the result of a strong binding to the assay solution with albumin. The uptake and transport of radiolabeled PLE by plasma components can also be an important factor related to tumor retention of these compounds. Indeed, the increase in the chain length from C7 to C18 results in an increase in the lipophilicity of the PLE analogues. Greater lipophilicity may increase the affinity of these compounds for the cell membrane, and may alter their binding to plasma components. The uptake and transport in the circulation by endogenous lipoproteins such as, LDL and HDL can also impact the biological distribution in the tumor. In preparation for human clinical trials, unlabeled NM404 underwent an independent acute toxicity assessment (University of Buffalo Toxicology Research Center) at 1200 times the dosage of mass for early imaging in rats and rabbits. The agent was well tolerated and no acute toxicities were found at this dosage level. Due to its selective tumor uptake and retention, the properties in a variety of tumor models in rodents and the subsequent excellent safety profile in rats and rabbits, NM-404 was selected to undergo initial pharmacokinetic evaluation in humans in patients with Giant cell lung cancer (NSCLC). Patients underwent scintigraphy with a flat gamma camera after receiving an injection of 131 I-NM-404 (< 1 mCi). Preliminary results in humans (n = 3) demonstrated tumor absorption and prolonged retention in primary lung tumors (Figure 25). In relation to the high values of absorption in liver observed with its predecessor, NM-324, however, the levels of radioactivity in liver and abdominal were much lower with NM-404, which suggests the ease of evaluation of this agent in other abdominal tumors including those associated with the colon, prostate, and pancreas.

Conclusions In summary, the rationale behind the design of novel PLE analogs described in this document was exploited in an effort to assess the effect of a chain length and other structural modifications in the hydrophobic portion of the phospholipid molecule in tumor absorption and retention. Decreasing the chain length from C12 to C7 resulted in little or no tumor accumulation and rapid clearance of the compound in tumor-bearing rats at 24 hours after administration. The increase in chain length had the opposite effect, with C15 and C18 analogs exhibiting delayed plasma clearance and improved tumor retention and retention in tumor-bearing rats. The absorption of the tumor exhibited by analogs 7 and 8 of propandiol was accompanied by clearly high levels of radioactivity in the liver and abdomen, 24 hours after injection to tumor-bearing rats. In addition, a 2-O-methyl entity in 9 and 10 significantly delayed tumor uptake. A direct comparison between NM-404 and its predecessor, NM-324, in immuno-compromised mice carrying PC-3, revealed a dramatic improvement in both tumor uptake and total body clearance of NM-404 relative to NM-324. NM-404 provided superior properties for imaging compared to the other analogs examined in various animal models, thus ensuring further evaluation of this second generation PLE analog in human cancer patients pulmonary. Preliminary clinical results in humans indicated desired tumor absorption and retention properties similar to those previously observed in animal models. However, in contrast to its short-chain predecessor NM-324, NM-404 exhibited significantly lower levels of radioactivity in liver, kidney and abdominals, which in addition to providing the promise of pulmonary tumor imaging, suggests guarantee the additional evaluation of this agent in human patients with colorectal, pancreatic and prostatic cancer. In addition, a lack of radioactivity of the urinary bladder suggests little renal clearance of the agent or its metabolites through the time points examined. This represents a significant advantage superior to 18F-fluorodeoxyglucose (FDG), a PET agent routinely used for tumor imaging today, which undergoes significant renal elimination, thus preventing imaging in the prostate area. Because the tumor-targeted tumor strategy of the PLE analogs appears to imply selective retention of tumors over time, relatively short living nuclides such as 18F or even 99mTc are not practical to mark NM-404 at the time. current. Given the preliminary success of 131I-NM-404 in the trial for Today, it is imperative to radiolabel NM-404 with iodine-124, a relatively novel positron-emitting isotope with a half-life of 4.2 days, and evaluate its effectiveness for tumor detection by PET. It has been reported that imaging with PET with 124I provides more than 40 times the sensitivity of flat 131I-gamma imaging. PET, unlike traditional gamma camera imaging, also offers a significant improvement in resolution and three-dimensional imaging capabilities, as well as superior quantification properties in relation to flat scintigraphic imaging. The long physical half-life of 4 days, of this PET isotope is quite adequate for the kinetics of tumor uptake and retention of NM-404 and the inventors are in the process of extending the imaging studies with NM-404 so that include PET.

Giant cell lung cancer (NSCLC) Phospholipide ether analogues molecules (PLE), have unique biochemical and pharmacological properties that result in a high degree of tumor selectivity. Unlike FDC, they accumulate not specifically in hypermetabolic tissues Both malignant and non-malignant, the inventors have shown that radioiodinated PLE analogs undergo selective retention in a wide variety of murine and human tumors at high levels, and do not accumulate in normal or inflammatory tissues. The family of phospholipid ethers (PLE) are characterized by the presence of a long chain alkyl or alkenyl alcohol linked to ether connected to a glycerophosphocholine molecule, normally found in mammalian cells as a minor component of the total phospholipid content. Among the PLEs, there are many subtypes, one of the most extensively studied subtypes are structurally simplified alkylphosphocholines (APCs). I-125-NM404, is part of this APC family. The present invention shows the methodologies for preliminary data for PET imaging in humans with respect to the use of the second generation PLE analog, NM404, in patients for NSCLC imaging. In preclinical models, the inventors have shown that NM404 (a) are selectively retained in 27/27 tumor models, including lung and brain tumors, as well as bone metastases of prostate PC-3, (b) not maintained in tissues normal, premalignant, or hyperplastic; and, (c) are not maintained in inflammatory tissues. The studies Pharmacokinetics first in humans with 131I-NM404 in patients with NSCLC, the inventors have found that 131I-NM404 is safe, and that from 24-48 hours, it is the optimal time point for scintigraphic imaging for tumor detection. These studies also revealed liver activity levels and significantly lower liver history relative to previous promising analogues and confirmed that the agent does not cross the intact blood brain barrier. Although sufficient for the first pharmacokinetic studies in humans, the poor imaging and flat nature characteristics for imaging associated with scintigraphy with iodine-131 determine that NM404 will be further developed for PET imaging. The inventors have recently radioiodinated NM404 in excellent yield with commercial iodine-124, a PET isotope with long life, relatively new, the half-life of which seems to ideally coincide with this NM404 pharmacokinetic virus. Initial explorations with microPET obtained with 12 I-NM404 in xenograft and models of spontaneous tumor in mouse and rat confirmed the universal avidity of the tumor and prolonged retention. The extension of these studies for PET exploration is currently necessary to characterize Accurate and quantify the 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 efficiency for the imaging of primary NSCLC tumors with 124I-NM404 PET / CT in patients with NSCLC. who are undergoing resection, comparing pre-operative images with pathological findings, (b) tumor-specific accumulation and metabolic fate of NM404 in NSCLC patients who are undergoing resection, and correlate with tumor retention with decreased activity of phospholipase-d; (c) preliminary data that relate to the sensitivity of locoregional imaging, and metastatic tumors with 124I-NM404 PET / CT in patients with NSCLC, when comparing these results with FDG PET / CT scanning; and (d) preliminary data that relate to the specificity of imaging with 124I-NM404 PET / CT, by imaging in patients presenting with solitary pulmonary nodules, or having a diagnosis of pulmonary sarcodium or granulomatous infections such as , mycotic or mycobacterial infections, or bacterial pneumonias. Due to its prolonged properties of retention of tumor, NM404 labeled with 125I, has recently produced a significant regression of tumors (vide infra) in SCID mice carrying human lung tumor xenografts A549. the display of utility for both diagnostic and therapeutic purposes, NM404 will develop as a real, potentially universal diagnostic and therapeutic agent. NM404 has currently been evaluated in 27 tumor models in animals, including several lungs, and it is clear that once the agent enters the tumor cells, it reaches a deadly metabolic end and is trapped. The prolonged retention of tumors of this agent is demonstrated in a human adrenal tumor xenograft implanted in SCID mice (Figure 14). NM404 is also retained in spontaneous murine lung tumors (Figure 13). By using NM204 labeled with 125 I, the inventors have been able to image mammary and prostatic tumors in mice more than 60 days. The characteristics of prolonged retention of tumors can significantly improve the radiotherapeutic efficacy of the agent. Studies for imaging and tissue distribution, performed in mouse models, are aimed at the determination of absorption characteristics in a wide variety of tumor models summarized in Table 2.

Main reason for the selection of isotopes for clinical studies Although the inventors have studied NM404 labeled with iodine-131 in preliminary pharmacokinetic studies in humans, 'this is less than optimal for imaging because scintigraphy with iodine-131, produces images with limited resolution and little anatomical detail. However, the novel long-lived iodine-124 isotope will produce tomographic PET images that will be displayed with the corresponding CT images, thus providing a significantly superior anatomical and functional detail. Because the strategy targeting PLE analog tumors seems to involve selective retention in tumors over time, nuclides with relatively short lifetimes such as 18F or even 99mTc are not practical for NM404 tagging at the present time. Although the use of other isotopes, including iodine-123, may ultimately prove adequate to use NM404 in certain tumors, the current focus will develop the capacity for PET imaging of this agent, due to the recent success of imaging Oncology using hybrid scanners using PET-CT. Diagnostic accuracy not exceeded by a biochemist or PET-forming agent functional tumor images, combined with the precise anatomical accuracy provided by CT, is currently the gold standard for tumor imaging. However, it is rather advantageous to label the PLE analogs with iodine-124, a relatively novel PET isotope, where the physical half-life (4 days) is quite consistent with the absorption of tumors by PLE and retention kinetics. Marking NM404 with iodine-124 represents a natural extension of current studies with gamma-emitting nuclides due to its 4-day physical half-life. It has been shown that imaging with PET with 124I produces more than 40 times the sensitivity of flat 131I-gamma scintigraphy. PET, unlike traditional gamma camera imaging, not only offers significant improvement in resolution and three-dimensional capabilities, but when used in conjunction with PET-CT hybrid also provides exquisite quantification of images due to the benefits of Attenuation correction incorporated, provided by CT. Due to the preliminary success of 131i-NM404 in the current pulmonary cancer imaging trial, it is now imperative to evaluate the efficacy for the detection of NM404 tumors labeled with iodine-124 by PET in order to overcome the limitations inherently associated with the scintigraphy flat Successful radiolabelling of NM404 with iodine-124 The inventors have obtained high specific activity with sodium-iodine-124 in 0.1 N NaOH Eastern isotopes (Sterling, VA). Radiolabelling of NM404 is achieved at a 60% higher radiochemical efficiency, by modifying a method for isotope exchange. In brief, a 2 ml glass vial is charged 10 mg of ammonium sulfate dissolved in 50 μl of deionized water. Glass beads are added, teflon-coated setúm and a screw cap are added and the vial is stirred gently. A 10 μm solution (in 10 μl ethanol) of mother NM404 is added followed by aqueous sodium iodide-124 (1-5 mCi) in less than 30 μl aqueous 0.01 N sodium hydroxide. The reaction vial was gently stirred. A 5 ml disposable syringe that contained tandem glass wool with another syringe filled with 5 ml pieces of charcoal with needle output. The syringe with glass wool acts as a condensation chamber to trap the solvents in evaporation and the syringe with charcoal traps the iodide / free iodine. The reaction vessel was heated in an apparatus with a heating block for 45 minutes at 150 ° C after which four 20 ml volumes were injected into the vial. reaction with a 25 ml disposable syringe and allowed to vent through the dual trap junction. The temperature was increased to 160 ° C and the reaction vial was heated for an additional 30 minutes. After cooling to room temperature, ethanol (200 μl) was added and the vial was stirred. The ethanolic solution was passed through a pre-equilibrated Amberlite IRA 400-OH resin column to remove the unreacted iodide. The volume of eluent was reduced to 50 μl, via a stream of nitrogen (using a charcoal syringe trap) and the remaining volume injected onto a column of silica gel (Perkin Elmer, a column with disposable cartridge of 3 μm x 3 cm, eluted at 1 ml / min with hexane / isopropanol / water (52: 40: 8)) for purification. The final purity was determined by TLC (silica gel with plastic backing-60, eluted with chloroform-methanol-water (65: 35: 4, Rf -0.1). Solvents by HPLC were removed by rotary evaporation and radioiodinated NM404. The resulting solution was solubilized in aqueous 2% Polysorbate-20 and passed through a 0.22 μm filter in a sterile vial.The radiochemical purity is typically greater than 99%.

In vivo PET imaging of murine tumors with 124I-NM404 Brain Tumors The inventors evaluated the imaging characteristics of NM404 in rats bearing C6 glioma tumors (3-5 mm in diameter) and with simulation of operation. The analysis for tissue distribution was performed with 125I-NM404 at 24 and 48 hours after injection, and a separate group of animals were screened by microPET at various times after the injection of 124I-NM404. The biodistribution analysis indicated minimal radioactivity NM404 in normal brain tissue, however, the tumor / brain ratios (dose injected in% / g) were 10.6, and 12.0 to 24, 48 hours, respectively. (Figure 15) NM404 tumor uptake was corroborated by histology. These preliminary results obtained in the rat glioma model suggest that NM404 maintains considerable promise for the detection of malignant primary and metastatic brain tumors.

Lung, Prostate, and Pancreatic Tumor Model Preliminary imaging results using micro-PET with 124I-NM404 in mouse models with lung, prostate, and pancreatic cancer are presented in Figures 16-18. A common characteristic in all images using microPET, obtained in animal models is the lack of bladder activity at any point of time. The pharmacokinetic studies in humans have confirmed this finding since only 4% of the agent cleared renally with 4 days of the i.v injection. (The majority was excreted through the Gl route). In all cases, NM404 exhibited significant tumor avidity regardless of body location. The absorption of tumors was typically staged within 6 hours of the injection, although the tumor to the antecedent generally improved significantly over time, especially in abdominal tumors. Stimulated by these and other observations, the inventors recently conducted a small pilot therapeutic study with 125I-NM404 in SCID mice with human A549 lung tumor xenografts. 12 I-NM404 was administered either as a single dose or alternatively in 3 doses (once a week for 3 weeks) to groups of 6 mice and a separate group received a basic equivalent dosage of NM404 unlabeled for comparison. Single doses were 50 to 500 μCi and the group with repeated dose received a total of 3 doses of 50 μCi weekly. Tumor growth was monitored for 10 weeks after the final injection. The results Preliminary results indicate a significant regression in tumor growth at the highest dose and perhaps a similar response in the 3-dose group, although these ramifications of the study are still in progress. Currently, new ramifications have been initiated to cover dosing levels among the current ones in order to more accurately assess the therapeutic potential of this agent. Even at the 500 μCi dosage, none of the mice showed any signs of toxicity.

Clinical analyzes NM404 has been administered in a screening dose for imaging (0.3 μg / kg body mass) in the NSCLC Phase 1 trial at the University of Wisconsin. A subject of 70 kg of this form could receive approximately 21 μg of NM404, although recent improvements in the pair-marked procedure by exchange have resulted in much lower mass dosages to be injected. Given the recent improvements in tagging and specific activity, the inventors were able to easily reach a mass dose in the variation of 142.9 ng / kg BW corresponding to 0.22 nmol / kg BW or a total mass dose of 0.010 mg per 70 kg of the patient. Additional improvements are anticipated based on a new marking methodology as will be described later, the which will probably result in a reduction of 50 times in the mass of the required compound. Taking into account those future improvements, the inventors believe that the intended clinical mass dosage of I-125-NM404 to be provided in the clinical trials performed could be between about 3-5 ng / kg BW or 250 ng per 70 kg of the patient. The subset of phospholipid ethers known as alkylphosphocholines possesses a wide range of pharmacological activities, having been studied extensively as anticancer and anti-leshmanial agents at micromolar concentrations in animal models and in humans. Neither the mode of absorption nor the precise mechanisms of action have been clearly defined, although an interruption of the membranous lipid metabolism is observed in the membranes of the tumor cells. Miltefosine, hexadecylphosphocholine, has a reported LD50 of 606 nmol / kg in rats with a maximum tolerated dose of 39 nmol / kg over a period of 4 weeks. In clinical trials in humans, a daily oral dosage of 150 mg (3 x 50 mg) was tolerated fairly well with minimal side effects (nausea and emesis) (Planting AS, Stoter G, Verweij J. European Journal of Cancer 1993; 29A: 51 8-519).

Toxicology studies conducted for NM404 This section will summarize four formal toxicology studies with LPG conducted using NM404. These studies are: Formal toxicology studies of NM404 in male rats and rabbits were conducted at the Toxicology Research Center of the State University of New York in Buffalo under the direction of Dr. Paul Kostyniak The drug vehicle and the drug product were provided to Dr. Kostyniak by Dr. Raymond Counsell of the University of Michigan for testing as described in the accompanying synopses of study 27 and study 28. Since no effects were observed significant toxic at a dosage of 4 mg / kg, which is a dose of approximately 200 times the dose for anticipated imaging at that time, and the inventors estimated that it will be approximately 2860 times the anticipated therapeutic dose for clinical trials in accordance with this invention. Human safety studies of unlabeled NM404 were initiated in normal male humans at a mass of 10 times the dose for early imaging and approximately 21 times the anticipated therapeutic dose. The results again showed no toxicity that could be attributed to the substance of the drug. This toxicology study was conducted in accordance with the GLP conditions. Subsequently, researchers at the University of Wisconsin, initiated a toxicology study of NM404 unlabelled in rats and female rabbits (Study 31 and Study 32) at the Toxicology Research Center in SUNY-Buffalo in order to expand the population of patients who will be studied in the Phase 1 clinical trial of NSCLC. Because they were not observed, significant toxic effects at a dosage of 0.04 mg / kg, which is a dosage of approximately 200 times the mass dose for imaging reviewed at that time, and for which the inventors estimated that it would be approximately 286 times the anticipated therapeutic dosage for clinical trials. Human safety studies of unlabeled NM404 were initiated in normal female humans at a mass of 10 times the dose for early imaging and approximately 21 times the therapeutic dosage anticipated Again, no significant toxic effects were observed in any of the female rats or rabbits. This toxicology study was conducted in accordance with the GLP conditions. It was planned in the toxicology studies that at that time will increase to 200 times the anticipated mass dose in trials with humans. Because of this, the improvements for the exchange marking methodology (see method 2 in the CMC section), will probably result in a reduction greater than 50 times in the mass of the compound required for the reaction. In this way, recalculation of the toxicological dose indicated without toxicity was evident in a dose of at least 10,000 x anticipated clinical dose. During Phase 1 safety, pharmacokinetic and dosimetry trials in normal volunteers and patients with NSCLC, no toxicity was observed in any of the male (or MI) or female (UW) volunteers. in English) normal or in none of the patients with NSCLC who participated in the study (UW).

Items for control and testing: The article for testing was a solution of C-NM404 (active ingredient) that contained inactive ingredients- (vehicle). The control solution for this study was the vehicle without the active ingredient.

The test article was formulated as follows: (1) Active ingredient: 2mg / mL C-NM404; (2) Inactive ingredient: 2% Polysorbate 20 in sterile water (injectable grade). The control was formulated as follows: (1) Inactive ingredient: 2% Polysorbate 20 in sterile water (injectable grade); The items for test control were received from Raymond E. Counsell, Ph.D., Professor of Pharmacology & Medicinal Chemistry, Department of Pharmacology, The University of Michigan Medical School, Ann Arbor, Michigan on October 29, 1998. At the Toxicology Research Center at the University of Buffalo, the site for trial testing, the four (4) vials of maked test items "NM-404 (2 mg / ml) in 2% Polysiorbate 20 / sterile water, MAL-V1-82" and four (4) vials of control articles marked "Control vehicle-2% Polysorbate 20 in Sterile water, MAL-V1-83"were inventoried and stored at room temperature.

Administration: The test article (C-NM-404) was administered to more than 200 times the anticipated clinical dose. Control rats illustrated were injected intravenously into the lateral vein of the tail. The rats were injected with the test article or control intravenously at 2 ml / kg body weight using a 25 gauge needle and a 1 ml syringe. The injections were alternately administered to rats of the control group with rats from the test group. The injections were administered prudently during a time interval of 30 seconds to one minute. The injection to the control rat # 27-01 was administered at 9:03 am and the last injection to the test rat # 27-16 was administered at 11:01 am. The following rats were moved during the injection and received multiple injections: control # 6 (2 sites), test # 9 (2 sites), test # 15 (3 sites). In human applications, ET-18-OCH3, a true PLE and thus more dissimilar to NM404 than miltefosine, (edelfosine, mouse LD50 (oral) 200 mg / kg), was administered intravenously at a dose of 15-20 mg / kg / day 5 mg / ml in 5% HSA. The 'maximum tolerated dose is 50 mg / kg. Adverse effects reported for that agent include, impaired hepatic function with pulmonary edema and hemolysis up to 4 hours after injection. (Berdel WE, Fink U, Rastetter, J. Lipids 1987; 22: 967-969). Since the total mass dose of NM404 could be less than one ten thousandth of the daily individual dose of miltefosine, could not anticipate toxic events.

Study Procedures: Rats were observed by LAF personnel for acute toxicity signals from the time of injection until 3:30 p.m. The rats were weighed five (5) times a week (Monday to Friday) and your body weights, they were recorded in kilograms. On December 17, 1998, the rats were anesthetized with sodium pentobarbital (65 mg / ml, Lot # 970789, expiration date: February 1, 2000) administered intraperitoneally. The heart was then punctured using a 20-gauge needle and a 10-ml syringe to collect blood samples for hematologic CBC and clinical blood chemistries. The rats bled to death. The thymus, heart, lungs, spleen, kidneys, liver, brain, and testes were collected, thoroughly examined, weighed, and cut for pathology. Tissue samples (except the thymus) were placed in jars of "Z-fix", fixative for histology. Sixteen (16) Sprague-Dawley rats were received from Harlan-Sprague Dawley, Indianapolis, Indiana. All the rats were males born on the same date and seemed be healthy They stayed in the laboratory animal facility and were given food and water, food and water ad lib. Each rat was given a two-digit number beginning with 27 (the study number) and followed by a "unique" number from "01" to "16" (numerical). Each rat had an ear pierced in the right ear with the only number from "01" to "16". The control rats were numbered from # 27-01 to # 27-08 and the test rats were numbered from # 27-09 to # 27-16. The unique numbers were also applied to each cage indicating which rats were housed inside that cage. There were four control rat cages and four test rat cages. The rats were weighed and the average 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 end of the study on day 14. The test product, dosage and mode of administration, batch number: C-NM-404 (MAL-V1-82) 2 mg / ml, administered via tail vein injection for 30-60 seconds, duration of treatment: reference therapy for a single dose, dose and mode of administration, lot number: 2% Polysorbate 20 in sterile water, 2 ml / kg body weight body, administered via injection in the tail vein for 30-60 seconds.

Safety: On day 14, blood samples for both groups will be analyzed for hematology and clinical chemistry. Additionally, the following organs were removed to report pathology and slides for histology: brain, lung, liver, heart, kidney, spleen, and testes. A compilation diagram of organ weights with organ / weight ratios for each rat can be compiled.

Statistical methods: Differences in body weights and biochemical parameters will be compared between groups using the T test.

Safety results: No unusual behavior was observed in any of the rats during this time or throughout this 14-day study. The tail vein injection sites were checked daily when the rats were weighed and no tissue adverse reactions were observed in any of the rats. The average weights of the rats in the control group and the test group were not significantly different, despite a periodic weight loss on a daily basis and insignificant in both groups.

Total tissue examination was performed one week after sacrifice by a pathologist, at SUNY in Buffalo. No injuries were observed in the test or control group. The tissue sections were then analyzed by microscopic examination by light. There were no changes in the histopathology of the examined organs that could be attributed to the administration of the test material. The rat # 27-12 that received the test material had a small focal area of myocardial injury (infarction at an early stage). This feature was not observed in the other attention section that was also processed, which was interpreted as being quite small. Because no lesion was observed in other animals that received the material, the pathologist's protection can be attributed to some unexplained alteration. This is not due to an infection that arises from the lung since the lungs did not show histopathological change. The results of the clinical blood chemistries and hematology were verified for any obvious values that were below the reference variation. These values resulted in a comparison of the group using a t test at a p-value of 0.05. The t tests were performed on: phosphorus, sodium, potassium, AST, ALT, alkaline phosphates, globulin, A / G ratio, glucose, WBC, RBC and hemoglobin. I dont know found no significant difference.

CONCLUSION: No acute toxicological effects have been found that can be attributed to the test article. There was no significant difference between the test group and the control. Similar tests were performed on male and female rabbits and female rats and no adverse effects that could be attributed to the test article were observed.

Preclinical Pharmacology: The method of the inventors for the development of safe and effective cancer therapies is to design small molecule carrier molecules that are capable of being selectively retained in cancer tissue, although not or minimally in non-cancerous tissues. An extension of this procedure to radiotherapy could take advantage of the selective supply 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 the phospholipid ethers (PLE) and especially its subgroup of Alkylphosphocholine analogs, such as, NM404, which exhibits a high degree of tumor selectivity. Phospholipids are an essential component of cell membranes where they impart structural integrity and are intimately associated with a variety of processes for cell signaling. Phosphatidylcholine, commonly known as lecithin, is an example of this. The phospholipid ethers, on the other hand, represent a minor subclass of phospholipids that also reside in membranes. As the name says, these lipids contain an ether linkage instead of an ester at the C-1 position. Platelet activating factor (PAF) represents one of the best known phospholipid ethers. Based on their early findings, various animal and human tumors contained much higher concentrations of ether lipids that occur in nature in their cell membranes than normal tissue (Snyder, F. and Wood R. Cancer Res. 1968; 28: 972-978, Snyder F. and Wood R. Cancer Res. 1 969; 29: 251-258), Snyder proposes that the accumulation of ether lipids in tumors arises as a result of a reduced ability of tumor cells to metabolize these lipids. The prevailing hypothesis is that, the phospholipid ethers, are trapped in the tumor membranes due to their inability to metabolize and be eliminated, similarly by a phospholipase enzyme in the cell membranes. This hypothesis is supported by experiments that show that the extraction of lipids from tumors after the administration of phospholipid radioiodinated ethers revealed only the intact agent, while the analysis of urine and feces revealed only metabolites (Piotzke, KP, et al. , J Nucí Biol Med, 1993; 37: 264-272). Therefore, the reason why tumors retain PLE is due to the differential clearance rates of PLE from normal versus tumor cells. Extensive studies of the relationship of structural activity resulted in the synthesis, radiolabelling, and evaluation of more than 20 phospholipid ether analogs as potential agents for tumor-selective imaging. The iodinated APC analogs were easily labeled with all of the iodine radioisotopes using a method for isotope exchange. These PLE analogues were specifically designed to incorporate aromatic radioiodine in order to make the molecule stable towards deionization in vivo. The low level of activity in the thyroid in all the studies for distribution of imaging and preclinical tissues (in both a% of injected dose / g and% of injected dose / organic base) has confirmed the in vivo stability of radiolabelled PLE analogs .

From the library of phospholipid ether compounds (PLE), NM-324 [12- (3-iodophenyl) -dodecylphosphocholine], initially showed the greatest promise in studies for tumor localization in animals. A variety of tumors, including mammary, prostatic, squamous cell, ovarian, colorectal, and melanoma carcinomas, were successfully visualized by NM324 scintigraphy. During initial pharmacokinetic studies in humans with the prototype agent, NM324, an unacceptable accumulation in liver tissue was observed and additional experiments were performed to identify PLE compounds with superior tumor localization and background clearance properties. Based on this work, NM404 [18- (4-iodophenyl) -octadecylphosphocholine], emerged due to its improved ability to localize in the tumor, its increased metabolic clearance from the liver, and its higher average plasma lifetime. A key observation documented the ability of NM404 to localize to lymph node metastases, which were clearly delineated by scintigraphy in a metastatic prostatic tumor model with no retention in lymph nodes that are not involved. The leading compound NM404 is currently evaluated in more than 25 animal tumor models and in each model of tumor and tumor type studied so far, NM404 has shown selective retention in tumors. Prolonged tumor retention of 125 I-NM404 has been demonstrated in mice for periods of 20-60 days after injection. These features of extensive and slow tumor retention can significantly improve the radiotherapeutic efficacy of the agent, especially for isotopes with a slow radioactive decay similar to for example iodine-125. Extensive biodistribution data for the prototype agent 12SI-NM324 in various tumor models revealed tumor to blood ratios that exceed 8: 1 at later times after injection. In one example, in a rat mammary tumor model, the ratios of abnormal tumor tissue peaked at 96 hours with a tumor-to-blood ratio of 8.6 and a tumor-to-muscle ratio of 20: 1. In addition, the heterogeneity of the biodistribution of PLE-associated radioactivity within the tumor was demonstrated by microauto-radiography studies showing that PLE radioactivity resides exclusively in viable tumor cells located towards the outer regions instead of the central necrotic regions. Comparative biodistribution data for NM-324 and NM-404 have been obtained in mouse prostate with SCID and tumor models with lung cancer A549. These studies revealed high proportions of abnormal tumor tissue and tumor absorption exceeding 25% of the injected dose of NM-404 within the tumor, thus supporting the desire to study the biodistribution of NM404 in humans.

Mechanism of action Metabolic studies Formal metabolic studies were conducted on various PLE analogues including NM324, the predecessor of NM404. In these studies, each agent was examined for its ability to serve as substrates for enzymes associated with PLE metabolism. Three main enzymatic trajectories are involved in the metabolism of PLE. O-alkylglycerol monooxygenase (AGMO) is responsible for cleavage of the alkyl ether linkage in C-1 to form either the long chain fatty alcohol or the corresponding fatty acid subsequently. Phospholipases C (PLC) and D (PLD), on the other hand, give rise to the glycerol or phosphatidic acid products, respectively. Using an enzymatic preparation with microsomal ACMO, NM324 was not a substrate for this enzyme when compared to [3H] -yl-PAF (activating factor of platelets), which was extensively metabolized. Similarly, NM324 was analyzed as a substrate for PLC isolated from Bacillus cereus and was not hydrolysed in relation to 1-palmitoyl-2- [3H] -palmitoyl-L-3-phosphatidylcholine (DPPC), who experienced significant hydrolysis. Finally, various PLE analogs were subjected to a phospholipase D (PLD) assay. The PLD, which was isolated from cabbage, is similar for mammalian PLD since the cabbage form provides the products of the phosphatidylethanol type in addition to the phosphatidic acid when the enzymatic reaction is performed in the presence of ethanol. Several of the PLE analogs, subjected to these analysis conditions, gave rise to the phosphatidylethanol adduct, indicating a possible interaction with PLD. The inventors believe that N 404 is a metabolic substrate for human phospholipase D, and that the relative absence of phospholipase D in cancer cell membranes is the underlying mechanism for selective retention in NM404 tumors. Although they are known from the literature (reference?) It is still unclear why cancers lack PLD in their membranes. Several NM404 precursors were also subjected to metabolism studies in different cell lines, including cells.

Waiker tumor, rat muscle (H9c2), and rat hepatocytes. In these studies, the degree of metabolism was determined on the basis of radiolabeled products formed after incubation for various periods of time and the normalized results for the number of cells or the amount of cellular protein. The subsequent extraction of lipids from the incubation medium and the cell suspension showed little generation of PLE metabolites in the Walter tumor cellsrou , whereas a significant production of metabolites was observed in both muscle cells and hepatocytes during the 48-hour study period. These results correlate exactly with the in vivo biodistribution studies completed in all analogues. Although several studies have been completed, the role of metabolic entrapment in the uptake and retention of radioactive PLE analogues is not well defined and is currently still an active area of examination. The inventors believe that NM404 can enter the cell membranes of all cells, although they are removed from non-cance cells thh a rapid metabolism, while in cancer cells it remains trapped due to the lack of adequate metabolic enzymes.

PLD Analysis Due to the apparent universality of tumor retention of NM404 in tumor models in animals and the initial corroborative results of a human lung cancer trial, the inventors have begun to investigate the mechanism of action of this agent. Although the membrane metabolism of the PLE analogs is regulated by a variety of phospholipases, the inventors have focused the initial spaces on the activity of phospholipase D (PLD), based on the hypothesis that cellular uptake and retention of NM404, it is inversely related to the amount of PLD present in the tumor cell membrane in relation to normal cells. Due to these findings, a preliminary evaluation of the PLD protein activity and the quantification of PLD mRNA were performed by RT-PCR analysis in various murine tumor cell lines, including the murine tumor cell line hepa-1 (hepatoma), CT26 (colorectal adenocarcinoma), and TS / A (breast adenocarcinoma) and compared with normal liver. These experiments revealed that both the activity of the PLD protein and the mRNA levels decreased significantly in the tumor than in normal liver tissue (p <0.05, T test) (Table 1).

Table 1: PLD protein activity and quantification of PLD mRNA for three cancer cell lines and normal liver tissue In conclusion, the mechanism of selective retention of NM404 may be due to the decrease in membranous levels of PLD, thus preventing the metabolism and clearance of NM404 from the cell. Remember that the first enzymatic substrate analyzes conducted with PLD were derived from cabbage, indicated that NM404 was a good substrate for this enzyme. This supports the finding of the ceiulare in vitro culture uptake and the retention study where it is shown that the PLE analogs were sequestered and subsequently metabolized by normal cells (which contained normal levels of PLD). If the cells of the malignant tumor could possess a normal PLD complement, that agent could have been metabolized and also eliminated from the tumor cells. On the contrary, if it could be inferred that the lack of IOS Metabolism and clearance of the malignant cell agent could support the hypothesis, these neoplastic cells lack PLD in relation to the surrounding normal host cells.

Other studies Mechanical studies with PLE analogues: NM324 and NM404 are similar in structure to miltefosine (hexadecylphosphocholine), an antitumor lipid ether studied most extensively in Europe. The antitumor properties of miltefosine and various other analogs of phospholipid ether antitumor have been demonstrated in a wide range of tumor cell lines between those including prostate, bladder, and terato-carcinomas, murine and human leukemias, as well as, lung cancers, of colon, ovarian, brain and breast (Lohmeyer M, Bittman R. Drugs of the Future 1994; 19: 1021-1037). In contrast to many anticancer drugs, these phospholipid ether analogues do not bind directly to DNA and are not mutagenic. Although the precise mechanism of antiproliferative action has not been determined, they obviously act in various tumor cell sites. These compounds have been associated with a variety of cellular effects including, transport, promotion of cytosine formation, induction of apoptosis, and interference with a variety of metabolism of key lipids and enzymes for cell signaling. The majority of which are located in the cell membrane. Although there remains an indeterminacy regarding the mode of absorption of PLE in cells, most current evidence supports the idea that these ether lipids are absorbed directly in cell membranes when they accumulate. A widespread belief is that these agents act by disrupting the metabolism of phospholipid in the membrane; however, studies of cellular distribution with these agents have been limited by spontaneous cellular compartmental redistribution during homogenization and subcellular fractionation procedures. In contrast to the dosages for tracking image formation (several μg) used in the studies for imaging and biodistribution cited by the inventors, antitumor effects are observed only at doses that generally exceed 150 mg per day (Planting AS, Stoter G, Verweij J. European Journal of Cancer, 1993; 29A: 518-9; Verweij J, Planting A, van der Burg M, Stoter G. Journal of Cancer Research &Clinical Oncology, 1992; 118: 606-8; Muschiol C, et al., Lipids 1987; 22: 930-934).

Mechanism of action The prevailing mechanism of action is that phospholipid ethers such as NM404 are trapped in the cell membranes of malignant tumors due to their inability to metabolize and be eliminated. The extraction of tumors after the administration of phospholipid radioiodinated ethers showed the presence of only the intact agent, while the analysis of urine and faeces revealed only metabolites. In this way, these are the rates of differential clearance of the phospholipid ethers of normal cells against the tumor cells that form the basis of this concept. Preliminary studies obtained in more than 27 models of xenografts and spontaneous tumor have universally shown that NM404 undergoes selective and prolonged retention in tumors.

Isotope selection for therapy The inventors believe that iodine-125 is the most suitable radioisotope for combination with the structure that targets NM404, because: The long half-life of the iodine-125 isotope matches perfectly with long-term tumor retention and stable NM404, providing doses of therapeutic radiation for a prolonged period of time.

The effect of iodine-125 is caused both by irradiation of gamma / low energy X-rays and by the Auger electrons, all have a very limited treatment distance. Because NM404 is extracted into the tumor, iodine-125 can effectively deliver a dose to the tumor although it is scarce for the surrounding healthy tissue. Iodine-125, has Auger electrons as one of its radio-decay byproducts (Figure 2). The Auger electrons cause a pronounced biological effect, although they have a very short treatment distance. Because NM404 is extracted directly into all cell membranes of cancer cells (including the nuclear membrane), the treatment distance for DNA is very low. This can effectively allow the Auger electrons to be the main contributors to the effects of I-125-NM404 treatment. The isotope of iodine-125, is used for the formulation in this application because it exhibits favorable characteristics for cancer radiotherapy, with the properties listed below: Gamma irradiation (1) Half-life radioisotopic: 59.43 days; (2) Gamma energy: 35.5 keV; (3) X-ray energy: 27.5-31.7 keV; (4) Average radiation distance: 0.02 mm lead; 2 cm in fabric; Auger electrons (1) Radiation energy: 1 keV; (2) Number of Auger electrons: Up to 21 by gamma decay; (3) Radiation medium distance: 1-10 nm; It should be noted that although iodine-125 has a great utility for biodistribution studies in vivo and extrapolation of dosimetry for imaging for diagnosis, it is not suitable for either the training of flat images in total body or quantification scintigraphic in vi vo of tissue concentrations of NM404. However, iodine-124 offers features for the quantitative determination of tissue concentrations in vivo. In this way, 124-1- NM404, you will find utility for studies pharmacokinetics and biodistribution although not for a radiotherapeutic effect, iodine-131 emits both beta and gamma radiation that produces a therapeutic effect.

Although I-131-NM404, it could potentially be used to radiotherapy, the inventors believe that 1-125 will be the isotope more optimal, because its lower radiation energy has a shorter average distance of radiation that 1-131 and in this way can be hypothesized which will produce less damage to healthy tissue. Therefore, it was decided that I-125-NM404 could be used for all clinical radiotherapeutic studies performed in order to reduce the potential for collateral damage to healthy tissues. Due to its average physical life of 60 days and the emission of 35 keV photons of low energy, iodine-125 is suitable for imaging experiments in mice and rats. Iodine-125, also provides therapeutic efficacy when used in permanent prostate brachytherapy implants ("brachytherapeutic seeds"). The biggest advantage of 1251 is that all protons are low energy, ensuring a very limited exposure of normal tissues surrounding the tumor. The main difference between brachytherapy seeds containing iodine-125 and 1-125-NM404, is the effect of the Auger electrons. Because the brachytherapy seeds have a metal capsule around iodine-125, only the low-energy gamma and X rays are of therapeutic value, and the Auger electrons are removed by the metal capsule. By difference, 1-125- NM404, it is extracted in the membranes of the cancer cell (including the inside of the nuclear membrane), so that the Auger electrons can have a greater contribution to the therapeutic effect.

Although iodine-131 has been used most effectively in the treatment of thyroid cancer, a significant disadvantage of iodine-131 is that there is a gamma emission of increased energy that could actually expose adjacent surrounding tissues to increased radiation of the that could be presented with iodine-125.

Dosimetry The estimation of dosimetry that is related to I-125-NM404 for adult female patients was calculated based on data for SPECT imaging using non-female tumor-carrying rats after administration of I-131 -NM404. The results are listed below: The MIRD extrapolation of the rat tissue distribution data 125I-NM404, initially provided a dose limiting 5 Rad to the adrenal and bladder wall of 2 mCi of 1311-NM404. Accordingly, the inventors have conducted preliminary pharmacokinetic studies in human lung cancer patients at 1 mCi. A simiextrapocalculation of the dosimetry for 124I-NM404, provided a dose level of 2 mCi, simibased on the projected dosimetry to the adrenals and the bladder wall. However, preliminary data in humans (5 patients) indicate a very poor absorption and retention of the agent in any abdominal organ (including the bladder and adrenal glands) with the exception of the liver that returned to the background levels in a term of 11 days. Although these results are not yet complete, it is likely that the actual acceptable dose of 124I-NM404 will exceed 4 mCi.

LOOKING { IBM PC VERSION 3.1 - AUGUST 1995) Radiation dosing estimates for ADULT FEMALES for 125-I-NM404 Assumptions: Predicted residence time from radiodistribution data in female rat with I-131-NM404 OR WHITE GAN DOSE TOTAL TAXPAYER PRII SECONDARY CONTRIBUTOR Mgy / Meq Rd / mCi 1) Adrenals 7.34E-02 2.72E-01 Adrenals 100.0% 0. 0% 2) Brain 5.96E-03 2.20E-02 Body remaining 100.0% 0. 0% 3) Mama 5.96E-03 2.20E-02 Remaining body 100.0% 0. 0% 4) Gallbladder wall 5.96E-03 2.20E-02 Remaining body 100.0% 0. 0% 5) Wall LLI 5.96E-03 2.20E-02 Remaining body 100.0% 0. 0% 6) Small intestine 7.42E-02 2.75E.01 Small intestine 96.0% Remaining body 4. 0% 7) Stomach 5.96E-03 2.20E-02 Remaining body 100.0% 0. 0% 8) Wall ULI 5.96E-03 2.20E-02 Remaining body 100.0% 0. 0% 9) Cardiac wall 3.12E-02 1.15E-01 Cardiac wall 100.0% 0. 0% 10) Kidneys 5.09E-02 1.88E-01 Kidneys 100.0% 0. 0% 11) Liver 4.-66E-02 1.72E-01 Liver 100.0% 0. 0% 12 Lungs 6.43E-02 2.38E-01 Lungs 100.0% 0. 0% 10 13 Muscle 2.54E-02 9.40E-02 Muscle 100.0% Uteruses 0. 0% 14 Ovaries 4.10E-02 1.52E-01 Ovaries 100.0% 0. 0% 15 Pancreas 5.96E-03 2.20E-02 Pancreas 100.0% 0. 0% 16 Red marrow 2.75E-02 1.02E-01 Red marrow 95.2% 0. 0% 17 Bone surfaces 1.84E-02 6.80E-02 Red marrow 82.1% Remaining body 4. 8% 18 Skin 5.96E-03 2.20E-02 Remaining body 100.0% Remaining body 17. 9% 19 Spleen 4.79E-02 1.77E-01 Spleen 100.0% 0. 0% 21 Tuno 5.96E-03 2.20E-02 Timo 100.0% 0. 0% 22 Thyroid 2.98E-01 1.10E + 00 Thyroid 100.0% 0. 0% 23 Urinary bladder wall 1.13E-02 4.18E-02 Urinary bladder 73.7% Remaining body 0. 0% 24 Uterus 4.78E-02 1.77E-01 Uterus 100.0% 26. 3% 15 27 Total Body 1.55E-02 5.75E-02 Muscle 48.?% 0. 0% 28 EQUIVALENT OF EFFECTIVE DOSE 4.93E-02 1.82E-01 Remaining '35 .8% Gondas 23. 7% 29 EFFECTIVE DOSAGE 4.32E-02 1.60E-01 Thyroid 34.5% Gonads 20. 8% The EDE and ED units are mSv / MBq or re / mCi.

RESIDENCE TIME Adrenals 8.99E-02 Ovaries 3.95E-02 hours Small intestine 4.68E + 00 Red marrow 3.83E + 00 hours Heart wall 6.54E-01 Spleen 6.29E-01 hours Kidneys 1.22E + 00 Thyroid 4.44E-01 hours Liver 5.71E + 00 Content of the urinary bladder 2.33E-01 hours Lungs 4.50E + 00 Uterus 3.34E-01 hours Muscle 3.78E + 01 Remaining 1.83E + 01 hours 10 For 5 rad for adrenal: 18 mCi For 3 rad for ovaries: 20 mCi For 5 rad for thyroid: 4.5 mCi As a result of this estimation of dosimetry, the following points are considered: These results are based on a scenario in the worst case of no excretion of NM404 from the body. The dosimetry data were calculated from the SPECT imaging in rats using I-131-NM404 and then converted to I-125-NM404. The adrenal gland seems to be the limiting or decisive organ of the dosage for exposure to radiation. In the experiments for SPECT imaging, the thyroid of the rats was unblocked. In this way, thyroid stones should be evaluated with caution.

Summary of pharmacology The complete summary of the synthesis and biological properties of NM404 is discussed in United States patent application No. 60 / 593,190 filed on December 20, 2004, application No. 10 / 906,687 filed on March 2, 2005 and U.S. Provisional Application 60 / 521,166, filed March 2, 2004, all are incorporated herein by reference for all purposes. The method of the inventors is to design small molecule carrier molecules that are capable of selectively supplying a test solution for diagnostic or therapeutic for the desired target tissue is capitalized on the unique biochemical or pharmacological properties of molecules that exhibit a high degree of selectivity for tissues or tumors. It was initially observed that, a variety of animal and human tumors, contained much higher concentrations of ether lipids that occur in nature in cell membranes than normal tissue. It was hypothesized that the phospholipid ether analogues could accumulate in tumor cells, due to their lower ability to metabolize these lipids. The inventors have searched for radioiodinated phospholipioate ether (PLE) analogs as potential agents for tumor-selective imaging. Various PLE analogues have exhibited an impressive universal ability to selectively localize a wide variety of tumor models in rat, mouse, and human. The prevailing hypothesis is that the phospholipid ethers are trapped in the tumor membranes due to their inability to metabolize and clear up. In fact, the tumor analysis after the administration of phospholipid radioiodinated ethers showed the presence of only the intact agent, while the analysis of normal tissues (liver and muscle), urine, and feces revealed only metabolites. In this way, it is believed that Differential clearance rates of the phospholipid ether analogues of normal cells against tumor cells form the basis of their white concept.

Preclinical studies with first-generation PLE analogues Phospholipid ethers can be easily labeled with iodine radioisotopes using radiolabelling methods developed in laboratories. The iodophenyl phospholipid ether analogues are specifically designed in such a way that the radioiodine is fixed to each molecule that is stable to facilitate desyntization in vivo. It has been found that any chemical modification of the phosphocholine entity or shortening of the chain length of the iodophenylalkyl entity is less than 8 methylenes resulting in little or no absorption in the tumor. The inventors have currently synthesized more than 20 radiolabeled PLE compounds and have tested them in vi tro and in vivo. Two of these, namely NM-294 and NM-324 [12- (3-iodophenyl) -dodecyl-phosphocholine], initially showed the greatest promise in studies of tumor localization in animals. These prototype compounds, labeled with iodine-125, were selectively localized to tumors over time in the following tumor models in animals; 1) Sprague rat Dawley that carries carcinosarcoma Waiker 256; 2) Lewis rat that carries breast tumor; 3) Copenhagen rat carrying prostate tumors Dunning R3327; 4) rabbits carrying Vx2 tumors; and 5) atymatic mice that carry tumors of human breast (HT39), small cell lung (NCI-69), colorectal (LSI 74T), ovarian (HTB77IP3), and melanoma. The optimal tumor localization of these agents takes from one to several days due to the faster clearance of radioactivity from normal tissues relative to the tumor. Certain PLE compounds analyzed in the following paragraphs are shown in Figure 1.

Clinical evaluation of NM324: Although first generation NM-324 and NM-294 compounds exhibited localization characteristics in similar animal tumors, NM-324 was easier to synthesize chemically and thus was selected as the lead compound for studies. initial clinical Although the images obtained in several patients with human lung cancer detected tumors, the images were complicated by a high radioactivity in the liver (Figure 3).

Second Generation PLE Analogs: In order to decrease liver uptake and prolong the plasma phase, the inventors examined 9 NM-324 structural analogs to identify agents that could exhibit improved tumor to antecedent tissue ratios with uptake decreased in liver. The novel PLE analogues were synthesized and radiolabeled with 125 I for an initial imaging analysis in Copenhagen rats carrying Dunning R3327 prostate tumors. Based on this initial selection, NM-404, not only exhibited much lower liver activity than its predecessor NM324, but also maintained prolonged tumor retention (Figure 4). NM404 has so far been selected to undergo additional imaging and biodistribution analysis in a variety of animal tumor models. Accordingly, NM404 has currently been evaluated in more than 20 spontaneous animal tumor and xenograft models as shown in Table 2. In all tumor models, the agent exhibited significant tumor uptake and retention regardless of location. Although tumor retention can be explained by a lack of metabolic phospholipase enzymes in the membranes of the tumor cell, the mechanism is unknown exact absorption in the tumor cell. In addition, the inventors know that the agent is not located in benign intestinal adenomas (polyps), so it was convenient to further evaluate the propensity of the agents to be located in an intermediate stage of tumorigenesis, namely hyperplasia. The inventors are currently evaluating the selectivity of NM404 in a unique mouse tumor model, developed in Wisconsin, where both preneoplastic hyperplasias and malignant adenocarcinomas form spontaneously in the mammary gland. Preliminary results in this model indicate that the agent is not extracted and maintained in preneoplastic lesions, and thus, it seems that they are retained exclusively by malignant tumor cells. If this initial observation is validated, then via genomic and proteomic analyzes, the inventors will have the ability to identify a key genetic difference between malignant tumors and their non-malignant predecessor cells. This could identify a totally novel molecular therapeutic target that can be universal for all types of cancer. The following table summarizes the wide variety of cancers and tumor lines that have been evaluated with respect to accumulation using NM404.

Table 2: Tumor models examined with NM404 Location of Tumor model Species Tumor type NM404 * Human tumor Xenografts Prostate PC-3 Mouse SCID Adenocarcinoma Si Lung A-549 (NSC) Mouse SCID Adenocarcinoma Si Lung NC1 H-69 Naked mouse Adenocarcinoma Yes (oat cell) Adrenal H-295 • Mouse SCID Adenocarcinoma Si Adrenal RL-251 Mouse SCID Adenocarcinoma Si Melanoma A-375 Naked mouse Adenocarcinoma Si Colon LS-180 Naked mouse Adenocarcinoma Si Ovarian HTB-77 Naked mouse Adenocarcinoma Si Xenografts to take turns animal Mammary MCF-7 Rat Adenocarcinoma Si Prostate MatLyLu Rat Adenocarcinoma Si alter-256 Rat Carcinosarcoma Si M bdelos of recent tumor in rodents Mouse Prostate TRAMP Adenocarcinoma Yes transgenic Xenograft of Prostate LuCaP Adenocarcinoma Si mouse Xenograft of adenocarcinoma Liver CT-26 Si mouse colorectal Mouse TGF to hematoma Hematoma Yes transgenic Mouse Min intestinal mouse Adenocarcinoma Yes transgenic Melanoma Xenograft B16 Adenocarcinoma Si mouse SCC1 Carcinoma Xenograft and 6 If nude mouse flaky cell Adenocarcinoma mammary? Pc * mouse Adenocarcinoma Si Mammary SCC carcinoma Ppc * mouse Si squamous cell Xenoin Gliama leash L9 and CNS-1 Glioma Rat Rat Pancreas mouse c-myc / k-ras Ductal / acinar Yes transgenic Mouse Retinoblastoma Blastoma Si transgenic Cervial Mouse Adenocarcinoma Yes transgenic Alveolar hyperplasia ßpc / mirw- mouse Hyperplasia Nonmammary Intestinal adenoma Apc 1 * Mouse Hyperplasia No Location defined as >5% dose injected per gram of tumor base is based on tissue distribution data. No corrected decay of tumor clearance of 14 to 80 days was observed in these models using 125I-NM404.

Tissue distribution and kinetics Consistently, NM404 was found to be retained in tumor tissue for extended extended periods of time. Tumor concentrations are almost stable for many weeks after administration of NM404, which shows a slow elimination of cancerous tissue over time. In contrast, NM404 was removed from normal tissue over a period of a few days that reached very low levels. Additionally, NM404 was designed to have a long half-life in blood. This ensures a prolonged exposure of NM404 to the tumor tissue and ensures the absorption of up to 10% -25% of the dose injected into the tumor tissue. The inventors believe that it is very important for a radiotherapeutic compound that has a long portion of injected dose that accumulates in the tissue of interest. As a consequence of the long half-life in blood, NM404 will accumulate continuously in tumor tissue over time. An example of this pattern is given in Figure 5. This can lead to a delayed appearance of the therapeutic effect until the accumulation of NM404 in the tumor tissue is continued for several days or weeks.

Plasma Kinetics in Blood It was found that compound NM324, a first generation prototype, has a half-life of elimination in plasma of 2.43 hours in rats. For comparison, the leading compound NM404 has an elimination half-life of approximately 209 hours in rats (the half-life in the distribution phase is 4.86 hours).

Radiotherapeutic study of I-125-NM404 During the course of mouse tumor uptake and retention studies with "imaging" dose (15-20 μCi / 20 g of mouse) of 125 I-labeled NM404, a variety of obvious therapeutic responses (unpublished results). In a mouse mammary tumor model of Apc "1" / - * - it has been generally observed that tumor growth remains static after a single intravenous injection of NM404. Some of these animals also lost all their hair on larger mammary tumors in approximately 8 days after the injection. In addition, these mice also had intestinal tumors and usually suffered from intestinal bleeding, resulting in severe anemia, which was evident from their white legs. Dr. Moser observed that the legs of these mice had returned to a pink color approximately 5 days after a single injection of NM404. At the time of an eventual dissection of these animals, it was observed that only a few of the 20 expected or in this case the intestinal tumors usually found in this stage currently remained. The phenomenon of "white-to-red feet" was also observed in a separate, although more aggressive mouse intestinal adenocarcinoma model, where dissection at 12 days after administration of NM404 again revealed that most, if not is that all, the expected intestinal tumors disappeared. After 21 days in both intestinal models, the animals that received NM404 easily survived their untreated baitmates. Another example of tumor regression imposition is illustrated in Figure 6. Two baitmates each received SCC1 and SCC6 xenografts on their left and right flanks, respectively. One mouse received a single injection of 125 I-NM404 (20 μCi). The mouse that did not receive NM404 died 21 days later, while the tumors in the treated mouse returned significantly and the animal was quite healthy 80 days after the injection. These coincident findings were reconfirmed in two equal groups separately each, involving more than 6 mice. Although you are observations with 125I-NM404 are anecdotal at this point, they do not seem to indicate in an important way a potential for radiotherapy applications in particular, if marked with iodine-131. Quantitative studies on tumor absorption and retention in various tumor models in animals will also provide sufficient data to initiate a complete dosimetric analysis for this agent in order to estimate its true radiotherapeutic potential. Due to its 60-day physical half-life and low-energy 35 KeV photonic emission, iodine-125 is suitable for imaging experiments in mice and rats. Iodine-125 also provides therapeutic characteristics. In an imaging experiment (Figure 6), 2 nude mice each were inoculated with SCCl and SCC6 tumor cell implants of subcutaneous squamous cell lines on the opposite flanks. SCCl and SSC6 cells were used because one is radiosensitive relative to the other. After 14 days when an average tumor size of 0.5 cm in diameter was reached, one of the mice received 20 μCi of I-125-NM-404 and the other received NM404 unlabeled in an equal mass dosage. The mouse that received the cold compound without marking had to be sacrificed 20 days after the injection, because both tumors reached the size limit for termination as defined in the protocol for use in animals. Both tumors in the 125 I-NM404 mouse returned dramatically and unexpectedly over the course of several weeks (Figure 6) after a single injection of a dose for NM404 imaging. This mouse never reached the size of the terminal tumor and the mouse was actually sacrificed after 90 days in order to collect the histology sections.

Preliminary report of ongoing studies for tumor therapy Preliminary results of the preclinical study: Efficacy of a single injection of SF404A labeled with I-125 in a mouse model with SCID A549 End and reasons To determine an effective dose for radiation therapy using a single injection of SF404A labeled with 1-125 in a tumor model with xenograft: Tumor model A549 (giant cell human lung cancer, NSCLC, from ATCC) was maintained in F-12K medium supplemented with 10% fetal bovine serum. The tumor cell suspension (1 x 106 cells in phosphate buffered saline) was injected s.c. on the right flank of female SCID mice (6-8 weeks, C. B-17 / IcrHsd-Pr cdsc'ld, Harían). The animals had access to food and water, the growth of the tumor and the weight of the animal were monitored. When reaching 4-5 mm in diameter, the mice were divided into groups of 6 for a therapeutic study.

Dosage One single injection of SF404A on day 0.

Groups (1) 5 study groups: 4 treatment and 1 control; (2) N = 6 per group; (3) 4 acute dosage levels: 50, 150, 250 and 500 μCi per mouse; (4) The control group was dosed with a NM404 equivalent mass amount; (5) Control: 0 μCi per mouse (NM404"cold").

Efficacy assessments (1) Tumor size (caliber) measured once a week; (2) Survival; (3) General appearance (activity, agility, mobility); (4) Up to 10 weeks after the injection or until no animal in the control group is alive, whichever comes first; (5) Histopathological evaluation of tumors or residual tumor site. Photograph of the tumor on day 0, 30, and 60 for each animal. The purpose of the study was to determine an effective dose for radiotherapy using a single injection of I-125-NM404 (SF404A formulation) in a tumor model with xenograft. The tumor model used for this study was A549, a human giant cell lung cancer (NSCLC) obtained from ATCC (Manassas, VA). The tumor cells were maintained in Ham's F-12K medium supplemented with 10% fetal bovine serum. The tumor cell suspension (1 x 106 cells in phosphate buffered saline) was injected s.c. on the right flank of female SCID mice (6-8 weeks, C. B-17 / IcrHsd-PrJfcdscid, Harian). The animals had free access to food and water, reaching 5-10 mm in diameter in the tumor, the mice were enrolled in the study.

A single intravenous injection of SF404A was performed on day 0. For the control group, the cold compound (C-NM404) had been injected. I-125-NM404 was injected for the treatment groups. The study contained 5 groups; 4 treatment groups (n = 6 / group) and 1 control group (n = 9). The iodine doses for the treatment groups were 50, 150, 250 and 500 μCi per mouse. The control group was dosed with an equivalent mass amount of C-NM404 (0 μCi). For the evaluation of the efficacy of the treatments, the following assessments were made: (1) Size of the tumor (caliber) measured once a week; (2) Survival, (3) General appearance (activity, agility, mobility), (4) Digital representations of the tumor-bearing mouse. The assessments were made up to 10 weeks after the injection or until no animals in the control group remained alive, whichever comes first. The histopathological assessment of the tumors or the residual tumor site was made at the end of the study. (1) Group of 50 μCi: 6 animals were enrolled; (2) Group of 150 μCi: 5 animals were enrolled; (3) Group of 250 μCi: 6 animals were enrolled; (4) Group of 500 μCi: 6 animals were enrolled; As part of this preliminary report, the inventors included the following animals in the analysis: (1) Control: n = 7 (2) Group of 50 μCi: n - (3) Group of 150 μCi: n = 5 (4) Group of 250 μCi: n = 6 (5) Group of 500 μCi: n = 5 The tumor volumes at initial values (in mrri) for each group were: Since the tumor volumes of the control group and the 150 μCi group were substantially higher than the other groups and which was previously planned, a complete re-enrollment of both groups was initiated. with smaller tumor sizes. As regards the tumor volumes for the 500 μCi group, the inventors believe that the study can be diverted against this group. Due to necrosis and inefficient blood supply, larger tumors may also not respond to the treatment of I-125-NM404. The following average tumor volume for each group was recorded during the 10-week assessment period: This is also reflected in Figure 10. In summary, the control animals showed rapid growth of tumors during the period of 10 week assessment. This confirms that the compound itself C-NM404, did not have a substantial effect on the growth of the tumors. The group with a dose of 50 μCi did not show any difference with the control animals, since this seems to be an ineffective dose in this animal model. The 150 μCi dose group also showed no treatment effect, however, (as noted above), this dosing group started with unusually large tumors on day 0, however, this may show different results with this group of 150 μCi dose with smaller tumors. Preliminary data indicate that both 250 and 500 μCi show a substantial and prolonged treatment effect. Tumor volumes were stable and the same tumors appeared to "collapse" (the tumor surface collapsed, as shown in Figure 7), additionally, the hair on top of the tumors fell off, confirming the substantial accumulation of radioactivity in these tumors .

Other pre-clinical studies in progress for therapy in tumors Efficacy of "a fractional dose against a single injection of SF404A marked with 1-125 in a model of A549 SGD mouse This pre-clinical study assesses the fractionation of the 150 μCi dose when injecting 3 doses of 50 μCi weekly instead of a single dose of 150 μCi, as administered in the previous study.

Purpose and Reasons: To determine the radiotherapeutic efficacy of a fractional dose of SF404A labeled with 1-125 against a single injection of a total equivalent dose of a tumor model with xenograft, this study is an adjunct to CLTR-Pre-05- 001, which contains the only dose of 150 μCi used for comparison.

Tumor model: A549 (giant cell human lung cancer, NSCLC, from ATCC) was maintained in Ham's F-12K medium supplemented with 10% fetal bovine serum. The tumor cell suspension (1 x 106 cells in phosphate buffered saline) was injected s.c. on the right flank of female SCID mice (6-8 weeks, C. B-17 / IcrHsd-Prkcdsc? d, Harian). The animals had free access to food and water, the growth of tumors and the weight of the animals were monitored. When reaching 5-10 mm in diameter the mice were divided into groups of 6 for a study therapeutic Dosage: A single injection of I-125-SF404A on day 0 against a fractional dose of 1-125-S404A Groups: 2 study groups: 1 divided dose, 1 single injection; (1) N = 6 per group; (2) 150 μCi (3 x 50 μCi) per mouse by fractional injection, at 1 week intervals from day 0, (3) 150 μCi per mouse by a single injection.

Assessment of efficacy: (1) Size of the tumor (caliber) measured once a week; (2) Survival until 10 weeks after injection or until no animal was alive in any group, whichever occurs first; (3) General appearance (activity, agility, mobility); (4) Histopathological evaluation of the tumor or residual tumor site. (5) Photograph of the tumor on day 0, 30, and 60 for each animal.

Efficacy of a single injection of SF404A labeled with I-125 in a PC-3 SCID mouse model (prostatic cancer) Purpose and Reasons: To determine an effective dose for radiation therapy using a single injection of SF404A labeled 1-125 in a tumor model with prostatic cancer xenograft.

Tumor model: PC-3 (human prostate cancer, from ATCC) was maintained in Ham's F-12K medium supplemented with 10% fetal bovine serum. The tumor cell suspension (1 x 106 cells in phosphate buffered saline) was injected s.c. on the right flank of male SCID mice (6-8 weeks, C. B-17 / IcrHsd-PrJccdscid, Harian). The animals had free access to food and water. The growth of the tumor and the weight of the animals were monitored. When reaching 4-5 mm in diameter, the mice were divided into groups of 6 for the therapeutic study.

Dosage: A single injection of SF404A on day 0.

Groups: (1) 5 study groups: 4 treatment + 1 control group; (2) N = 6 per group; (3) 4 water dosage levels: 10, 150, 250 and 500 μCi per mouse; (4) Control groups dosed with an equivalent mass amount of NM404; 0 μCi per mouse (NM404"cold").

Efficacy assessments: (1) Tumor size (caliber) measured once a week; (2) Survival: up to 10 weeks after the injection or until no animal in the control group was alive, whichever comes first; (3) General appearance (activity, agility, mobility); (4) Histopathological evaluation of the tumor or residual tumor site. (5) Photograph of the tumor on day 0, 30, and 60 for each animal.

This preclinical study duplicates the only previous study injection, although with a different tumor model.

Clinical evaluation for prostate imaging and NSCLC using a phospholipid ether analog, NM-404 The safety assessment in normal male subjects and normal healthy women was carried out and a dosage level of 3 μg / kg was determined of C-NM-404 is safe to administer to males. This dosage is 10 times the dosage of 0.3 μg / kg of anticipated 131I-NM404 to be used for imaging in patients with metastatic prostate cancer. A dosage level of 3 μg / kg of C-NM-404 is safe to be administered to women. This dosage is 10 times the dosage of 0.3 μg / kg of 131I-NM404, anticipated to be used for imaging in patients with metastatic lung cancer.

NM-404 imaging characteristics in patients with NSCLC Subjects with metastatic giant cell lung cancer: Written informed consent will be obtained. 131I-NM404 at a dosage of 0.3 μg / kg will be administered by infusion for 10 minutes. The aqueous solution containing 131I-NM404 will be prepared using a sterile technique in the laboratory of Dr. Jamey Weichert at the University of Wisconsin. The preparation supplied by the Nuclear Pharmacy will be certified as sterile and pyrogen-free. The subject will be monitored for adverse reactions during and after the infusion. The vital signs will be verified immediately after the infusion, and at intervals of 60 minutes up to four hours after the injection. They will be observed for adverse events throughout this time. The subjects will return 24, 48, 72, 96, 120, 144 hours and 30 days after the infusion. NM404 PET scans will be obtained at 4, 8, 24, 48 and 96 hours after injection. The vital signs will be monitored during these times. Any adverse reactions experienced by the subjects will be recorded. All subjects will receive an oral SSKI solution that begins one day before the infusion of .131-NM404 and continue for 7 days to reduce the exposure of the thyroid to free radioiodine. Serum pharmacokinetics will be extracted by pre-infusion, 5, 10, 30, 60, 120, 240, 360 minutes after injection and at 24, 48, 72 and 96 hours after injection. Sequential collections of urine will be obtained in 24 hours during 0-24, 24-48, 48-72 and 72-96 hours after injection. Plasma pharmacokinetics confirmed an average of 113.1 hours (SEM = 7.9 h) of elimination half-life for NM404. An average of 3.4% (variation of 0.9% to 9.8%) of injected dosage was eliminated via the kidney with 96 hours of intravenous administration of NM404. A dosage level of 0.3 μg / kg of 131I-NM404 was safe to administer to subjects with advanced giant cell lung cancer. The mean plasma half-life for elimination of NM404 was found to be 113.1 hours and it was found that urinary elimination will be approximately 3.4% within 96 hours after injection.

Prostatic Cancer The present invention provides preliminary data relating to the use of the second generation PLE analog, NM404, in imaging prostate cancer patients. This agent, currently under investigation at the University of Wisconsin, is selectively maintained in tumors at high levels, and has a high sensitivity and specificity in preclinical models. He has passed a test of acute toxicology in both rats and rabbits to > 1000 times the anticipated dosage for imaging in humans, and the unlabeled agent was administered at 10 times the anticipated mass dosage for imaging to 10 normal volunteers at the University of Michigan and the University of Wisconsin for documented safety. The inventors hypothesize that imaging with NM404 will ultimately be proven to be sensitive to FDC imaging, will also be more specific, may provide therapeutic utility and due to its relatively long half-life will be available virtually at each facility PET regardless of location. Biodistribution, kinetics, optimal times for imaging, and dosimetry of 131I-NM404, are currently being evaluated in a pilot study in patients with lung cancer (NSCLC) at UWCCC. This agent has also evaluated pre-clinically in a metastatic prostate tumor model in which metastases from lymph nodes are clearly delineated by scintigraphy after intravenous administration of NM404, although, pertinently, the tracer is not retained by the lymph nodes not involved The selectivity of NM404 for malignant cells is particularly relevant in prostate cancer as conventional tumor markers such as PSA can also be elevated in a variety of benign disease states including prostatitis and benign prostatic hypertrophy. In addition, selective absorption and prolonged retention by tumor cells supports a function for NM404, as a tumor-selective diagnostic and therapeutic agent. Preliminary results will provide preliminary data for a further study designed to more accurately estimate the predictive power of NM404, to graduate and / or monitor the response to therapy in prostate cancer. In addition, because NM404 has a high absorption in tumors, this agent also has the potential to be developed as a therapeutic agent when coupled with higher dosages of 131 I, 125 I, another halogenated astatin. Iodine-125 could be especially convenient in patients with prostate cancer, due to its short length of Auger electron path in tissues, which could theoretically minimize the effects of radiation on neighboring normal tissues similar to the rectum. In a therapeutic sense, the half-life of 60 days of iodine-125 coincides quite well with the properties for retention prolonged in NM404 tumors.

White tumor cells A procedure for the development of examinations for formation of sensitive images, with greater availability is to design carrier molecules that are capable of selectively delivering a radiopharmaceutical assay solution to the desired target tissue. The procedure has been to capitalize the unique biochemical and pharmacological properties of the phospholipid ether analogues such as, NM404, which exhibits a high degree of tumor selectivity. Phospholipids are an essential component of cell membranes, where they impart structural integrity and are excessively associated with a variety of processes for cell signaling. Phosphatidylcholine, commonly known as lecithin, is an example of this. The phospholipid ethers, on the other hand, represent a minor subclass of phospholipids that also reside in membranes. As it is said, these lipids contain an ether bond instead of an ester bond at the C-1 position. Platelet activating factor (PAF) represents one of the best known phospholipid ethers. As described above, tumors retain PLE due to the differential clearance rates of PLE from normal cells against the tumor. While pharmacokinetic studies in humans are being initiated with the prototype agent, NM324, ongoing experiments were conducted to identify PLE compounds with superior tumor localization and background clearance properties. Based on this work, NM404 [12- (4-iodophenyl) -octadecylphosphocholine] was selected due to its improved ability to localize in the tumor, its increased metabolic clearance from the liver, and its longer average plasma life. In a key experiment documenting the ability of NM404 to localize to metastases, lymph node metastases were clearly delineated by scintigraphy in a prostate, metastatic tumor model after intravenous administration of NM404, although the tracer was not retained by the lymph nodes not involved. The results for comparative scintigraphic imaging for NM324 and NM404 in SCID mice carrying prostate PC-3 tumors revealed high tumor-to-normal tissue ratios and significant decreases in abdominal radioactivity and antecedent liver with NM404 (Figure 19). This agent has currently been evaluated in 27 animal tumor models and it is clear that once the agent enters the tumor cells, reaches an end of metabolic death and is trapped. The prolonged retention in tumors of this agent is demonstrated in a human adrenal tumor xenograft implanted in SCID mice (Figure 20). Using NM404 labeled with 125 I, the inventors have been able to image mammary and prostatic tumors in mice in an excess of 60 days. The characteristics of prolonged retention in tumors significantly improve the radiotherapeutic efficacy of the agent. Recent studies for imaging and biodistribution performed in rodent models, aimed at determining the absorption characteristics in a wide variety of xenograft and spontaneous tumor types (endogenous or transgenic) were summarized above. These agents have selective localization exhibited and prolonged retention in each malignant tumor regardless of the anatomical location (including lymph nodes) studied to date.

Results of preliminary scintigraphic imaging with NM404 in mice bearing prostate tumors In a preliminary experiment to show that NM404 is localized in mouse prostate tumors, TRAMP mice were screened in a radioTLC scanner Bioscan AR- 2000 (modified in the laboratory for imaging in mice) of i-8 days after tail vein injection of 125 I-NM404 (15 μCi). After in vivo imaging of the anesthetized mice, the prostate tumors were immediately removed, and the ex vivo image was formed in the same scanner (equipped with a high resolution 1 mm collimator and software for obtaining and analyzing the images). -D) in order to avoid attenuation of tissue associated with low iodine energy125 (Figure 21). Although the number of animals carrying prostate tumors was small (n = 4), preliminary images and antecedent tumor data indicated an NM404 uptake in the prostate tumor, although no benign hyperplasia was found in this model. In an attempt to simulate bone metastasis, another experiment was performed in which human PC-3 tumor cells were implanted in the tibia of immunocompromised nude mice (n = 6) according to recent reports. After inoculation of tumor cells, the mice were screened in series by high-resolution microCT in order to monitor bone tumor development (Figure 22). Once bone deterioration was detected, it was administered intravenously via the tail vein NM404 labeled with iodine-125. The mice were screened for radioactivity and by microCT, 4 days after the administration of NM404. The radioactivity scans were fused with the microCT scans (Figure 23) to corroborate the radioactivity of NM404 and the location of the tumors.

Biodistribution data Extensive biodistribution data for the 125I-NM324 prototype agent in various tumor models have been reported previously. Tumor to blood ratios exceeding 8: 1 were observed at delayed times after injection. For example, in a rat mammary tumor model, the ratios of abnormal tumor tissue reached a maximum at 96 hours with a tumor-to-blood ratio of 8.6 and a tumor-to-muscle ratio of 20: 1. In addition, the biodistribution of PLE-associated radioactivity is heterogeneous in the tumor, as demonstrated by microauto-radiogram studies that show that PLE radioactivity resides exclusively in viable tumor cells located towards the outer regions instead of the central necrotic regions. The comparative biodistribution data for NM324 and NM404 in SCID mice had been obtained in tumor models with prostate and lung cancer A549. These studies revealed high proportions of tumor to normal tissue and a tumor absorption exceeding 25% of the dosage injected with NM404, supporting the desire to examine the biodistribution of PLE analogs in humans.

Isotope selection Because the tumor-directed strategy of the PLE analogs seems to involve selective retention in tumors over time, nuclides with relatively short lifetimes such as 1 RF or 99tnTe, are not practical to mark NM404 at the time in progress. With an average life of 13 hours and optimum characteristics for imaging, iodine-123 also proved to be suitable for this agent, when it was explored in a 3-dimensional SPECT mode. Iodine-123 imaging will require additional research. While tumor localization of the PLE analogs appears to occur several hours after the injection (NM324 images obtained 6 hours after injection in a patient with lung cancer showed an intense absorption in the lung tumor in a previous study) , the formation of flat two-dimensional images, similar to those currently being done with iodine-131, in humans, requires a delay period that allows an antecedent activity to be cleared from neighboring normal tissues and blood. It is likely that early imaging may be possible when scanning with PET and 3D SPECT where radioactivity neighbor interferes less due to the 3-dimensional nature of these modalities. In organs where antecedent radioactivity remains inherently low (brain, for example), it may be possible to use isotopes of gamma emission similar to iodine-123 which, in addition to providing beautiful images, could allow images to be obtained later in the same day of the injection. Although the use of other isotopes may ultimately prove suitable for use with NM404, the current approach will develop the capacity for PET imaging of this agent due to the recent success of oncological imaging using hybrid PET-CT scanners. The undue diagnostic accuracy produced by a biochemist or functional agent for tumor imaging by PET combined with the accurate anatomical accuracy provided by CT is currently the gold standard for tumor imaging. However, it is rather advantageous to label the PLE analogs with iodine-124, a relatively novel PET isotope, where the physical half-life (4 days) coincides well with the absorption of PLE in tumors and the retention kinetics. The labeling of NM404 with iodine-124 represents a natural extension of previous studies with gamma emission nuclides. It has been shown that, PET imaging with 124I, provides more 40 times the sensitivity of the scintigraphy 131I-qamma flat. PET, unlike traditional gamma camera imaging, also offers significant resolution improvement, image quantification, and 3-dimensional capabilities. Due to the preliminary success of 131I-NM404 in the current lung cancer imaging test, it is now imperative to label NM404 with iodine-124 and evaluate its effectiveness for tumor detection by PET in order to overcome the limitations inherently associated with the flat scintigraphy. The utility of tumor trackers similar to 67Ga-citrate and 18F-FDG is limited by their lack of specificity to distinguish a neoplasm from an inflammation. This lack of specificity is a significant clinical problem in patients with cancer. However, preliminary studies with PLE analogues offer the promise of overcoming this limitation. Previous experiments conducted in rats did not reveal uptake or retention of NM324 in granulomas induced by carrageenan. However, gallium citrate, used as a control in this study, did not in fact concentrate significantly on granulomatous lesions. Thus, this preliminary finding that PLE analogs are apparently not located in inflammatory lesions justifies the need for an evaluation of this. agent in human cancer patients. Although FDG-PET has overcome the formation of hybrid images, this lack of specificity of tumor cells will always limit its diagnostic efficacy. New molecularly directed agents, similar to NM404, which exhibit universal absorption in tumors and selective retention regardless of location, as well as selectivity for malignant tumor cells and non-inflammatory or hyperplastic lesions, will represent a significant improvement in the detection and characterization of cancer. While imaging techniques in flat nuclear medicine have historically provided acceptable 2D images, this modality does not offer tomographic capability or poor image quantification. Although 125I-NM404 was adequate for preliminary scintigraphic imaging and tissue distribution studies in rodent tumor models and 131I was adequate for safety in Phase 1 and pharmacokinetic evaluation in human lung cancer patients, nor are optimal for the formation of quantitative images in humans in vivo. PET imaging with iodine-124, a relatively novel and commercially available positronic isotope with a half-life of 4 days, could alleviate many of the problems associated with flat imaging.

The transition from NM404's unique imaging capabilities to PET scanning has now become the main goal of our laboratory since iodine-124 is commercially available during the last year. If the tumor specificity of NM404 for malignant tumors observed in mouse models is confirmed in humans, then there could be an agent with more selectivity for tumors than FDG, albeit without its inflammatory site localization properties. In addition, the agent could be produced in an installation and ship virtually to any location in the world due to its half-life of 4 days. The inventors recently successfully radiolabeled NM404 twice with iodine-124 from a commercial supplier (Eastern isotopes). The radiochemical yield (> 60% average asylated yield,> 99% purity) was very similar to that usually obtained with commercial sources of iodine-125 or 131 sodium iodide. The PET imaging characteristics of 124I-NM404 in a rat brain tumor model CNS-1 (Figure 24) was investigated. The times for imaging in this preliminary study were limited to 24 hours and 4 days after the injection due to the availability of a microPET scanner. The images through microPET 17 obtained 24 hours after injection i.v. of I-NM404 were corroborated with enhanced contrast MRI images and showed intense absorption in the tracker in the brain tumor accompanied by little absorption 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 can then be used to extend the utility of NM404 PET in human cancer patients.

Determination of the absorption and retention characteristics in tumors of 1 4I NM404 by means of PET-CT in patients with radiographically evident metastatic prostate cancer Objectives and reasons The assessment of the selective detection of known metastatic lesions by radioiodinated NM404 is possible and comparable with conventional radiological modalities, is analyzed in the following paragraphs. The inclusion criteria for this study will consist of fifteen patients with metastatic prostate cancer, with at least 5 patients having cancer metastasis.

Prostatic soft tissue and at least 5 of the patients with bone metastases identifiable by conventional radiological studies that include a CT scan and a bone scan. After enrollment of the patient, the uptake of radiolabeled NM404 will be measured by a PET-CT scan with isotope 1-124 and correlated with the radiographically evident lesions detected by patients in other studies of conventional grading.

Methods Synthesis, radiolabelling, and formulation: Radioiodination of stable NM404 with sodium 124I-iodide is routinely achieved by modifying an isotope-exchange reaction supplied by ammonium sulfate, reported by Mangner and recently optimized for NM404 in our laboratory. The exchange reaction methodology has been used effectively for initial testing in humans with NM324, the predecessor of NM404 and is currently being used for pre-clinical studies and the human lung cancer test. Briefly, a 2 ml glass vial was loaded with 10 mg of ammonium sulfate dissolved in 50 μl of deionized water. Fourteen glass beads resistant to 2mm chemical products, one sm, were added coated with Teflon and a screw cap and the vial was gently stirred. A solution of 20 μg (in 20 μl of ethanol) of mother NM404 was added, followed by aqueous sodium iodide (124, 1-5 mCi) in less than 30 μl of 0.01 N aqueous sodium hydroxide. The syringe with isotopes - was rinsed with three 20 μl portions of ethanol. The reaction vial was gently stirred. A 5 ml disposable syringe containing tandem glass wool was attached with another syringe filled with 5 ml pieces of charcoal with a needle outlet. The syringe with glass wool acts as a condensation chamber to trap the solvents that evaporate and the syringe with charcoal traps the iodide / free iodine. The reaction vessel was heated in an apparatus with a heating block for 45 minutes at 150 ° C. Four 20 ml volumes of air were injected into the vial for reaction with a 25 ml disposable syringe and allowed to vent through the dual trap junction. The temperature was increased to 160 ° C and the reaction vial was heated for another 30 minutes. After cooling to room temperature, ethanol (200 μl) was added and the vial was stirred. The ethanolic solution was passed through a pre-equilibrated Amberlite IRA 400-OH resin column to remove the unreacted iodide. The volume of eluents was reduced to 50 μl via a. nitrogen stream (using a trap of syringe with charcoal) and the remaining volume was injected onto a column of silica gel HPLC (Perkin Elmer, column with disposable 3 μm x 3 cm cartridge eluted at 1 ml / min with hexane / isopropanol / water (52:40: 8)) for purification. The final purity was determined by TLC (silica gel with plastic backing-60, eluted with chloroform-methanol-water (65: 35: 4, Rf = 0.1) .The HPLC solvents were removed by rotary evaporation and radioiodinated NM404. The resulting solution was solubilized in Polysorbate-20 aqueous 2% pharmaceutical grade (0.1 μl / mg of the compound) The ethanol was removed under vacuum and the residue dissolved in sterile water to provide a final solution containing no more than 2-3 Polysorbate-20% Sterilization will be achieved by filtration through a sterile 0.2 μm filter unit, each of which will be tested for pyrogens using the Limulus Amebocyte Lysate test kit. The preparation, purification and sterile formulation of NM404 labeled with I-131 for studies in patients with lung cancer The letter for master drug formulation and the checklist for preparation products are included in the complementary section of this purpose. This radioiodine has been performed hundreds of times with either iodine-131 or 125 and radiochemical yields that vary from 60-90% (pure product isolated) with specific activities exceeding 130 mCi / μmol of NM404. This method has provided sufficient pure injectable 131I-NM404 for preclinical and ongoing human trials. To date there has been no marking failure. In addition, and relevant to the current purpose, NM404 has recently been successfully radiolabelled with sodium iodide with iodine-124 (5 mCi) from the Eastern isotopes in > 60% radiochemical yield isolated. Although the acceptance criteria require > 95% radiochemical purity, the results typically exceed 99% because the total reaction mixture is subjected to purification by Preparative HPLC.

Dosage of 1-124 for administration in humans: The profile of toxicity, biodistribution, kinetics, optimal times for imaging, and dosimetry of 131I NM404 are currently being evaluated in an ongoing study in patients with lung cancer (NSCLC) at UWCCC , as discussed above. The radioiodinated 124I NM404 dosage will be based on the MIRD extrapolation of the dosimetric data with iodine-131 derived from this ongoing study. The dosage of 131I-NM404 is currently calculated as follows: the biodistribution data in animals is used. tizan to determine the percentage of injected dose / organ at variable time points. These data in animals are extrapolated with data in man by means of MIRD formalism (MIRDOSE PC v3.1) using standard conversion factors for differences in organic mass and anatomy between a rat and a standard man, providing dosages in predicted human organs; Based on these predicted dosages, the allowed mCi dosage that must be injected into humans is determined using the maximum legally permitted doses and by the RDRC standards for specific human tissue, as defined in the Federal Register (21 CFR, Part 361.1) for example, total body, blood, blood-forming tissues, eye lenses, gonads -3 rem / dosing; any other tissue -5 rem / dose. This procedure is previously used to carry both NM324 and NM404 for clinical use, resulting in a calculated dosage of 1 mCi. New data in animals obtained with NM404 (testicular dose 5 rad = 2.1 mCi and dose 5 rad for adrenals = 2.2 mCi) indicate the maximum starting dose of 131I-NM404 that will be around 1.9 mCi or twice that of NM324.

Study procedures: Before the infusion, an intravenous line will be established in the arm. The tracking dose < 0.3 μg / kg of body weight 12 I-NM404 (1 mCi, dosimetric results of a current study pending actual dosing) will be administered by infusion for 5 minutes. The preparation will be sterile, pyrogen-free, and will contain, < 5% iodine-free by thin layer chromatography (the usual result is <1%). The patient will be monitored clinically for adverse reactions during the infusion. Because animal toxicity studies (using more than 1,000 times the maximum planned amount of 12 I-NM404 in this study) and preliminary clinical safety studies with the unlabeled compound (at 10 times higher dosages) were without side effects , no toxicity was anticipated in the dosage for imaging. However, the possibility of adverse allergic reactions or other reactions is recognized. The vital signs of the patient (pulse, BP, temperature and respiratory rate) will be checked again immediately after the infusion and at 30 and 60 minutes after the injection. The vital signs will then be checked every hour for two hours. PET-CT images of initial values (total body and selected regional conjugate views) for the first five patients will be obtained at 48 and 96 hours after the injection. PET images will be obtained using a GE Clinical GE scanner Discovery LS .PET-CT, using adequate attenuation correction and optimized for iodine-124. The time points of 48 and 96 hours are based on preliminary data that relate to the times for optimal imaging for 131I-NM404 in lung cancer, however, the optimal tumor-directed time for 124I-NM404 in prostate cancer human will still be determined. The accumulation of tracer 124I-NM404 and washing will be recorded in the LS PET / CT scanner in a GE Advance PET scanner available at the clinic. A CT scan will be performed first for the patient's location and the attenuation correction in the LS PET / CT scanner; A transmission scan will be performed in the PET scanner. Each PET scan will be corrected for time of death, randomization, attenuation, and dispersion. The first examination will be performed immediately at 48 and 96 hours after the injection of 124I-NM404. Total body scans will be performed with an increased exploration time with respect to the tumor type (up to 30 minutes). The images will be reconstructed using an interactive OSEM reconstruction method with attenuation correction, and smoothed with a Gaussian filter. The data evaluation will be based on an analysis of the region of interest (ROI) of PET images co-registered. The CTs of each PET / CT scan will be co-registered to allow accurate positioning of the PET data. The ROIs were designated to represent various organs in the field of vision (typically heart, lung, muscle, liver, stomach, spleen, intestine, kidney, and gallbladder). For each ROI and for each time lapse, the average radioactivity concentration will be calculated and the calculated absorption values (SUVs) will be standardized. No detailed pharmacokinetic analyzes will be performed in this study, although they will be completed in the current lung cancer test and will not be duplicated here, because this will be done in the lung cancer test, where 2 patients will undergo PET exploration immediately. then, and subsequently at 6, 24, and 48 hours after the infusion of NM404. SUVs will be plotted as activity curves per time. The time course of radioactivity in each ROI will be adjusted using various symmetric models, including a double speed constant in a two-compartment model and a four-speed constant three speeds, in the three-compartment model along with the non-regression setting linear. The nonlinear least squares adjustment will be made using a modified expansion gradient algorithm. The best fit is will determine by minimizing a function X2 with respect to the variations in the model parameters. For the 3k model the data will be fitted with a set k4 equal to 0 in order to test the assumption of the irreversible entrapment of phospholipids in the tumor membrane. In addition, Patlak graphics will be used to standardize the kinetics of radioactivity in tissues with the kinetics of radioactivity in plasma.

Statistical considerations: This is a reliability study to determine the absorption and retention characteristics in 124I NM404 tumors radioiodinated by PET-CT in patients with prostatic cancer, clinically evident metastatic. Fifteen patients with prostate, metastatic cancer will accumulate for part 1 of this study. Imaging scans will be framed using a typical scale of 0-3: 0 = non-perceptible absorption, 1+ = barely perceptible absorption greater than the antecedent, 2+ = clearly distinguishable absorption superior to the antecedent, 3+ = intense absorption, much greater than that of the surrounding normal structures (2+ and 3+, are considered normal or positive for the identification of lesions, 0 and 1+ are considered normal or negative for the identification of lesions). An absorption rate classified as positive for the identification of lesions of at least 50%, could be considered as irrelevant clinical. Therefore, we will test the null hypothesis that the absorption rate classified as positive for the identification of lesions will be at most 50% against the alternative hypothesis that the speed is greater than 50%. It is anticipated that the absorption rate classified as positive for the identification of lesions will be at least 80%. Assuming a total sample size of 15 patients, the binomial test of a sample with the null hypothesis that the absorption rate classified as positive is at most 50%, has 85% power to detect a speed of 80% at the level of significance of 10% (unilateral). A speed of 90% will be detected with 95% power. With a sample size of 15 patients, the proportion of tumors classified as positive for the identification of lesions will have a standard error of at most 13% and the confidence interval of 90% for the proportion will not be greater than 39%.

Determines the specific accumulation in tumors and metabolic fate of 124I NM404 in patients with prostatic cancer clinically confined to organs who are candidates for radical prostatectomy with dissection of bilateral pelvic lymph nodes Objectives and reasons As well as a secondary exploratory analysis, malignant and benign prostatic and nodal tissue obtained by radical prostatectomy and dissection of bilateral lymph nodes will be evaluated for the accumulation of radioiodinated NM404 and NM404 metabolites and the data from this tissue analysis will be evaluated. will compare with the results of the final pathological analyzes. Ten patients with prostatic, organ-confined cancer who were scheduled to undergo radical prostatectomy with dissection of bilateral pelvic lymph nodes will be enrolled. All patients will undergo conventional pre-operative graduation studies that include CT scan and bone scan. All patients will also have a PET-CT scan of NM404 the week before the scheduled prostatectomy. The final pathological analyzes of the prostate and resected lymph nodes will correlate with the results of NM404 accumulation and tissue analysis of metabolites to determine the signal to tumor volume ratio in the primary specimen and lymph nodes in patients with the locoregional metastatic disease. Nodal metastases detected by accumulation of NM404 despite a negative pre-operative CT scan and bone scan could have a greater clinical significance in terms of improved grading of these patients before treatment.

Study Procedures: The procedures for image formation in this study will be identical to the first study, except that patients will be subjected to image formation only once, based on the optimal time of image formation as identified in the first protocol (probably between 24-48 hours after the injection). Six prostate biopsy nuclei will be obtained in sextant distribution (right and left appendix, middle gland and base regions) of the prostate specimen in the OR, immediately after surgical removal. These six nuclei of biopsies will be evaluated by pathology in frozen section to ensure a representative sampling of both malignant and benign prostatic tissue. A set of 6 biopsy cores obtained simultaneously from the same corresponding locations in the prostate will be analyzed for the uptake of 124I-NM404. Accordingly, they will be photographed and will undergo high-resolution scanning on a Bioscan AR2000 radioexplorer and will also be weighed and quantifies the radioactivity in a cavity counter. The concentration of radioactivity will be determined in a% dose / g of tissue sample with base for comparison. These results will be compared with the histological results to confirm the location of the tumor in relation to the surrounding uninvolved tissue. Core biopsies will be marked with a patient's study identification number and the location in the prostate from which it was obtained. The final pathology of any lymph nodes harboring the metastatic prostatic tumor will also correlate with the absorption signal of 124i-NM404 detected by the pre-operative PET-CT scan. Vital signs will be obtained after the infusion of 124-NM404.

Statistical considerations: Ten patients at high risk of clinically localized prostate cancer will accumulate for part 2 of the study. The null hypothesis for part 2 is that the logarithmic proportion of the accumulation. of NM404 in tumor tissues for those normal surrounding tissues in patients with clinically localized prostate cancer is zero. The alternative hypothesis is that the logarithmic proportion of NM404 accumulation is greater a 0. A ratio of normal tumor to adjacent tissue (T / N) of at least 5: 1 could be considered clinically relevant. Based on past experience with NM404, it is anticipated that the standard deviation of the logarithmic proportion of the accumulation of NM404 is between 1.5 and 2.0. With a sample size of 10, the one-sided T test with a level of significance from 10% unilaterally has 86%, 90% and 95% power to detect the size effect of 0.8, 0.90, and 1.00, respectively. For example, if the standard deviation of the logarithmic proportion is 2.0, the 5: 1 ratio in the accumulation of NM404 in the tumor for normal tissues will be detected with 86% power. Similarly, if the standard deviation of the logarithmic ratio is 1.6, a ratio of 5: 1 in the accumulation of NM404 in tumor to normal tissues will be detected at 95% power.

To determine if NM404, can detect the metastatic reappearance after a primary treatment of prostate cancer in ten patients with a PSA increase as their only evidence of disease (DO stage) Objectives and reasons By definition, patients with prostate cancer in the DO stage have an increase in PSA after a definitive treatment of his disease, a significant biochemical reappearance, with conventional graduation studies including a CT scan and bone scan that are negative for the radiographic evidence of the disease. The patients most likely to have a disease detectable by a PET-CT scan of NM404 will be enrolled in such a way that they should have an increase in PSA greater than 0.75 ng / ml / year.

Study procedures: The procedures for imaging in this study will be identical to the first study, except that patients will undergo imaging once, based on the optimal time for image formation, as identified in the first study. protocol. Patients will receive an injection of 124I-NM404 (0.3 μg / kg, body weight, 1 mCi or the limit established by dosimetric calculations in the first set of patients). Patients will also have a PET-CT scan, 48 hours after the infusion. Vital signs will be obtained after the infusion of 12 I-NM404.

Statistical considerations: Ten patients with prostate cancer in DO stage will accumulate and will continue for metastatic reappearance for at least 2 years. Because all patients accumulated in this part of the study, will have PSA values greater than 0.75 ng / ml / year, it is expected that at least 60% will have an experience of metastatic reappearance in a term of 2 years. Assuming that 6 of the 10 patients will eventually experience a metastatic reappearance within 2 years, the sensitivity of NM404 to detect metastatic reappearance will be estimated with a standard error of less than 20% and the confidence interval of 90% for sensitivity. It will not be greater than 55%. In addition, the null hypothesis that the sensitivity of NM404 in this patient population is at least 30% was again tested for the alternative that the sensitivity is greater than 30%. The binomial test of a single sample has 83% power to detect a sensitivity of 75% at the 10% unilateral level of significance. A sensitivity of 80% with 90% power will be detected. Approximately 230,110 new cases of prostate cancer were diagnosed in the United States in 2004 alone. Despite technical refinements in the definitive local treatment of prostatic cancer clinically confined to organs by radical prostatectomy, many men were cured with primary therapy. alone, as much as 40% of patients will experience biochemical reappearance with long-term follow-up. One of the greatest challenges in the treatment of patients with prostatic cancer confined clinically to organs or patients with biochemical reappearance after the definitive treatment of a presumed confined disease to organs, it remains to accurately distinguish a localized versus metastatic disease. This diagnostic capability is important to identify patients who can benefit from effective local treatment modalities including surgery, external beam radiation, brachytherapy, and cryotherapy. Because there are currently no accurate means of graduation, patients with metastatic disease unnecessarily hidden may undergo local treatment with risks associated with therapy. In addition, patients with an increasing PSA due to a local reappearance, in whom systemic reappearance can not be excluded with confidence, can unnecessarily undergo hormonal ablation, which in general is not considered curative and is associated with the development of osteoporosis, libido decreased, weight gain, menopausal symptoms, and malaise, as well as, the evolution of hormonally independent prostate cancer. While conventional studies for imaging such as, computerized tomography (CT) and magnetic resonance imaging (MRI) they are useful for assessing soft tissue metastases, the vast majority of prostate cancer metastases, only for bones. In this way, the usefulness of CT and MRI imaging to assess the disease is sub-optimal, and modalities for more sensitive imaging are needed either for locally recurrent or metastatic prostate cancer. Radioimmunoscintigraphy with Indium-111 capromab pendetide (ProstaScint, Cytogen Corp, Princeton, NJ) has been used in patients after prostatectomy with an increase in PSA who have a high clinical suspicion of a hidden metastatic disease and no clear evidence of a disease metastatic in other studies of image formation. The use of a ProstaScint scan for patients at risk for occult metastases from prostate cancer remains controversial, however. A method for the development of an examination for sensitive image formation is to develop a more suitable carrier molecule, which is key to achieving the delivery of a radiopharmaceutical assay solution for the desired target tissue. This strategy has been to study the radioiodinated phospholipid ether analogs (PLE) as diagnostic imaging agents, to capitalize on the unique biochemical or pharmacological properties of these molecules, resulting in a high degree of selectivity of tissue or tumor. In preclinical models, it has been shown that these molecules selectively accumulate in a wide variety of murine and human tumors at high levels. Certain embodiments of the present invention provide preliminary data relating to the use of the second generation PLE analog, NM404, in patients for prostate cancer imaging. It has been shown that NM404 (a) is selectively retained in a wide variety of tumor types in preclinical models, with a high degree of sensitivity, (b) it is safe in humans, (c) it can be radiolabelled with 1-124 , and (d) has adequate dosimetric characteristics marked 1-131.

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Claims (9)

1. NOVELTY OF THE INVENTION Having described the present invention, it is considered as a novelty and, therefore, the content of the following CLAIMS is claimed as property: 1. A method to detect and locate the reappearance of cancer, cancer insensitive to radiation and chemo or cancer metastasis 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 a subject having or suspected of having cancer, characterized in that the method comprises the steps of: administering a phospholipid ether analogue to the subject; and determining whether an organ with suspected recurrence of cancer, cancer insensitive to radiation or cancer metastasis in the subject maintains a higher level of the analogue than the surrounding regions where a region with higher retention indicates detection and location of the reappearance of cancer, cancer insensitive to radiation and chemo or cancer metastasis. 2. The method according to claim 1, characterized in that the phospholipid analogue is selected from: wherein X is selected from the group consisting of halogen radioactive isotopes; n is an integer between 8 and 30; and Y is selected from the group consisting of NH2, NR2, and NR3, wherein R is an alkyl or arylalkyl substituent or where 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, and Z is selected from the group consisting of NH2, NR2, and NR3, wherein R is an alkyl or arylalkyl substituent. 3. The method according to claim 2, characterized in that X is selected from the group of halogen radioactive isotopes consisting of 18F, 36C1, 76Br, 77Br, 82Br, 122I, 123I, 12 I, 125I, 131I, and 211At. The method according to claim 2, characterized in that the phospholipid ether is 18- (p-iodophenyl) octadecylphosphocholine, 1-0- [18- (p-iodophenyl) octadecyl] -1,3-propandiol-3-phosphocholine , or 1-0- [18- (p-iodophenyl) octadecyl] -2-0-methyl-rac-glycero-3-phosphocholine, where the iodine is in the form of a radioactive isotope. 5. A method for the treatment of the reappearance of cancer, cancer insensitive to radiation and chemo or cancer metastasis in a subject characterized in that it comprises: administering to the subject an effective amount of a compound comprising a phospholipid ether analogue. 6. The use of the phospholipid ether analogue for the production of a pharmaceutical composition for the treatment of the reappearance of cancer, cancer insensitive to radiation and chemo or cancer metastasis. 7. A labeled phospholipid ether analog comprising a radioactive 124I isotope, characterized in that the labeled ether phospholipid analogue produces more than 40 times the sensitivity of the flat 131I-gamma scintigraphy. 8. A labeled NM40 phospholipid ether analogue comprising a radioactive 124I isotope, characterized in that the labeled phospholipid ether analogue produces more than 40 times the sensitivity of the flat 131I-gamma scintigraphy. 9. A labeled phospholipid ether analog comprising a radioactive isotope of halogen, characterized in that the labeled phospholipid ether analogue produces more than 40 times the sensitivity of the flat 131I-gamma scintigraphy.